WorldWideScience

Sample records for polyhydroxyalkanoate pha-specific acetoacetyl

  1. Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Tippmann, Stefan; Ferreira, Raphael; Siewers, Verena

    2017-01-01

    to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible...... functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain...

  2. Bacterial polyhydroxyalkanoates: Still fabulous?

    Science.gov (United States)

    Możejko-Ciesielska, Justyna; Kiewisz, Robert

    2016-11-01

    Bacterial polyhydroxyalkanoates (PHA) are polyesters accumulated as carbon and energy storage materials under limited growth conditions in the presence of excess carbon sources. They have been developed as biomaterials with unique properties for the past many years being considered as a potential substitute for conventional non-degradable plastics. Due to the increasing concern towards global climate change, depleting petroleum resource and problems with an utilization of a growing number of synthetic plastics, PHAs have gained much more attention from industry and research. These environmentally friendly microbial polymers have great potential in biomedical, agricultural, and industrial applications. However, their production on a large scale is still limited. This paper describes the backgrounds of PHAs and discussed the current state of knowledge on the polyhydroxyalkanoates. Ability of bacteria to convert different carbon sources to PHAs, the opportunities and challenges of their introduction to global market as valuable renewable products have been also discussed. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. Current trends in biodegradable polyhydroxyalkanoates.

    Science.gov (United States)

    Chanprateep, Suchada

    2010-12-01

    The microbial polyesters known as polyhydroxyalkanoates (PHAs) positively impact global climate change scenarios by reducing the amount of non-degradable plastic used. A wide variety of different monomer compositions of PHAs has been described, as well as their future prospects for applications where high biodegradability or biocompatibility is required. PHAs can be produced from renewable raw materials and are degraded naturally by microorganisms that enable carbon dioxide and organic compound recycling in the ecosystem, providing a buffer to climate change. This review summarizes recent research on PHAs and addresses the opportunities as well as challenges for their place in the global market. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Polyhydroxyalkanoate (PHA) production from waste.

    Science.gov (United States)

    Rhu, D H; Lee, W H; Kim, J Y; Choi, E

    2003-01-01

    PHA (polyhydroxyalkanoate) production was attempted with SBRs from food waste. Seed microbes were collected from a sewage treatment plant with a biological nutrient removal process, and acclimated with synthetic substrate prior to the application of the fermented food waste. Laboratory SBRs were used to produce PHA with limited oxygen and nutrients. The maximum content of 51% PHA was obtained with an anaerobic/aerobic cycle with P limitation, and the yield was estimated to be about 0.05 gPHA(produced)/gCOD(applied) or 25 kg PHA/dry ton of food waste, assuming more than 40% of the PHA contents were recoverable. PHB/PHA ratios were 0.74 to 0.77 due to the higher acetate concentrations. Economical analysis seemed to suggest the PHA produced from the food waste could be an alternative material to produce the biodegradable plastic to be used for the collection bags for solid waste.

  5. Synthetic Biology of Polyhydroxyalkanoates (PHA).

    Science.gov (United States)

    Meng, De-Chuan; Chen, Guo-Qiang

    Microbial polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters which have been extensively studied using synthetic biology and metabolic engineering methods for improving production and for widening its diversity. Synthetic biology has allowed PHA to become composition controllable random copolymers, homopolymers, and block copolymers. Recent developments showed that it is possible to establish a microbial platform for producing not only random copolymers with controllable monomers and their ratios but also structurally defined homopolymers and block copolymers. This was achieved by engineering the genome of Pseudomonas putida or Pseudomonas entomophiles to weaken the β-oxidation and in situ fatty acid synthesis pathways, so that a fatty acid fed to the bacteria maintains its original chain length and structures when incorporated into the PHA chains. The engineered bacterium allows functional groups in a fatty acid to be introduced into PHA, forming functional PHA, which, upon grafting, generates endless PHA variety. Recombinant Escherichia coli also succeeded in producing efficiently poly(3-hydroxypropionate) or P3HP, the strongest member of PHA. Synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate and 3-hydroxypropionate were assembled respectively to allow their synthesis from glucose. CRISPRi was also successfully used to manipulate simultaneously multiple genes and control metabolic flux in E. coli to obtain a series of copolymer P3HB4HB of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB). The bacterial shapes were successfully engineered for enhanced PHA accumulation.

  6. Advances in cyanobacterial polyhydroxyalkanoates production.

    Science.gov (United States)

    Singh, Akhilesh Kumar; Mallick, Nirupama

    2017-11-01

    Polyhydroxyalkanoates (PHAs) have received much attention in the current scenario due to their attractive material properties, namely biodegradability, biocompatibility, thermoplasticity, hydrophobicity, piezoelectricity and stereospecificity. All these properties make them highly competitive for various industrial applications similar to non-degradable conventional plastics. In PHA biosynthesis, PHA synthase acts as a natural catalyst for PHA polymerization process using the (R)-hydroxyacyl-CoA as substrate. Cyanobacteria can accumulate PHAs under photoautotrophic and/or mixotrophic growth conditions with organic substrates such as acetate, glucose, propionate, valerate, and so on. The natural incidence of PHA accumulation by the cyanobacteria is known since 1966. Nevertheless, PHA accumulation in cyanobacteria based on the cell biomass and volumetric productivity is critically lower than the heterotrophic bacteria. Consequently, cyanobacteria are nowadays not considered for commercial production of PHAs. Thus, strain improvements by genetic modification, new cultivation and harvesting techniques, advanced photobioreactor development, efficient and sustainable downstream processes, alternate economical carbon sources and usage of various metabolic inhibitors are suggested for enhancing cyanobacterial PHA accumulation. In addition, identification of transcriptional regulators like RNA polymerase sigma factor (SigE) and a response regulator (Rre37) together with the recent major scientific breakthrough on the existence of complete Krebs cycle in cyanobacteria would be helpful in taking PHA production from cyanobacteria to a new-fangled height in near future. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Enhanced poly(3-hydroxybutyrate) production in transgenic tobacco BY-2 cells using engineered acetoacetyl-CoA reductase.

    Science.gov (United States)

    Yokoo, Toshinori; Matsumoto, Ken'ichiro; Ooba, Takashi; Morimoto, Kenjiro; Taguchi, Seiichi

    2015-01-01

    Highly active mutant of NADPH-dependent acetoacetyl-CoA reductase (PhaB) was expressed in Nicotiana tabacum cv. Bright Yellow-2 cultured cells to produce poly(3-hydroxybutyrate) [P(3HB)]. The mutated PhaB increased P(3HB) content by three-fold over the control, indicating that the mutant was a versatile tool for P(3HB) production. Additionally, the PhaB-catalyzed reaction was suggested to be a rate-limiting step of P(3HB) biosynthesis in tobacco BY-2 cells.

  8. Production of polyhydroxyalkanoates (PHAs) in transgenic potato

    NARCIS (Netherlands)

    Romano, A.

    2002-01-01

    Polyhydroxyalkanoates (PHAs) represent a large class of microbial polyesters which are widely distributed in prokaryotes. Because of the current environmental concerns related to the use of mineral-oil-based plastics, PHAs gained a considerable interest for

  9. Mechanical Properties of Polyhydroxyalkanoate Bioceramic Nanocomposites

    Institute of Scientific and Technical Information of China (English)

    江涛; 胡平; 柳林; 李亚栋

    2002-01-01

    New composites prepared containing nanoscale hydroxyapatite or monetite uniformly distributed in a polyhydroxyalkanoate (polyhydroxybutyrate or polyhydroxybutyrate-hydroxyvalerate) matrix with mass fractions of 1%5% were then injected into dumb-like specimens. The results show that the tensile strength and the tensile modulus of the composites are improved. The microstructures of the composites were observed using transmission electron microscopy (TEM).

  10. [Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria].

    Science.gov (United States)

    Boiandin, A N; Kalacheva, G S; Rodicheva, E K; Volova, T G

    2008-01-01

    The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDSM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40-70% of the cell dry mass weight) were determined. The capacity of synthesizing two- and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.

  11. Synthetic biology of microbes synthesizing polyhydroxyalkanoates (PHA

    Directory of Open Access Journals (Sweden)

    Guo-Qiang Chen

    2016-12-01

    Full Text Available Microbial polyhydroxyalkanoates (PHA have been produced as bioplastics for various purposes. Under the support of China National Basic Research 973 Project, we developed synthetic biology methods to diversify the PHA structures into homo-, random, block polymers with improved properties to better meet various application requirements. At the same time, various pathways were assembled to produce various PHA from glucose as a simple carbon source. At the end, Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions. The synthetic biology will advance the PHA into a bio- and material industry.

  12. Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production

    DEFF Research Database (Denmark)

    Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos

    2017-01-01

    Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility...

  13. Isolation and recovery of microbial polyhydroxyalkanoates

    Directory of Open Access Journals (Sweden)

    2011-07-01

    Full Text Available The deleterious environmental impacts caused by plastic wastes have attracted worldwide concern. The biobased and biodegradable polyhydroxyalkanoate (PHA appears to be one of the potential candidates to replace some conventional plastics. However, high production cost of PHAs has limited their market penetration. The major cost absorbing factors are the upstream fermentation processes and the downstream PHA recovery technologies. The latter significantly affects the overall process economics. Various recovery technologies have been proposed and studied in small scales in the laboratory as well as in industrial scales. These include solvent extraction, chemical digestion, enzymatic treatment and mechanical disruption, supercritical fluid disruption, flotation techniques, use of gamma irradiation and aqueous two-phase system. This paper reviews all the recovery methods known to date and compares their efficiency and the quality of the resulting PHA. Some of the large-scale production of PHA and the strategies employed to reduce the production cost are also discussed.

  14. Bacillus subtilis as potential producer for polyhydroxyalkanoates

    Directory of Open Access Journals (Sweden)

    Patel Sanjay KS

    2009-07-01

    Full Text Available Abstract Polyhydroxyalkanoates (PHAs are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB, the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.

  15. Bacillus subtilis as potential producer for polyhydroxyalkanoates.

    Science.gov (United States)

    Singh, Mamtesh; Patel, Sanjay Ks; Kalia, Vipin C

    2009-07-20

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process - for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.

  16. Biodegradability of Poly(hydroxyalkanoate Materials

    Directory of Open Access Journals (Sweden)

    Keiji Numata

    2009-08-01

    Full Text Available Poly(hydroxyalkanoate (PHA, which is produced from renewable carbon resources by many microorganisms, is an environmentally compatible polymeric material and can be processed into films and fibers. Biodegradation of PHA material occurs due to the action of extracellular PHA depolymerase secreted from microorganisms in various natural environments. A key step in determining the overall enzymatic or environmental degradation rate of PHA material is the degradation of PHA lamellar crystals in materials; hence, the degradation mechanism of PHA lamellar crystals has been studied in detail over the last two decades. In this review, the relationship between crystal structure and enzymatic degradation behavior, in particular degradation rates, of films and fibers for PHA is described.

  17. Polyhydroxyalkanoates: bioplastics with a green agenda.

    Science.gov (United States)

    Keshavarz, Tajalli; Roy, Ipsita

    2010-06-01

    Production of polyhydroxyalkanoates (PHAs) has been investigated for more than eighty years but recently a number of factors including increase in the price of crude oil and public awareness of the environmental issues have become a notable driving force for extended research on biopolymers. The versatility of PHAs has made them good candidates for the study of their potential in a variety of areas from biomedical/medical fields to food, packaging, textile and household material. While production costs are still a drawback to wider usage of these biopolymers, their application as low volume high cost items is becoming a reality. The future trend is to focus on the development of more efficient and economical processes for PHA production, isolation, purification and improvement of PHA material properties. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Studium biodegradace polyhydroxyalkanoátů.

    OpenAIRE

    Wurstová, Agáta

    2014-01-01

    Diplomová práce je zaměřena na studium biodegradace polyhydroxyalkanoátů, konkrétně polymeru polyhydroxybutyrátu. První část práce se zabývá studiem biodegradace polyhydroxybutyrátu ve formě PHB prášku a PHB fólie pomocí vybraných druhů mikroorganismů z řad bakterií, kvasinek a plísní. Jako zástupce bakterií byl vybrán mikroorganismus Delftia acidovorans, z řad kvasinek Aureobasidium pullulans a ze zástupců plísní Aspergillus fumigatus. Aktivita extracelulární PHB depolymerázy byla analyzován...

  19. Clonorchis sinensis acetoacetyl-CoA thiolase: identification and characterization of its potential role in surviving in the bile duct.

    Science.gov (United States)

    Lin, Jinsi; Qu, Hongling; Chen, Guishan; He, Lei; Xu, Yanquan; Xie, Zhizhi; Ren, Mengyu; Sun, Jiufeng; Li, Shan; Chen, Wenjun; Chen, Xueqing; Wang, Xiaoyun; Li, Xuerong; Liang, Chi; Huang, Yan; Yu, Xinbing

    2015-02-25

    Clonorchis sinensis (C. sinensis) inhabits in bile duct of the host. However, the mechanisms involved in why C. sinensis can survive in the bile environment containing lipids have not yet been explored. In this study, C. sinensis acetoacetyl-CoA thiolase (CsACAT), a member of the thiolase family which has a key role in the beta oxidation pathway of fatty acid production, was identified and characterized to understand its potential role in adapting to the bile environment. The encoding sequence, conserved domains and spatial structure of CsACAT were identified and analyzed by bioinformatic tools. Recombinant CsACAT (rCsACAT) was obtained using a procaryotic expression system. The expression pattern of CsACAT was confirmed by quantitative real-time PCR, western blotting, and immunofluorescence. Gradients of lecithin were then set to culture C. sinensis adults in vitro and the survival rate of C. sinensis was analyzed, as well as the expression level and enzymatic activity of CsACAT in different lipid environments. Hypercholesteremia rabbit models were established by feeding with a hyperlipidemic diet and then infected intragastrically with C. sinensis. One and a half months later, the worm burdens and the expression level of CsACAT was detected. CsACAT was confirmed to be a member of the thiolase family and present in the excretory/secretory proteins of C. sinensis. CsACAT was specifically localized at the vitellarium and sub-tegumental muscle layer in adult worms. The mRNA level of CsACAT in eggs was higher than those in adult worms and metacercariae. When adult worms were cultured with higher concentration of lecithin, the expression level and enzyme activity of CsACAT were up-regulated. The survival rate of adult worms was higher than control group. More adult worms were recovered from hypercholesteremia rabbit models. The expression level of CsACAT in these worms was higher than control group. Our results implied that C. sinensis might sense lipid levels and

  20. Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production.

    Science.gov (United States)

    Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos; Burniol-Figols, Anna; Varrone, Cristiano; Gavala, Hariklia N; Reis, Maria A M

    2017-06-11

    Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing.

  1. Bacterial production of the biodegradable plastics polyhydroxyalkanoates.

    Science.gov (United States)

    Urtuvia, Viviana; Villegas, Pamela; González, Myriam; Seeger, Michael

    2014-09-01

    Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life cycle assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Engineering bacteria for enhanced polyhydroxyalkanoates (PHA biosynthesis

    Directory of Open Access Journals (Sweden)

    Guo-Qiang Chen

    2017-09-01

    Full Text Available Polyhydroxyalkanoates (PHA have been produced by some bacteria as bioplastics for many years. Yet their commercialization is still on the way. A few issues are related to the difficulty of PHA commercialization: namely, high cost and instabilities on molecular weights (Mw and structures, thus instability on thermo-mechanical properties. The high cost is the result of complicated bioprocessing associated with sterilization, low conversion of carbon substrates to PHA products, and slow growth of microorganisms as well as difficulty of downstream separation. Future engineering on PHA producing microorganisms should be focused on contamination resistant bacteria especially extremophiles, developments of engineering approaches for the extremophiles, increase on carbon substrates to PHA conversion and controlling Mw of PHA. The concept proof studies could still be conducted on E. coli or Pseudomonas spp. that are easily used for molecular manipulations. In this review, we will use E. coli and halophiles as examples to show how to engineer bacteria for enhanced PHA biosynthesis and for increasing PHA competitiveness.

  3. Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

    Science.gov (United States)

    Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

    2016-01-01

    Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

  4. Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate (PHA) production

    NARCIS (Netherlands)

    Jiang, Y.; Hebly, M.; Kleerebezem, R.; Muyzer, G.; van Loosdrecht, M.C.M.

    2011-01-01

    Polyhydroxyalkanoate (PHA) production by mixed microbial communities can be established in a two-stage process, consisting of a microbial enrichment step and a PHA accumulation step. In this study, a mathematical model was constructed for evaluating the influence of the carbon substrate composition

  5. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    Science.gov (United States)

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

  6. Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan.

    Science.gov (United States)

    Liu, Xinyu; Walsh, Christopher T

    2009-09-15

    The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily. By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.

  7. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

    OpenAIRE

    Anderson, A J; Dawes, E A

    1990-01-01

    Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence. They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell. The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago. Copolymers of C4 (3-hydroxybutyrate [3HB]) and C5 (3-hydroxyvalerate [3HV]) monomer unit...

  8. Activation of liver carnitine palmitoyltransferase-1 and mitochondrial acetoacetyl-CoA thiolase is associated with elevated ketone body levels in the elasmobranch Squalus acanthias.

    Science.gov (United States)

    Treberg, Jason R; Crockett, Elizabeth L; Driedzic, William R

    2006-01-01

    Elasmobranch fishes are an ancient group of vertebrates that have unusual lipid metabolism whereby storage lipids are mobilized from the liver for peripheral oxidation largely as ketone bodies rather than as nonesterified fatty acids under normal conditions. This reliance on ketones, even when feeding, implies that elasmobranchs are chronically ketogenic. Compared to specimens sampled within 2 d of capture (recently captured), spiny dogfish Squalus acanthias that were held for 16-33 d without apparent feeding displayed a 4.5-fold increase in plasma concentration of d- beta -hydroxybutyrate (from 0.71 to 3.2 mM) and were considered ketotic. Overt activity of carnitine palmitoyltransferase-1 in liver mitochondria from ketotic dogfish was characterized by an increased apparent maximal activity, a trend of increasing affinity (reduced apparent K(m); P=0.09) for l-carnitine, and desensitization to the inhibitor malonyl-CoA relative to recently captured animals. Acetoacetyl-CoA thiolase (ACoAT) activity in isolated liver mitochondria was also markedly increased in the ketotic dogfish compared to recently captured fish, whereas no difference in 3-hydroxy-3-methylglutaryl-CoA synthase activity was found between these groups, suggesting that ACoAT plays a more important role in the activation of ketogenesis in spiny dogfish than in mammals and birds.

  9. Biosynthesis and composition of bacterial poly(hydroxyalkanoates).

    Science.gov (United States)

    Anderson, A J; Haywood, G W; Dawes, E A

    1990-04-01

    It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA. Various other bacteria produce similar copolymers from propionic acid and/or valeric acid. A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols.

  10. Advances in the Applications of Polyhydroxyalkanoate Nanoparticles for Novel Drug Delivery System

    Directory of Open Access Journals (Sweden)

    Anupama Shrivastav

    2013-01-01

    Full Text Available Drug delivery technology is emerging as an interdisciplinary science aimed at improving human health. The controlled delivery of pharmacologically active agents to the specific site of action at the therapeutically optimal rate and dose regimen has been a major goal in designing drug delivery systems. Over the past few decades, there has been considerable interest in developing biodegradable drug carriers as effective drug delivery systems. Polymeric materials from natural sources play an important role in controlled release of drug at a particular site. Polyhydroxyalkanoates, due to their origin from natural sources, are given attention as candidates for drug delivery materials. Biodegradable and biocompatible polyhydroxyalkanoates are linear polyesters produced by microorganisms under unbalanced growth conditions, which have emerged as potential polymers for use as biomedical materials for drug delivery due to their unique physiochemical and mechanical properties. This review summarizes many of the key findings in the applications of polyhydroxyalkanoates and polyhydroxyalkanoate nanoparticles for drug delivery system.

  11. Process optimization for polyhydroxyalkanoate (PHA) production from waste via microbial enrichment cultures

    NARCIS (Netherlands)

    Korkakaki, E.

    2017-01-01

    Polyhydroxyalkanoates (PHA) are compounds naturally produced by microorganisms, with many industrial applications, either as bioplastics or as precursors for production of chemicals. Until now, industrial PHA production was conducted with pure strains of bacteria fed with well-defined feedstocks,

  12. Potential of Diverse Prokaryotic Organisms for Glycerol-based Polyhydroxyalkanoate Production

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2015-06-01

    Full Text Available The potential and performance of various Gram-negative, Gram-positive and archaeal wild type microorganisms, and bacterial mixed cultures, as well as the application of genetically engineered strains as whole-cell biocatalysts for glycerol-based polyhydroxyalkanoate production are analyzed and assessed. This encompasses the comparison of growth and polyhydroxyalkanoate accumulation kinetics, thermo-mechanical properties of isolated glycerol-based polyhydroxyalkanoate of different composition on the monomeric level, and the presentation of mathematical models developed to describe glycerol-based polyhydroxyalkanoate production processes. For all these aspects, the article provides a detailed compilation of the contemporary state of knowledge, and gives an outlook to expected future developments.

  13. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    Science.gov (United States)

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J.

    2012-03-31

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT‐encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T‐DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol‐localized, mevalonate‐derived isoprenoid biosynthetic pathway.

  15. Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135

    OpenAIRE

    Haywood, Geoffrey W.; Anderson, Alistair J.; Ewing, David F.; Dawes, Edwin A.

    1990-01-01

    A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C2 to C6); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer sy...

  16. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry.

    Science.gov (United States)

    Chen, Guo-Qiang

    2009-08-01

    Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).

  17. Polyhydroxyalkanoates: A way to sustainable development of bioplastics

    Directory of Open Access Journals (Sweden)

    Roopesh Jain

    2010-01-01

    Full Text Available This paper reviews role of polyhydroxyalkanoates (PHAs for sustainable development of bioplastics. PHAs are polyesters of hydroxyalkanoates with the general structural and have been investigated by biologists, chemists, engineers and other experts over the past many years. Bio-based and biodegradable plastics can form the basis for environmentally referable and sustainable alternative to current materials based exclusively on petroleum feed stocks. The result of the efforts made by scientist sought solution in form of biopolymer obtained either from microorganism or plants source and likely to replace currently used synthetic polymers as bioplastic. Applications of PHA as bioplastics, fine chemicals, implant biomaterials, and medicines have been developed. The PHA polymers promise to extend significantly the range of biomaterials as suitable alternative of plastic.

  18. Recovery of polyhydroxyalkanoates from municipal secondary wastewater sludge.

    Science.gov (United States)

    Kumar, Manish; Ghosh, Pooja; Khosla, Khushboo; Thakur, Indu Shekhar

    2018-05-01

    In the current study, the feasibility of utilizing municipal secondary wastewater sludge for Polyhydroxyalkanoate (PHA) extraction was improved by optimization of various parameters (temperature, duration and concentration of sludge solids). Optimized process parameters resulted in PHA recovery of 0.605 g, significantly higher than un-optimized conditions. The characterization of PHA was carried out by GC-MS, FT-IR and NMR ( 1 H and 13 C) spectroscopy. The PHA profile was found to be dominated by mcl PHA (58%) along with other diverse PHA. The results of the present study show rich diversity of PHA extracted from a raw material which is readily available at minimal cost. In conclusion, exploring the potential of wastes for production of bioplastics not only reduces the cost of bioplastic production, but also provides a sustainable means for waste management. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Improved detergent-based recovery of polyhydroxyalkanoates (PHAs).

    Science.gov (United States)

    Yang, Yung-Hun; Brigham, Christopher; Willis, Laura; Rha, ChoKyun; Sinskey, Anthony

    2011-05-01

    Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process. © Springer Science+Business Media B.V. 2011

  20. Low frequency sonic waves assisted cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator.

    Science.gov (United States)

    Murugesan, Sivananth; Iyyaswami, Regupathi

    2017-08-15

    Low frequency sonic waves, less than 10kHz were introduced to assist cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator present within the crude broth. Process parameters including surfactant system variables and sonication parameters were studied for their effect on extraction efficiency. Introduction of low frequency sonic waves assists in the dissolution of microbial cell wall by the surfactant micelles and release of cellular content, polyhydroxyalkanoate granules released were encapsulated by the micelle core which was confirmed by crotonic acid assay. In addition, sonic waves resulted in the separation of homogeneous surfactant and broth mixture into two distinct phases, top aqueous phase and polyhydroxyalkanoate enriched bottom surfactant rich phase. Mixed surfactant systems showed higher extraction efficiency compared to that of individual Triton X-100 concentrations, owing to increase in the hydrophobicity of the micellar core and its interaction with polyhydroxyalkanoate. Addition of salts to the mixed surfactant system induces screening of charged surfactant head groups and reduces inter-micellar repulsion, presence of ammonium ions lead to electrostatic repulsion and weaker cation sodium enhances the formation of micellar network. Addition of polyethylene glycol 8000 resulted in increasing interaction with the surfactant tails of the micelle core there by reducing the purity of polyhydroxyalkanoate. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Emerging bone tissue engineering via Polyhydroxyalkanoate (PHA)-based scaffolds.

    Science.gov (United States)

    Lim, Janice; You, Mingliang; Li, Jian; Li, Zibiao

    2017-10-01

    Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers derived from microorganisms. On top of their biodegradability and biocompatibility, different PHA types can contribute to varying mechanical and chemical properties. This has led to increasing attention to the use of PHAs in numerous biomedical applications over the past few decades. Bone tissue engineering refers to the regeneration of new bone through providing mechanical support while inducing cell growth on the PHA scaffolds having a porous structure for tissue regeneration. This review first introduces the various properties PHA scaffold that make them suitable for bone tissue engineering such as biocompatibility, biodegradability, mechanical properties as well as vascularization. The typical fabrication techniques of PHA scaffolds including electrospinning, salt-leaching and solution casting are further discussed, followed by the relatively new technology of using 3D printing in PHA scaffold fabrication. Finally, the recent progress of using different types of PHAs scaffold in bone tissue engineering applications are summarized in intrinsic PHA/blends forms or as composites with other polymeric or inorganic hybrid materials. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate.

    Science.gov (United States)

    Guzik, Maciej W; Kenny, Shane T; Duane, Gearoid F; Casey, Eoin; Woods, Trevor; Babu, Ramesh P; Nikodinovic-Runic, Jasmina; Murray, Michael; O'Connor, Kevin E

    2014-05-01

    A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.

  3. Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates.

    Science.gov (United States)

    Yu, P H; Chua, H; Huang, A L; Ho, K P

    1999-01-01

    Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, respectively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.

  4. Potential and Prospects of Continuous Polyhydroxyalkanoate (PHA Production

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2015-05-01

    Full Text Available Together with other so-called “bio-plastics”, Polyhydroxyalkanoates (PHAs are expected to soon replace established polymers on the plastic market. As a prerequisite, optimized process design is needed to make PHAs attractive in terms of costs and quality. Nowadays, large-scale PHA production relies on discontinuous fed-batch cultivation in huge bioreactors. Such processes presuppose numerous shortcomings such as nonproductive time for reactor revamping, irregular product quality, limited possibility for supply of certain carbon substrates, and, most of all, insufficient productivity. Therefore, single- and multistage continuous PHA biosynthesis is increasingly investigated for production of different types of microbial PHAs; this goes for rather crystalline, thermoplastic PHA homopolyesters as well as for highly flexible PHA copolyesters, and even blocky-structured PHAs consisting of alternating soft and hard segments. Apart from enhanced productivity and constant product quality, chemostat processes can be used to elucidate kinetics of cell growth and PHA formation under constant process conditions. Furthermore, continuous enrichment processes constitute a tool to isolate novel powerful PHA-producing microbial strains adapted to special environmental conditions. The article discusses challenges, potential and case studies for continuous PHA production, and shows up new strategies to further enhance such processes economically by developing unsterile open continuous processes combined with the application of inexpensive carbon feedstocks.

  5. Synthesis of polyhydroxyalkanoate from palm oil and some new applications.

    Science.gov (United States)

    Sudesh, Kumar; Bhubalan, Kesaven; Chuah, Jo-Ann; Kek, Yik-Kang; Kamilah, Hanisah; Sridewi, Nanthini; Lee, Yan-Fen

    2011-03-01

    Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world's second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.

  6. Carbon Sources for Polyhydroxyalkanoates and an Integrated Biorefinery

    Directory of Open Access Journals (Sweden)

    Guozhan Jiang

    2016-07-01

    Full Text Available Polyhydroxyalkanoates (PHAs are a group of bioplastics that have a wide range of applications. Extensive progress has been made in our understanding of PHAs’ biosynthesis, and currently, it is possible to engineer bacterial strains to produce PHAs with desired properties. The substrates for the fermentative production of PHAs are primarily derived from food-based carbon sources, raising concerns over the sustainability of their production in terms of their impact on food prices. This paper gives an overview of the current carbon sources used for PHA production and the methods used to transform these sources into fermentable forms. This allows us to identify the opportunities and restraints linked to future sustainable PHA production. Hemicellulose hydrolysates and crude glycerol are identified as two promising carbon sources for a sustainable production of PHAs. Hemicellulose hydrolysates and crude glycerol can be produced on a large scale during various second generation biofuels’ production. An integration of PHA production within a modern biorefinery is therefore proposed to produce biofuels and bioplastics simultaneously. This will create the potential to offset the production cost of biofuels and reduce the overall production cost of PHAs.

  7. Polyhydroxyalkanoate biosynthesis by oxalotrophic bacteria from high Andean soil

    Directory of Open Access Journals (Sweden)

    Roger David Castillo-Arteaga

    2018-02-01

    Full Text Available Oxalate is a highly oxidized organic acid anion used as a carbon and energy source by oxalotrophic bacteria. Oxalogenic plants convert atmospheric CO2 into oxalic acid and oxalic salts. Oxalate-salt formation acts as a carbon sink in terrestrial ecosystems via the oxalate-carbonate pathway (OCP. Oxalotrophic bacteria might be implicated in other carbon-storage processes, including the synthesis of polyhydroxyalkanoates (PHAs. More recently, a variety of bacteria from the Andean region of Colombia in Nariño have been reported for their PHA-producing abilities. These species can degrade oxalate and participate in the oxalate-carbonate pathway. The aim of this study was to isolate and characterize oxalotrophic bacteria with the capacity to accumulate PHA biopolymers. Plants of the genus Oxalis were collected and bacteria were isolated from the soil adhering to the roots. The isolated bacterial strains were characterized using biochemical and molecular biological methods. The consumption of oxalate in culture was quantified, and PHA production was monitored in batch fermentation. The polymeric composition was characterized using gas chromatography. Finally, a biosynthetic pathway based on our findings and on those from published sources is proposed. Strains of Bacillus spp. and Serratia sp. were found to metabolize calcium oxalate and synthesize PHA.

  8. Start a Research on Biopolymer Polyhydroxyalkanoate (PHA: A Review

    Directory of Open Access Journals (Sweden)

    Giin-Yu Amy Tan

    2014-03-01

    Full Text Available With the impending fossil fuel crisis, the search for and development of alternative chemical/material substitutes is pivotal in reducing mankind’s dependency on fossil resources. One of the potential substitute candidates is polyhydroxyalkanoate (PHA. PHA is a carbon-neutral and valuable polymer that could be produced from many renewable carbon sources by microorganisms, making it a sustainable and environmental-friendly material. At present, PHA is not cost competitive compared to fossil-derived products. Encouraging and intensifying research work on PHA is anticipated to enhance its economic viability in the future. The development of various biomolecular and chemical techniques for PHA analysis has led to the identification of many PHA-producing microbial strains, some of which are deposited in culture collections. Research work on PHA could be rapidly initiated with these ready-to-use techniques and microbial strains. This review aims to facilitate the start-up of PHA research by providing a summary of commercially available PHA-accumulating microbial cultures, PHA biosynthetic pathways, and methods for PHA detection, extraction and analysis.

  9. [Preparation and characterization of polyhydroxyalkanoate bioplastics with antibacterial activity].

    Science.gov (United States)

    Lou, Qiuli; Ma, Yiming; Che, Xuemei; Zhong, Jin; Sun, Xiaoxia; Zhang, Haoqian

    2016-08-25

    Polyhydroxyalkanoates (PHAs), as a novel class of biopolymer, are attracting more attention due to their diverse material properties and environment-independent biodegradability. Here we report the preparation of PHA exhibiting efficient antibacterial activity by embedding Nisin, a food additive generally recognized as safe, into poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a type of PHA with high biocompatibility. We first prepared Nisin-containing PHBHHx films using solvent casting method. Confocal laser scanning microscopy analysis showed that a well-mixed integrated structure of the films with an even distribution of the Nisin particles in the PHBHHx matrices. Then the antimicrobial activity of PHBHHx/Nisin films against Micrococcus luteus was quantified on agar plate by measuring the size of inhibition zone. Cultivation in liquid media further confirmed the releasing of Nisin from the films and the long-time antibacterial activity. Results showed that the threshold of Nisin concentration for long-time and effective inhibition against bacteria growth is 25 μg/g. These results altogether establish a technological foundation for the application of PHA in biomedicine and food industry.

  10. Carbon Sources for Polyhydroxyalkanoates and an Integrated Biorefinery.

    Science.gov (United States)

    Jiang, Guozhan; Hill, David J; Kowalczuk, Marek; Johnston, Brian; Adamus, Grazyna; Irorere, Victor; Radecka, Iza

    2016-07-19

    Polyhydroxyalkanoates (PHAs) are a group of bioplastics that have a wide range of applications. Extensive progress has been made in our understanding of PHAs' biosynthesis, and currently, it is possible to engineer bacterial strains to produce PHAs with desired properties. The substrates for the fermentative production of PHAs are primarily derived from food-based carbon sources, raising concerns over the sustainability of their production in terms of their impact on food prices. This paper gives an overview of the current carbon sources used for PHA production and the methods used to transform these sources into fermentable forms. This allows us to identify the opportunities and restraints linked to future sustainable PHA production. Hemicellulose hydrolysates and crude glycerol are identified as two promising carbon sources for a sustainable production of PHAs. Hemicellulose hydrolysates and crude glycerol can be produced on a large scale during various second generation biofuels' production. An integration of PHA production within a modern biorefinery is therefore proposed to produce biofuels and bioplastics simultaneously. This will create the potential to offset the production cost of biofuels and reduce the overall production cost of PHAs.

  11. Characterisation of polyhydroxyalkanoate copolymers with controllable four-monomer composition.

    Science.gov (United States)

    Dai, Yu; Lambert, Lynette; Yuan, Zhiguo; Keller, Jurg

    2008-03-20

    Polyhydroxyalkanoate (PHA) copolymers comprising the four monomers 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-2-methylbutyrate (3HMB) were generated using the recently discovered Defluviicoccus vanus-related glycogen accumulating organisms (DvGAOs) under anaerobic conditions without applying any nutrient limitations. The composition could be manipulated in a defined range by modifying the ratio of propionate and acetate provided in the feed stream. The PHAs produced were characterised as random copolymers (from propionate alone) or a mixture of random copolymers (from mixture of propionate and acetate) through microstructure analysis using 13C NMR spectroscopy. The sequence distribution of all eight comonomer pairs in the carbonyl region of 3HB and 3HV was identified and assigned with confidence utilising two-dimensional heteronuclear multiple bond coherence (HMBC) spectroscopy. Weight average molecular weights were in the range 390-560 kg/mol. Differential scanning calorimetry (DSC) traces showed that the melting temperature (Tm) varied between 70 and 161 degrees C and glass transition temperature (Tg) ranged from -8 to 0 degrees C. The incorporation of considerable amounts of 3HMV and 3HMB monomer units introduced additional "defects" into the PHBV copolymer structure and hence greatly lowered the crystallinity. The data indicate the potential of these four-monomer PHAs to be employed for practical applications, considering their favourable properties and the cost-effective production process using a mixed culture and simple carbon sources.

  12. Renewable alkenes from the hydrothermal treatment of polyhydroxyalkanoates-containing sludge

    NARCIS (Netherlands)

    Torri, Cristian; Detert Oude Weme, Tom; Samorì, Chiara; Kiwan, Alisar; Brilman, Derk W.F.

    2017-01-01

    Polyhydroxyalkanoates (PHA) are a key constituent of excess sludge produced by Aerobic Sewage Sludge Treatment plants. The accumulation of significant amount of PHA inside aerobic microbial cells occurs when a surplus of an easily degradable carbon source (e.g., volatile fatty acids, VFA) is found

  13. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates.

    Science.gov (United States)

    Anderson, A J; Dawes, E A

    1990-12-01

    Polyhydroxyalkanoates (PHAs), of which polyhydroxybutyrate (PHB) is the most abundant, are bacterial carbon and energy reserve materials of widespread occurrence. They are composed of 3-hydroxyacid monomer units and exist as a small number of cytoplasmic granules per cell. The properties of the C4 homopolymer PHB as a biodegradable thermoplastic first attracted industrial attention more than 20 years ago. Copolymers of C4 (3-hydroxybutyrate [3HB]) and C5 (3-hydroxyvalerate [3HV]) monomer units have modified physical properties; e.g., the plastic is less brittle than PHB, whereas PHAs containing C8 to C12 monomers behave as elastomers. This family of materials is the centre of considerable commercial interest, and 3HB-co-3HV copolymers have been marketed by ICI plc as Biopol. The known polymers exist as 2(1) helices with the fiber repeat decreasing from 0.596 nm for PHB to about 0.45 nm for C8 to C10 polymers. Novel copolymers with a backbone of 3HB and 4HB have been obtained. The native granules contain noncrystalline polymer, and water may possibly act as a plasticizer. Although the biosynthesis and regulation of PHB are generally well understood, the corresponding information for the synthesis of long-side-chain PHAs from alkanes, alcohols, and organic acids is still incomplete. The precise mechanisms of action of the polymerizing and depolymerizing enzymes also remain to be established. The structural genes for the three key enzymes of PHB synthesis from acetyl coenzyme A in Alcaligenes eutrophus have been cloned, sequenced, and expressed in Escherichia coli. Polymer molecular weights appear to be species specific. The factors influencing the commercial choice of organism, substrate, and isolation process are discussed. The physiological functions of PHB as a reserve material and in symbiotic nitrogen fixation and its presence in bacterial plasma membranes and putative role in transformability and calcium signaling are also considered.

  14. Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida.

    Science.gov (United States)

    Li, Shi Yan; Dong, Cui Ling; Wang, Shen Yu; Ye, Hai Mu; Chen, Guo-Qiang

    2011-04-01

    Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ(Ac) cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ(A.c)) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T(g)), one melting temperature (T(m)) and one cool crystallization temperature (T(c)). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young's modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community. © Springer-Verlag 2010

  15. Effect of process variables on the production of Polyhydroxyalkanoates by activated sludge

    Directory of Open Access Journals (Sweden)

    Mokhtarani Nader

    2012-09-01

    Full Text Available Abstract Polyhydroxyalkanoates are known to be temporarily stored by microorganisms in activated sludge, especially in anaerobic-aerobic processes. Due to the problems resulted from the disposals of plastic wastes and excess sludge of wastewater treatment plants, the production of polyhydroxyalkanoates by treating activated sludge and determining the effect of process variables were the main issues of this paper. In this research, an anaerobic-aerobic sequencing batch reactor was used to make microorganism adapted and a batch aerobic reactor was used for enriching them. The variables affecting polyhydroxyalkanoates production including aeration time, sludge retention time, and volatile fatty acids concentration of the influent in sequencing batch reactor, and also carbon to nitrogen ratio and cultivation time in polymer production reactor, were investigated using Taguchi statistical approach to determine optimum conditions. The maximum polymer production of 29% was achieved at sludge retention time of 5–10 days, aeration time of 2 hours, supplementation of 40% of volatile fatty acids in the influent and increasing of carbon to nitrogen ratio of polymer production reactor to above 25 g/g. Based on the results, in optimum conditions, the volatile fatty acids concentration which increased the production of polyhydroxyalkanoates up to 49% was the most effective variable. Carbon to nitrogen ratio, sludge retention time and aeration time were ranked as the next affecting parameters. Although the polyhydroxyalkanoates content achieved in present study is much lower than that by pure culture, but the proposed method may still serve well as an environmental friendly means to convert waste into valuable product.

  16. Effect of Process Variables on the Production of Polyhydroxyalkanoates by Activated Sludge

    Directory of Open Access Journals (Sweden)

    Nader Mokhtarani

    2012-09-01

    Full Text Available Polyhydroxyalkanoates are known to be temporarily stored by microorganisms in activated sludge, especially in anaerobic-aerobic processes. Due to the problems resulted from the disposals of plastic wastes and excess sludge of wastewater treatment plants, the production of polyhydroxyalkanoates by treating activated sludge anddetermining the effect of process variables were the main issues of this paper. In this research, an anaerobic-aerobic sequencing batch reactor was used to make microorganism adapted and a batch aerobic reactor was used for enriching them. The variables affecting polyhydroxyalkanoates production including aeration time, sludge retention time, and volatile fatty acids concentration of the influent in sequencing batch reactor, and also carbon to nitrogenratio and cultivation time in polymer production reactor, were investigated using Taguchi statistical approach to determine optimum conditions. The maximum polymer production of 29% was achieved at sludge retention time of 5–10 days, aeration time of 2 hours, supplementation of 40% of volatile fatty acids in the influent and increasing of carbon to nitrogen ratio of polymer production reactor to above 25 g/g. Based on the results, in optimum conditions, the volatile fatty acids concentration which increased the production of polyhydroxyalkanoates up to 49% was the most effective variable. Carbon to nitrogen ratio, sludge retention time and aeration time were ranked as the next affecting parameters. Although the polyhydroxyalkanoates content achieved in present study is muchlower than that by pure culture, but the proposed method may still serve well as an environmental friendly means to convert waste into valuable product.

  17. Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135.

    Science.gov (United States)

    Haywood, G W; Anderson, A J; Ewing, D F; Dawes, E A

    1990-11-01

    A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C(2) to C(6)); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer synthesis occurred in batch culture after cessation of growth due to exhaustion of nitrogen. In continuous culture under nitrogen limitation up to 16.9% (wt/wt) polyhydroxyalkanoate was synthesized from glucose as the carbon source. The monomer units are mainly of the R-(-) configuration. Nuclear magnetic resonance studies confirmed the composition of the polymer. Differential scanning calorimetry suggested that the solvent-extracted polymer contained a significant proportion of crystalline material. The weight-average molecular weight of the polymer from glucose-grown cells was 143,000.

  18. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava

    OpenAIRE

    Dietrich, Diane; Illman, Barbara; Crooks, Casey

    2013-01-01

    Background The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose. Findings We examined the sensitivity of seven polyhydroxyalkanoate producing ba...

  19. Metabolic modelling of polyhydroxyalkanoate copolymers production by mixed microbial cultures

    Directory of Open Access Journals (Sweden)

    Reis Maria AM

    2008-07-01

    Full Text Available Abstract Background This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. Results The model was validated with experimental data collected in a sequencing batch reactor (SBR operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments, and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichment strategies were studied: (i either with acetate or (ii with propionate as carbon source material. Metabolic flux analysis (MFA was performed for the different feeding conditions and culture enrichment strategies. Flux balance analysis (FBA was used to calculate optimal feeding scenarios for high quality PHA polymers production, where it was found that a suitable polymer would be obtained when acetate is fed in excess and the feeding rate of propionate is limited to ~0.17 C-mol/(C-mol.h. The results were compared with published pure culture metabolic studies. Conclusion Acetate was more conducive toward the enrichment of a microbial culture with higher PHA storage fluxes and yields as compared to propionate. The P/O ratio was not only influenced by the selected microbial culture, but also by the carbon substrate fed to each culture, where higher P/O ratio values were consistently observed for acetate than propionate. MFA studies suggest that when mixtures of

  20. Production of functionalized polyhydroxyalkanoates by genetically modified Methylobacterium extorquens strains

    Directory of Open Access Journals (Sweden)

    Miguez Carlos B

    2010-09-01

    Full Text Available Abstract Background Methylotrophic (methanol-utilizing bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB, the best known polyhydroxyalkanoate (PHA. With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications. Results Our proprietary M. extorquens ATCC 55366 was found unable to yield functionalized PHAs when fed methanol and selected unsaturated carboxylic acids as secondary substrates. However, cloning of either the phaC1 or the phaC2 gene from P. fluorescens GK13, using an inducible and regulated expression system based on cumate as inducer (the cumate switch, yielded recombinant M. extorquens strains capable of incorporating modest quantities of C-C double bonds into PHA, starting from either C6= and/or C8=. The two recombinant strains gave poor results with C11=. The strain containing the phaC2 gene was better at using C8= and at incorporating C-C double bonds into PHA. Solvent fractioning indicated that the produced polymers were PHA blends that consequently originated from independent actions of the native and the recombinant PHA synthases. Conclusions This work constitutes an example of metabolic engineering applied to the construction of a methanol-utilizing bacterium capable of producing functionalized PHAs containing C-C double bonds. In this regard, the PhaC2 synthase

  1. Polyhydroxyalkanoate production by a novel bacterium Massilia sp UMI-21 isolated from seaweed, and molecular cloning of its polyhydroxyalkanoate synthase gene

    OpenAIRE

    Han, Xuerong; Satoh, Yasuharu; Kuriki, Yumi; Seino, Teruyuki; Fujita, Shinji; Suda, Takanori; Kobayashi, Takanori; Tajima, Kenji

    2014-01-01

    We successfully isolated one microorganism (UMI-21) from Ulva, a green algae that contains starch. The strain UMI-21 can produce polyhydroxyalkanoate (PHA) from starch, maltotriose, or maltose as a sole carbon source. Taxonomic studies and 16S rDNA sequence analysis revealed that strain UMI-21 was phylogenetically related to species of the genus Massilia. The PHA content under the cultivation condition using a 10-L jar fermentor was 45.5% (w/w). This value was higher than that obtained after ...

  2. Application of Poly(hydroxyalkanoate) In Food Packaging: Improvements by Nanotechnology

    OpenAIRE

    Khosravi-Darani, K.; Bucci, D. Z.

    2015-01-01

    The environmental impact of plastic usage is of critical concern and too great to repair. A shift toward biodegradable food packaging is one option. The aim of this review paper is the study of the potential of biodegradable materials for food packaging. The main characteristics in relation to food usage can be narrowed down to mass transfer (gas and water vapor), thermal and mechanical properties. Among several kinds of biodegradable polymers, poly(hydroxyalkanoate) is one of the favorable c...

  3. Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria.

    Science.gov (United States)

    Akiyama, Hideo; Okuhata, Hiroshi; Onizuka, Takuo; Kanai, Shozo; Hirano, Masahiko; Tanaka, Satoshi; Sasaki, Ken; Miyasaka, Hitoshi

    2011-12-01

    A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Quantitative Raman Spectroscopy Analysis of Polyhydroxyalkanoates Produced by Cupriavidus necator H16

    Czech Academy of Sciences Publication Activity Database

    Samek, Ota; Obruča, S.; Šiler, Martin; Sedláček, P.; Benešová, P.; Kučera, D.; Márová, I.; Ježek, Jan; Bernatová, Silvie; Zemánek, Pavel

    2016-01-01

    Roč. 16, č. 11 (2016), 1808:1-7 ISSN 1424-8220 R&D Projects: GA ČR(CZ) GA15-20645S; GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : Raman spectroscopy * Cupriavidus necator H16 * polyhydroxyalkanoates Subject RIV: BH - Optics, Masers, Lasers Impact factor: 2.677, year: 2016

  5. Processes for producing polyhydroxybutyrate and related polyhydroxyalkanoates in the plastids of higher plants

    Science.gov (United States)

    Somerville, C.R.; Nawrath, C.; Poirier, Y.

    1997-03-11

    The present invention relates to a process for producing poly-D-(-)-3-hydroxybutyric acid (PHB) and related polyhydroxyalkanoates (PHA) in the plastids of plants. The production of PHB is accomplished by genetically transforming plants with modified genes from microorganisms. The genes encode the enzymes required to synthesize PHB from acetyl-CoA or related metabolites and are fused with additional plant sequences for targeting the enzymes to the plastid. 37 figs.

  6. Characterization of a polyhydroxyalkanoate obtained from pineapple peel waste using Ralsthonia eutropha.

    Science.gov (United States)

    Vega-Castro, Oscar; Contreras-Calderon, Jose; León, Emilson; Segura, Almir; Arias, Mario; Pérez, León; Sobral, Paulo J A

    2016-08-10

    Agro-industrial waste can be the production source of biopolymers such as polyhydroxyalkanoates. The aim of this study was to produce and characterize Polyhydroxyalkanoates produced from pineapple peel waste fermentation processes. The methodology includes different pineapple peel waste fermentation conditions. The produced biopolymer was characterized using FTIR, GC-MS and NMR. The best fermentation condition for biopolymer production was obtained using pH 9, Carbon/Nitrogen 11, carbon/phosphorus 6 and fermentation time of 60h. FTIR analyzes showed PHB group characteristics, such as OH, CH and CO. In addition, GC-MS showed two monomers with 4 and 8 carbons, referred to PHB and PHBHV. H(1) NMR analysis showed 0.88-0.97 and 5.27ppm signals, corresponding to CH3 and CH, respectively. In conclusion, polyhydroxyalkanoate production from pineapple peels waste is an alternative for the treatment of waste generated in Colombia's fruit industry. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Exploration of Global Trend on Biomedical Application of Polyhydroxyalkanoate (PHA): A Patent Survey.

    Science.gov (United States)

    Ponnaiah, Paulraj; Vnoothenei, Nagiah; Chandramohan, Muruganandham; Thevarkattil, Mohamed Javad Pazhayakath

    2018-01-30

    Polyhydroxyalkanoates are bio-based, biodegradable naturally occurring polymers produced by a wide range of organisms, from bacteria to higher mammals. The properties and biocompatibility of PHA make it possible for a wide spectrum of applications. In this context, we analyze the potential applications of PHA in biomedical science by exploring the global trend through the patent survey. The survey suggests that PHA is an attractive candidate in such a way that their applications are widely distributed in the medical industry, drug delivery system, dental material, tissue engineering, packaging material as well as other useful products. In our present study, we explored patents associated with various biomedical applications of polyhydroxyalkanoates. Patent databases of European Patent Office, United States Patent and Trademark Office and World Intellectual Property Organization were mined. We developed an intensive exploration approach to eliminate overlapping patents and sort out significant patents. We demarcated the keywords and search criterions and established search patterns for the database request. We retrieved documents within the recent 6 years, 2010 to 2016 and sort out the collected data stepwise to gather the most appropriate documents in patent families for further scrutiny. By this approach, we retrieved 23,368 patent documents from all the three databases and the patent titles were further analyzed for the relevance of polyhydroxyalkanoates in biomedical applications. This ensued in the documentation of approximately 226 significant patents associated with biomedical applications of polyhydroxyalkanoates and the information was classified into six major groups. Polyhydroxyalkanoates has been patented in such a way that their applications are widely distributed in the medical industry, drug delivery system, dental material, tissue engineering, packaging material as well as other useful products. There are many avenues through which PHA & PHB could be

  8. Synthetic routes to degradable copolymers deriving from the biosynthesized polyhydroxyalkanoates: A mini review

    Directory of Open Access Journals (Sweden)

    Y. Ke

    2016-01-01

    Full Text Available Polyhydroxyalkanoates are a family of natural polyesters being produced as intracellular carbon and energy reserves by a wide variety of microorganisms. They have developed rapidly in both research and development efforts globally in the last 15 years. Till now, over 100 different types of PHAs have been successfully biosynthesized using both genetic engineering and fermentation techniques. Their unique biodegradable, biocompatible and thermoplastic characteristics make PHAs promising candidates for the commodity and biomedical applications. This review focused on the chemical synthesis of the derivatives of the biosynthesized PHAs.

  9. Production of Polyhydroxyalkanoates from Sludge Palm Oil Using Pseudomonas putida S12.

    Science.gov (United States)

    Kang, Du-Kyeong; Lee, Cho-Ryong; Lee, Sun Hee; Bae, Jung-Hoon; Park, Young-Kwon; Rhee, Young Ha; Sung, Bong Hyun; Sohn, Jung-Hoon

    2017-05-28

    Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

  10. Cybernetic structured modeling of the production of polyhydroxyalkanoates by Alcaligenes Eutrophus

    Directory of Open Access Journals (Sweden)

    L. FERRAZ

    1999-06-01

    Full Text Available This paper presents a cybernetic structured mathematical model developed for the fermentation step of the process of production of the copolymer of polyhydroxyalkanoates by the bacteria Alcaligenes eutrophus. This process is performed in two different fermentation stages. The first emphasizes the growth of the microorganism in a batch operation without substrates limitations, while in the second, the focus is on copolymer production by a fed-batch operation in the absence of the nitrogen source. This paper presents the results of the treatment of experimental data and of preliminary parameter estimation. The fitting of the proposed model to the experimental data of a standard experiment showed a good agreement.

  11. Specific character of bacterial biodegradation of polyhydroxyalkanoates with different chemical structure in soil.

    Science.gov (United States)

    Prudnikova, S V; Vinogradova, O N; Trusova, M Y

    2017-03-01

    The study addresses the influence of the physicochemical properties of the reserve cellular macromolecules (polyhydroxyalkanoates, PHA) with different chemical composition on their biodegradation in the agro-transformed field soil of the Siberian region (Krasnoyarsk Territory, Russia). It was shown that the degradation of the PHA samples depends on the degree of polymer crystallinity (C x ). For the first time, it was shown that the range of PHA-degrading microorganisms differs for each of PHA types. The study defines the primary degraders specific to each PHA type and common to all types of examined polymers.

  12. Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics

    Science.gov (United States)

    Witholt; Kessler

    1999-06-01

    Medium chain length (mcl) poly(hydroxyalkanoic acids) (PHAs) are polyesters accumulated by fluorescent Pseudomonads and other bacteria. Work on the genetics of mcl-PHA formation has led to polymer synthesis in recombinant bacteria and plants. Several high and medium cost applications are now emerging. With optimized bacterial mcl-PHA synthesis on inexpensive agro-substrates and the development of plant-based mcl-PHAs in the next decade, the production economics of these bioplastics will ultimately permit their sustainable production for bulk applications.

  13. Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

    Science.gov (United States)

    Mizuno, Kouhei; Kihara, Takahiro; Tsuge, Takeharu; Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa; Nomura, Christopher T

    2017-01-01

    Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

  14. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava

    Science.gov (United States)

    Diane Dietrich; Barbara Illman; Casey Crooks

    2013-01-01

    The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides...

  15. Statistical media design for efficient polyhydroxyalkanoate production in Pseudomonas sp. MNNG-S.

    Science.gov (United States)

    Saranya, V; Rajeswari, V; Abirami, P; Poornimakkani, K; Suguna, P; Shenbagarathai, R

    2016-07-03

    Polyhydroxyalkanoate (PHA) is a promising polymer for various biomedical applications. There is a high need to improve the production rate to achieve end use. When a cost-effective production was carried out with cheaper agricultural residues like molasses, traces of toxins were incorporated into the polymer, which makes it unfit for biomedical applications. On the other hand, there is an increase in the popularity of using chemically defined media for the production of compounds with biomedical applications. However, these media do not exhibit favorable characteristics such as efficient utilization at large scale compared to complex media. This article aims to determine the specific nutritional requirement of Pseudomonas sp. MNNG-S for efficient production of polyhydroxyalkanoate. Response surface methodology (RSM) was used in this study to statistically design for PHA production based on the interactive effect of five significant variables (sucrose; potassium dihydrogen phosphate; ammonium sulfate; magnesium sulfate; trace elements). The interactive effects of sucrose with ammonium sulfate, ammonium sulfate with combined potassium phosphate, and trace element with magnesium sulfate were found to be significant (p production more than fourfold (from 0.85 g L(-1) to 4.56 g L(-1)).

  16. Polyhydroxyalkanoate-based natural-synthetic hybrid copolymer films: A small-angle neutron scattering study

    International Nuclear Information System (INIS)

    Foster, L. John R.; Knott, Robert; Sanguanchaipaiwong, Vorapat; Holden, Peter J.

    2006-01-01

    Polyhydroxyalkanoates have attracted attention as biodegradable alternatives to conventional thermoplastics and as biomaterials. Through modification of their biosynthesis using Pseudomonas oleovorans, we have manipulated the material properties of these biopolyesters and produced a natural-synthetic hybrid copolymer of polyhydroxyoctanoate-block-diethylene glycol (PHO-b-DEG). A mixture of PHO and PHO-DEG were solvent cast from analytical grade chloroform and analysed using small-angle neutron scattering. A scattering pattern, easily distinguished above the background, was displayed by the films with a diffraction ring at q∼0.12 A -1 . This narrow ring of intensity is suggestive of a highly ordered system. Analysis of the diffraction pattern supported this concept and showed a d-spacing of approximately 50 A. In addition, conformation of the hybrid polymer chains can be manipulated to support their self-assembly into ordered microporous films

  17. Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis.

    Science.gov (United States)

    Tribelli, Paula M; Di Martino, Carla; López, Nancy I; Raiger Iustman, Laura J

    2012-09-01

    Diesel is a widely distributed pollutant. Bioremediation of this kind of compounds requires the use of microorganisms able to survive and adapt to contaminated environments. Pseudomonas extremaustralis is an Antarctic bacterium with a remarkable survival capability associated to polyhydroxyalkanoates (PHAs) production. This strain was used to investigate the effect of cell growth conditions--in biofilm versus shaken flask cultures--as well as the inocula characteristics associated with PHAs accumulation, on diesel degradation. Biofilms showed increased cell growth, biosurfactant production and diesel degradation compared with that obtained in shaken flask cultures. PHA accumulation decreased biofilm cell attachment and enhanced biosurfactant production. Degradation of long-chain and branched alkanes was observed in biofilms, while in shaken flasks only medium-chain length alkanes were degraded. This work shows that the PHA accumulating bacterium P. extremaustralis can be a good candidate to be used as hydrocarbon bioremediation agent, especially in extreme environments.

  18. A techno-economic analysis of polyhydroxyalkanoate and hydrogen production from syngas fermentation of gasified biomass.

    Science.gov (United States)

    Choi, DongWon; Chipman, David C; Bents, Scott C; Brown, Robert C

    2010-02-01

    A techno-economic analysis was conducted to investigate the feasibility of a gasification-based hybrid biorefinery producing both hydrogen gas and polyhydroxyalkanoates (PHA), biodegradable polymer materials that can be an attractive substitute for conventional petrochemical plastics. The biorefinery considered used switchgrass as a feedstock and converted that raw material through thermochemical methods into syngas, a gaseous mixture composed mainly of hydrogen and carbon monoxide. The syngas was then fermented using Rhodospirillum rubrum, a purple non-sulfur bacterium, to produce PHA and to enrich hydrogen in the syngas. Total daily production of the biorefinery was assumed to be 12 Mg of PHA and 50 Mg of hydrogen gas. Grassroots capital for the biorefinery was estimated to be $55 million, with annual operating costs at $6.7 million. With a market value of $2.00/kg assumed for the hydrogen, the cost of producing PHA was determined to be $1.65/kg.

  19. Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Minglian; Li, Zhenguo; Zheng, Wei; Lou, Zhiyong [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Chen, Guo-Qiang, E-mail: chengq@stu.edu.cn [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong (China)

    2006-08-01

    The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

  20. Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria from Pulp, Paper, and Cardboard Industry Wastes

    Directory of Open Access Journals (Sweden)

    Anish Kumari Bhuwal

    2013-01-01

    Full Text Available Background. Polyhydroxyalkanoates (PHAs are storage materials that accumulate by various bacteria as energy and carbon reserve materials. They are biodegradable, environmentally friendly, and also biocompatible bioplastics. Unlike petrochemical-based plastics that take several decades to fully degrade, PHAs can be completely degraded within a year by variety of microorganisms into CO2 and water. In the present study, we aim to utilize pulp, paper, and cardboard industry sludge and waste water for the isolation and screening of polyhydroxyalkanoates (PHAs accumulating bacteria and production of cost-effective PHB using cardboard industry waste water. Results. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminary screening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining, a more specific dye for PHA granules. The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% with polymer concentration of 5.236 g/L and 4.042 g/L with cardboard industry waste water. Both of the selected isolates, NAP11 and NAC1, were classified up to genus level by studying their morphological and biochemical characteristics and were found to be Enterococcus sp., Brevundimonas sp. and, respectively. Conclusion. The isolates Enterococcus sp. NAP11 and Brevundimonas sp. NAC1 can be considered as good candidates for industrial production of PHB from cardboard industry waste water. We are reporting for the first time the use of cardboard industry waste water as a cultivation medium for the PHB production.

  1. Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria from Pulp, Paper, and Cardboard Industry Wastes

    Science.gov (United States)

    Bhuwal, Anish Kumari; Singh, Gulab; Aggarwal, Neeraj Kumar; Goyal, Varsha; Yadav, Anita

    2013-01-01

    Background. Polyhydroxyalkanoates (PHAs) are storage materials that accumulate by various bacteria as energy and carbon reserve materials. They are biodegradable, environmentally friendly, and also biocompatible bioplastics. Unlike petrochemical-based plastics that take several decades to fully degrade, PHAs can be completely degraded within a year by variety of microorganisms into CO2 and water. In the present study, we aim to utilize pulp, paper, and cardboard industry sludge and waste water for the isolation and screening of polyhydroxyalkanoates (PHAs) accumulating bacteria and production of cost-effective PHB using cardboard industry waste water. Results. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminary screening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining, a more specific dye for PHA granules. The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% with polymer concentration of 5.236 g/L and 4.042 g/L with cardboard industry waste water. Both of the selected isolates, NAP11 and NAC1, were classified up to genus level by studying their morphological and biochemical characteristics and were found to be Enterococcus sp., Brevundimonas sp. and, respectively. Conclusion. The isolates Enterococcus sp. NAP11 and Brevundimonas sp. NAC1 can be considered as good candidates for industrial production of PHB from cardboard industry waste water. We are reporting for the first time the use of cardboard industry waste water as a cultivation medium for the PHB production. PMID:24288534

  2. Enhancing the Production of Polyhydroxyalkanoate Biopolymer by Azohydromonas Australica Using a Simple Empty and Fill Bioreactor Cultivation Strategy

    OpenAIRE

    G. Gahlawat; A. K. Srivastava

    2018-01-01

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, widespread use of these polymers is still hampered due to their high cost of production. This shortcoming could partly be resolved by obtaining high yields and productivity. In the present study, a drain-and-fill strategy of repeated-batch cultivation was adopted for the enhanced production of p...

  3. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava.

    Science.gov (United States)

    Dietrich, Diane; Illman, Barbara; Crooks, Casey

    2013-06-04

    The aim of this study is determine the relative sensitivity of a panel of seven polyhydroxyalkanoate producing bacteria to a panel of seven lignocellulosic-derived fermentation inhibitors representing aliphatic acids, furans and phenolics. A further aim was to measure the polyhydroxybutyrate production of select organisms on lignocellulosic-derived monosaccharides arabinose, xylose, glucose and mannose. We examined the sensitivity of seven polyhydroxyalkanoate producing bacteria: Azohydromonas lata, Bacillus megaterium, Bacillus cereus, Burkholderia cepacia, Pseudomonas olevorans, Pseudomonas pseudoflava and Ralstonia eutropha, against seven fermentation inhibitors produced by the saccharification of lignocellulose: acetic acid, levulinic acid, coumaric acid, ferulic acid, syringaldehyde, furfural, and hyroxymethyfurfural. There was significant variation in the sensitivity of these microbes to representative phenolics ranging from 0.25-1.5 g/L coumaric and ferulic acid and between 0.5-6.0 g/L syringaldehyde. Inhibition ranged from 0.37-4 g/L and 0.75-6 g/L with acetic acid and levulinic acid, respectively. B. cepacia and P. pseudoflava were selected for further analysis of polyhydroxyalkanoate production. We find significant differences in sensitivity to the fermentation inhibitors tested and find these variations to be over a relevant concentration range given the concentrations of inhibitors typically found in lignocellulosic hydrolysates. Of the seven bacteria tested, B. cepacia demonstrated the greatest inhibitor tolerance. Similarly, of two organisms examined for polyhydroxybutyrate production, B. cepacia was notably more efficient when fermenting pentose substrates.

  4. Polyhydroxyalkanoate production by a novel bacterium Massilia sp. UMI-21 isolated from seaweed, and molecular cloning of its polyhydroxyalkanoate synthase gene.

    Science.gov (United States)

    Han, Xuerong; Satoh, Yasuharu; Kuriki, Yumi; Seino, Teruyuki; Fujita, Shinji; Suda, Takanori; Kobayashi, Takanori; Tajima, Kenji

    2014-11-01

    We successfully isolated one microorganism (UMI-21) from Ulva, a green algae that contains starch. The strain UMI-21 can produce polyhydroxyalkanoate (PHA) from starch, maltotriose, or maltose as a sole carbon source. Taxonomic studies and 16S rDNA sequence analysis revealed that strain UMI-21 was phylogenetically related to species of the genus Massilia. The PHA content under the cultivation condition using a 10-L jar fermentor was 45.5% (w/w). This value was higher than that obtained after cultivation in a flask, suggesting the possibility of large-scale PHA production by UMI-21 from starch. A major issue for the industrial production of microbial PHAs is the very high production cost. Starch is a relatively inexpensive substrate that is also found in abundant seaweeds such as Ulva. Therefore, the strain isolated in this study may be very useful for producing PHA from seaweeds containing polysaccharides such as starch. In addition, a 3.7-kbp DNA fragment containing the whole PHA synthase gene (phaC) was obtained from the strain UMI-21. The results of open reading frame (ORF) analysis suggested that the DNA fragment contained two ORFs, which were composed of 1740 (phaC) and 564 bp (phaR). The deduced amino acid sequence of PhaC from strain UMI-21 shared high similarity with PhaC from Ralstonia eutropha, which is a representative PHA-producing bacterium with a class I PHA synthase. This is the first report for the cloning of the PHA synthase gene from Massilia species. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity.

    Science.gov (United States)

    Manso Cobos, Isabel; Ibáñez García, María Isabel; de la Peña Moreno, Fernando; Sáez Melero, Lara Paloma; Luque-Almagro, Víctor Manuel; Castillo Rodríguez, Francisco; Roldán Ruiz, María Dolores; Prieto Jiménez, María Auxiliadora; Moreno Vivián, Conrado

    2015-06-10

    Cyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN(-)) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required. In this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha(-), deleted in putative mcl-PHA synthases; Spha(-), deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC-MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha(-) and Spha(-), respectively. The scl-PHAs were identified as homopolymers of 3

  6. Perspectives on the production, structural characteristics and potential applications of bioplastics derived from polyhydroxyalkanoates.

    Science.gov (United States)

    Albuquerque, Priscilla B S; Malafaia, Carolina B

    2018-02-01

    Since the last two decades, the use of synthetic materials has increased and become more frequent in this capitalist system. Polymers used as raw materials are usually disposed very rapidly and considered serious damages when they return to the environment. Because of this behaviour, there was an increasing in the global awareness by minimizing the waste generated, in addition to the scientific community concern for technological alternatives to solve this problem. Alternatively, biodegradable polymers are attracting special interest due to their inherent properties, which are similar to the ones of the conventional plastics. Bioplastics covers plastics made from renewable resources, including plastics that biodegrade under controlled conditions at the end of their use phase. Polyhydroxyalkanoates (PHAs) are polyesters composed of hydroxy acids, synthesized by a variety of microorganisms as intracellular carbon and energy storage. These environmentally friendly biopolymers have excellent potential in domestic, agricultural, industrial and medical field, however their production on a large scale is still limited. This review considered the most recent scientific publications on the production of bioplastics based on PHAs, their structural characteristics and the exploitation of different renewable sources of raw materials. In addition, there were also considered the main biotechnological applications of these biopolymers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Microbial production of polyhydroxyalkanoates (PHAs) and its copolymers: A review of recent advancements.

    Science.gov (United States)

    Anjum, Anbreen; Zuber, Mohammad; Zia, Khalid Mahmood; Noreen, Aqdas; Anjum, Muhammad Naveed; Tabasum, Shazia

    2016-08-01

    Traditional mineral oil based plastics are important commodity to enhance the comfort and quality of life but the accumulation of these plastics in the environment has become a major universal problem due to their low biodegradation. Solution to the plastic waste management includes incineration, recycling and landfill disposal methods. These processes are very time consuming and expensive. Biopolymers are important alternatives to the petroleum-based plastics due to environment friendly manufacturing processes, biodegradability and biocompatibility. Therefore use of novel biopolymers, such as polylactide, polysaccharides, aliphatic polyesters and polyhydroxyalkanoates is of interest. PHAs are biodegradable polyesters of hydroxyalkanoates (HA) produced from renewable resources by using microorganisms as intracellular carbon and energy storage compounds. Even though PHAs are promising candidate for biodegradable polymers, however, the production cost limit their application on an industrial scale. This article provides an overview of various substrates, microorganisms for the economical production of PHAs and its copolymers. Recent advances in PHAs to reduce the cost and to improve the performance of PHAs have also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Marine sponge-associated bacteria as a potential source for polyhydroxyalkanoates.

    Science.gov (United States)

    Sathiyanarayanan, Ganesan; Saibaba, Ganesan; Kiran, George Seghal; Yang, Yung-Hun; Selvin, Joseph

    2017-05-01

    Marine sponges are filter feeding porous animals and usually harbor a remarkable array of microorganisms in their mesohyl tissues as transient and resident endosymbionts. The marine sponge-microbial interactions are highly complex and, in some cases, the relationships are thought to be truly symbiotic or mutualistic rather than temporary associations resulting from sponge filter-feeding activity. The marine sponge-associated bacteria are fascinating source for various biomolecules that are of potential interest to several biotechnological industries. In recent times, a particular attention has been devoted to bacterial biopolymer (polyesters) such as intracellular polyhydroxyalkanoates (PHAs) produced by sponge-associated bacteria. Bacterial PHAs act as an internal reserve for carbon and energy and also are a tremendous alternative for fossil fuel-based polymers mainly due to their eco-friendliness. In addition, PHAs are produced when the microorganisms are under stressful conditions and this biopolymer synthesis might be exhibited as one of the survival mechanisms of sponge-associated or endosymbiotic bacteria which exist in a highly competitive and stressful sponge-mesohyl microenvironment. In this review, we have emphasized the industrial prospects of marine bacteria for the commercial production of PHAs and special importance has been given to marine sponge-associated bacteria as a potential resource for PHAs.

  9. Increasing polyhydroxyalkanoate (PHA) yields from Cupriavidus necator by using filtered digestate liquors.

    Science.gov (United States)

    Passanha, Pearl; Esteves, Sandra R; Kedia, Gopal; Dinsdale, Richard M; Guwy, Alan J

    2013-11-01

    The production of polyhydroxyalkanoates (PHAs) using digestate liquor as culture media is a novel application to extend the existing uses of digestates. In this study, two micro-filtered digestates (0.22 μm) were evaluated as a source of complex culture media for the production of PHA by Cupriavidus necator as compared to a conventional media. Culture media using a mixture of micro-filtered liquors from food waste and from wheat feed digesters showed a maximum PHA accumulation of 12.29 g/l PHA, with 90% cell dry weight and a yield of 0.48 g PHA/g VFA consumed, the highest reported to date for C. necator studies. From the analysis of the starting and residual media, it was concluded that ammonia, potassium, magnesium, sulfate and phosphate provided in the digestate liquors were vital for the initial growth of C. necator whereas copper, iron and nickel may have played a significant role in PHA accumulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Perspectives on the production of polyhydroxyalkanoates in biorefineries associated with the production of sugar and ethanol.

    Science.gov (United States)

    Silva, Luiziana Ferreira; Taciro, Marilda Keico; Raicher, Gil; Piccoli, Rosane Aparecida Moniz; Mendonça, Thatiane Teixeira; Lopes, Mateus Schreiner Garcez; Gomez, José Gregório Cabrera

    2014-11-01

    Polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bacterial thermoplastic polymers that can be obtained from renewable resources. The high impact of the carbon source in the final cost of this polymer has been one of the major limiting factors for PHA production and agricultural residues, mainly lignocellulosic materials, have gained attention to overcome this problem. In Brazil, production of 2nd generation ethanol from the glucose fraction, derived from sugarcane bagasse hydrolysate has been studied. The huge amounts of remaining xylose will create an opportunity for the development of other bioprocesses, generating new products to be introduced into a biorefinery model. Although PHA production from sucrose integrated to a 1G ethanol and sugar mill has been proposed in the past, the integration of the process of 2G ethanol in the context of a biorefinery will provide enormous amounts of xylose, which could be applied to produce PHA, establishing a second-generation of PHA production process. Those aspects and perspectives are presented in this article. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Syngas obtained by microwave pyrolysis of household wastes as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum.

    Science.gov (United States)

    Revelles, Olga; Beneroso, Daniel; Menéndez, J Angel; Arenillas, Ana; García, J Luis; Prieto, M Auxiliadora

    2017-11-01

    The massive production of urban and agricultural wastes has promoted a clear need for alternative processes of disposal and waste management. The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHA) by a process known as syngas fermentation is considered herein as an attractive bio-economic strategy to reduce these wastes. In this work, we have evaluated the potential of Rhodospirillum rubrum as microbial cell factory for the synthesis of PHA from syngas produced by microwave pyrolysis of the MSW organic fraction from a European city (Seville). Growth rate, uptake rate, biomass yield and PHA production from syngas in R. rubrum have been analysed. The results revealed the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. Microwave-induced pyrolysis is a tangible alternative to standard pyrolysis, because it can reduce cost in terms of energy and time as well as increase syngas production, providing a satisfactory PHA yield. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  12. Biodegradable and Biocompatible Polyhydroxy-alkanoates (PHA: Auspicious Microbial Macromolecules for Pharmaceutical and Therapeutic Applications

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2018-02-01

    Full Text Available Polyhydroxyalkanoates (PHA are bio-based microbial biopolyesters; their stiffness, elasticity, crystallinity and degradability are tunable by the monomeric composition, selection of microbial production strain, substrates, process parameters during production, and post-synthetic processing; they display biological alternatives for diverse technomers of petrochemical origin. This, together with the fact that their monomeric and oligomeric in vivo degradation products do not exert any toxic or elsewhere negative effect to living cells or tissue of humans or animals, makes them highly stimulating for various applications in the medical field. This article provides an overview of PHA application in the therapeutic, surgical and tissue engineering area, and reviews strategies to produce PHA at purity levels high enough to be used in vivo. Tested applications of differently composed PHA and advanced follow-up products as carrier materials for controlled in vivo release of anti-cancer drugs or antibiotics, as scaffolds for tissue engineering, as guidance conduits for nerve repair or as enhanced sutures, implants or meshes are discussed from both a biotechnological and a material-scientific perspective. The article also describes the use of traditional processing techniques for production of PHA-based medical devices, such as melt-spinning, melt extrusion, or solvent evaporation, and emerging processing techniques like 3D-printing, computer-aided wet-spinning, laser perforation, and electrospinning.

  13. Biomechanics of Polyhydroxyalkanoate Mesh-Augmented Single-Row Rotator Cuff Repairs.

    Science.gov (United States)

    Tashjian, Robert Z; Kolz, Christopher W; Suter, Thomas; Henninger, Heath B

    Polyhydroxyalkanoate (PHA) mesh is a bioresorbable scaffold used to reinforce the suture-tendon interface in rotator cuff repairs (RCRs). We conducted a study of cyclic and ultimate failure properties of PHA mesh-augmented single-row RCRs and nonaugmented RCRs. Eight pairs of fresh-frozen cadaver humeri (6 male, 2 female) were tested. Mean (SD) age was 61 (9) years. The supraspinatus tendon was resected and reattached in a single-row configuration using 2 triple-loaded suture anchors and 6 simple stitches. The opposite humerus underwent RCR augmented with 2 strips of 13-mm × 23-mm PHA mesh. Humeri were mounted in an Instron load frame, cycled 1000 times to 1.0 MPa of effective stress, and loaded to failure. Construct gapping and ultimate failure loads/displacements were recorded. Paired t tests compared augmented and nonaugmented RCRs (P ≤ .05 was significant). There was no difference in gapping over 1000 cycles (P = .879). Mean (SD) failure load was higher for PHA mesh-augmented RCRs, 571 (173) N, than for nonaugmented (control) RCRs, 472 (120) N (P = .042), and failures were consistent within pairs because of tissue failure at the knots or anchor pullout. This technique for arthroscopic augmentation can be used to improve initial biomechanical repair strength in tears at risk for failure.

  14. Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

    Science.gov (United States)

    Riedel, Sebastian L; Jahns, Stefan; Koenig, Steven; Bock, Martina C E; Brigham, Christopher J; Bader, Johannes; Stahl, Ulf

    2015-11-20

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Production of co-polymers of polyhydroxyalkanoates by regulating the hydrolysis of biowastes.

    Science.gov (United States)

    Kumar, Prasun; Ray, Subhasree; Kalia, Vipin C

    2016-01-01

    Production of polyhydroxyalkanoate (PHA) co-polymers by Bacillus spp. was studied by feeding defined volatile fatty acids (VFAs) obtained through controlled hydrolysis of various wastes. Eleven mixed hydrolytic cultures (MHCs) each containing 6 strains could generate VFA from slurries of (2% total solids): pea-shells (PS), potato peels (PP), apple pomace (AP) and onion peels (OP). PS hydrolysates (obtained with MHC2 and MHC5) inoculated with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45 produced co-polymers of PHA at the rate of 15-60mg/L with a 3HV content of 1%w/w. An enhancement in PHA yield of 3.66-fold, i.e. 205-550mg/L with 3HV content up to 7.5%(w/w) was observed upon addition of OP hydrolysate and 1% glucose (w/v) to PS hydrolysates. This is the first demonstration, where PHA co-polymer composition, under non-axenic conditions, could be controlled by customizing VFA profile of the hydrolysate by the addition of different biowastes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Polyhydroxyalkanoate (PHA: Review of synthesis, characteristics, processing and potential applications in packaging

    Directory of Open Access Journals (Sweden)

    E. Bugnicourt

    2014-11-01

    Full Text Available Polyhydroxyalkanoates (PHAs are gaining increasing attention in the biodegradable polymer market due to their promising properties such as high biodegradability in different environments, not just in composting plants, and processing versatility. Indeed among biopolymers, these biogenic polyesters represent a potential sustainable replacement for fossil fuel-based thermoplastics. Most commercially available PHAs are obtained with pure microbial cultures grown on renewable feedstocks (i.e. glucose under sterile conditions but recent research studies focus on the use of wastes as growth media. PHA can be extracted from the bacteria cell and then formulated and processed by extrusion for production of rigid and flexible plastic suitable not just for the most assessed medical applications but also considered for applications including packaging, moulded goods, paper coatings, non-woven fabrics, adhesives, films and performance additives. The present paper reviews the different classes of PHAs, their main properties, processing aspects, commercially available ones, as well as limitations and related improvements being researched, with specific focus on potential applications of PHAs in packaging.

  17. Development and Characterization of a Metal Injection Molding Bio Sourced Inconel 718 Feedstock Based on Polyhydroxyalkanoates

    Directory of Open Access Journals (Sweden)

    Alexandre Royer

    2016-04-01

    Full Text Available The binder plays the most important role in the metal injection molding (MIM process. It provides fluidity of the feedstock mixture and adhesion of the powder to keep the molded shape during injection molding. The binder must provide strength and cohesion for the molded part and must be easy to remove from the molded part. Moreover, it must be recyclable, environmentally friendly and economical. Also, the miscibility between polymers affects the homogeneity of the injected parts. The goal of this study is to develop a feedstock of superalloy Inconel 718 that is environmentally friendly. For these different binders, formulations based on polyethylene glycol (PEG, because of his water solubility property, and bio sourced polymers were studied. Polyhydroxyalkanoates (PHA were investigated as a bio sourced polymer due to its miscibility with the PEG. The result is compared to a standard formulation using polypropylene (PP. The chemical and rheological behavior of the binder formulation during mixing, injection and debinding process were investigated. The feedstock was characterized in the same way as the binders and the interactions between the powder and the binders were also studied. The results show the well adapted formulation of polymer binder to produce a superalloy Inconel 718 feedstock.

  18. Production and optimization of polyhydroxyalkanoates from non-edible Calophyllum inophyllum oil using Cupriavidus necator.

    Science.gov (United States)

    Arumugam, A; Senthamizhan, S G; Ponnusami, V; Sudalai, S

    2018-06-01

    Polyhydroxyalkanoates (PHA) are biodegradable polymers found in the cellular masses of a wide range of bacterial species and the demand for PHA is steadily growing. In this work we have produced PHA from a low-cost substrate, Calophyllum inophyllum oil, using Cupriavidus necator. Effects of various process parameters such as Oil concentration, Nitrogen source and inoculum size on the production of PHA were studied using Response Surface Methodology. A quadratic equation was used in the model to fit the experimental data. It was found that the model could satisfactorily predict the PHA yield (R 2 =99.17%). Linear, quadratic and interaction terms used in the model were found to be statistically significant. Maximum PHA yield of 10.6gL -1 was obtained under the optimized conditions of oil concentration - 17.5%, inoculum concentration - 50mL/L and nitrogen content - 1.125gL -1 , respectively. The product obtained was characterized using FTIR and NMR to confirm that it was PHA. The results demonstrate that C. inophyllum oil, a non-edible oil, can be potentially used as a low-cost substrate for the production of PHA. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Novel approach for productivity enhancement of polyhydroxyalkanoates (PHA) production by Cupriavidus necator DSM 545.

    Science.gov (United States)

    Berezina, Nathalie

    2013-01-25

    In the global context of increased concerns for our environment, the use of bioplastics as a replacement for existing petroleum-based polymers is an important challenge. Indeed, bioplastics hardly meet economical and technical constraints. One, of the most promising among currently studied bioplastics, is the polyhydroxyalkanoate (PHA). To circumvent the economical issue for this particular biopolymer one solution can be the enhancement of the overall productivity by the improvement of the nutritional medium of the microorganism producing the biopolymer. Thus, several nutrition media, supplemented or not with sodium glutamate, were tested for the growth and the PHA production by Cupriavidus necator DSM 545 strain. The most efficient for the biomass and the PHA production improvement were found to be the Luria broth (LB) and the Bonnarme's media, both supplemented with 10 g/L sodium glutamate. Hence the overall productivity was 33 times enhanced comparing to traditional cultivation methods. These results open a new route for the PHA production by C. necator which appears to be more suitable on a rich, or enriched, medium with no limiting factors. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

    Science.gov (United States)

    Matavulj, M; Molitoris, H P

    1992-12-01

    The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

    Science.gov (United States)

    Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

    2017-11-01

    Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  2. Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai

    Directory of Open Access Journals (Sweden)

    Aneesh Balakrishna Pillai

    Full Text Available Abstract This study was focused on the polyhydroxybutyrate (PHB accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

  3. Recycling of Waste Streams of the Biotechnological Poly(hydroxyalkanoate Production by Haloferax mediterranei on Whey

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2015-01-01

    Full Text Available For manufacturing “bioplastics” such as poly(hydroxyalkanoates (PHA, the combination of utilization of inexpensive carbon sources with the application of robust microbial production strains is considered a decisive step to make this process more cost-efficient and sustainable. PHA production based on surplus whey from dairy industry was accomplished by the extremely halophile archaeon Haloferax mediterranei. After fermentative production of PHA-rich biomass and the subsequent cell harvest and downstream processing for PHA recovery, environmentally hazardous, highly saline residues, namely spent fermentation broth and cell debris, remain as residues. These waste streams were used for recycling experiments to assess their recyclability in subsequent production processes. It was demonstrated that spent fermentation broth can be used to replace a considerable part of fresh saline fermentation medium in subsequent production processes. In addition, 29% of the expensive yeast extract, needed as nitrogen and phosphate source for efficient cultivation of the microorganism, can be replaced by cell debris from prior cultivations. The presented study provides strategies to combine the reduction of costs for biomediated PHA production with minimizing ecological risks by recycling precarious waste streams. Overall, the presented work shall contribute to the quick economic success of these promising biomaterials.

  4. Biosynthesis and Lipase-Catalysed Hydrolysis of 4-Hydroxybutyrate-Containing Polyhydroxyalkanoates from Delftia acidovorans

    Directory of Open Access Journals (Sweden)

    Diana Hooi-Ean Ch’ng

    2012-09-01

    Full Text Available Aims: Polyhydroxyalkanoates (PHA having various molar fractions of 4-hydroxybutyrate has been successfully synthesized by Delftia acidovorans.Methodology and results: The monomer compositions of the PHA were varied by cultivating the bacterium in a mixture of 1,4-butanediol and sodium valerate, γ-butyrolactone and sodium valerate as well as 4-hydroxybutyric acid and sodium valerate, which resulted in the production of PHA terpolymers. Although the highest terpolymer content achieved was only 57 wt% of the dry cell weight, the 4HB molar fractions can be regulated from 2-50 mol% when culture conditions such as initial pH, inoculum concentration and aeration were varied. The in vitro degradation of [P(3HB-co-50 % 4HB] synthesized by D. acidovorans were also studied by monitoring the erosion rate of the copolymer in aqueous solutions of lipases (Lipase A ‘Amano’ 12 and Newlase F. Results have shown that the types of lipases, concentration of lipase solution and pH of the buffer solution influenced the degradation rate of the PHA copolymer.Conclusion, significance and impact of the study: The overall results have shown that D. acidovorans is a very promising strain for the production of 4HB containing PHAs with specific compositions which are very suitable to be tailor made into biodegradable and biocompatible materials for medical applications.

  5. Production and Characterization of Polyhydroxyalkanoates and Native Microorganisms Synthesized from Fatty Waste

    Directory of Open Access Journals (Sweden)

    Javier Ricardo Gómez Cardozo

    2016-01-01

    Full Text Available Polyhydroxyalkanoates (PHAs are biodegradable and biocompatible plastics. They are synthesized by a wide variety of microorganisms (i.e., fungi and bacteria and some organisms such as plants, which share characteristics with petrochemical-based plastics. The most recent studies focus on finding inexpensive substrates and extraction strategies that allow reducing product costs, thus moving into a widespread market, the market for petroleum-based plastics. In this study, the production of polyhydroxybutyrate (PHB was evaluated using the native strains, Bacillus megaterium, Bacillus sp., and Lactococcus lactis, and glycerol reagent grade (GRG, residual glycerol (RGSB byproduct of biodiesel from palm oil, Jatropha oil, castor oil, waste frying oils, and whey as substrates. Different bacteria-substrate systems were evaluated thrice on a laboratory scale under different conditions of temperature, pH, and substrate concentration, employing 50 mL of broth in 250 mL. The bacterial growth was tested in all systems; however, the B. megaterium GRG system generated the highest accumulation of PHA. The previous approach was allowed to propose a statistical design optimization with RGSB (i.e., RGSB, 15 g/L, pH 7.0, and 25°C. This system reached 2.80 g/L of PHB yield and was the optimal condition tested; however, the optimal biomass 5.42 g/L occurs at pH 9.0 and 25°C, with a substrate concentration of 22 g/L.

  6. Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons

    Directory of Open Access Journals (Sweden)

    Saiqa Tufail

    Full Text Available ABSTRACT Polyhydroxyalkanoates (PHA are efficient, renewable and environment friendly polymeric esters. These polymers are synthesized by a variety of microbes under stress conditions. This study was carried out to check the suitability of waste frying oil in comparison to other oils for economical bioplastic production. Six bacterial strains were isolated and identified as Bacillus cereus (KF270349, Klebsiella pneumoniae (KF270350, Bacillus subtilis (KF270351, Brevibacterium halotolerance (KF270352, Pseudomonas aeruginosa (KF270353, and Stenotrophomonas rhizoposid (KF270354 by ribotyping. All strains were PHA producers so were selected for PHA synthesis using four different carbon sources, i.e., waste frying oil, canola oil, diesel and glucose. Extraction of PHA was carried out using sodium hypochlorite method and maximum amount was detected after 72 h in all cases. P. aeruginosa led to maximum PHA production after 72 h at 37 °C and 100 rpm using waste frying oil that was 53.2% PHA in comparison with glucose 37.8% and cooking oil 34.4%. B. cereus produced 40% PHA using glucose as carbon source which was high when compared against other strains. A significantly lesser amount of PHA was recorded with diesel as a carbon source for all strains. Sharp Infrared peaks around 1740-1750 cm-1 were present in Fourier Transform Infrared spectra that correspond to exact position for PHA. The use of waste oils and production of poly-3hydroxybutyrate-co-3hydroxyvalerate (3HB-co-3HV by strains used in this study is a good aspect to consider for future prospects as this type of polymer has better properties as compared to PHBs.

  7. Poly(hydroxyalkanoates for Food Packaging: Application and Attempts towards Implementation

    Directory of Open Access Journals (Sweden)

    M. Koller

    2014-09-01

    Full Text Available Plastics are well-established for convenient and safe packaging and distribution of food and feed goods. At present, this special sector of the plastic market displays remarkably increasing quantities of its annual production. Caused by the ongoing limitation and strongly fluctuating prices of fossil feedstocks, classically used for plastic production, there is an evident trend to switch towards so-called “bio-plastics”. Especially for bulk applications such as food packaging, a broad implementation of “bio-plastics” constitutes a future-oriented strategy to restrict the dependence of global industry on fossil feedstocks, and to diminish current problematic environmental issues arising from plastic disposal. However, food packaging demands a great deal of the utilized packaging material. This encompasses tailored mechanical properties such as low brittleness and adequate tensile strength, a sufficient barrier for oxygen, CO2, and aromatic flavors, high UV-resistance, and high water retention-capacity to block the food´s moisture content, or to prevent humidity, respectively. Due to their hydrophobic character and the broad flexibility of their mechanical features, prokaryotic poly(hydroxyalkanoates (PHAs are considered as promising materials to compete with petro-plastics on the food-packaging market. Nevertheless, short-comings in particular aspects of their material performance and economics of their biosynthesis and purification constitute stumbling blocks on the long way towards broad implementation of PHAs for food packaging. This article discusses advantages and drawbacks of PHAs as food packaging materials, and demonstrates how desired properties can be improved by the designing of novel composite materials, and also encompassing techniques by applying nanoparticles.

  8. Evaluation of Factors Affecting Polyhydroxyalkanoates Production by Comamonas sp. EB172 Using Central Composite Design

    Directory of Open Access Journals (Sweden)

    Noor Azman Mohd Johar

    2012-09-01

    Full Text Available Aims: Statistical approach, central composite design (CCD was used to investigate the complex interaction among temperature (25-37 °C, initial medium pH (5-9, inoculum size (4-10 % (v/v, concentration of (NH42SO4 (0-1 g/L and concentration of mixed organic acids (5-10 g/L in the production of polyhydroxyalkanoates by Comamonas sp. EB172.Methodology and Results: Mixed organic acids derived from anaerobically treated palm oil mill effluent (POME containing acetic:propionic:butyric (ratio of 3:1:1 were used as carbon source in the batch culture of Comamonas sp. EB172 to produce polyhydoxyalkanoates (PHAs. The analysis of variance (ANOVA showed that all five factors were significantly important in the batch fermentation by shake flask with a P value of less than 0.001. The optimal temperature, initial medium pH, inoculum size, concentration of (NH42SO4 and concentration of mixed organic acids were 30 °C, 7.04, 4.0 % (v/v, 0.01 g/L and 5.05 g/L respectively.Conclusion, significance and impact of study: Optimization of the production medium containing mixed organic acids has improved the PHA production for more than 2 folds. Under optimal condition in the shake flask fermentation, the predicted growth is 2.98 g/L of dry cell weight (DCW with 47.07 wt % of PHA content. The highest yield of PHA was 0.28 g of PHA per g mixed organic acids.

  9. Intracellular organisation of polyhydroxyalkanoate inclusion bodies: a role for small angle neutron scattering?

    International Nuclear Information System (INIS)

    Foster, L.J.R.; Holden, P.J.; Garvey, C.J.; Russell, R.A.; Stone, D.J.M.

    2003-01-01

    Full text: Polyhydroxyalkanoates (PHAs) are a diverse family of bacterially produced biopolyesters. Their biodegradability, and in some cases biocompatibility, suggest applications ranging from bioplastics to biomedical implantation devices. Despite extensive interest in their production and potential applications, little is known about their intracellular organisation. Microbial PHAs are synthesised by microorganisms under conditions of nutrient stress and can comprise up to 90% of the dry cell mass. The formation and organisation of these PHA inclusion bodies requires clarification. Such investigations have important implications for the biotechnological production of PHAs in microbes and other organisms, for downstream processing and in vitro precision polymerisation. Morphological and biochemical evidence supports two different models for the intracellular organisation of PHAs. Steinbuchel and coworkers propose a simple model of amorphous PHA enclosed by a single protein membrane consisting of structural proteins (PHAsins) and enzymes responsible for synthesis and degradation. In contrast, Fuller and coworkers have theorised a more complex system of PHA encompassed by a PHAsin bilayer separated by phospholipid. The polymerase and depolymerase enzymes are proposed to be associated with an incomplete inner PHAsin layer. It may be that such models are genera or species specific, since both proposals were derived from research on different species producing different types of PHA. Our initial investigations have focussed on in vivo deuteration of polyhydroxyoctanoate, produced by Pseudomonas oleovorans, both in fermentation on natural and deuterated substrates and during Small Angle Neutron Scattering by whole cells using AUSANS. The nature of the structural questions and our preliminary findings including contrast variation data will be discussed

  10. Commercialization of bacterial cell factories for the sustainable production of polyhydroxyalkanoate thermoplastics: progress and prospects.

    Science.gov (United States)

    Kumar, Abhishek; Srivastava, Janmejai K; Mallick, Nirupama; Singh, Akhilesh K

    2015-01-01

    Ubiquitous conventional plastics, generally manufactured from finite, nonsustainable fossil fuels are non-biodegradable wonder entities but their ill effect on Mother Nature has subsequently raised major environmental concerns like their safe disposal, solid waste management and several potential hazards. Such concerns have fuelled initiatives for research globally towards development of sustainable and eco-friendly bioplastics. The new generation of plastics called 'bioplastics' are polymers of long chain of repeating monomer units that are classified as photodegradable, semi-biodegradable, chemically synthesized and polyhydroxyalkanoates (PHAs). The commonly emerged novel bioplastics are polyesters of hydroxyalkanoates (HAs) called PHAs, which are lipoidic storage materials found in the cytosol of vast and diverse forms of bacteria. Among 150 different PHAs known so far, poly- 3-hydroxybutyrate is the most common and comprehensively characterized PHA. Interestingly, PHAs are only completely biodegradable plastics with material properties comparable to conventional plastics that can be achieved by regulating the co-monomers incorporation into PHAs backbone. PHA bioplastics are exploited in the form of user-friendly goods viz. films, absorbable sutures, bone plates, drug carriers, etc. Besides advantages, such useful entity(s) has major shortcomings as well like high production cost compared to conventional plastics. Precisely, in PHAs production, about fifty percent of the overall price is due to the carbon substrates. Consequently, exploring novel cost-effective substrates is a major compulsion for successful commercialization of this bioplastic, which is anticipated to reduce the cost of production as a result of advancing and intensifying research work. This review presents an insight and patent developments in the field of PHAs bioplastics.

  11. Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans.

    Science.gov (United States)

    Alva Munoz, Luis Esteban; Riley, Mark R

    2008-08-01

    Utilization of wastes from agriculture is becoming increasingly important due to concerns of environmental impact. The goals of this work were to evaluate the ability of an unusual organism, Saccharophagus degradans (ATCC 43961), to degrade the major components of plant cell walls and to evaluate the ability of S. degradans to produce polyhydroxyalkanoates (PHAs, also known as bioplastics). S. degradans can readily attach to cellulosic fibers, degrade the cellulose, and utilize this as the primary carbon source. The growth of S. degradans was assessed in minimal media (MM) containing glucose, cellobiose, avicel, and bagasse with all able to support growth. Cells were able to attach to avicel and bagasse fibers; however, growth on these insoluble fibers was much slower and led to a lower maximal biomass production than observed with simple sugars. Lignin in MM alone did not support growth, but did support growth upon addition of glucose, although with an increased adaptation phase. When culture conditions were switched to a nitrogen depleted status, PHA production commences and extends for at least 48 h. At early stationary phase, stained inclusion bodies were visible and two chronologically increasing infrared light absorbance peaks at 1,725 and 1,741 cm(-1) confirmed the presence of PHAs. This work demonstrates for what we believe to be the first time, that a single organism can degrade insoluble cellulose and under similar conditions can produce and accumulate PHA. Additional work is necessary to more fully characterize these capabilities and to optimize the PHA production and purification. (c) 2008 Wiley Periodicals, Inc.

  12. Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate).

    Science.gov (United States)

    Kenny, Shane T; Runic, Jasmina Nikodinovic; Kaminsky, Walter; Woods, Trevor; Babu, Ramesh P; Keely, Chris M; Blau, Werner; O'Connor, Kevin E

    2008-10-15

    The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting

  13. Impact of nitrogen feeding regulation on polyhydroxyalkanoates production by mixed microbial cultures.

    Science.gov (United States)

    Silva, Fernando; Campanari, Sabrina; Matteo, Stefania; Valentino, Francesco; Majone, Mauro; Villano, Marianna

    2017-07-25

    A sequencing batch reactor (SBR) is typically used for selecting mixed microbial cultures (MMC) for polyhydroxyalkanoate (PHA) production. Since many waste streams suitable as process feedstock for PHA production are nitrogen-deficient, a nutrient supply in the SBR is typically required to allow for efficient microbial growth. The scope of this study was to devise a nitrogen feeding strategy which allows controlling the nitrogen levels during the feast and famine regime of a lab-scale SBR, thereby selecting for PHA-storing microorganisms. At the beginning of the cycle the reactor was fed with a synthetic mixture of acetic and propionic acids at an overall organic load rate of 8.5gCODL -1 d -1 (i.e. 260CmmolL -1 d -1 ), whereas nitrogen (in the form of ammonium sulphate) was added either simultaneously to the carbon feed (coupled feeding strategy) or after the end of the feast phase (uncoupled feeding strategy). As a main result, PHA production was more than doubled (up to about 1300±64mgCODL -1 ) when carbon and nitrogen were separately fed and the higher PHA production also corresponded to an 82% increase in the polymer HV content (up to 20±1%, wtwt -1 ). Three SBR runs were performed with the uncoupled carbon and nitrogen feeding at different carbon to nitrogen (C/N) ratios (of 14.3, 17.9, and 22.3CmolNmol -1 , respectively) which were varied by progressively reducing the concentration of the nitrogen feeding. In spite of a comparable PHA storage yield at 14.3 and 17.9CmolNmol -1 (0.41±0.05 gCOD PHA gCOD VFA -1 and 0.38±0.05 gCOD PHA gCOD VFA -1 , respectively), the storage response of the selected MMC significantly decreased when the C/N ratio was set at the highest investigated value. Notably, an increase in this parameter also resulted in a change in the HV content in the polymer regardless the composition of the organic acids solution. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126.

    Science.gov (United States)

    Haywood, G W; Anderson, A J; Williams, D R; Dawes, E A; Ewing, D F

    1991-04-01

    A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.

  15. Production of Medium Chain Length Polyhydroxyalkanoates From Oleic Acid Using Pseudomonas putida PGA1 by Fed Batch Culture

    Directory of Open Access Journals (Sweden)

    Sidik Marsudi

    2010-10-01

    Full Text Available Bacterial polyhydroxyalkanoates (PHAs are a class of p0lymers currently receiving much attention because of their potential as renewable and biodegradable plastics. A wide variety of bacteria has been reported to produce PHAs including Pseudomonas strains. These strains are known as versatile medium chain length PHAs (PHAs-mcl producers using fatty acids as carbon source. Oleic acid was used to produce PHAs-mcl using Pseudomonas putida PGA 1 by continuous feeding of both nitrogen and carbon source, in a fed batch culture. During cell growth, PHAs also accumulated, indicating that PHA production in this organism is growth associated. Residual cell increased until the nitrogen source was depleted. At the end of fermentation, final cell concentration, PHA content, and roductivity were 30.2 g/L, 44.8 % of cell dry weight, and 0.188 g/l/h, respectively.

  16. Enhancing the Production of Polyhydroxyalkanoate Biopolymer by Azohydromonas Australica Using a Simple Empty and Fill Bioreactor Cultivation Strategy

    Directory of Open Access Journals (Sweden)

    G. Gahlawat

    2018-01-01

    Full Text Available Polyhydroxyalkanoates (PHAs are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, widespread use of these polymers is still hampered due to their high cost of production. This shortcoming could partly be resolved by obtaining high yields and productivity. In the present study, a drain-and-fill strategy of repeated-batch cultivation was adopted for the enhanced production of polyhydroxybutyrate PHB using Azohydromonas australica. In this strategy, 20 % (v/v of the culture broth was removed from the reactor and supplemented with an equal volume of fresh medium. This strategy demonstrated a 3.3 fold and 1.8 fold increase in PHB concentration and productivity, respectively, as compared to batch cultivation. Repeated cultivation had also the benefit of avoiding non-productive time required for cleaning, refilling and sterilization of bioreactor during batch, thereby increasing the overall volumetric productivity and industrial importance of the process.

  17. Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

    2014-07-01

    Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

  18. Investigation on ultrasonication mediated biosurfactant disintegration method in sludge flocs for enhancing hydrolytic enzymes activity and polyhydroxyalkanoates.

    Science.gov (United States)

    Sethupathy, A; Sivashanmugam, P

    2018-06-04

    In this study, a novel biosurfactant potential bacterial strain Pseudomonas pachastrellae RW43 was isolated from pulp and paper sludge and the biosurfactant namely rhamnolipid produced by Pseudomonas pachastrellae RW43 was investigated by varying pH and incubation time in batch liquid fermentation process. The maximal yield of rhamnolipid was found to be 12.1 g/L at an optimized condition of pH 7 and incubation time of 168 h. NMR analysis was performed for identification of molecular structure of produced rhamnolipid and its results concluded that the product was identified as di rhamnolipid. Then, statistically the global optimum conditions for hydrolytic enzymes extraction parameters (sonication power (100 W), extraction time (15 min) and rhamnolipid dosage (2% v/v)) were established. At 30,456 kJ/kg TS specific energy, ultrasonication with rhamnolipid disintegration method extracted maximal consortium activity of hydrolytic enzymes from mixed sludge (municipal and pulp & paper sludge) and the maximum observed were found to be 42.22, 51.75, 34.26, 24.21, 11.35 Units/g VSS respectively for protease, α-amylase, cellulase, lipase and α-glucosidase. Polyhydroxyalkanoates was recovered from enzymes extracted sludge using various solvents namely chloroform, sodium hypochlorite with chloroform and sodium lauryl sulfate with sodium hypochlorite. The maximum recovery was found to be 74 g/kg using sodium hypochlorite and chloroform extraction solvents.

  19. Production of polyhydroxyalkanoates (PHA) by bacterial consortium from excess sludge fermentation liquid at laboratory and pilot scales.

    Science.gov (United States)

    Jia, Qianqian; Xiong, Huilei; Wang, Hui; Shi, Hanchang; Sheng, Xinying; Sun, Run; Chen, Guoqiang

    2014-11-01

    The generation of polyhydroxyalkanoates (PHA) from excess sludge fermentation liquid (SFL) was studied at lab and pilot scale. A PHA-accumulated bacterial consortium (S-150) was isolated from activated sludge using simulated SFL (S-SFL) contained high concentration volatile fatty acids (VFA) and nitrogen. The maximal PHA content accounted for 59.18% in S-SFL and dropped to 23.47% in actual SFL (L-SFL) of the dry cell weight (DCW) at lab scale. The pilot-scale integrated system comprised an anaerobic fermentation reactor (AFR), a ceramic membrane system (CMS) and a PHA production bio-reactor (PHAR). The PHA content from pilot-scale SFL (P-SFL) finally reached to 59.47% DCW with the maximal PHA yield coefficient (YP/S) of 0.17 g PHA/g COD. The results indicated that VFA-containing SFL was suitable for PHA production. The adverse impact of excess nitrogen and non-VFAs in SFL might be eliminated by pilot-scale domestication, which might resulted in community structure optimization and substrate selective ability improvement of S-150. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Production of medium-chain-length polyhydroxyalkanoate by Pseudomonas oleovorans grown in sugary cassava extract supplemented with andiroba oil

    Directory of Open Access Journals (Sweden)

    Diego Aires da Silva

    2014-12-01

    Full Text Available Pseudomonas oleovorans were grown on sugary cassava extracts supplemented with andiroba oil for the synthesis of a mediumchain- length polyhydroxyalkanoate (PHA MCL. The concentration of total sugars in the extract was approximately: 40 g/L in culture 1, 15 g/L in cultures 2 and 3, and 10 g/L in culture 4. Supplementation with 1% andiroba oil and 0.2 g/L of (NH42HPO4 was performed 6.5 hours after growth in culture 3, and supplementation with the same amount of andiroba oil and 2.4 g/L of (NH42HPO4 was performed at the beginning of growth in culture 4. The synthesis resulted mainly in 3-hydroxy-decanoate and 3-hydroxy-dodecanoate units; 3-hydroxy-butyrate, 3-hydroxy-hexanoate; and 3-hydroxy-octanoate monomers were also produced but in smaller proportions. P. oleovorans significantly accumulated PHA MCL in the deceleration phase of growth with an oxygen limitation but with sufficient nitrogen concentration to maintain cell growth. The sugary cassava extract supplemented with andiroba oil proved to be a potential substrate for PHA MCL production.

  1. The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

    Science.gov (United States)

    La Rosa, Ruggero; de la Peña, Fernando; Prieto, María Axiliadora; Rojo, Fernando

    2014-01-01

    Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Mixed culture polyhydroxyalkanoates production from sugar molasses: the use of a 2-stage CSTR system for culture selection.

    Science.gov (United States)

    Albuquerque, M G E; Concas, S; Bengtsson, S; Reis, M A M

    2010-09-01

    Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers. The use of mixed microbial cultures (MMC) and low cost feedstocks have a positive impact on the cost-effectiveness of the process. It has typically been carried out in Sequencing Batch Reactors (SBR). In this study, a 2-stage CSTR system (under Feast and Famine conditions) was used to effectively select for PHA-storing organisms using fermented molasses as feedstock. The effect of influent substrate concentration (60-120 Cmmol VFA/L) and HRT ratio between the reactors (0.2-0.5h/h) on the system's selection efficiency was assessed. It was shown that Feast reactor residual substrate concentration impacted on the selective pressure for PHA storage (due to substrate-dependent kinetic limitation). Moreover, a residual substrate concentration coming from the Feast to the Famine reactor did not jeopardize the physiological adaptation required for enhanced PHA storage. The culture reached a maximum PHA content of 61%. This success opens new perspectives to the use of wastewater treatment infrastructure for PHA production, thus valorizing either excess sludge or wastewaters. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR.

    Science.gov (United States)

    Lane, Courtney E; Benton, Michael G

    2015-12-01

    A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result - amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. A screening method for the isolation of polyhydroxyalkanoate-producing purple non-sulfur photosynthetic bacteria from natural seawater

    Directory of Open Access Journals (Sweden)

    Mieko Higuchi-Takeuchi

    2016-09-01

    Full Text Available Polyhydroxyalkanoates (PHAs are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2 showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions.

  5. Optimization of critical factors to enhance polyhydroxyalkanoates (PHA) synthesis by mixed culture using Taguchi design of experimental methodology.

    Science.gov (United States)

    Venkata Mohan, S; Venkateswar Reddy, M

    2013-01-01

    Optimizing different factors is crucial for enhancement of mixed culture bioplastics (polyhydroxyalkanoates (PHA)) production. Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was applied to evaluate the influence and specific function of eight important factors (iron, glucose concentration, VFA concentration, VFA composition, nitrogen concentration, phosphorous concentration, pH, and microenvironment) on the bioplastics production. Three levels of factor (2(1) × 3(7)) variation were considered with symbolic arrays of experimental matrix [L(18)-18 experimental trails]. All the factors were assigned with three levels except iron concentration (2(1)). Among all the factors, microenvironment influenced bioplastics production substantially (contributing 81%), followed by pH (11%) and glucose concentration (2.5%). Validation experiments were performed with the obtained optimum conditions which resulted in improved PHA production. Good substrate degradation (as COD) of 68% was registered during PHA production. Dehydrogenase and phosphatase enzymatic activities were monitored during process operation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Analysis and optimization of process parameters for production of polyhydroxyalkanoates along with wastewater treatment by Serratia sp. ISTVKR1.

    Science.gov (United States)

    Gupta, Asmita; Kumar, Madan; Thakur, Indu Shekhar

    2017-10-01

    A previously reported biodegrading bacterial strain Serratia sp. ISTVKR1 was studied for polyhydroxyalkanoate (PHA) production along with wastewater contaminant removal. Nile red fluorescence, GC-MS, FT-IR, NMR and TEM confirmed the accumulation of homopolymer poly-3-hydroxyvalerate (PHV) within the bacterial cells. Analysis of culture after 72h of bacterial treatment showed maximum COD removal (8.4-fold), non-detection of organic contaminants such as 1H-Cyclopropa [a] naphthalene (R.T.=10.12) using GC-MS and increased proportion of elements like Cr, Mn, Fe, Ni, Cu, Cd and Pb in the bacterial cell pellets by SEM-EDX analysis. Optimization of process parameters for enhanced PHA production along with wastewater treatment done using Response Surface Methodology (RSM) showed 5% and 0.74% increase in the PHA production (0.3368±0.13gL -1 ) and % COD reduction (88.93±2.41) of wastewater, respectively. The study, thus established the production of PHA along with wastewater contaminant removal by Serratia sp. ISTVKR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The influence of substrate source on the growth of Ralstonia eutropha, aiming at the production of polyhydroxyalkanoate

    Directory of Open Access Journals (Sweden)

    Marangoni C.

    2001-01-01

    Full Text Available With the aim of producing polyhydroxyalkanoates, a study of the influence of culture conditions (nitrogen and carbon sources and temperature on the growth of Ralstonia eutropha in stirred flasks was carried out and the use of some low-cost sources (hydrolyzed lactose, inverted sugar and corn steep liquor as evaluated. The best specific growth rate was obtained when inverted sugar was utilized as the substrate (mumax = 0.26 h-1. Two different phases in the assimilation of the carbon source were observed when hydrolyzed lactose was present, suggesting the assimilation first of glucose and then of galactose. To confirm the growth of Ralstonia eutropha using galactose as the only carbon source, experiments were carried out and the results showed that this bacterium is able to grow in the presence of this sugar at a growth rate of 0.13 h-1. The use of galactose by Ralstonia eutropha for its growth has not been reported in the literature until now. Corn steep liquor was found to be a viable alternative nitrogen source to ammonium sulfate. The results of experiments carried out at 30°C and 34°C were similar.

  8. Microbial Community Composition of Polyhydroxyalkanoate-Accumulating Organisms in Full-Scale Wastewater Treatment Plants Operated in Fully Aerobic Mode

    Science.gov (United States)

    Oshiki, Mamoru; Onuki, Motoharu; Satoh, Hiroyasu; Mino, Takashi

    2013-01-01

    The removal of biodegradable organic matter is one of the most important objectives in biological wastewater treatments. Polyhydroxyalkanoate (PHA)-accumulating organisms (PHAAOs) significantly contribute to the removal of biodegradable organic matter; however, their microbial community composition is mostly unknown. In the present study, the microbial community composition of PHAAOs was investigated at 8 full-scale wastewater treatment plants (WWTPs), operated in fully aerobic mode, by fluorescence in situ hybridization (FISH) analysis and post-FISH Nile blue A (NBA) staining techniques. Our results demonstrated that 1) PHAAOs were in the range of 11–18% in the total number of cells, and 2) the microbial community composition of PHAAOs was similar at the bacterial domain/phylum/class/order level among the 8 full-scale WWTPs, and dominant PHAAOs were members of the class Alphaproteobacteria and Betaproteobacteria. The microbial community composition of α- and β-proteobacterial PHAAOs was examined by 16S rRNA gene clone library analysis and further by applying a set of newly designed oligonucleotide probes targeting 16S rRNA gene sequences of α- or β-proteobacterial PHAAOs. The results demonstrated that the microbial community composition of PHAAOs differed in the class Alphaproteobacteria and Betaproteobacteria, which possibly resulted in a different PHA accumulation capacity among the WWTPs (8.5–38.2 mg-C g-VSS−1 h−1). The present study extended the knowledge of the microbial diversity of PHAAOs in full-scale WWTPs operated in fully aerobic mode. PMID:23257912

  9. Whole genome amplification approach reveals novel polyhydroxyalkanoate synthases (PhaCs) from Japan Trench and Nankai Trough seawater.

    Science.gov (United States)

    Foong, Choon Pin; Lau, Nyok-Sean; Deguchi, Shigeru; Toyofuku, Takashi; Taylor, Todd D; Sudesh, Kumar; Matsui, Minami

    2014-12-24

    Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.

  10. Synthesis of Medium-Chain-Length Polyhydroxyalkanoate Homopolymers, Random Copolymers, and Block Copolymers by an Engineered Strain of Pseudomonas entomophila.

    Science.gov (United States)

    Wang, Ying; Chung, Ahleum; Chen, Guo-Qiang

    2017-04-01

    Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), widely used in medical area, are commonly synthesized by Pseudomonas spp. This study tries to use β-oxidation pathways engineered P. entomophila to achieve single source of a series of mcl-monomers for microbial production of PHA homopolymers. The effort is proven successful for the first time to obtain a wide range of mcl-PHA homopolymers from engineered P. entomophila LAC23 grown on various fatty acids, respectively, ranging from poly(3-hydroxyheptanoate) to poly(3-hydroxytetradecanoate). Effects of a PHA monomer chain length on thermal and crystallization properties including the changes of T m , T g , and T d5% are investigated. Additionally, strain LAC23 is used to synthesize random copolymers of 3-hydroxyoctanoate (3HO) and 3-hydroxydodecanoate (3HDD) or 3-hydroxytetradecanoates, their compositions could be controlled by adjusting the ratios of two related fatty acids. Meanwhile, block copolymer P(3HO)-b-P(3HDD) is synthesized by the same strain. It is found for the first time that even- and odd number mcl-PHA homopolymers have different physical properties. When the gene of the PHA synthase in the engineered P. entomophila is replaced by phaC from Aeromonas hydrophila 4AK4, poly(3-hydroxybutyrate-co-30 mol%-3-hydroxyhexanoate) is synthesized. Therefore, P. entomophila can be used to synthesize the whole range of PHA (C7-C14) homopolymers, random- and block copolymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Structure reveals regulatory mechanisms of a MaoC-like hydratase from Phytophthora capsici involved in biosynthesis of polyhydroxyalkanoates (PHAs).

    Science.gov (United States)

    Wang, Huizheng; Zhang, Kai; Zhu, Jie; Song, Weiwei; Zhao, Li; Zhang, Xiuguo

    2013-01-01

    Polyhydroxyalkanoates (PHAs) have attracted increasing attention as "green plastic" due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R)-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic. We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R)-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC. The data in our study reveal the regulatory mechanism of an (R)-hydratase, providing information on enzyme engineering to produce low cost PHAs.

  12. The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate.

    Science.gov (United States)

    Nikodinovic, Jasmina; Kenny, Shane T; Babu, Ramesh P; Woods, Trevor; Blau, Werner J; O'Connor, Kevin E

    2008-09-01

    Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers--polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.

  13. How Does Poly(hydroxyalkanoate) Affect Methane Production from the Anaerobic Digestion of Waste-Activated Sludge?

    Science.gov (United States)

    Wang, Dongbo; Zhao, Jianwei; Zeng, Guangming; Chen, Yinguang; Bond, Philip L; Li, Xiaoming

    2015-10-20

    Recent studies demonstrate that, besides being used for production of biodegradable plastics, poly(hydroxyalkanoate) (PHA) that is accumulated in heterotrophic microorganisms during wastewater treatment has another novel application direction, i.e., being utilized for enhancing methane yield during the anaerobic digestion of waste-activated sludge (WAS). To date, however, the underlying mechanism of how PHA affects methane production remains largely unknown, and this limits optimization and application of the strategy. This study therefore aims to fill this knowledge gap. Experimental results showed that with the increase of sludge PHA levels from 21 to 184 mg/g of volatile suspended solids (VSS) the methane yield linearly increased from 168.0 to 246.1 mL/g of VSS (R(2) = 0.9834). Compared with protein and carbohydrate (the main components of a cell), PHA exhibited a higher biochemical methane potential on a unit VSS basis. It was also found that the increased PHA not only enhanced cell disruption of PHA cells but also benefited the soluble protein conversion of both PHA- and non-PHA cells. Moreover, the reactor fed with higher PHA sludge showed greater sludge hydrolysis and acidification than those fed with the lower PHA sludges. Further investigations using fluorescence in situ hybridization and enzyme analysis revealed that the increased PHA enhanced the abundance of methanogenic Archaea and increased the activities of protease, acetate kinase, and coenzyme F420, which were consistent with the observed methane yield. This work provides insights into PHA-involved WAS digestion systems and may have important implications for future operation of wastewater treatment plants.

  14. Structure reveals regulatory mechanisms of a MaoC-like hydratase from Phytophthora capsici involved in biosynthesis of polyhydroxyalkanoates (PHAs.

    Directory of Open Access Journals (Sweden)

    Huizheng Wang

    Full Text Available Polyhydroxyalkanoates (PHAs have attracted increasing attention as "green plastic" due to their biodegradable, biocompatible, thermoplastic, and mechanical properties, and considerable research has been undertaken to develop low cost/high efficiency processes for the production of PHAs. MaoC-like hydratase (MaoC, which belongs to (R-hydratase involved in linking the β-oxidation and the PHA biosynthetic pathways, has been identified recently. Understanding the regulatory mechanisms of (R-hydratase catalysis is critical for efficient production of PHAs that promise synthesis an environment-friendly plastic.We have determined the crystal structure of a new MaoC recognized from Phytophthora capsici. The crystal structure of the enzyme was solved at 2.00 Å resolution. The structure shows that MaoC has a canonical (R-hydratase fold with an N-domain and a C-domain. Supporting its dimerization observed in structure, MaoC forms a stable homodimer in solution. Mutations that disrupt the dimeric MaoC result in a complete loss of activity toward crotonyl-CoA, indicating that dimerization is required for the enzymatic activity of MaoC. Importantly, structure comparison reveals that a loop unique to MaoC interacts with an α-helix that harbors the catalytic residues of MaoC. Deletion of the loop enhances the enzymatic activity of MaoC, suggesting its inhibitory role in regulating the activity of MaoC.The data in our study reveal the regulatory mechanism of an (R-hydratase, providing information on enzyme engineering to produce low cost PHAs.

  15. Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway.

    Science.gov (United States)

    Hoffmann, N; Steinbüchel, A; Rehm, B H

    2000-11-01

    Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6-C14), when grown on simple carbon sources such as, for example, gluconate or acetate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG. Southern hybridization experiments with digoxigenin-labeled phaG(Pp) from P. putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads. Although P. oleovorans does not accumulate PHA(MCL) from non-related carbon sources, its genomic DNA reveals a strong hybridization signal. We employed PCR to amplify this phaG homologue. The respective PCR product comprising the coding region of phaG(Po) was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaG(Po) from P. oleovorans exhibited about 95% amino acid sequence identity to PhaG(Pp) from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG(Po) was not transcribed even tinder inducing conditions, i.e. in the presence of gluconate as carbon source, whereas induction of phaG(Pp) transcription was obtained in P. putida. When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR84 complemented the phaG-negative mutant PhaG(N)-21 from P. putida. Interestingly, reintroduction of phaG(Po) under lac promoter control into the natural host P. oleovorans established PHA(MCL) synthesis from non-related carbon sources in this bacterium. These data indicated that phaG(Po) in P. oleovorans is not functionally expressed and does not exert its original function.

  16. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

    Directory of Open Access Journals (Sweden)

    Mariela V Catone

    Full Text Available Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB, a short chain length polyhydroxyalkanoate (sclPHA infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA. All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC in comparison with the mclPHA core genome genes (phaC1 and phaC2 indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

  17. Community proteomics provides functional insight into polyhydroxyalkanoate production by a mixed microbial culture cultivated on fermented dairy manure.

    Science.gov (United States)

    Hanson, Andrea J; Guho, Nicholas M; Paszczynski, Andrzej J; Coats, Erik R

    2016-09-01

    Polyhydroxyalkanoates (PHAs) are bio-based, biodegradable polyesters that can be produced from organic-rich waste streams using mixed microbial cultures (MMCs). To maximize PHA production, MMCs are enriched for bacteria with a high polymer storage capacity through the application of aerobic dynamic feeding (ADF) in a sequencing batch reactor (SBR), which consequently induces a feast-famine metabolic response. Though the feast-famine response is generally understood empirically at a macro-level, the molecular level is less refined. The objective of this study was to investigate the microbial community composition and proteome profile of an enriched MMC cultivated on fermented dairy manure. The enriched MMC exhibited a feast-famine response and was capable of producing up to 40 % (wt. basis) PHA in a fed-batch reactor. High-throughput 16S rRNA gene sequencing revealed a microbial community dominated by Meganema, a known PHA-producing genus not often observed in high abundance in enrichment SBRs. The application of the proteomic methods two-dimensional electrophoresis and LC-MS/MS revealed PHA synthesis, energy generation, and protein synthesis prominently occurring during the feast phase, corroborating bulk solution variable observations and theoretical expectations. During the famine phase, nutrient transport, acyl-CoA metabolism, additional energy generation, and housekeeping functions were more pronounced, informing previously under-determined MMC functionality under famine conditions. During fed-batch PHA production, acetyl-CoA acetyltransferase and PHA granule-bound phasin proteins were in increased abundance relative to the SBR, supporting the higher PHA content observed. Collectively, the results provide unique microbial community structural and functional insight into feast-famine PHA production from waste feedstocks using MMCs.

  18. Secondary Structural Models (16S rRNA of Polyhydroxyalkanoates Producing Bacillus Species Isolated from Different Rhizospheric Soil: Phylogenetics and Chemical Analysis

    Directory of Open Access Journals (Sweden)

    Swati Mohapatra

    2016-09-01

    Full Text Available Polyhydroxyalkanoates (PHAs producing bacterial isolates are gaining more importance over the world due to the synthesis of a biodegradable polymer which is extremely desirable to substitute synthetic plastics. PHAs are produced by various microorganisms under certain stress conditions. In this study, sixteen bacterial isolates characterized previously by partial 16S rRNA gene sequencing (NCBI Accession No. KF626466 to KF626481 were again stained by Nile red after three years of preservation in order to confirm their ability to accumulate PHAs. Also, phylogenetic analysis carried out in the present investigation evidenced that the bacterial species belonging to genus Bacillus are the dominant flora of the rhizospheric region, with a potentiality of biodegradable polymer (PHAs production. Again, RNA secondary structure prediction hypothesized that there is no direct correlation between RNA folding pattern stability with a rate of PHAs production among the selected isolates of genus Bacillus.

  19. Influence of sludge retention time on tolerance of copper toxicity for polyphosphate accumulating organisms linked to polyhydroxyalkanoates metabolism and phosphate removal.

    Science.gov (United States)

    Tsai, Yung-Pin; Chen, Hsiu-Ting

    2011-12-01

    This study explored the influence of sludge retention time (SRT) on tolerance of copper invasion for polyphosphate accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR). The experimental data showed the anaerobic polyhydroxyalkanoates (PHA) storage for the sludge at 10d SRT was less influenced by copper invasion than those at 5d and 15d SRTs. The reaction of PAOs aerobically taking up phosphate for the sludge at 5d or 15d SRT almost ceased at 2 mg Cu L(-1), whereas PAOs in the sludge at 10d SRT retained half of the ability to take up phosphate. Both the PHAs degradation and synthesis rates decreased with increasing copper concentration, regardless of the SRTs. However, the copper inhibition of the former was greater than that of the later. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Mesorhizobium bacterial strains isolated from the legume Lotus corniculatus are an alternative source for the production of polyhydroxyalkanoates (PHAs) to obtain bioplastics.

    Science.gov (United States)

    Marcos-García, Marta; García-Fraile, Paula; Filipová, Alena; Menéndez, Esther; Mateos, Pedro F; Velázquez, Encarna; Cajthaml, Tomáš; Rivas, Raúl

    2017-07-01

    Polyhydroxyalkanoic acids (PHAs) are natural polyesters that can be used to produce bioplastics which are biodegradable. Numerous microorganisms accumulate PHAs as energy reserves. Combinations of different PHAs monomers lead to the production of bioplastics with very different properties. In the present work, we show the capability of strains belonging to various phylogenetic lineages within the genus Mesorhizobium, isolated from Lotus corniculatus nodules, to produce different PHA monomers. Among our strains, we found the production of 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxydodecanoate, and 3-hydroxyhexadecanoate. Most of the PHA-positive strains were phylogenetically related to the species M. jarvisii. However, our findings suggest that the ability to produce different monomers forming PHAs is strain-dependent.

  1. Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil.

    Science.gov (United States)

    Phukon, Pinkee; Saikia, Jyoti Prasad; Konwar, Bolin Kumar

    2011-09-01

    Polyhydroxyalkanoate (PHA) was produced by growing Bacillus circulans (MTCC 8167) in the specific detection medium. The identification of the polymer as PHA was confirmed by fluorescence microscopy. The PHA was purified and characterized using FT-IR. The silver nanoparticles (SNP) were synthesized from AgNO3 in the dispersed colloids of PHA (0.085%) using NaBH4 (sodium borohydrate as reducing agent). The stability was tested using wave length scanning with a UV-Vis spectrophotometer and finally with transmission electron microscopy. The PHA stabilized solution was found to be stable for 30 days as against the low stability of silver nanoparticles (SNP) solution alone. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. [Physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, Ralstonia eutropha B8562].

    Science.gov (United States)

    Volova, T G; Kozhevnikov, I V; Dolgopolova, Iu B; Trusova, M Iu; Kalacheva, G S; Aref'eva, Iu V

    2005-01-01

    The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO2, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, beta-hydroxybutyrate was predominant (over 99 mol %); beta-hydroxyvalerate (0.25-0.72 mol %) and beta-hydroxyhexanoate (0.008-1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.

  3. The Molecular Level Characterization of Biodegradable Polymers Originated from Polyethylene Using Non-Oxygenated Polyethylene Wax as a Carbon Source for Polyhydroxyalkanoate Production.

    Science.gov (United States)

    Johnston, Brian; Jiang, Guozhan; Hill, David; Adamus, Grazyna; Kwiecień, Iwona; Zięba, Magdalena; Sikorska, Wanda; Green, Matthew; Kowalczuk, Marek; Radecka, Iza

    2017-08-28

    There is an increasing demand for bio-based polymers that are developed from recycled materials. The production of biodegradable polymers can include bio-technological (utilizing microorganisms or enzymes) or chemical synthesis procedures. This report demonstrates the corroboration of the molecular structure of polyhydroxyalkanoates (PHAs) obtained by the conversion of waste polyethylene (PE) via non-oxygenated PE wax (N-PEW) as an additional carbon source for a bacterial species. The N-PEW, obtained from a PE pyrolysis reaction, has been found to be a beneficial carbon source for PHA production with Cupriavidus necator H16. The production of the N-PEW is an alternative to oxidized polyethylene wax (O-PEW) (that has been used as a carbon source previously) as it is less time consuming to manufacture and offers fewer industrial applications. A range of molecular structural analytical techniques were performed on the PHAs obtained; which included nuclear magnetic resonance (NMR) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Our study showed that the PHA formed from N-PEW contained 3-hydroxybutyrate (HB) with 11 mol% of 3-hydroxyvalerate (HV) units.

  4. Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Shailesh S. Sawant

    2017-02-01

    Full Text Available Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs in pure cultures of marine bacterium, Saccharophagus degradans 2-40 (Sde 2-40, its contaminant, Bacillus cereus, and a co-culture of these bacteria (Sde 2-40 and B. cereus degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight. The present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.

  5. Agro-industrial residues and starch for growth and co-production of polyhydroxyalkanoate copolymer and α-amylase by Bacillus sp. CFR-67

    Directory of Open Access Journals (Sweden)

    T. R. Shamala

    2012-09-01

    Full Text Available Polyhydroxyalkanoates (PHA and α-amylase (α-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1 were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH or rice bran (RBH individually or in combination (5-20 g L-1, based on weight of soluble substrates-SS. In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L-1 of SS along with ammonium acetate (1.75 g L-1 and corn starch (30 g L-1 produced maximum quantity of biomass (10 g L-1 and PHA (5.9 g L-1. The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L-1 in the medium enhanced fermentative yield of α-amylase (2-40 U mL-1 min-1. The enzyme was active in a wide range of pH (4-9 and temperature (40-60ºC. This is the first report on simultaneous production of copolymer of bacterial PHA and α-amylase from unhydrolysed corn starch and agro-industrial residues as substrates.

  6. Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process

    Science.gov (United States)

    Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

    2016-08-01

    Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types.

  7. Polyhydroxyalkanoates (PHA) production from fermented crude glycerol: Study on the conversion of 1,3-propanediol to PHA in mixed microbial consortia

    DEFF Research Database (Denmark)

    Burniol Figols, Anna; Varrone, Cristiano; Daugaard, Anders Egede

    2018-01-01

    Crude glycerol, a by-product from the biodiesel industry, can be converted by mixed microbial consortia into 1,3-propanediol (1,3-PDO) and volatile fatty acids. In this study, further conversion of these main products into polyhydroxyalkanoates (PHA) was investigated with the focus on 1,3-PDO. Two...... different approaches for the enrichment of PHA accumulating microbial consortia using an aerobic dynamic feeding strategy were applied. With the first approach, where nitrogen was present during the whole cycle, no net production of PHA from 1,3-PDO was observed in the fermented effluent, not even...... the storage response. Nitrogen was still supplied during the famine phase. With the latter strategy, a net production of PHA from 1,3-PDO was observed at a yield of 0.24 Cmol PHA/Cmol 1,3-PDO. The overall yield from the fermented effluent was 0.42 Cmol PHA/Cmol substrate. Overall, the PHA yield from 1,3-PDO...

  8. Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

    Science.gov (United States)

    Franz, A; Rehner, R; Kienle, A; Grammel, H

    2012-01-01

    The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics. © 2011 The Authors. Letters in Applied Microbiology ©2011 The Society for Applied Microbiology.

  9. Bio-plastic (poly-hydroxy-alkanoate) production from municipal sewage sludge in the Netherlands: a technology push or a demand driven process?

    Science.gov (United States)

    Bluemink, E D; van Nieuwenhuijzen, A F; Wypkema, E; Uijterlinde, C A

    Valorisation of components from municipal 'waste' water and sewage sludge gets more and more attention in order to come to a circular economy by developing an efficient 'waste' to value concept. On behalf of the transition team 'Grondstoffenfabriek' ('Resource factory') a preliminary research was performed for all the Dutch water boards to assess the technical and economical feasibility of poly-hydroxy-alkanoate (PHA)-production from sewage sludge, a valuable product to produce bio-plastics. This study reveals that the production of bio-plastics from sewage sludge is feasible based on technical aspects, but not yet economically interesting, even though the selling price is relatively close to the actual PHA market price. (Selling price is in this particular case the indicative cost effective selling price. The cost effective selling price covers only the total production costs of the product.) Future process optimization (maximizing the volatile fatty acids production, PHA storage capacity, etc.) and market developments are needed and will result in cost reductions of the various sub-processes. PHA-production from sewage sludge at this stage is just a technology; every further research is needed to incorporate the backward integration approach, taking into account the market demand including associated product quality aspects.

  10. Insights into Feast-Famine polyhydroxyalkanoate (PHA)-producer selection: Microbial community succession, relationships with system function and underlying driving forces.

    Science.gov (United States)

    Huang, Long; Chen, Zhiqiang; Wen, Qinxue; Zhao, Lizhi; Lee, Duu-Jong; Yang, Lian; Wang, Yao

    2017-12-18

    The Feast-Famine (FF) process has been frequently used to select polyhydroxyalkanoate (PHA)-accumulating mixed cultures (MCs), but there has been little insight into the ecophysiology of the microbial community during the selection process. In three FF systems with well-defined conditions, synchronized variations in higher-order properties of MCs and complicate microbial community succession mainly including enrichment and elimination of non-top competitors and unexpected turnover of top competitors, were observed. Quantification of PHA-accumulating function genes (phaC) revealed that the top competitors maintained the PHA synthesis by playing consecutive roles when the highly dynamic turnover occurred. Due to its specific physiological characteristics during the PHA-accumulating process, Thauera strain OTU 7 was found to be responsible for the fluctuating SVI, which threatened the robustness of the FF system. This trait was also responsible for its later competitive exclusion by the other PHA-producer, Paracoccus strain OTU 1. Deterministic processes dominated the entire FF system, resulting in the inevitable microbial community succession in the acclimation phase and maintenance of the stable PHA-accumulating function in the maturation phase. However, neutral processes, likely caused by predation from bacterial phages, also occurred, which led to the unpredictable temporal dynamics of the top competitors. Copyright © 2017. Published by Elsevier Ltd.

  11. Selecting optimal feast-to-famine ratio for a new polyhydroxyalkanoate (PHA) production system fed by valerate-dominant sludge hydrolysate.

    Science.gov (United States)

    Hao, Jiuxiao; Wang, Hui; Wang, Xiujin

    2018-04-01

    The feast-to-famine ratio (F/F) represents the extent of selective pressure during polyhydroxyalkanoate (PHA) culture selection. This study evaluated the effects of F/F on a new PHA production system by an enriched culture with valerate-dominant sludge hydrolysate and selected the optimal F/F. After the original F/F 1/3 was modified to 1/1, 1/2, 1/4, and 1/5, F/F did not affect their lengths of feast phase, but affected their biomass growth behaviors during the famine phase and PHA-producing abilities. The optimal F/F was 1/2, and compared with 1/3, it increased the maximal PHA content and the fraction of 3-hydroxyvalerate (3HV) and 3-hydroxy-2-methylvalerate (3H2MV) monomers, with higher productivity and better polymer properties. Although F/F 1/2 impaired the advantage of the dominant genus Delftia, it improved the PHA production rate while decreased biomass growth rate, meanwhile enhancing the utilization and conversion of valerate. These findings indicate that in contrast to previous studies using acetate-dominant substrate for PHA production, the new system fed by valerate-dominant substrate can adopt a higher F/F.

  12. Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Le Meur Sylvaine

    2012-08-01

    Full Text Available Abstract Background Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs. To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. Results The genes encoding xylose isomerase (XylA and xylulokinase (XylB from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. Conclusions Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

  13. Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB.

    Science.gov (United States)

    Löwe, Hannes; Hobmeier, Karina; Moos, Manuel; Kremling, Andreas; Pflüger-Grau, Katharina

    2017-01-01

    One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low. Here, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO 2 . The co-culture consists of two bio - modules : Bio - module I , in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO 2 , converts it to sucrose, and exports it into the culture supernatant; and bio - module II , where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement. These results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida . The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.

  14. Microbial bio-based plastics from olive-mill wastewater: Generation and properties of polyhydroxyalkanoates from mixed cultures in a two-stage pilot scale system.

    Science.gov (United States)

    Ntaikou, I; Valencia Peroni, C; Kourmentza, C; Ilieva, V I; Morelli, A; Chiellini, E; Lyberatos, G

    2014-10-20

    The operational efficiency of a two stage pilot scale system for polyhydroxyalkanoates (PHAs) production from three phase olive oil mill wastewater (OMW) was investigated in this study. A mixed anaerobic, acidogenic culture derived from a municipal wastewater treatment plant, was used in the first stage, aiming to the acidification of OMW. The effluent of the first bioreactor that was operated in continuous mode, was collected in a sedimentation tank in which partial removal of the suspended solids was taking place, and was then forwarded to an aerobic reactor, operated in sequential batch mode under nutrient limitation. In the second stage an enriched culture of Pseudomonas sp. was used as initial inoculum for the production of PHAs from the acidified waste. Clarification of the acidified waste, using aluminium sulphate which causes flocculation and precipitation of solids, was also performed, and its effect on the composition of the acidified waste as well as on the yields and properties of PHAs was investigated. It was shown that clarification had no significant qualitative or quantitative effect on the primary carbon sources, i.e. short chain fatty acids and residual sugars, but only on the values of total suspended solids and total chemical oxygen demand of the acidified waste. The type and thermal characteristics of the produced PHAs were also similar for both types of feed. However the clarification of the waste seemed to have a positive impact on final PHAs yield, measured as gPHAs/100g of VSS, which reached up to 25%. Analysis of the final products via nuclear magnetic resonance spectroscopy revealed the existence of 3-hydroxybutyrate (3HB) and 3-hydroxyoctanoate (HO) units, leading to the conclusion that the polymer could be either a blend of P3HB and P3HO homopolymers or/and the 3HB-co-3HO co-polymer, an unusual polymer occurring in nature with advanced properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico

    Directory of Open Access Journals (Sweden)

    Carolina A. Martínez-Gutiérrez

    2018-05-01

    Full Text Available Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs, which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

  16. Polyhydroxyalkanoate Production on Waste Water Treatment Plants: Process Scheme, Operating Conditions and Potential Analysis for German and European Municipal Waste Water Treatment Plants

    Directory of Open Access Journals (Sweden)

    Timo Pittmann

    2017-06-01

    Full Text Available This work describes the production of polyhydroxyalkanoates (PHA as a side stream process on a municipal waste water treatment plant (WWTP and a subsequent analysis of the production potential in Germany and the European Union (EU. Therefore, tests with different types of sludge from a WWTP were investigated regarding their volatile fatty acids (VFA production-potential. Afterwards, primary sludge was used as substrate to test a series of operating conditions (temperature, pH, retention time (RT and withdrawal (WD in order to find suitable settings for a high and stable VFA production. In a second step, various tests regarding a high PHA production and stable PHA composition to determine the influence of substrate concentration, temperature, pH and cycle time of an installed feast/famine-regime were conducted. Experiments with a semi-continuous reactor operation showed that a short RT of 4 days and a small WD of 25% at pH = 6 and around 30 °C is preferable for a high VFA production rate (PR of 1913 mgVFA/(L×d and a stable VFA composition. A high PHA production up to 28.4% of cell dry weight (CDW was reached at lower substrate concentration, 20 °C, neutral pH-value and a 24 h cycle time. A final step a potential analysis, based on the results and detailed data from German waste water treatment plants, showed that the theoretically possible production of biopolymers in Germany amounts to more than 19% of the 2016 worldwide biopolymer production. In addition, a profound estimation regarding the EU showed that in theory about 120% of the worldwide biopolymer production (in 2016 could be produced on European waste water treatment plants.

  17. Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

    Directory of Open Access Journals (Sweden)

    Kim Tae

    2011-06-01

    Full Text Available Abstract Background Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO2 and H2 as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level. Results We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(--3hydroxybutyrate] (PHB under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16. Conclusion The

  18. A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

    Directory of Open Access Journals (Sweden)

    Zinn Manfred

    2011-04-01

    Full Text Available Abstract Background The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. Results To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate, PHA-unrelated (gluconate and poor PHA substrate (citrate. The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. Conclusions The study illustrates that the recruitment of a pleiotropic mutation, whose effects might

  19. Polyhydroxyalkanoate synthesis by mixed microbial consortia cultured on fermented dairy manure: Effect of aeration on process rates/yields and the associated microbial ecology.

    Science.gov (United States)

    Coats, Erik R; Watson, Benjamin S; Brinkman, Cynthia K

    2016-12-01

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with q PHA ranging from 0.10 to 0.19 CmmolPHA gVSS -1 min -1 ; VFA uptake rates exhibited similar statistical indifferences. PHA production assessments on the enriched MMC resulted in maximum intracellular concentrations ranging from 22.5 to 90.7% (mgPHA mgVSS -1 ); at maximum concentration, the mean hydroxyvalerate mol content was 73 ± 0.6%. While a typical feast-famine dissolved oxygen (DO) pattern was observed at maximum aeration, less resolution was observed at decreasing aeration rates, suggesting that DO may not be an optimal process monitoring parameter. At lower aeration states, nitrogen cycling patterns, supported by molecular investigations targeting AOBs and NOBs, indicate that NO 2 and NO 3 sustained feast-famine PHA synthesis. Next-generation sequencing analysis of the respective MMC revealed numerous and diverse genera exhibiting the potential to achieve PHA synthesis, suggesting functional redundancy embedded in the diverse

  20. Characterization of Polyhydroxyalkanoates Produced by ...

    African Journals Online (AJOL)

    Contaminated Soil Bacteria using Wastewater and Glucose as Carbon ... compare the use of wastewater as a carbon source for PHA production. .... The microorganisms were identified using ... the polymer production with respect to substrate.

  1. Polyhydroxyalkanoates (PHA) Bioplastic Packaging Materials

    Science.gov (United States)

    2010-05-01

    the non-isothermal melt-crystallization kinetics of the subject PHB copolymers. In this test, the specimen (cut from a disc compression molded at...of PHB was determined to be about 1.10 g/cm3 at 160 °C; this value was used in all the calculations. Specifically, the specimen disc was placed

  2. The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

    Directory of Open Access Journals (Sweden)

    Poblete-Castro Ignacio

    2012-03-01

    Full Text Available Abstract Background Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs, which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. Results We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h-1 was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. Conclusion This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

  3. Identificación Molecular de Bacterias Productoras de Polihidroxialcanoatos en Subproductos de Lácteos y Caña de Azúcar / Molecular Identification of Polyhydroxyalkanoate-Producing Bacteria Isolated from Dairy and Sugarcane Residues

    Directory of Open Access Journals (Sweden)

    Ana Carolina Cardona Echavarría

    2013-12-01

    Full Text Available Los polihidroxialcanoatos (PHAs son bioplásticostermoestables sintetizados por algunas bacterias, que losacumulan como reservas de carbono en forma de inclusiones citoplasmáticas. Estos compuestos se constituyen en una opción para la sustitución de polímeros sintéticos no biodegradables. En este trabajo se evaluó la presencia de bacterias productoras de PHAs en lactosueros derivados de la producción de quesos, y en melaza, cachaza y bagazo de caña de azúcar. El aislamiento bacteriano se realizó en medio mínimo de sales suplementadocon glucosa al 2% y 1 μL mL-1 de rojo Nilo (0,1%. Las colonias que presentaron fluorescencia a 340 nm en este medio, se evaluaron nuevamente mediante microscopía de fluorescenciacon azul Nilo. Aquellas cepas que resultaron positivas para ambaspruebas fueron consideradas como potenciales productoras de PHAs e identificadas por secuenciación de la región 16S del ADN ribosomal. Seguidamente se evaluó, en algunas de éstas, la presencia del gen phaC mediante PCR con cebadores específicos. Se detectaron 38 cepas productoras de PHAs, representando 18morfotipos bacterianos. Fueron identificadas en los sustratosde lactosuero cepas pertenecientes a los géneros Lactococcus, Klebsiella, Pseudomonas, Enterobacter y Enterococcus; mientras que en los subproductos de caña de azúcar se encontraron cepas de los géneros Bacillus, Enterobacter, Pantoea, Klebsiellay Gluconobacter. El gen phaC se detectó por PCR en 16 bacterias que presentaron los arreglos genéticos I y IV. Este trabajo abre la posibilidad de emplear las bacterias obtenidas en procesos alternativos, ambientalmente sostenibles y generadores de valoragregado, para la disposición final de subproductos y residuos agroindustriales. / Polyhydroxyalkanoates (PHAs are thermostable bioplastics produced by bacteria and stored as inclusion bodies to serve as a reserve carbon source. These compounds are a goodalternative to non-biodegradable synthetic plastics

  4. Production of Polyhydroxyalkanoates, a bacterial biodegradable ...

    African Journals Online (AJOL)

    African Journal of Biotechnology. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 3, No 1 (2004) >. Log in or Register to get access to full text downloads.

  5. Exobiopolymer from polyhydroxyalkanoate-producing transgenic yeast

    African Journals Online (AJOL)

    Subsequently, produced exopolymer was subject for further identification, characterization and analysis. Partial purification of exopolymer was performed and characterized as glycoprotein. HPLC analysis of the polymer revealed the presence of a fructose chain. The functional group analysis by FT-IR spectroscopy showed ...

  6. Polyhydroxyalkanoate production from unexplored sugar substrates

    Directory of Open Access Journals (Sweden)

    Alejandro Salazar

    2014-01-01

    Full Text Available La producción de biopolímeros a escala industrial es restringida por los elevados costos de producción, en comparación con aquellos asociados a polímeros sintéticos (no biodegradables y no biocompatibles. En este estudio evaluamos por primera vez dos sustratos de carbono de bajo costo (i.e. pulpa de algarrobo y jugo de fique para la producción a escala de laboratorio de polyhydroxyalcanoato (PHA con Bacillus megaterium. La detección e identificación de PHA se hizo mediante cromatografía de gases con detector selectivo de masas operado en el modo de Monitoreo de Ion Selectivo (GC-MS/SIM. Los resultados sugieren que la producción de PHA a partir de pulpa de algarrobo (de Hymenaea courbaril puede ser tan alta como con melaza de caña. Más aún, puede servir para la síntesis del tipo de PHA más comercializado (i.e. polihidroxibutirato; PHB y/o de otras variedades (e.g. polihidroxi-butirato-co-valerato; PHBV con diferentes propiedades y posibles aplicaciones.

  7. Synthesis, Characterization and Application of Thermoresponsive Polyhydroxyalkanoate-graft-Poly(N-isopropylacrylamide)

    DEFF Research Database (Denmark)

    Ma, Yi-Ming; Wei, Dai-Xu; Yao, Hui

    2016-01-01

    (3HDD-co-3H10U), a random copolymer of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-10-undecylenate (3H10U), via a thiol–ene click reaction. Enhanced hydrophilicity and thermoresponsive property of the resulted PHA-g-PNIPAm were confirmed by water contact angle studies. The biocompatibility of PHA...

  8. Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

    Science.gov (United States)

    Gangoiti, Joana; Santos, Marta; Prieto, María Auxiliadora; de la Mata, Isabel; Llama, María J.

    2012-01-01

    Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases. PMID:22865072

  9. Analysis of polyhydroxyalkanoates granuales in bacillus Sp. MFD11 and enterobacter Sp. SEL2

    International Nuclear Information System (INIS)

    Naheed, N.; Jamil, N.

    2016-01-01

    Bacillus sp. MFD11 (JF901809) and Enterobacter sp. SEL2 (JF901810) were isolated from agriculture waste contaminated sites. When fed with 2% glucose as a carbon source, these bacteria produced 75.26±0.45% and 76.61±0.28% PHA of their wet weight respectively. The accumulated PHA was extracted by direct addition of sodium dodysyl sulphate in the culture medium, which yielded 52.3±0.56 micro g/l and 136.21±0.45 micro g/l PHA respectively when assayed with Crotonic acid. The PHA detection medium (PDM) provided nutrient limitation condition which favored accumulation of PHA granules. A tremendous increase in cell size was observed when strain MFD11 was grown in PDM. The size of the granules as revealed by TEM micrographs spanned from 0.1 to 1.5 micro m which is quite large as compared to the size reported in the literature 0.2 to 0.5 micro m 18). (PHA polymer was analyzed by FTIR, GC/MS and proton Nuclear magnetic resonance. The intense absorption band in the spectrum at 1724-1740 cm -1 and 1215 cm -1 to 1280 corresponding to C=O and C-O stretching group, respectively, indicated that the both strains were PHA producers. GC/MS analysis indicated that the polymer produced were copolymers of PHB-co-PHV. NMR also suggested that the extracted PHA was not a homopolymer but was the blend of copolymers with 3HV in lower abundance. Differential calorimetric thermal analysis showed melting temperature of 163 and 169 degree C for PHA produced by both strains, respectively. However, the observed melting temperature was found to be lower than the standard PHB (Signa-aldrich). (author)

  10. Comparative life cycle assessment and financial analysis of mixed culture polyhydroxyalkanoate production.

    Science.gov (United States)

    Gurieff, Nicholas; Lant, Paul

    2007-12-01

    A life cycle assessment and financial analysis of mixed culture PHA (PHA(MC)) and biogas production was undertaken based on treating an industrial wastewater. Internal rate of return (IRR) and non-renewable CO(2)eq emissions were used to quantify financial viability and environmental impact. PHA(MC) was preferable to biogas production for treating the specified industrial effluent. PHA(MC) was also financially attractive in comparison to pure culture PHA production. Both PHA production processes had similar environmental impacts that were significantly lower than HDPE production. A large potential for optimisation exists for the PHA(MC) process as financial and environmental costs were primarily due to energy use for downstream processing. Under the conditions used in this work PHA(MC) was shown to be a viable biopolymer production process and an effective industrial wastewater treatment technology. This is the first study of its kind and provides valuable insight into the PHA(MC) process.

  11. Characterization and optimization of antibiotic resistant bacterial strains for polyhydroxyalkanoates (phas) production

    International Nuclear Information System (INIS)

    Rehman, S. U.; Jamil, N.; Hussain, S.

    2005-01-01

    In this investigation, sugarcane soil, sewage water and soil containing long chain hydrocarbons was screened to obtain bacterial strains that were able to synthesize poly-beta-hydroxyalkanoates (PHA). The potential to synthesize PHA was tested qualitatively by Sudan Black staining of colonies growing in glucose and sucrose. Sixteen bacterial strains were isolated, purified and characterized for Gram reaction, biochemical analysis and PHA production. Isolates showed a wide range of tolerance to different commonly used antibiotics. PHA extraction was done by solvent extraction and hypochlorite digestion method. PHA production was optimized for different nitrogen concentrations. (author)

  12. Stabilization of antimicrobial silver nanoparticles by a polyhydroxyalkanoate obtained from mixed bacterial culture.

    Science.gov (United States)

    Castro-Mayorga, J L; Martínez-Abad, A; Fabra, M J; Olivera, Catarina; Reis, M; Lagarón, J M

    2014-11-01

    The incorporation of antimicrobials into polymer matrices is a promising technology in the food packaging and biomedical areas. Among the most widely used antimicrobials, silver nanoparticles (AgNPs) have emerged as one of the most researched technologies to prevent microbial outbreaks. However, it is known that AgNPs are rather unstable and present patterns of agglomeration that might limit their application. In this work, AgNPs were produced by chemical reduction in suspensions of an unpurified poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) which was previously obtained from a mixed culture fermentation using a synthetic medium mimicking fermented cheese whey. The synthesis of AgNPs was carried out within the unpurified PHBV suspension (in situ) and by physical mixing (mix). The stability of crystalline and spherical nanoparticles (7±3nm) obtained in situ was found to be stable during at least 40 days. The results suggest that the unpurified PHBV appears to be a very efficient capping agent, preventing agglomeration and, thereby, stabilizing successfully the silver nanoparticles. The in situ obtained AgNP-PHBV materials were also found to exhibit a strong antibacterial activity against Salmonella enterica at low concentration (0.1-1ppm). Copyright © 2014 Elsevier B.V. All rights reserved.

  13. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

    NARCIS (Netherlands)

    Poblete-Castro, I.; Binger, D.; Rodrigues, A.; Becker, J.; Martins Dos Santos, V.A.P.; Wittmann, C.

    2013-01-01

    Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism

  14. A tuneable switch for controlling environmental degradation of bioplastics: addition of isothiazolinone to polyhydroxyalkanoates.

    Directory of Open Access Journals (Sweden)

    Catherine Anne Woolnough

    Full Text Available Controlling the environmental degradation of polyhydroxybutyrate (PHB and polyhydroxyvalerate (P(HB-co-HV bioplastics would expand the range of their potential applications. Combining PHB and P(HB-co-HV films with the anti-fouling agent 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOI, <10% w/w restricted microbial colonisation in soil, but did not significantly affect melting temperature or the tensile strength of films. DCOI films showed reduced biofouling and postponed the onset of weight loss by up to 100 days, a 10-fold increase compared to unmodified films where the microbial coverage was significant. In addition, the rate of PHA-DCOI weight loss, post-onset, reduced by about 150%; in contrast a recorded weight loss of only 0.05% per day for P(HB-co-HV with a 10% DCOI loading was observed. This is in stark contrast to the unmodified PHB film, where a recorded weight loss of only 0.75% per day was made. The 'switch' that initiates film weight loss, and its subsequent reduced rate, depended on the DCOI loading to control biofouling. The control of biofouling and environmental degradation for these DCOI modified bioplastics increases their potential use in biodegradable applications.

  15. A tuneable switch for controlling environmental degradation of bioplastics: addition of isothiazolinone to polyhydroxyalkanoates.

    Science.gov (United States)

    Woolnough, Catherine Anne; Yee, Lachlan Hartley; Charlton, Timothy Stuart; Foster, Leslie John Ray

    2013-01-01

    Controlling the environmental degradation of polyhydroxybutyrate (PHB) and polyhydroxyvalerate (P(HB-co-HV)) bioplastics would expand the range of their potential applications. Combining PHB and P(HB-co-HV) films with the anti-fouling agent 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOI, bioplastics increases their potential use in biodegradable applications.

  16. ACCUMULATION OF POLYHYDROXYALKANOIC ACIDS BY AZOTOBACTER CHROOCOCCUM MAL-201 FROM ORGANIC WASTE

    Directory of Open Access Journals (Sweden)

    Soma Pal Saha

    2013-08-01

    Full Text Available Azotobacter chroococcum MAL-201 (MTCC 3853, a free-living nitrogen-fixing bacterium accumulated intracellular poly(3-hydroxybutyric acid [P(3HB] accounting 69% of cell dry weight (CDW when grown in nitrogrn-free Stockdale medium containing 2% (w/v glucose. It also produced copolymer of poly(3-hydroxybutyrate co-3-hydroxyvalerate [P(3HB-co-3HV] using glucose as primary carbon source and valerate cas cosubstrate. To make the polymer production cost effective four types of waste material of different origin were tested for growth and polymer production. Stockdale medium supplemented with 1% (w/v waste materials failed to yield good growth and polymer accumulation. Two–step cultivation was adopted for better growth and enhanced polymer accumulation. The candy factory waste was most suitable for synthesis of P(3HB accounting 17.8 and 40.58% using single and two-step cultivation conditions respectively. Wastes of domestic and poultry origin produced P(3HB-co-3HV with 3HV content 28.8 and 21.5 mol% respectively in two-step cultivation. Increase concentration of these wastes resulted in further upliftment of 3HV content of polymer with reduced growth and polymer accumulation. However, at optimum incubation the strain MAL-201 cells accumulated P(3HB 48.5% of CDW (at 40h from candy factory waste and P(3HB-co-3HV 24.75 % of CDW with 3HV 34.65 mol % from domestic waste. Intrinsic viscosity, molecular weight and thermal degradation of the polymers accumulated in the cells grown in glucose, glucose with valerate and glucose with waste were compared.

  17. Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2.

    Science.gov (United States)

    Yezza, A; Fournier, D; Halasz, A; Hawari, J

    2006-11-01

    A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at alpha concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.

  18. A biodegradable rubber by crosslinking poly(hydroxyalkanoate) from Pseudomonas oleovorans

    NARCIS (Netherlands)

    DEKONING, GJM; VANBILSEN, HMM; LEMSTRA, PJ; HAZENBERG, W; Witholt, B.; Preusting, H.; VANDERGALIEN, JG; SCHIRMER, A; JENDROSSEK, D

    1994-01-01

    Poly((R)-3-hydroxyalkanoate)s (PHAs) are bacterial storage polyesters, currently receiving much attention because of their potential application as biodegradable and biocompatible plastics. Among them are the PHAs from Pseudomonas oleovorans, which are semicrystalline elastomers. Their applicability

  19. Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them

    Energy Technology Data Exchange (ETDEWEB)

    Bohmert-Tatarev, Karen; McAvoy, Susan; Peoples, Oliver P.; Snell, Kristi D.

    2015-08-04

    Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.

  20. Microbial synthesis of functional homo-, random, and block polyhydroxyalkanoates by β-oxidation deleted Pseudomonas entomophila

    DEFF Research Database (Denmark)

    Li, Shijun; Cai, Longwei; Wu, Linping

    2014-01-01

    -link with other PHA polymer chains. However, it has been very difficult to obtain structurally controllable functional homo-, random, or block PHA. For the first time, a β-oxidation deleted Pseudomonas entomophila was used to successfully synthesize random copolymers of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9...... be controlled to meet various requirements....

  1. Benzene containing polyhydroxyalkanoates homo- and copolymers synthesized by genome edited Pseudomonas entomophila

    DEFF Research Database (Denmark)

    Shen, Rui; Cai, Longwei; Meng, Dechuan

    2014-01-01

    Microbial synthesis of functional polymers has become increasingly important for industrial biotechnology. For the first time, it became possible to synthesize controllable composition of poly(3-hydroxyalkanoate) (P3HA) consisting of 3-hydroxydodecanoate (3HDD) and phenyl group on the side......HPhV) and 3-hydroxydodecanoate (3HDD) were synthesized when the strain was grown on mixtures of PVA and dodecanoic acid (DDA). Compositions of 3HPhV in P(3HPhV-co-3HDD) were controllable ranging from 3% to 32% depending on DDDA/PVA ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated...

  2. Development of a bioprocess to convert PET derived terephthalic acid and biodiesel derived glycerol to medium chain length polyhydroxyalkanoate

    Energy Technology Data Exchange (ETDEWEB)

    Kenny, Shane T.; Nikodinovic Runic, Jasmina; O' Connor, Kevin E. [University College Dublin (Ireland). School of Biomolecular and Biomedical Sciences; Kaminsky, Walter [Hamburg Univ. (Germany). Inst. of Technical and Macromolecular Chemistry; Woods, Trevor; Babu, Ramesh P. [Dublin Univ. (Ireland). Materials Ireland Polymer Research Center

    2012-08-15

    Sodium terephthalate (TA) produced from a PET pyrolysis product and waste glycerol (WG) from biodiesel manufacture were supplied to Pseudomonas putida GO16 in a fed-batch bioreactor. Six feeding strategies were employed by altering the sequence of TA and WG feeding. P. putida GO16 reached 8.70 g/l cell dry weight (CDW) and 2.61 g/l PHA in 48 h when grown on TA alone. When TA and WG were supplied in combination, biomass productivity (g/l/h) was increased between 1.3- and 1.7-fold and PHA productivity (g/l/h) was increased 1.8- to 2.2-fold compared to TA supplied alone. The monomer composition of the PHA accumulated from TA or WG was predominantly composed of 3-hydroxydecanoic acid. PHA monomers 3-hydroxytetradeeanoic acid and 3-hydroxytetradecenoic acid were not present in PHA accumulated from TA alone but were present when WG was supplied to the fermentation. When WG was either the sole carbon source or the predominant carbon source supplied to the fermentation the molecular weight of PHA accumulated was lower compared to PHA accumulated when TA was supplied as the sole substrate. Despite similarities in data for the properties of the polymers, PHAs produced with WG present in the PHA accumulation phase were tacky while PHA produced where TA was the sole carbon substrate in the polymer accumulation phase exhibited little or no tackiness at room temperature. The co-feeding of WG to fermentations allows for increased utilisation of TA. The order of feeding of WG and TA has an effect on TA utilisation and polymer properties. (orig.)

  3. Novel extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Streptomyces exfoliatus K10 DSMZ 41693

    DEFF Research Database (Denmark)

    Martinez, Virginia; de Santos, Patricia Gómez; García-Hidalgo, Javier

    2015-01-01

    reaction product. Markedly, PhaZSex2 is able to degrade functionalized polymers containing thioester groups in the side chain (PHACOS), releasing functional thioester-based monomers and oligomers demonstrating the potentiality of this novel biocatalyst for the industrial production of enantiopure (R)-3...

  4. Isolation of Bacillus sp Producing Polyhydroxyalkanoate (PHA from Isfahan Refinery Wastewater and Qualification of Production in Submerged Fermentation

    Directory of Open Access Journals (Sweden)

    Mahsa Keshavarz Azam

    2015-12-01

    Full Text Available Introduction: The aim of present study was isolation of polyhydroxybutyrate producing Bacillus species from oil refinery waste water, Isfahan, Iran and primarily optimization of production condition. Petroleum wastes are rich of carbon sources and have low amounts of nitrogen and phosphorus sources. AS the most important factor in production of intracellular inclusions is increasing the C/N ratio, it seemed that polyhydroxybutyrate producing microorganisms will be found in these wastes. Materials and methods: Bacillus species were isolated and purified from oil refinery wastewater. The polymer was verified using different staining procedures. Polymer was extracted by digestion method and the optimum production conditions were investigated in minimal salt medium with the organic carbon source by submerged fermentation. Production of polyhydroxybutyrate was studied using dry weight and optical density measurement. Results: Between various isolated Bacillus strains, two of them (B1 and B2 were polyhydroxybutyrate producers. Maximum PHA production based on dry weight and concentration were obtained for strain B1 after 72 hours incubation, at 31°C, in the presence of glucose as carbon source and yeast extract as nitrogen source, pH=7, and aeration in 120 rpm; and for strain B2 in the same condition, except optimal temperature which was 32°C. The most production amounts were 367 mg.ml-1 for B1 and 473 mg.ml-1 for B2 isolates. Also the most polymer percentage was 52/16 and 58.43 for B1 and B2 isolates respectively. Discussion and conclusion: The results showed that the production of polyhydroxybutyrate was increased by optimization of the conditions in both isolates. Using petroleum wastes as well as production of biodegradable plastics, leads to decontamination of theses wastes.

  5. Biosynthesis and characterization of polyhydroxyalkanoates produced by an extreme halophilic bacterium, Halomonas nitroreducens, isolated from hypersaline ponds.

    Science.gov (United States)

    Cervantes-Uc, J M; Catzin, J; Vargas, I; Herrera-Kao, W; Moguel, F; Ramirez, E; Rincón-Arriaga, S; Lizama-Uc, G

    2014-10-01

    Morphological, biochemical and genotypic characterization of a halophilic bacterium isolated from hypersaline ponds located at Las Coloradas (Río Lagartos, Yucatán, Mexico). Characterization of polymer produced by this strain was also performed. Twenty strains were isolated from water samples of salt ponds and selected based on both morphological features and their PHA storage capacity, which were determined by SEM and staining methods with Nile red and Nile blue, respectively; strains were also analysed by the fluorescence imaging technique. Among them, JCCOL25.8 strain showed the highest production of PHA's reason why phenotypic and genotypic characterization was performed; this strain was identified as Halomonas nitroreducens. Polymer produced by this strain was characterized by FTIR, DSC, GPC and EDX spectroscopy. Results indicated that the biosynthesized polymer was polyhydroxybutyrate (PHB) which had a melting peak at 170°C and a crystallinity percentage of about 36%. Based on phenotypic and genotypic aspects, JCCOL25.8 strain was identified as H. nitroreducens and it was capable to accumulate PHB. To our knowledge, there is only one study published on the biosynthesis of PHA's by H. nitroreducens strains, although the characterization of the obtained polymer was not reported. © 2014 The Society for Applied Microbiology.

  6. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun-Jung; Son, Hyeoncheol Francis [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); Kim, Sangwoo [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); School of Nono-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Ahn, Jae-Woo [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of); Kim, Kyung-Jin, E-mail: kkim@knu.ac.kr [Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701 (Korea, Republic of)

    2014-02-14

    Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

  7. Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Directory of Open Access Journals (Sweden)

    Ahmad Mohammed Gumel

    Full Text Available The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW basis were observed when fatty acids ranging from octanoic acid (C(8:0 to oleic acid (C(18:1 were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d of 264.6 to 318.8 (± 0.2 (oC, melting temperature (T(m of 43. (± 0.2 (oC, glass transition temperature (T(g of -1.0 (± 0.2 (oC and apparent melting enthalpy of fusion (ΔH(f of 100.9 (± 0.1 J g(-1.

  8. Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Science.gov (United States)

    Gumel, Ahmad Mohammed; Annuar, Mohamad Suffian Mohamad; Heidelberg, Thorsten

    2012-01-01

    The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C(8:0)) to oleic acid (C(18:1)) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1)H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d)) of 264.6 to 318.8 (± 0.2) (o)C, melting temperature (T(m)) of 43. (± 0.2) (o)C, glass transition temperature (T(g)) of -1.0 (± 0.2) (o)C and apparent melting enthalpy of fusion (ΔH(f)) of 100.9 (± 0.1) J g(-1).

  9. Pseudomonas putida KT2442 as a platform for the biosynthesis of polyhydroxyalkanoates with adjustable monomer contents and compositions

    DEFF Research Database (Denmark)

    Tripathi, Lakshmi; Wu, Lin-Ping; Dechuan, Meng

    2013-01-01

    of P(3HHx-co-3HD) consisting of 3-hydroxyhexanoate (3HHx) and 3-hydroxydecanoate (3HD), the monomer fraction of 3HHx ranged from 16 mol% to 63 mol%. The comonomer compositions were easily regulated by varying the fatty acid concentrations. P. putida KTQQ20 produced a novel diblock copolymer P3HHx-b-P(3......6), random copolymers of P(3HB-co-3HHx) consisting of 3-hydroxybutyrate (3HB), 3-hydroxyhexanoate (3HHx), were accumulated with 3HHx content ranged from 19 mol% to 75 mol%. While recombinant P. putida KTQQ20 grown on mixtures of sodium hexanoate and decanoic acid (C6:C10), produced random copolymers......HD-co-3HDD) consisting of 49 mol% P3HHx and 51 mol% P(3HD-co-3HDD) [35.25 mol% 3HDD (3-hydroxydodecanoate)], which was characterized by (13)C NMR, HMBC NMR, DSC, GPC and universal testing machine....

  10. Microbial community-based polyhydroxyalkanoates (PHAs) production from wastewater : Techno-economic analysis and ex-ante environmental assessment

    NARCIS (Netherlands)

    Fernandez Dacosta, Cora; Posada, John A.; Kleerebezem, Robbert; Cuellar, Maria C.; Ramirez, Andrea

    2015-01-01

    This work investigates the potential for polyhydroxybutyrate (PHB) production from wastewater, from a techno-economic and an environmental perspective, examining scale-up opportunities and bottlenecks prior to commercialisation. Conceptual process design, economic, environmental impacts and

  11. Comb-like temperature-responsive polyhydroxyalkanoate-graft-poly(2-dimethylamino-ethylmethacrylate) for controllable protein adsorption

    DEFF Research Database (Denmark)

    Yao, Hui; Wei, Daixu; Che, Xuemei

    2016-01-01

    %. Graft copolymer PHA-g-PDMAEMA showed enhanced thermal stability, biocompatibility and controllable hydrophilicity compared with non-grafted P(3HDD-co-3H9D). The grafted material presented an obvious tendency of increasing protein adsorptions over the lower critical solution temperature (LCST 47.5 °C...... controllable protein adsorption for biomedical usages....

  12. Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid.

    Science.gov (United States)

    Ashby, Richard D; Solaiman, Daniel K Y; Nuñez, Alberto; Strahan, Gary D; Johnston, David B

    2018-04-01

    Burkholderia sacchari was used to produce poly-3-hydroxybutyrate-co-3-hydroxyvalerate block copolymers from xylose and levulinic acid. Levulinic acid was the preferred substrate resulting in 3-hydroxyvalerate (3HV) contents as high as 95 mol% at 24 h. The 3HB:3HV ratios were controlled by the initial levulinic acid media concentration and fermentation length. Higher levulinic acid concentrations and longer durations, resulted in polymers with two glass transition temperatures, each approximating those associated with poly-3HB and poly-3HV. 13 C NMR confirmed the presence of high concentrations of 3HB-3HB and 3HV-3HV homopolymeric dyads, while mass spectrometry of the partial hydrolysis products did not conform to Bernoullian statistics for randomness, confirming block sequences. MS/MS analysis of specific oligomers showed the mass-loss of 86 amu (a 3HB unit) and 100 amu (a 3HV unit) attesting to some randomness within the polymers. This study verifies the potential for producing Poly-3HB-block-3HV copolymers from inexpensive biorenewable feedstocks without sequential addition of carbon sources. Published by Elsevier Ltd.

  13. In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process

    DEFF Research Database (Denmark)

    Liu, W.-T.; Nielsen, Alex Toftgaard; Wu, JH

    2001-01-01

    , electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI......, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA, No cellular inclusions were detected in most members of the alpha -Proteobacteria...

  14. Combined polyhydroxyalkanoates (PHA) and 1,3-propanediol production from crude glycerol: Selective conversion of volatile fatty acids into PHA by mixed microbial consortia

    DEFF Research Database (Denmark)

    Burniol Figols, Anna; Varrone, Cristiano; Le, Simone Balzer

    2018-01-01

    in the supernatant by means of mixed microbial consortia selection strategies. The process showed highly reproducible results in terms of PHA yield, 0.99 ± 0.07 Cmol PHA/Cmol S (0.84 g COD PHA/g COD S), PHA content (76 ± 3.1 g PHA/100 g TSS) and 1,3-PDO recovery (99 ± 2.1%). The combined process had an ultimate...

  15. Polyhydroxyalkanoate as a slow-release carbon source for in situ bioremediation of contaminated aquifers: From laboratory investigation to pilot-scale testing in the field.

    Science.gov (United States)

    Pierro, Lucia; Matturro, Bruna; Rossetti, Simona; Sagliaschi, Marco; Sucato, Salvatore; Alesi, Eduard; Bartsch, Ernst; Arjmand, Firoozeh; Papini, Marco Petrangeli

    2017-07-25

    A pilot-scale study aiming to evaluate the potential use of poly-3-hydroxy-butyrate (PHB) as an electron donor source for in situ bioremediation of chlorinated hydrocarbons in groundwater was conducted. Compared with commercially available electron donors, PHB offers a restricted fermentation pathway (i.e., through acetic acid and molecular hydrogen) by avoiding the formation of any residual carbon that could potentially spoil groundwater quality. The pilot study was carried out at an industrial site in Italy, heavily contaminated by different chlorinated aliphatic hydrocarbons (CAHs). Prior to field testing, PHB was experimentally verified as a suitable electron donor for biological reductive dechlorination processes at the investigated site by microcosm studies carried out on site aquifer material and measuring the quantitative transformation of detected CAHs to ethene. Owing to the complex geological characteristics of the aquifer, the use of a groundwater circulation well (GCW) was identified as a potential strategy to enable effective delivery and distribution of electron donors in less permeable layers and to mobilise contaminants. A 3-screened, 30-m-deep GCW coupled with an external treatment unit was installed at the site. The effect of PHB fermentation products on the in situ reductive dechlorination processes were evaluated by quantitative real-time polymerase chain reaction (qPCR). The results from the first 4 months of operation clearly demonstrated that the PHB fermentation products were effectively delivered to the aquifer and positively influenced the biological dechlorination activity. Indeed, an increased abundance of Dehalococcoides mccartyi (up to 6.6 fold) and reduced CAH concentrations at the installed monitoring wells were observed. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

    Energy Technology Data Exchange (ETDEWEB)

    Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

    2012-10-22

    Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

  17. Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid

    Science.gov (United States)

    Burkholderia sacchari DSM 17165 was used as a biocatalyst for the production of poly-3-hydroxybutyrate-co-3-hydroxyvalerate block copolymers (Poly-3HB-block-3HV) from xylose and levulinic acid. Among the carbon source mixtures, levulinic acid was preferred and was consumed early in the fermentations...

  18. Infrared and Raman spectroscopy and quantum chemistry calculation studies of C-H...O hydrogen bondings and thermal behavior of biodegradable polyhydroxyalkanoate

    Czech Academy of Sciences Publication Activity Database

    Sato, H.; Dybal, Jiří; Murakami, R.; Noda, I.; Ozaki, Y.

    744-747, - (2005), s. 35-46 ISSN 0022-2860 R&D Projects: GA AV ČR IAA4050208 Keywords : infrared and Raman spectroscopy * quantum chemical calculation * C-H...O hydrogen bonding Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.440, year: 2005

  19. Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA▿

    OpenAIRE

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-01-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in...

  20. Presidential Green Chemistry Challenge: 2005 Small Business Award

    Science.gov (United States)

    Presidential Green Chemistry Challenge 2005 award winner, Metabolix, used biotechnology to develop microorganisms that produce polyhydroxyalkanoates: natural, biodegradable plastics with a range of environmental benefits.

  1. Functional Expression of the Thiolase Gene thl from Clostridium beijerinckii P260 in Lactococcus lactis and Lactobacillus buchneri

    Science.gov (United States)

    The first step of the butanol pathway involves an acetyl-CoA acetyltransferase (ACoAAT), which controls the key branching point from acetyl-CoA to butanol. ACoAAT, also known as thiolase (EC 2.3.1.9), is encoded by the thl gene and catalyzes ligation of 2 acetyl-CoA into acetoacetyl-CoA. Bioinform...

  2. A life cycle assessment of poly-hydroxybutyrate extraction from microbial biomass using dimethyl carbonate

    DEFF Research Database (Denmark)

    Righi, Serena; Baioli, Filippo; Samorì, Chiara

    2017-01-01

    Poly-hydroxyalkanoates are an example of biodegradable and biocompatible polymers, produced from renewable raw materials. With respect to other bioplastics the market share of poly-hydroxyalkanoates is still limited because of their commercial costs. To develop more cost-effective processes, a mu...... by evaporation appears to be the most promising in terms of environmental sustainability performance....

  3. Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Seker, Tamay; Møller, Kasper; Nielsen, Jens

    2005-01-01

    The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl Co......A, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we...... used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase...

  4. GenBank blastx search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available droxybutyrate biosynthetic beta-ketothiolase (phbA), and PHB synthase (phbC) genes, complete cds.|BCT BCT 1e-56 +3 ... ...AK061948 001-042-E07 AF267243.2 Azotobacter vinelandii PhbF (phbF), putative phasin protein PhbP (phbP), putative transcription activator PhbR (phb...R), acetoacetyl-CoA reductase (phbB), polyhy

  5. GenBank blastx search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available droxybutyrate biosynthetic beta-ketothiolase (phbA), and PHB synthase (phbC) genes, complete cds.|BCT BCT 3e-82 +1 ... ...AK059654 001-031-C10 AF267243.2 Azotobacter vinelandii PhbF (phbF), putative phasin protein PhbP (phb...P), putative transcription activator PhbR (phbR), acetoacetyl-CoA reductase (phbB), polyhy

  6. Comamonas sp. EB172 isolated from digester treating palm oil mill ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-11-19

    Nov 19, 2008 ... palm oil mill effluent as potential polyhydroxyalkanoate ... Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, ... industry is actively looking for ways to minimize the ... methane gas.

  7. Recovery and characterization of poly(3-Hydroxybutyric acid ...

    African Journals Online (AJOL)

    Darshan

    Polyhydroxyalkanoates (PHA) are biodegradable polyesters accumulated intracellularly as energy resources by .... 20 min and filtered through Whatman No.1 filter paper and ... heating rate of 20°C air/min from ambient to a final temperature of.

  8. Comamonas sp. EB172 isolated from digester treating palm oil mill ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-11-19

    Nov 19, 2008 ... palm oil mill effluent as potential polyhydroxyalkanoate. (PHA) producer ... modifications to old ones to reduce the environmental impact of ... In this study, sludge was obtained from open digester tank (ODT) treating POME at ...

  9. A life-cycle assessment of poly-hydroxybutyrate extraction from microbial biomass using dimethylcarbonate

    DEFF Research Database (Denmark)

    Righi, Serena; Baioli, Filippo; Samorì, Chiara

    2016-01-01

    Plastic materials have wide commercial applicability. However, they are made from non-renewable resources and are characterised by resistance to degradation. Poly-hydroxyalkanoates (PHAs) provides one example of a polymer biodegradable, biocompatible and produced from renewable raw materials...

  10. Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8

    OpenAIRE

    Pettinari, M. Julia; Vázquez, Gustavo J.; Silberschmidt, Daniel; Rehm, Bernd; Steinbüchel, Alexander; Méndez, Beatriz S.

    2001-01-01

    Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucos...

  11. Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches.

    Science.gov (United States)

    Fox, Ana Romina; Soto, Gabriela; Mozzicafreddo, Matteo; Garcia, Araceli Nora; Cuccioloni, Massimiliano; Angeletti, Mauro; Salerno, Juan Carlos; Ayub, Nicolás Daniel

    2014-01-01

    Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route. © 2013.

  12. Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC.

    Science.gov (United States)

    Oosterkamp, Margreet J; Boeren, Sjef; Atashgahi, Siavash; Plugge, Caroline M; Schaap, Peter J; Stams, Alfons J M

    2015-06-01

    Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a β-hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to β-hydroxybutyrate that may be converted to the energy and carbon storage compound, poly-β-hydroxybutyrate. Accordingly, we confirmed the formation of poly-β-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Mixed Culture PHA Production With Alternating Feedstocks

    DEFF Research Database (Denmark)

    Oliveira, C.S.S.; Duque, A.F.; Carvalho, Gilda

    Polyhydroxyalkanoates (PHA) are a sustainable alternative to conventional plastics that can be obtained from industrial wastes/by-products using mixed microbial cultures (MMC). MMC PHA production is commonly carried out in a 3-stage process consisting of an acidogenic stage, a PHA producing cultu...

  14. (R)-3-hydroxyacyl-ACP:CoA transacylase of Pseudomonas chlororaphis: gene cloning, characterization and knock-out on PHA and rhamnolipid syntheses

    Science.gov (United States)

    Pseudomonas chlororaphis is a useful microorganism capable of producing polyhydroxyalkanoate (PHA) biopolymer and rhamnolipid (RL) biosurfactants by using carbon- and nitrogen-sources derived from renewable feedstocks as substrates of fermentation. We are interested in increasing the yield of RL pr...

  15. Enhanced alpha-galactosidase expression in pseudomonas chlororaphis

    Science.gov (United States)

    Pseudomonas chlororaphis is a non-pathogenic bacterium useful for fermentative production of biopolymer (i.e., poly(hydroxyalkanoates); PHA) and biosurfactant (i.e., rhamnolipid; RhL). In order to enable P. chlororaphis to better fermentatively utilize the residual soy sugars in soy molasses – a lo...

  16. Composite scaffolds for cartilage tissue engineering based on natural polymers of bacterial origin, thermoplastic poly(3-hydroxybutyrate) and micro-fibrillated bacterial cellulose

    Czech Academy of Sciences Publication Activity Database

    Akaraonye, E.; Filip, J.; Šafaříková, Miroslava; Salih, V.; Keshavarz, T.; Knowles, J.C.; Roy, I.

    2016-01-01

    Roč. 65, č. 7 (2016), s. 780-791 ISSN 0959-8103 Institutional support: RVO:60077344 Keywords : polyhydroxyalkanoates * poly(3-hydroxybutyrate) * bacterial cellulose * micro-fibrillated cellulose * tissue engineering scaffold * composite materials Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.070, year: 2016

  17. Characterization of the promising poly(3-hydroxybutyrate) producing halophilic bacterium Halomonas halophila

    Czech Academy of Sciences Publication Activity Database

    Kučera, D.; Pernicová, I.; Kovalčik, A.; Koller, M.; Müllerová, L.; Sedláček, P.; Mravec, F.; Nebesářová, Jana; Kalina, M.; Márová, I.; Krzyžánek, Vladislav; Obruča, S.

    2018-01-01

    Roč. 256, May (2018), s. 552-556 ISSN 0960-8524 R&D Projects: GA MŠk(CZ) LM2015062; GA ČR(CZ) GA15-20645S Institutional support: RVO:68081731 Keywords : Halomonas halophila * halophiles * lignocellulose hydrolysates * morphology of bacterial cells * polyhydroxyalkanoates Impact factor: 5.651, year: 2016

  18. Bacteria from contaminated urban and hilly areas as a source of ...

    African Journals Online (AJOL)

    Polyhydroxyalkanoates (PHA) production and extraction of different bacterial strains isolated from contaminated urban and hilly areas was conducted. The 30 bacterial isolates were Gram negative and belonged to Pseudomonas, Citrobacter, Klebsiella, Escherichia and Enterobacter genera. Bacterial level of resistance ...

  19. Isolation and characterization of two novel polyhydroxybutyrate ...

    African Journals Online (AJOL)

    Bioplastics are naturally occurring biodegradable polymers made from polyhydroxyalkanoates (PHA) of which poly 3-hydroxy butyric acid (PHB) is the most common. PHB serves as an energy storage molecule and accumulates intracellularly as storage granules in microbes. This work utilized the biodiversity of bacteria to ...

  20. African Journal of Biotechnology - Vol 10, No 34 (2011)

    African Journals Online (AJOL)

    ... microcephaly associated (ASPM) gene mutation is a major cause of primary ... Optimization of somatic embryogenesis induction in Iranian melon (Cucumis melo cv. ... related gene expression in response to enhanced UV-B radiation in Arabidopsis ... Exobiopolymer from polyhydroxyalkanoate-producing transgenic yeast ...

  1. Draft Genome Sequence of Halostagnicola sp. A56, an Extremely Halophilic Archaeon Isolated from the Andaman Islands

    Science.gov (United States)

    Kanekar, Sagar P.; Saxena, Neha; Pore, Soham D.; Arora, Preeti; Kanekar, P. P.

    2015-01-01

    The first draft genome of Halostagnicola sp. A56, isolated from the Andaman Islands is reported here. The A56 genome comprises 3,178,490 bp in 26 contigs with a G+C content of 60.8%. The genome annotation revealed that A56 could have potential applications for the production of polyhydroxyalkanoate or bioplastics. PMID:26564049

  2. Resource recovery from organic waste streams by microbial enrichment cultures

    NARCIS (Netherlands)

    Tamis, J.

    2015-01-01

    Polyhydroxyalkanoate (PHA) is a natural product that can potentially replace a part of the chemicals and plastics derived from fossil sources. One of the main barriers for market entry of PHA is its relatively high price compared to conventional (fossil) feedstocks. This high price is related to

  3. Biodegradable Polymeric Substances Produced by a Marine Bacterium from a Surplus Stream of the Biodiesel Industry

    DEFF Research Database (Denmark)

    Bhattacharya, Sourish; Dubey, Sonam; Singh, Priyanka

    2016-01-01

    epsilon-polylysine and 64.6% (w/w) intracellular polyhydroxyalkanoate (PHA) in the same fermentation broth (1 L shake flask) utilizing Jatropha biodiesel waste residues as carbon rich source by marine bacterial strain (Bacillus licheniformis PL26), isolated from west coast of India. The synthesized...

  4. African Journal of Biotechnology - Vol 11, No 31 (2012)

    African Journals Online (AJOL)

    Polyploidy levels of Chinese large-flower chrysanthemum determined by flow ... Segregation and expression of transgenes in the progenies of Bt transgenic rice ... Impact of crop residues on seed germination of native desert plants grown as ... Screening of root nodule bacteria for the production of polyhydroxyalkanoate ...

  5. PHA Production in Aerobic Mixed Microbial Cultures

    NARCIS (Netherlands)

    Johnson, K.

    2010-01-01

    Polyhydroxyalkanoate (PHA) is a common intracellular energy and carbon storage material in bacteria, which is considered as a bioplastic due to its plastic like properties. PHAs are versatile materials which are biodegradable and made from renewable resources. Commercial production of PHAs is

  6. Metal based pharmacologically active complexes of Cu(II), Ni(II) and Zn(II): synthesis, spectral, XRD, antimicrobial screening, DNA interaction and cleavage investigation.

    Science.gov (United States)

    Raman, Natarajan; Mahalakshmi, Rajkumar; Arun, T; Packianathan, S; Rajkumar, R

    2014-09-05

    The present contribution reports a thorough characterization of newly obtained metallointercalators incorporating Schiff bases, formed by the condensation of N-acetoacetyl-o-toluidine with 1-amino-4-nitrobenzene (L(1))/1-amino-4-chlorobenzene (L(2)) as main ligand and 1,10-phenanthroline as co-ligand respectively. The characterization of newly formed metallointercalators has been done by (1)H NMR, UV-Vis, IR, EPR spectroscopy and molar conductivity studies. X-ray powder diffraction illustrates that they are crystalline nature. Binding interaction of these complexes with calf thymus (CT-DNA) has been investigated by emission, absorption, viscosity, cyclic voltammetry and differential pulse voltammetry. DNA binding experiments results reveal that the synthesized complexes interact with DNA through intercalative mode. The in vitro antibacterial and antifungal assay indicate that these complexes are good antimicrobial agents against various pathogens. The DNA cleavage exhibits that they act as efficient cleaving agents. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity.

    Science.gov (United States)

    Subbaiah, P V; Chen, C H; Bagdade, J D; Albers, J J

    1985-01-01

    The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.

  8. Parallel and four-step synthesis of natural-product-inspired scaffolds through modular assembly and divergent cyclization

    Directory of Open Access Journals (Sweden)

    Hiroki Oguri

    2012-06-01

    Full Text Available By emulating the universal biosynthetic strategy, which employs modular assembly and divergent cyclizations, we have developed a four-step synthetic process to yield a collection of natural-product-inspired scaffolds. Modular assembly of building blocks onto a piperidine-based manifold 6, having a carboxylic acid group, was achieved through Ugi condensation, N-acetoacetylation and diazotransfer, leading to cyclization precursors. The rhodium-catalyzed tandem cyclization and divergent cycloaddition gave rise to tetracyclic and hexacyclic scaffolds by the appropriate choice of dipolarophiles installed at modules 3 and 4. A different piperidine-based manifold 15 bearing an amino group was successfully applied to demonstrate the flexibility and scope of the unified four-step process for the generation of structural diversity in the fused scaffolds. Evaluation of in vitro antitrypanosomal activities of the collections and preliminary structure–activity relationship (SAR studies were also undertaken.

  9. Obtención de polihidroxialcanoatos (PHA a partir de cultivos mixtos microbianos usando efluentes ricos en ácidos grasos volátiles como sustrato

    Directory of Open Access Journals (Sweden)

    Rolando Calero

    2015-10-01

      The aim of this work is about the study of bioreactors in obtaining biopolymers (polyhydroxyalkanoate by mixed microbial sources using cheese whey from the dairy industry as a substrate. To obtain polyhydroxyalkanoates (PHAs is necessary to start with  an enrichment process of the bacterial strain in a sequential batch reactor (SBR where the microbial culture is subjected to a selection process in order to achieve the highest levels of polymer reserve. The substrate used is mainly compound of volatile short chain fatty obtained in a preliminary stage of treatment in an anaerobic reactor wherein the sugar content of the whey is subjected to a process of acids fermentation. Once enriched the bacterial cultures with great capacity of storage of PHAs, we proceed in other reactor process known as a Fed-Batch in order to obtain a maximum production of biopolymer. In this experiment the PHA obtaining was about 42% compared with  the activate biomass.

  10. Tissue engineering of heart valves: in vitro experiences.

    Science.gov (United States)

    Sodian, R; Hoerstrup, S P; Sperling, J S; Daebritz, S H; Martin, D P; Schoen, F J; Vacanti, J P; Mayer, J E

    2000-07-01

    Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced

  11. Catabolismo de los polihidroxialcanoatos en la bacteria depredadora "Bdellovibrio bacteriovorus": apliaciones biotecnológicas y diseño de nuevos sistemas para la extracción de bioplástico en cultivos bacterianos

    OpenAIRE

    Martínez López, Virginia

    2013-01-01

    Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram-negative bacteria, including mcl-polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the ...

  12. Biodegradable Sonobuoy Decelerators

    Science.gov (United States)

    2015-06-01

    of Water Temperature and the Presence of Salt on the Disintegration Time of MonoSol A200 PVOH...polyhydroxyalkanoate (PHA). The proposed film would disintegrate , dissolve, and eventually biodegrade to prevent long-term effects on marine life. Ensuring no...Standard Specification for Non-Floating Biodegradable Plastics in the Marine Environment. Results showed that no PHA grades were toxic to the marine

  13. Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass

    Science.gov (United States)

    2014-01-01

    Background Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. Results The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. Conclusions In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants. PMID:25209261

  14. Integrated systems for biopolymers and bioenergy production from organic waste and by-products: a review of microbial processes.

    Science.gov (United States)

    Pagliano, Giorgia; Ventorino, Valeria; Panico, Antonio; Pepe, Olimpia

    2017-01-01

    Recently, issues concerning the sustainable and harmless disposal of organic solid waste have generated interest in microbial biotechnologies aimed at converting waste materials into bioenergy and biomaterials, thus contributing to a reduction in economic dependence on fossil fuels. To valorize biomass, waste materials derived from agriculture, food processing factories, and municipal organic waste can be used to produce biopolymers, such as biohydrogen and biogas, through different microbial processes. In fact, different bacterial strains can synthesize biopolymers to convert waste materials into valuable intracellular (e.g., polyhydroxyalkanoates) and extracellular (e.g., exopolysaccharides) bioproducts, which are useful for biochemical production. In particular, large numbers of bacteria, including Alcaligenes eutrophus , Alcaligenes latus , Azotobacter vinelandii , Azotobacter chroococcum , Azotobacter beijerincki , methylotrophs, Pseudomonas spp., Bacillus spp., Rhizobium spp., Nocardia spp., and recombinant Escherichia coli , have been successfully used to produce polyhydroxyalkanoates on an industrial scale from different types of organic by-products. Therefore, the development of high-performance microbial strains and the use of by-products and waste as substrates could reasonably make the production costs of biodegradable polymers comparable to those required by petrochemical-derived plastics and promote their use. Many studies have reported use of the same organic substrates as alternative energy sources to produce biogas and biohydrogen through anaerobic digestion as well as dark and photofermentation processes under anaerobic conditions. Therefore, concurrently obtaining bioenergy and biopolymers at a reasonable cost through an integrated system is becoming feasible using by-products and waste as organic carbon sources. An overview of the suitable substrates and microbial strains used in low-cost polyhydroxyalkanoates for biohydrogen and biogas

  15. Chicken feather hydrolysate as an inexpensive complex nitrogen source for PHA production by Cupriavidus necator on waste frying oils.

    Science.gov (United States)

    Benesova, P; Kucera, D; Marova, I; Obruca, S

    2017-08-01

    The chicken feather hydrolysate (FH) has been tested as a potential complex nitrogen source for the production of polyhydroxyalkanoates by Cupriavidus necator H16 when waste frying oil was used as a carbon source. The addition of FH into the mineral salt media with decreased inorganic nitrogen source concentration improved the yields of biomass and polyhydrohyalkanoates. The highest yields were achieved when 10 vol.% of FH prepared by microwave-assisted alkaline hydrolysis of 60 g l -1 feather was added. In this case, the poly(3-hydroxybutyrate) (PHB) yields were improved by more than about 50% as compared with control cultivation. A positive impact of FH was also observed for accumulation of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when sodium propionate was used as a precursor. The copolymer has superior processing and mechanical properties in comparison with PHB homopolymer. The application of FH eliminated the inhibitory effect of propionate and resulted in altered content of 3-hydroxyvalerate (3HV) in copolymer. Therefore, the hydrolysed feather can serve as an excellent complex source of nitrogen for the polyhydroxyalkanoates (PHA) production. Moreover, by the combination of two inexpensive types of waste, such as waste frying oil and feather hydrolysate, it is possible to produce PHA with substantially improved efficiency and sustainability. Millions of tons of feathers, important waste product of poultry-processing industry, are disposed off annually without any further benefits. Thus, there is an inevitable need for new technologies that enable ecologically and economically sensible processing of this waste. Herein, we report that alkali-hydrolysed feathers can be used as a complex nitrogen source considerably improving polyhydroxyalkanoates production on waste frying oil employing Cupriavidus necator. © 2017 The Society for Applied Microbiology.

  16. Estratégias de cultivo para produção dos plásticos biodegradáveis poli(3-Hidroxibutirato e poli(3-hidroxibutirato-co-3-hidroxivalerato por bactérias Cultivation strategies for production of the biodegradable plastics poly(3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate by bacteria

    Directory of Open Access Journals (Sweden)

    Cláudia Regina Squio

    2004-08-01

    Full Text Available Polyhydroxyalkanoates (PHAs are carbon and energy storage materials that are accumulated as intracellular granules in a variety of microorganisms during unbalanced growth. PHAs have drawn attention due to their properties similar to conventional plastics and complete biodegradability. They can be used for food and cosmetics packaging, and in medicine and agriculture. However, their applicability is reduced because of their high production cost compared to conventional plastics. An overview on production strategies of poly(3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyvalerate aiming at reducing the production costs is presented.

  17. Techno-economic feasibility of waste biorefinery

    DEFF Research Database (Denmark)

    Shahzad, Khurram; Narodoslawsky, Michael; Sagir, Muhammad

    2017-01-01

    elaborated a process for the production of polyhydroxyalkanoate (PHA) biopolymers starting from diverse waste streams of the animal processing industry. This article provides a detailed economic analysis of PHA production from this waste biorefinery concept, encompassing the utilization of low......-quality biodiesel, offal material and meat and bone meal (MBM). Techno-economic analysis reveals that PHA production cost varies from 1.41 €/kg to 1.64 €/kg when considering offal on the one hand as waste, or, on the other hand, accounting its market price, while calculating with fixed costs for the co...

  18. Water and vapor permeability at different temperatures of poly (3-Hydroxybutyrate dense membranes

    Directory of Open Access Journals (Sweden)

    Luiz H. Poley

    2005-03-01

    Full Text Available Polyhydroxyalkanoates (PHAs are polymers produced from renewable resources with biodegradability and biocompatibility, being therefore attractive for medical and pharmaceutical purposes. Poly (3-hydroxybutyrate (PHB is the most important polymer of this family by considering the biotechnology process of its synthesis. In the present study, dense films of PHB were prepared by casting from chloroform solutions (1% m/m. Permeability studies with water, methanol, ethanol and n-propanol were performed using the gravimetric method at different temperatures (from 50 ºC to 65 ºC. Results provide new data on permeability coefficients of PHB membranes.

  19. New tool for spreading proteins to the environment: Cry1Ab toxin immobilized to bioplastics.

    Science.gov (United States)

    Moldes, Cristina; Farinós, Gema P; de Eugenio, Laura I; García, Pedro; García, José L; Ortego, Félix; Hernández-Crespo, Pedro; Castañera, Pedro; Prieto, María A

    2006-08-01

    A new tool to provide an environmentally friendly way to deliver active proteins to the environment has been developed, based on the use of polyhydroxyalkanoate (PHA, bioplastic) granules. To illustrate this novel approach, a derived Cry1Ab insect-specific toxin protein was in vivo immobilized into PHA granules through the polypeptide tag BioF. The new toxin, named Fk-Bt1, was shown to be active against Sesamia nonagrioides (Lepidoptera: Noctuidae). The dose-mortality responses of the new toxin granule formulation (PFk-Bt1) and purified Cry1Ab have been compared, demonstrating the effectiveness of PFk-Bt1 and suggesting a common mode of action.

  20. Chain elongation and cyclization in type III PKS DpgA.

    Science.gov (United States)

    Wu, Hai-Chen; Li, Yi-San; Liu, Yu-Chen; Lyu, Syue-Yi; Wu, Chang-Jer; Li, Tsung-Lin

    2012-04-16

    Chain elongation and cyclization of precursors of dihydroxyphenylacetyl-CoA (DPA-CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di- and tri-ketidyl-CoA (DK- and TK-CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [(13)C(3)]malonyl-CoA (MA-CoA) was able to form partially (13)C-enriched DPA-CoA. By NMR and MS analysis, the distribution of (13)C atoms in the partially (13)C-enriched DPA-CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA-catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK-CoA, or one DK-CoA plus one acetoacetyl-CoA (AA-CoA), but not two molecules of AA-CoA can form one molecule of DPA-CoA. As a result, polyketidyl-CoA serves as both the starter and extender, whereas polyketone-CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Mechanistic studies on β-ketoacyl thiolase from Zoogloea ramigera: Identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

    International Nuclear Information System (INIS)

    Thompson, S.; Mayerl, F.; Walsh, C.T.; Peoples, O.P.; Masamune, S.; Sinskey, A.J.

    1989-01-01

    Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys 89 by acetylation with [ 14 C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degree C. Cys 89 has been converted to the alternate nucleophile Ser 89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser 89 enzyme retains 1% of the V max of the Cys 89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the V max in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [ 14 C]acetyl-CoA and isolation of the labeled Ser 89 -containing tryptic peptide. Comparisons of the Cys 89 and Ser 89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [ 32 P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA

  2. Culture engineering examination and metabolism flux distribution system analysis for madding to convert into poly {beta}- hydroxybutyric acid (PHB) using the hydrogen bacteria of organic acid got in liquid-phase oxidation of lignite; Kattan no ekiso sanka de erareru yukisan wo suiso saikin wo riyoshite pori {beta}-hidorokishi rakusan(PHB) ni henkan saseru tameno baiyo kogakuteki kento to taisha ryusoku bunpu shisutemu kaiseki

    Energy Technology Data Exchange (ETDEWEB)

    Tsujimoto, Kinko; Seki, Suito; Shimizu, Kazuyuki; Mae, Kazuhiro; Miura, Koichi

    1999-04-05

    The culture engineering examination for madding to convert into poly {beta} - hydroxy Wisteria (PHB) which glycolic acid. Acetic acid, ant acid, malonic acid got in liquid-phase oxidation of lignite are raw material of biodegradable plastic using hydrogen bacteria Alcaligenes eutrophus was carried out. It was proven that acetic acid was the most efficiently converted into the PHB as a result of cultivating these organic acid as a single carbon source. And, it was utilized to the bacterial cell at the order of ant acid, acetic acid, glycolic acid, when it was cultivated in mixing organic acid, and it was proven to convert into the PHB. Though the malonic acid was not utilized for the bacterial cell breeding, it was indicated that as the result which analyzed metabolism flow distribution by calculating using the culture data, the succinate dehydrogenase of the tricarboxylic acid (TCA) circuit received competitive inhibition, when this is added in culture middle point, and that the flux of griot lysyl acid route and gluconeogenesis route lowers. And, it was proven that it was utilized in the route which comes to the PHB synthesis from acetoacetyl CoA with the lowering of the ammonia concentration on NADPH produced from the isocitric acid, though it was prior consumed to the glutamic acid of tricarboxylic acid cycle in the route, if ammonia concentration is high. (translated by NEDO)

  3. Fermentation characteristics in conversion of organic acids obtained by oxidation of low-rank coals to poly({beta}-hydroxybutyrate) using A. eutrophus cells with some analysis on metabolic flux distribution; Kattan no ekisosanka de erareru yukisan wo suiso saikin wo riyoshite pori {beta}-hidorokishi rakusan (PHB) ni henkansaseru tameno baiyo kogakuteki kento to taisha ryusoku bunpu shisutemu kaiseki

    Energy Technology Data Exchange (ETDEWEB)

    Tsujimoto, Shoko.; Shin, Huidong.; Shimizu, Kazuyuki. [Kyushu Institute of Technology, Fukuoka (Japan). Department of Biochemical engineering and science; Mae, Kazuhiro.; Miura, Koichi. [Kyoto University, Kyoto (Japan). Department of Chemical Engineering

    1999-03-10

    Fermentation characteristics are investigated for the conversion of glycolate, acetate, formate, and malonate obtained by the oxidation of low-rank coals to poly ({beta}-hydrox butyrate) (PHB) using A. eutrophus cells. Based on the cultivation experiments using one of the organic acids as a sole carbon source, it is found that acetate is the most effectively converted to PHB. When mixed organic acids are used, formate is preferentially consumed, followed by acetate, and finally glycolate. Although malate can not be utilized, it is implied that it might change the pathway flux distributions based on the metabolic flux analysis. Namely, it shows competitive inhibition to succinate dehydrogenase so that its addition during fermentation results in flux reduction from succinate to maleic acid as well as glyoxylate flux and gluconeogenesis flux. It is also found that NADPH generated from isocitrate is preferentially utilized for the reaction from {alpha}-ketoglutarate to glutamate when NH{sub 3} concentration is high, while it is eventually used for the PHB production from acetoacetyl CoA as NH{sub 3} concentration decreases. (author)

  4. Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

    Science.gov (United States)

    Song, X Q; Fukao, T; Watanabe, H; Shintaku, H; Hirayama, K; Kassovska-Bratinova, S; Kondo, N; Mitchell, G A

    1998-01-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

  5. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

    Energy Technology Data Exchange (ETDEWEB)

    Steen, EricJ.; Chan, Rossana; Prasad, Nilu; Myers, Samuel; Petzold, Christopher; Redding, Alyssa; Ouellet, Mario; Keasling, JayD.

    2008-11-25

    BackgroundIncreasing energy costs and environmental concerns have motivated engineering microbes for the production of ?second generation? biofuels that have better properties than ethanol.Results& ConclusionsSaccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

  6. Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico.

    Science.gov (United States)

    McAnulty, Michael J; Yen, Jiun Y; Freedman, Benjamin G; Senger, Ryan S

    2012-05-14

    Genome-scale metabolic networks and flux models are an effective platform for linking an organism genotype to its phenotype. However, few modeling approaches offer predictive capabilities to evaluate potential metabolic engineering strategies in silico. A new method called "flux balance analysis with flux ratios (FBrAtio)" was developed in this research and applied to a new genome-scale model of Clostridium acetobutylicum ATCC 824 (iCAC490) that contains 707 metabolites and 794 reactions. FBrAtio was used to model wild-type metabolism and metabolically engineered strains of C. acetobutylicum where only flux ratio constraints and thermodynamic reversibility of reactions were required. The FBrAtio approach allowed solutions to be found through standard linear programming. Five flux ratio constraints were required to achieve a qualitative picture of wild-type metabolism for C. acetobutylicum for the production of: (i) acetate, (ii) lactate, (iii) butyrate, (iv) acetone, (v) butanol, (vi) ethanol, (vii) CO2 and (viii) H2. Results of this simulation study coincide with published experimental results and show the knockdown of the acetoacetyl-CoA transferase increases butanol to acetone selectivity, while the simultaneous over-expression of the aldehyde/alcohol dehydrogenase greatly increases ethanol production. FBrAtio is a promising new method for constraining genome-scale models using internal flux ratios. The method was effective for modeling wild-type and engineered strains of C. acetobutylicum.

  7. Carbonylation as a key reaction in anaerobic acetone activation by Desulfococcus biacutus.

    Science.gov (United States)

    Gutiérrez Acosta, Olga B; Hardt, Norman; Schink, Bernhard

    2013-10-01

    Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO2. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg(-1) protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.

  8. Fermentation and genomic analysis of acetone-uncoupled butanol production by Clostridium tetanomorphum.

    Science.gov (United States)

    Gong, Fuyu; Bao, Guanhui; Zhao, Chunhua; Zhang, Yanping; Li, Yin; Dong, Hongjun

    2016-02-01

    In typical acetone-butanol-ethanol (ABE) fermentation, acetone is the main by-product (50 % of butanol mass) of butanol production, resulting in a low yield of butanol. It is known that some Clostridium tetanomorphum strains are able to produce butanol without acetone in nature. Here, we described that C. tetanomorphum strain DSM665 can produce 4.16 g/L butanol and 4.98 g/L ethanol at pH 6.0, and 9.81 g/L butanol and 1.01 g/L ethanol when adding 1 mM methyl viologen. Butyrate and acetate could be reassimilated and no acetone was produced. Further analysis indicated that the activity of the acetate/butyrate:acetoacetyl-CoA transferase responsible for acetone production is lost in C. tetanomorphum DSM665. The genome of C. tetanomorphum DSM665 was sequenced and deposited in DDBJ, EMBL, and GenBank under the accession no. APJS00000000. Sequence analysis indicated that there are no typical genes (ctfA/B and adc) that are typically parts of an acetone synthesis pathway in C. tetanomorphum DSM665. This work provides new insights in the mechanism of clostridial butanol production and should prove useful for the design of a high-butanol-producing strain.

  9. Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly(3-hydroxybutyrate) (PHB) production.

    Science.gov (United States)

    Lin, Ji-Hong; Lee, Ming-Chieh; Sue, You-Sheng; Liu, Yung-Chuan; Li, Si-Yu

    2017-08-01

    PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD 600 , gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.

  10. A novel autolysis system controlled by magnesium and its application to poly (3-hydroxypropionate) production in engineered Escherichia coli.

    Science.gov (United States)

    Tamekou Lacmata, Stephen; Yao, Lan; Xian, Mo; Liu, Hui; Kuiate, Jules-Roger; Liu, Huizhou; Feng, Xinjun; Zhao, Guang

    2017-09-03

    The release of intracellular products, especially polyhydroxyalkanoates, is still a great challenge in industry. To solve this bottleneck, a novel autolysis system strictly controlled with magnesium was constructed and applied to poly(3-hydroxypropionate) production in engineered Escherichia coli. The autolysis system was constructed by inserting the 5'untranslated region (5'UTR) behind promoter PmgtA with lysis genes (S, R, and Rz, from E. coli) overexpressed. The autolysis system functioned well (lysis efficiency of more than 90%) in the P3HP producer with double plasmids containing lysis genes and P3HP biosynthesis genes, whereas the P3HP production was reduced due to plasmid losses. After the autolysis genes and P3HP biosynthesis genes were integrated into one plasmid, the P3HP content of 72.7% (2.4 times of the control) and the plasmid stability of 79.8 ± 3.1% were achieved in strain Q2646 with promoter PmgtA-UTR. However, the strain Q2647 with promoter PmgtA could not accumulate P3HP because of rapid cell lysis. The novel autolysis system activated in Mg 2+ -depleted conditions proves to be feasible for polyhydroxyalkanoates production, which may have great application potential for other intracellular products.

  11. Interface design of environmentally friendly carbon nanotube-filled polyester composites: Fabrication, characterisation, functionality and application

    Directory of Open Access Journals (Sweden)

    C. S. Wu

    2017-03-01

    Full Text Available Polyhydroxyalkanoate (PHA composites containing multi-walled carbon nanotubes (MWCNTs were prepared using a process of melt-blending three-dimensional (3D printing filaments. Maleic anhydride (MA-grafted polyhydroxyalkanoate (PHA-g-MA and chemically modified MWCNTs (MWCNTs-COOH were used to improve the compatibility and dispersibility of the MWCNTs within the PHA matrix. Structural, morphological, thermal and mechanical characterisations revealed dramatic enhancements in the thermal and mechanical properties of the PHA-g-MA/MWCNTs-COOH composites compared with PHA, because of the formation of ester carbonyl groups through the reaction between MA groups of PHA-g-MA and the carboxylic acid groups of the MWCNTs-COOH. For example, with an addition of 1.0 wt% of MWCNTs-COOH, the initial decomposition temperature and tensile strength at failure increased by 72 °C and 16.0 MPa, respectively. Moreover, MWCNTs or MWCNTs-COOH enhanced the antibacterial activity and static dissipative properties of the composites. Composites of PHA-g-MA or PHA containing MWCNTs or MWCNTs-COOH had better antibacterial activities and antistatic properties.

  12. Biodegradable Polymeric Substances Produced by a Marine Bacterium from a Surplus Stream of the Biodiesel Industry

    Directory of Open Access Journals (Sweden)

    Sourish Bhattacharya

    2016-11-01

    Full Text Available Crude glycerol is generated as a by-product during transesterification process and during hydrolysis of fat in the soap-manufacturing process, and poses a problem for waste management. In the present approach, an efficient process was designed for simultaneous production of 0.2 g/L extracellular ε-polylysine and 64.6% (w/w intracellular polyhydroxyalkanoate (PHA in the same fermentation broth (1 L shake flask utilizing Jatropha biodiesel waste residues as carbon rich source by marine bacterial strain (Bacillus licheniformis PL26, isolated from west coast of India. The synthesized ε-polylysine and polyhydroxyalkanoate PHA by Bacillus licheniformis PL26 was characterized by thermogravimetric analysis (TGA, differential scanning colorimetry (DSC, Fourier transform infrared spectroscopy (FTIR, and 1H Nuclear magnetic resonance spectroscopy (NMR. The PHA produced by Bacillus licheniformis was found to be poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV. The developed process needs to be statistically optimized further for gaining still better yield of both the products in an efficient manner.

  13. Bio-plastic (P-3HB-co-3HV) from Bacillus circulans (MTCC 8167) and its biodegradation.

    Science.gov (United States)

    Phukon, Pinkee; Saikia, Jyoti Prasad; Konwar, Bolin Kumar

    2012-04-01

    Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized by bacteria for carbon and energy storage and it has commercial potential as bioplastic. The bacterial species Bacillus circulans MTCC 8167, isolated from crude oil contaminated soil, can efficiently produce medium chain length polyhydroxyalkanoates (P-3HB-co-3HV) from cheap carbon sources like dextrose. The molecular mass of P-3HB-co-3HV was reported as 5.1×10(4)Da with polydispersity index of 1.21 by gel permeation chromatography. In the present investigation different bacteria and fungi species were used for testing the biodegradability of the extracted polymer. The FTIR spectra of the biodegraded PHBV film showed a decrease in the peak from 1735 cm(-1) (untreated film) to 1675 cm(-1), and disappearance of a peak present in the control at 2922 cm(-1) indicating the breakdown of ester (>C=O) or O-R group and -C=H bond, respectively. From biodegradability testing, the tested microorganisms were found to have decisive contribution to the biodegradation of P-3HB-co-3HV polymer. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules.

    Science.gov (United States)

    Sznajder, Anna; Jendrossek, Dieter

    2011-03-01

    A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

  15. Biodegradable plastics from Sinorhizobium meliloti as plastics compatible with the environment and human health

    Directory of Open Access Journals (Sweden)

    Mehrdad Hashemi Beidokhti

    2016-03-01

    Full Text Available Introduction: Polyhydroxyalkanoates (PHAs are natural polyesters and biodegradable plastics that are stored as intracellular inclusion bodies by a great variety of bacteria. The aim of this study was to extract polyhydroxyalkanoate from native Sinorhizobium meliloti in Iran. Materials and methods: Sinorhizobium meliloti isolates were collected from roots of alfalfa plants and were identified by Gram staining, biochemical experiments and amplification of 1500 bp fragment of 16Sr DNA gene. PHA granules were detected by microscopic examination. PHA production was evaluated in nutrient deficient medium and its amount was determined by conversion of PHA into crotonic acid by sulphuric acid treatment. The effect of various temperatures, agitation rate and carbon source (sucrose, mannitol, and maltose were evaluated on dry cell weight and polyhydroxybutyrate (PHB production. Results: The maximum amount of polymer production (43.10% was seen in basal mineral medium at 29°C, pH~7 and 215 revolutions per minute (rpm. The results of this research showed that the S5 isolate was capable to produce maximum poly3- hydroxybutyrate. The produced polymer was analyzed for its purity by GC- mass (gas chromatography- mass spectroscopy and confirmed to be PHB compared with the standard polymer. Discussion and conclusion: Native strains of Sinorhizobium can be used in the production of biodegradable plastics and the results of present study showed that S. meliloti S5 was capable to produce maximum PHB at 29°C, agitation rate of 215 rpm, and pH~7. 

  16. Metabolic pathways for ketone body production. 13C NMR spectroscopy of rat liver in vivo using 13C-multilabeled fatty acids

    International Nuclear Information System (INIS)

    Pahl-Wostl, C.; Seelig, J.

    1986-01-01

    The hormonal regulation of ketogenesis in the liver of living rat has been studied noninvasively with 13 C nuclear magnetic resonance. The spatial selection for the liver was better than 90%, with extrahepatic adipose tissue contribution only a very small amount of signal. The metabolic activities of the liver were investigated by infusion of 13 C-labeled butyrate in the jugular vein of the anesthetized rat. The rate of butyrate infusion was chosen to be close to the maximum oxidative capacity of the rat liver, and the 13 C signal intensities were enhanced by using doubly labeled [1,3- 13 C]butyrate as a substrate. Different 13 C NMR spectra and hence different metabolites were observed depending on the hormonal state of the animal. The 13 C NMR studies demonstrate that even when rate of acetyl-CoA production are high, the disposal of this compound is not identical in fasted and diabetic animals. This supports previous suggestions that the redox state of the mitochondrion represents the most important factor in regulation. For a given metabolic state of the animal, different signal intensities were obtained depending on whether butyrate was labeled at C-1, C-3, or C-1,3. From the ratios of incorporation of 13 C label into the carbons of 3-hydroxybutyrate, it could be estimated that a large fraction of butyrate evaded β-oxidation to acetyl-CoA but was converted directly to acetoacetyl-CoA. 13 C-labeled glucose could be detected in vivo in the liver of diabetic rats

  17. The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

    Science.gov (United States)

    Marcella, Aaron M; Barb, Adam W

    2017-12-01

    The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

  18. The metabolic network of Clostridium acetobutylicum: Comparison of the approximate Bayesian computation via sequential Monte Carlo (ABC-SMC) and profile likelihood estimation (PLE) methods for determinability analysis.

    Science.gov (United States)

    Thorn, Graeme J; King, John R

    2016-01-01

    The Gram-positive bacterium Clostridium acetobutylicum is an anaerobic endospore-forming species which produces acetone, butanol and ethanol via the acetone-butanol (AB) fermentation process, leading to biofuels including butanol. In previous work we looked to estimate the parameters in an ordinary differential equation model of the glucose metabolism network using data from pH-controlled continuous culture experiments. Here we combine two approaches, namely the approximate Bayesian computation via an existing sequential Monte Carlo (ABC-SMC) method (to compute credible intervals for the parameters), and the profile likelihood estimation (PLE) (to improve the calculation of confidence intervals for the same parameters), the parameters in both cases being derived from experimental data from forward shift experiments. We also apply the ABC-SMC method to investigate which of the models introduced previously (one non-sporulation and four sporulation models) have the greatest strength of evidence. We find that the joint approximate posterior distribution of the parameters determines the same parameters as previously, including all of the basal and increased enzyme production rates and enzyme reaction activity parameters, as well as the Michaelis-Menten kinetic parameters for glucose ingestion, while other parameters are not as well-determined, particularly those connected with the internal metabolites acetyl-CoA, acetoacetyl-CoA and butyryl-CoA. We also find that the approximate posterior is strongly non-Gaussian, indicating that our previous assumption of elliptical contours of the distribution is not valid, which has the effect of reducing the numbers of pairs of parameters that are (linearly) correlated with each other. Calculations of confidence intervals using the PLE method back this up. Finally, we find that all five of our models are equally likely, given the data available at present. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

    Science.gov (United States)

    Tsai, Meng-Tsz; Chen, Yu-Jen; Chen, Ching-Yi; Tsai, Mong-Hsun; Han, Chia-Li; Chen, Yu-Ju; Mersmann, Harry J; Ding, Shih-Torng

    2017-03-01

    Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model. Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis. Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 μM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined. Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S -transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition ( P steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro ( P < 0.05). Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic indicators for NAFLD in the future. © 2017 American Society for Nutrition.

  20. The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice

    Directory of Open Access Journals (Sweden)

    Weinberg Michael V

    2008-01-01

    Full Text Available Abstract Background Helicobacter pylori colonizes the human stomach and is the etiological agent of peptic ulcer disease. All three H. pylori strains that have been sequenced to date contain a potential operon whose products share homology with the subunits of acetone carboxylase (encoded by acxABC from Xanthobacter autotrophicus strain Py2 and Rhodobacter capsulatus strain B10. Acetone carboxylase catalyzes the conversion of acetone to acetoacetate. Genes upstream of the putative acxABC operon encode enzymes that convert acetoacetate to acetoacetyl-CoA, which is metabolized further to generate two molecules of acetyl-CoA. Results To determine if the H. pylori acxABC operon has a role in host colonization the acxB homolog in the mouse-adapted H. pylori SS1 strain was inactivated with a chloramphenicol-resistance (cat cassette. In mouse colonization studies the numbers of H. pylori recovered from mice inoculated with the acxB:cat mutant were generally one to two orders of magnitude lower than those recovered from mice inoculated with the parental strain. A statistical analysis of the data using a Wilcoxin Rank test indicated the differences in the numbers of H. pylori isolated from mice inoculated with the two strains were significant at the 99% confidence level. Levels of acetone associated with gastric tissue removed from uninfected mice were measured and found to range from 10–110 μmols per gram wet weight tissue. Conclusion The colonization defect of the acxB:cat mutant suggests a role for the acxABC operon in survival of the bacterium in the stomach. Products of the H. pylori acxABC operon may function primarily in acetone utilization or may catalyze a related reaction that is important for survival or growth in the host. H. pylori encounters significant levels of acetone in the stomach which it could use as a potential electron donor for microaerobic respiration.

  1. Binding of chlorfenvinphos and malathion with DNA and their detection using new sensitive luminescent Tb(III) complex probe

    Energy Technology Data Exchange (ETDEWEB)

    Azab, Hassan A. [Chemistry Department, Faculty of Science, Suez Canal University, 41522 Ismailia (Egypt); Kamel, Rasha M., E-mail: rashamoka@yahoo.com [Chemistry Department, Faculty of Science, Suez University, 43518 Suez (Egypt)

    2016-02-15

    In this work, the interaction of new luminescent terbium-N(acetoacetyl)-3-allyl-2hydroxybenzaldehyde hydrazone complex in a metal ligand ratio 1:2 in ethanol was studied with different pesticides, using fluorescence technique. The results indicate that the pesticides chlorfenvinphos and malathion exhibit quenching effect on the characteristics emission peak for Tb(III) at λ=545 nm. The luminescence variations values using the probe Tb(III)(HBH){sub 2} fit Stern–Volmer equation, where the detection limits are 4.53 and 9.59 µmol/L for chlorfenvinphos and malathion, respectively. The thermodynamic parameters associated with the interaction of the complex with the two pesticides were calculated, where the reaction is spontaneous through the obtained negative values of free energy change ΔG, and the process is of exothermic nature with decrease in entropy. Hence the nature of forces acting between the complex and pesticide molecules are Vander Val's and hydrogen bond. Also, it was found that the quenching mechanism is static type. Effect of some relevant interferents on the detection of pesticides has been investigated. The new sensing complex was applied to the determination of the pesticides in different real water samples (tap, river, and waste water) with precise recovery. Also, the effect of different concentrations of pesticides on the fluorescence spectrum of DNA was studied. - Highlights: • The method can be used for detection of pesticides in environmental samples. • The DL is 4.53 and 9.59 µmol L{sup −1} for chlorfenvinphos and malathion respectively. • Stern–Volmer studies at different temperatures were studied. • Effect of some interferents on the detection of pesticides has been investigated.

  2. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

    Science.gov (United States)

    Sznajder, Anna; Pfeiffer, Daniel; Jendrossek, Dieter

    2015-03-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Production of (R)-3-hydroxybutyric acid by Arxula adeninivorans.

    Science.gov (United States)

    Biernacki, Mateusz; Riechen, Jan; Hähnel, Urs; Roick, Thomas; Baronian, Kim; Bode, Rüdiger; Kunze, Gotthard

    2017-12-01

    (R)-3-hydroxybutyric acid can be used in industrial and health applications. The synthesis pathway comprises two enzymes, β-ketothiolase and acetoacetyl-CoA reductase which convert cytoplasmic acetyl-CoA to (R)-3-hydroxybutyric acid [(R)-3-HB] which is released into the culture medium. In the present study we used the non-conventional yeast, Arxula adeninivorans, for the synthesis enantiopure (R)-3-HB. To establish optimal production, we investigated three different endogenous yeast thiolases (Akat1p, Akat2p, Akat4p) and three bacterial thiolases (atoBp, thlp, phaAp) in combination with an enantiospecific reductase (phaBp) from Cupriavidus necator H16 and endogenous yeast reductases (Atpk2p, Afox2p). We found that Arxula is able to release (R)-3-HB used an existing secretion system negating the need to engineer membrane transport. Overexpression of thl and phaB genes in organisms cultured in a shaking flask resulted in 4.84 g L -1 (R)-3-HB, at a rate of 0.023 g L -1  h -1 over 214 h. Fed-batch culturing with glucose as a carbon source did not improve the yield, but a similar level was reached with a shorter incubation period [3.78 g L -1 of (R)-3-HB at 89 h] and the rate of production was doubled to 0.043 g L -1  h -1 which is higher than any levels in yeast reported to date. The secreted (R)-3-HB was 99.9% pure. This is the first evidence of enantiopure (R)-3-HB synthesis using yeast as a production host and glucose as a carbon source.

  4. A study on thermal properties of biodegradable polymers using photothermal methods

    Science.gov (United States)

    Siqueira, A. P. L.; Poley, L. H.; Sanchez, R.; da Silva, M. G.; Vargas, H.

    2005-06-01

    In this work is reported the use of photothermal techniques applied to the thermal characterization of biodegradable polymers of Polyhydroxyalkanoates (PHAs) family. This is a family of polymer produced by bacteria using renewable resources. It exhibits thermoplastic properties and therefore it can be an alternative product for engineering plastics, being also applied as packages for food industry and fruits. Thermal diffusivities were determined using the open photoacoustic cell (OPC) configuration. Specific heat capacity measurements were performed monitoring temperature of the samples under white light illumination against time. Typical values obtained for the thermal properties are in good agreement with those found in the literature for other polymers. Due to the incorporation of hydroxyvalerate in the monomer structure, the thermal diffusivity and thermal conductivity increase reaching a saturation value, otherwise the specific thermal capacity decreases as the concentration of the hydroxyvalerate (HV) increases. These results can be explained by polymers internal structure and are allowing new applications of these materials.

  5. Influence of gamma radiation on thermal properties and water vapor transmission of poly(3-hydroxybutyrate) (PHB) in blends

    Energy Technology Data Exchange (ETDEWEB)

    Forster, Pedro L.; Martins, Natalia A.; Parra, Duclerc F.; Egute, Nayara S.; Lugao, Ademar B., E-mail: dfparra@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2009-07-01

    Biodegradable polymers are a newly emerging field. A vast number of biodegradable polymers have been synthesized recently and some microorganisms and enzymes capable of degrading them have been identified. Polyesters such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs) have attracted commercial and academic interest as new biodegradable materials. In this work, we investigated the effect of gamma radiation on the thermal properties and biodegradation behavior of PHB in blend with poly(ethyleneglycol)(PEG). The samples were irradiated at gamma radiation of 5 and 10 kGy. The thermal behaviour was investigated by utilization of differential scanning calorimetry (DSC) changes in thermal stability, glass transition and melting point were reported. (author)

  6. Novel Essential Role of Ethanol Oxidation Genes at Low Temperature Revealed by Transcriptome Analysis in the Antarctic Bacterium Pseudomonas extremaustralis

    DEFF Research Database (Denmark)

    Tribelli, Paula Maria; Solar Venero, Esmeralda C.; Ricardi, Martiniano M

    2015-01-01

    Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures....... Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB...... with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures...

  7. A new player in the biorefineries field: phasin PhaP enhances tolerance to solvents and boosts ethanol and 1,3-propanediol synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Mezzina, Mariela P.; Álvarez, Daniela; Egoburo, Diego

    2017-01-01

    The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. Phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against...... as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and to other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which...... and chemicals production.Importance. This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the Pha...

  8. [Preface for special issue on bio-based materials (2016)].

    Science.gov (United States)

    Weng, Yunxuan

    2016-06-25

    Bio-based materials are new materials or chemicals with renewable biomass as raw materials such as grain, legume, straw, bamboo and wood powder. This class of materials includes bio-based polymer, biobased fiber, glycotechnology products, biobased rubber and plastics produced by biomass thermoplastic processing and basic biobased chemicals, for instance, bio-alcohols, organic acids, alkanes, and alkenes, obtained by bio-synthesis, bio-processing and bio-refinery. Owing to its environmental friendly and resource conservation, bio-based materials are becoming a new dominant industry taking the lead in the world scientific and technological innovation and economic development. An overview of bio-based materials development is reported in this special issue, and the industrial status and research progress of the following aspects, including biobased fiber, polyhydroxyalkanoates, biodegradable mulching film, bio-based polyamide, protein based biomedical materials, bio-based polyurethane, and modification and processing of poly(lactic acid), are introduced.

  9. Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes.

    Science.gov (United States)

    Matsumoto, Ken'ichiro; Taguchi, Seiichi

    2013-12-01

    The development of synthetic biology has transformed microbes into useful factories for producing valuable polymers and/or their precursors from renewable biomass. Recent progress at the interface of chemistry and biology has enabled the production of a variety of new biopolymers with properties that substantially differ from their petroleum-derived counterparts. This review touches on recent trials and achievements in the field of biopolymer synthesis, including chemo-enzymatically synthesized aliphatic polyesters, wholly biosynthesized lactate-based polyesters, polyhydroxyalkanoates and other unusual bacterially synthesized polyesters. The expanding diversities in structure and the material properties of biopolymers are key for exploring practical applications. The enzyme and metabolic engineering approaches toward this goal are discussed by shedding light on the successful case studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

    DEFF Research Database (Denmark)

    Kaartokallio, H.; Sogaard, D. H.; Norman, L.

    2013-01-01

    Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance...... and cell population properties (by flow cytometry) in subarctic sea ice in SW Greenland. Short-term temporal variability was moderate, and steep environmental gradients, typical for sea ice, were the main drivers of the variability in bacterial cell properties and activity. Low nucleic acid (LNA) bacteria...... and marine biofilm systems. Leu: TdR ratios were high (up to >300) in lowermost ice layers, and when compared to published respiration measurements, these results suggest non-specific Leu incorporation. There was evidence of polyhydroxyalkanoate (PHA)-containing bacteria in the sea ice, shown by brightly...

  11. Crystallization and preliminary X-ray analysis of the MaoC-like dehydratase from Phytophthora capsici

    International Nuclear Information System (INIS)

    Wang, Huizheng; Guo, Jiubiao; Pang, Hai; Zhang, Xiuguo

    2010-01-01

    The MaoC-like dehydratase from P. capsici was cloned, expressed and purified to homogeneity. Crystals were obtained that diffracted to 1.93 Å resolution. MaoC-like dehydratase (MaoC) plays an important role in supplying (R)-3-hydroxyacyl-CoA from the fatty-acid oxidation pathway to polyhydroxyalkanoate (PHA) biosynthetic pathways. PHAs have been attracting much attention as they can be used in the biosynthesis of synthetic plastics. Crystals of MaoC from Phytophora capsici were grown by the hanging-drop vapour-diffusion method at 289 K in a number of screening conditions. An MaoC crystal diffracted to 1.93 Å resolution using X-ray radiation and belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 81.458, b = 82.614, c = 124.228 Å, α = β = γ = 90°

  12. Methane to bioproducts: the future of the bioeconomy?

    Science.gov (United States)

    Pieja, Allison J; Morse, Molly C; Cal, Andrew J

    2017-12-01

    Methanotrophs have been studied since the 1970s, but interest has increased tremendously in recent years due to their potential to transform methane into valuable bioproducts. The vast quantity of available methane and the low price of methane as natural gas have helped to spur this interest. The most well-studied, biologically-derived products from methane include methanol, polyhydroxyalkanoates, and single cell protein. However, many other high-interest chemicals such as biofuels or high-value products such as ectoine could be made industrially relevant through metabolic engineering. Although challenges must be overcome to achieve commercialization of biologically manufactured methane-to-products, taking a holistic view of the production process or radically re-imagining pathways could lead to a future bioeconomy with methane as the primary feedstock. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Consolidated bioprocessing for production of polyhydroxyalkanotes from red algae Gelidium amansii.

    Science.gov (United States)

    Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

    2018-04-01

    Noncompetitive carbon sources such as algae are unconventional and promising raw material for sustainable biofuel production. The capability of one marine bacterium, Saccharophagus degradans 2-40 to degrade red seaweed Gelidium amansii for production of polyhydroxyalkanoates (PHA) was evaluated in this study. S. degradans can readily attach to algae, degrade algal carbohydrates, and utilize that material as main carbon source. Minimal media containing 8g/L G. amansii were used for the growth of S. degradans. The PHA content obtained was 17-27% of dry cell weight by pure culture of S. degradans and co-culture of S. degradans and Bacillus cereus, a contaminant found with S. degradans cultures. The PHA type was found to be poly(3-hydroxybutyrate) by gas chromatography and Fourier transform-infrared spectroscopy. This work demonstrates PHA production through consolidated bioprocessing of insoluble, untreated red algae by bacterial pure culture and co-culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Comparative Environmental Life Cycle Assessment of Alternative Uses of Wastewater Carbon Content

    DEFF Research Database (Denmark)

    Kroghsbo, Nena; Nicolaisen, Janna; Wenzel, Henrik

    Alternative scenarios for the wastewater and sludge treatment configurations in urban wastewater treatment were studied with the aim of comparing their environmental aspects. As the reference, a conventional activated sludge treatment was chosen including a primary settling and biogas made from...... the mixed primary and secondary sludge. This reference was then compared to an alternative use of the mixed sludge for the fermentative generation of polyhydroxyalkanoates, PHA and subsequent use of the PHA to substitute polypropylene on the polymer markets. This comparison allows for assessing...... the environmental priorities between biogas and PHA formation from the carbon content of the sludge. Further, the elimination of the primary settling with the aim of using the carbon content of the wastewater for enhanced nitrogen removal in the activated sludge process was studied. This comparison allows...

  15. Identifikace PHA produkujících bakterií pomocí nástrojů molekulární biologie

    OpenAIRE

    Gajdová, Barbora

    2017-01-01

    Tato diplomová práce se zabývá identifikací bakterií, které jsou schopny produkce polyhydroxyalkanoátů (PHA). Mezi testovanými bakteriemi byli převážně zástupci rodu Pseudomonas, Lactobacillus, Bifidobacterium, dále vzorky z termofilní kultury a vzorky z přírodních zdrojů. Bakterie byly testovány pomocí molekulárně biologické metody PCR. Byla analyzována amplifikace genu kódujícího PHA syntázu (phaC). V první reakci byl detekován jak phaC gen zodpovědný za syntézu PHA, tak současně i 16S rRNA...

  16. Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442

    DEFF Research Database (Denmark)

    Tripathi, Lakshmi; Wu, Lin-Ping; Chen, Jinchun

    2012-01-01

    ), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and -16.4°C, one melting temperature...... (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. CONCLUSIONS: It is possible to produce PHA......BACKGROUND: Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB...

  17. Decision Support For Digester Algae Integration For Improved Environmental And Economic Sustainability

    Energy Technology Data Exchange (ETDEWEB)

    2017-06-28

    The Idaho National Laboratory (INL) has teamed with University of Idaho and Boise State University to make the use of ADs more attractive by implementing a two-stage AD and coupling additional processes to the system. The addition of a polyhydroxyalkanoate (PHA) reactor, algae cultivation system, and a biomass treatment system such as fast-pyrolysis or hydrothermal liquefaction (HTL) would further sequester carbon and nutrients, as well as add valuable products that can be sold or used on-site to mitigate costs. The Decision-support for Digester-Algae IntegRation for Improved Environmental and Economic Sustainability (DAIRIEES) technoeconomic model will play a key role in evaluating the effectiveness and viability of this system to achieve economic and environmental sustainability by the dairy industry.

  18. The Evolution of Polymer Composition during PHA Accumulation: The Significance of Reducing Equivalents

    Directory of Open Access Journals (Sweden)

    Liliana Montano-Herrera

    2017-03-01

    Full Text Available This paper presents a systematic investigation into monomer development during mixed culture Polyhydroxyalkanoates (PHA accumulation involving concurrent active biomass growth and polymer storage. A series of mixed culture PHA accumulation experiments, using several different substrate-feeding strategies, was carried out. The feedstock comprised volatile fatty acids, which were applied as single carbon sources, as mixtures, or in series, using a fed-batch feed-on-demand controlled bioprocess. A dynamic trend in active biomass growth as well as polymer composition was observed. The observations were consistent over replicate accumulations. Metabolic flux analysis (MFA was used to investigate metabolic activity through time. It was concluded that carbon flux, and consequently copolymer composition, could be linked with how reducing equivalents are generated.

  19. The greenhouse gas emissions and fossil energy requirement of bioplastics from cradle to gate of a biomass refinery.

    Science.gov (United States)

    Yu, Jian; Chen, Lilian X L

    2008-09-15

    Polyhydroxyalkanoates (PHA) are promising eco-friendly bioplastics that can be produced from cellulosic ethanol biorefineries as value-added coproducts. A cradle-to-factory-gate life cycle assessment is performed with two important categories: the greenhouse gas (GHG)emissions and fossil energy requirement per kg of bioplastics produced. The analysis indicates that PHA bioplastics contribute clearly to the goal of mitigating GHG emissions with only 0.49 kg CO(2-e) being emitted from production of 1 kg of resin. Compared with 2-3 kg CO(2-e) of petrochemical counterparts, it is about 80% reduction of the global warming potential. The fossil energy requirement per kg of bioplastics is 44 MJ, lowerthan those of petrochemical counterparts (78-88 MJ/kg resin). About 62% of fossil energy is used for processing utilities and wastewater treatment, and the rest is required for raw materials in different life cycle stages.

  20. Engineered biosynthesis of biodegradable polymers.

    Science.gov (United States)

    Jambunathan, Pooja; Zhang, Kechun

    2016-08-01

    Advances in science and technology have resulted in the rapid development of biobased plastics and the major drivers for this expansion are rising environmental concerns of plastic pollution and the depletion of fossil-fuels. This paper presents a broad view on the recent developments of three promising biobased plastics, polylactic acid (PLA), polyhydroxyalkanoate (PHA) and polybutylene succinate (PBS), well known for their biodegradability. The article discusses the natural and recombinant host organisms used for fermentative production of monomers, alternative carbon feedstocks that have been used to lower production cost, different metabolic engineering strategies used to improve product titers, various fermentation technologies employed to increase productivities and finally, the different downstream processes used for recovery and purification of the monomers and polymers.

  1. [A systematic review of biosynthesis of poly (3-hydroxypropionate)].

    Science.gov (United States)

    Chang, Le; Zhan, Yuanlong; Liu, Changli

    2018-04-25

    Poly (3-hydroxypropionate) (P3HP), a new member of thermoplastic of family polyhydroxyalkanoates (PHAs), has excellent characteristics of biodegradability and biocompatibility. By now no reports can be found about wild-type bacteria that naturally synthesize P3HP, so the main way to produce P3HP is chemical and biological methods. Chemical method by adding high cost 3-HP monomers or their structural analogs as precursors, has the drawbacks of toxicity, low effectiveness and high cost. Biological method using engineered strain may utilize inexpensive and renewable carbon source to produce P3HP and has gradually become more and more popular. We systematically review here the biosynthesis of P3HP research progress. The advantages and disadvantages of biosynthesis pathways of glycerol pathway, malonyl-CoA pathway and β-alanine pathway were analyzed.

  2. Optical and mechanical properties of UV-weathered biodegradable PHBV/PBAT nanocomposite films containing halloysite nanotubes

    Science.gov (United States)

    Scarfato, P.; Avallone, E.; Acierno, D.; Russo, P.

    2014-05-01

    Recently, the increasing use of plastics, stringent environmental issues and the awareness of the progressive reduction of available petrochemical resources have ever more guided the research interest towards the investigation and development of innovative materials intrinsically biodegradable or derived from renewable sources, and generally known as bio-based polymers. Amongst the biobased and biodegradable polymers, many investigations were reported in literature about a family of polyesters known as poly(hydroxyalkanoate)s (PHAs), one of whose most prevalent is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In this context, here we report the results of a photo-degradation study performed on biodegradable blown film samples based on a commercial grade PHBV/PBAT formulation. The films, subjected to photo-oxidative weathering in a climatic chamber under UV exposure, were systematically analysed in order to check the chemico-physical changes induced by the aging protocol, taking the as-produced films as the reference materials.

  3. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    Directory of Open Access Journals (Sweden)

    L.P.S. Alves

    Full Text Available The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i cell permeabilization, ii Nile red staining, and iii analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99 compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  4. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

    Science.gov (United States)

    Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

    2017-01-16

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  5. Production and characterization of PHA from recombinant E. coli harbouring phaC1 gene of indigenous Pseudomonas sp. LDC-5 using molasses

    Directory of Open Access Journals (Sweden)

    V Saranya

    2011-09-01

    Full Text Available Polyhydroxyalkanoates (PHA are biodegradable and biocompatible green thermoplastics, synthesized by wide variety of bacteria as an intracellular carbon and energy storage intermediate. They are used as an alternative to nonrenewable petroleum derived plastics. The current interest in these biopolyesters is stimulated by the search for cost-effective capitalized production. This paper attempts to achieve maximized production rate from recombinant system using inexpensive substrate. Molasses from agro-industrial waste was used to produce PHA from recombinant E.coli in batch culture. PHA yield in molasses (3.06g/L ± 0.05-75.5% was higher than that of sucrose (2.5g/L ± 0.05 - 65.1%. Properties of the polymer produced from molasses and sucrose were analyzed by DSC, TGA, DTA, GC/MS, TLC and optical rotation studies. The findings suggested that molasses enhanced PHA production in recombinant E.coli.

  6. Bacillus and biopolymer: Prospects and challenges

    Directory of Open Access Journals (Sweden)

    Swati Mohapatra

    2017-12-01

    Full Text Available The microbially derived polyhydroxyalkanoates biopolymers could impact the global climate scenario by replacing the conventional non-degradable, petrochemical-based polymer. The biogenesis, characterization and properties of PHAs by Bacillus species using renewable substrates have been elaborated by many for their wide applications. On the other hand Bacillus species are advantageous over other bacteria due to their abundance even in extreme ecological conditions, higher growth rates even on cheap substrates, higher PHAs production ability, and the ease of extracting the PHAs. Bacillus species possess hydrolytic enzymes that can be exploited for economical PHAs production. This review summarizes the recent trends in both non-growth and growth associated PHAs production by Bacillus species which may provide direction leading to future research towards this growing quest for biodegradable plastics, one more critical step ahead towards sustainable development.

  7. Degradation of microbial polyesters.

    Science.gov (United States)

    Tokiwa, Yutaka; Calabia, Buenaventurada P

    2004-08-01

    Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics. Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA. Microorganisms degrading these polyesters are widely distributed in various environments. Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review. We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB.

  8. Production of polyhydroxybutyrate in switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Somleva, Mariya N.; Snell, Kristi D.; Beaulieu, Julie; Peoples, Oliver P.; Garrison, Bradley; Patterson, Nii

    2013-07-16

    Transgenic plants, plant material, and plant cells for synthesis of polyhydroxyalkanoates, preferably poly(3-hydroxybutyrate) (also referred to a as PHB) are provided. Preferred plants that can be genetically engineered to produce PHB include plants that do not normally produce storage products such as oils and carbohydrates, and plants that have a C.sub.4 NAD-malic enzyme photosynthetic pathway. Such plants also advantageously produce lignocellulosic biomass that can be converted into biofuels. An exemplary plant that can be genetically engineered to produce PHB and produce lignocellulosic biomass is switchgrass, Panicum virgatum L. A preferred cultivar of switchgrass is Alamo. Other suitable cultivars of switchgrass include but are not limited to Blackwell, Kanlow, Nebraska 28, Pathfinder, Cave-in-Rock, Shelter and Trailblazer.

  9. Advancing Renewable Materials by Light and X-ray Scattering

    Energy Technology Data Exchange (ETDEWEB)

    Akpalu, Yvonne A

    2014-03-26

    With the ultimate goal to design PHA polymer nanocomposites with tailored properties, we have completed systematic study of the influence of cooling rate [Xie et al, J. Appl. Poly. Sci., 2008] and nanofiller [Xie et al, Polymer 2009] characteristics on model bionanocomposites. Structure-property relationships for a model bionanocomposites system were investigated. These results yielded new fundamental knowledge that supports the discovery of cost-effective manufacturing technologies for a family of promising polyhydroxyalkanoates (PHAs) polyesters, with the potential to replace polyethylene and polypropylene (see Noda letter). Our results show that simple two-phase composite models do not account for the data. Although improvement of the mechanical properties (stiffness/modulus and toughness) must be due to alteration of the matrix by the nanoparticle filler, the observed improvement was not caused by the change of crystallinity or spherulitic morphology. Instead, improvement depends on the molecular weight of the polymer matrix and unknown filler-matrix interactions.

  10. Influence of gamma radiation on thermal properties and water vapor transmission of poly(3-hydroxybutyrate) (PHB) in blends

    International Nuclear Information System (INIS)

    Forster, Pedro L.; Martins, Natalia A.; Parra, Duclerc F.; Egute, Nayara S.; Lugao, Ademar B.

    2009-01-01

    Biodegradable polymers are a newly emerging field. A vast number of biodegradable polymers have been synthesized recently and some microorganisms and enzymes capable of degrading them have been identified. Polyesters such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs) have attracted commercial and academic interest as new biodegradable materials. In this work, we investigated the effect of gamma radiation on the thermal properties and biodegradation behavior of PHB in blend with poly(ethyleneglycol)(PEG). The samples were irradiated at gamma radiation of 5 and 10 kGy. The thermal behaviour was investigated by utilization of differential scanning calorimetry (DSC) changes in thermal stability, glass transition and melting point were reported. (author)

  11. Purification, immobilization, and characterization of nattokinase on PHB nanoparticles.

    Science.gov (United States)

    Deepak, Venkataraman; Pandian, Suresh babu Ram Kumar; Kalishwaralal, Kalimuthu; Gurunathan, Sangiliyandi

    2009-12-01

    In this study, nattokinase was purified from Bacillus subtilis using ion exchange chromatography and immobilized upon polyhydroxybutyrate (PHB) nanoparticles. A novel strain isolated from industrial dairy waste was found to synthesize polyhydroxyalkanoates (PHA) and the strain was identified as Brevibacterium casei SRKP2. PHA granules were extracted from 48 h culture and the FT-IR analysis characterized them as PHB, a natural biopolymer from B. casei. Nanoprecipitation by solvent displacement technique was used to synthesize PHB nanoparticles. PHB nanoparticles were characterized using transmission electron microscopy and particle size ranged from 100-125 nm. Immobilization of nattokinase upon PHB nanoparticles resulted in a 20% increase in the enzyme activity. Immobilization also contributed to the enhanced stability of the enzyme. Moreover, the activity was completely retained on storage at 4 degrees C for 25 days. The method has proven to be highly simple and can be implemented to other enzymes also.

  12. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    Science.gov (United States)

    Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

    2017-01-01

    The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582

  13. Production of novel biopolymers in plants: recent technological advances and future prospects.

    Science.gov (United States)

    Snell, Kristi D; Singh, Vijay; Brumbley, Stevens M

    2015-04-01

    The production of novel biopolymers in plants has the potential to provide renewable sources of industrial materials through agriculture. In this review we will highlight recent progress with plant-based production of polyhydroxyalkanoates (PHAs), silk, elastin, collagen, and cyanophycin with an emphasis on the synthesis of poly[(R)-3-hydroxybutyrate] (PHB), a renewable biodegradable PHA polymer with potential commercial applications in plastics, chemicals, and feed markets. Improved production of PHB has required manipulation of promoters driving expression of transgenes, reduction in activity of endogenous enzymes in competing metabolic pathways, insertion of genes to increase carbon flow to polymer, and basic plant biochemistry to understand metabolic limitations. These experiments have increased our understanding of carbon availability and partitioning in different plant organelles, cell types, and organs, information that is useful for the production of other novel molecules in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development.

    Science.gov (United States)

    Hanson, Andrea J; Paszczynski, Andrzej J; Coats, Erik R

    2016-03-01

    The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Magnesium Affects Poly(3-hydroxybutyrate-co-4-hydroxybutyrate Content and Composition by Affecting Glucose Uptake in Delftia acidovorans

    Directory of Open Access Journals (Sweden)

    Lee, W. H.

    2007-01-01

    Full Text Available Precise control of polyhydroxyalkanoate (PHA composition is necessary in order to synthesize polymers with specific properties. Among the various types of PHA that have been identified, those that contain 4-hydroxybutyrate (4HB monomers are especially useful in the medical and pharmaceutical fields as absorbable biomaterial. In this study, we have investigated the effect of magnesium concentration on the biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB] by Delftia acidovorans DS-17. Our results show that, magnesium affects the copolymer content and composition by affecting glucose uptake from the culture medium. Higher concentrations of magnesium resulted in lower molar fractions of 3HB in the copolymer and reduced uptake of glucose. The results show for the first time that magnesium may be used to achieve fine control of biologically synthesized PHA copolymer composition.

  16. Municipal wastewater biological nutrient removal driven by the fermentation liquid of dairy wastewater.

    Science.gov (United States)

    Liu, Hui; Chen, Yinguang; Wu, Jiang

    2017-11-01

    Carbon substrate is required by biological nutrient removal (BNR) microorganism, but it is usually insufficient in the influent of many municipal wastewater treatment plants. In this study the use of ethanol-enriched fermentation liquid, which was derived from dairy wastewater, as the preferred carbon substrate of BNR was reported. First, the application of dairy wastewater and food processing wastewater and their fermentation liquid as the carbon substrate of BNR was compared in the short-term tests. The fermented wastewater showed higher BNR performance than the unfermented one, and the fermentation liquid of dairy wastewater (FL-DW), which was obtained under pH 8 and fermentation time of 6 day, exhibited the highest phosphorus (95.5%) and total nitrogen (97.6%) removal efficiencies due to its high ethanol content (57.9%). Then, the long-term performance of FL-DW acting as the carbon substrate of BNR was compared with that of acetate and ethanol, and the FL-DW showed the greatest phosphorus and total nitrogen removal. Further investigation showed that the use of FL-DW caused the highest polyhydroxyalkanoates (PHAs) synthesis in BNR microbial cells, and more PHAs were used for phosphorus uptake and denitrification rather than glycogen synthesis and microbial growth. The FL-DW can be used as a preferred carbon substrate for BNR microbes. AB: aerobic end sludge active biomass; BNR: biological nutrient removal; DW: dairy wastewater; FL-DW: fermentation liquid of dairy wastewater; FPW: food processing wastewater; FL-FPW: fermentation liquid of food processing wastewater; PHAs: polyhydroxyalkanoates; PHB: poly-3-hydroxybutyrate; PHV: poly-3-hydroxyvalerate; PH2MV: poly-3-hydroxy-2- methylvalerate; PAOs: phosphorus accumulating organisms; SBR: sequencing batch reactor; SOP: soluble ortho-phosphorus; TN: total nitrogen; TSS: total suspended solids; VSS: volatile suspended solids; VFAs: volatile fatty acids; WWTPs: wastewater treatment plants.

  17. Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers.

    Science.gov (United States)

    Koutinas, Apostolis A; Vlysidis, Anestis; Pleissner, Daniel; Kopsahelis, Nikolaos; Lopez Garcia, Isabel; Kookos, Ioannis K; Papanikolaou, Seraphim; Kwan, Tsz Him; Lin, Carol Sze Ki

    2014-04-21

    The transition from a fossil fuel-based economy to a bio-based economy necessitates the exploitation of synergies, scientific innovations and breakthroughs, and step changes in the infrastructure of chemical industry. Sustainable production of chemicals and biopolymers should be dependent entirely on renewable carbon. White biotechnology could provide the necessary tools for the evolution of microbial bioconversion into a key unit operation in future biorefineries. Waste and by-product streams from existing industrial sectors (e.g., food industry, pulp and paper industry, biodiesel and bioethanol production) could be used as renewable resources for both biorefinery development and production of nutrient-complete fermentation feedstocks. This review focuses on the potential of utilizing waste and by-product streams from current industrial activities for the production of chemicals and biopolymers via microbial bioconversion. The first part of this review presents the current status and prospects on fermentative production of important platform chemicals (i.e., selected C2-C6 metabolic products and single cell oil) and biopolymers (i.e., polyhydroxyalkanoates and bacterial cellulose). In the second part, the qualitative and quantitative characteristics of waste and by-product streams from existing industrial sectors are presented. In the third part, the techno-economic aspects of bioconversion processes are critically reviewed. Four case studies showing the potential of case-specific waste and by-product streams for the production of succinic acid and polyhydroxyalkanoates are presented. It is evident that fermentative production of chemicals and biopolymers via refining of waste and by-product streams is a highly important research area with significant prospects for industrial applications.

  18. Employing a Recombinant Strain of Advenella mimigardefordensis for Biotechnical Production of Homopolythioesters from 3,3′-Dithiodipropionic Acid

    Science.gov (United States)

    Xia, Yongzhen; Wübbeler, Jan Hendrik; Qi, Qingsheng

    2012-01-01

    Advenella mimigardefordensis strain DPN7T was genetically modified to produce poly(3-mercaptopropionic acid) (PMP) homopolymer by exploiting the recently unraveled process of 3,3′-dithiodipropionic acid (DTDP) catabolism. Production was achieved by systematically engineering the metabolism of this strain as follows: (i) deletion of its inherent 3MP dioxygenase-encoding gene (mdo), (ii) introduction of the buk-ptb operon (genes encoding the butyrate kinase, Buk, and the phosphotransbutyrylase, Ptb, from Clostridium acetobutylicum), and (iii) overexpression of its own polyhydroxyalkanoate synthase (phaCAm). These measures yielded the potent PMP production strain A. mimigardefordensis strain SHX22. The deletion of mdo was required for adequate synthesis of PMP due to the resulting accumulation of 3MP during utilization of DTDP. Overexpression of the plasmid-borne buk-ptb operon caused a severe growth repression. This effect was overcome by inserting this operon into the genome. Polyhydroxyalkanoate (PHA) synthases from different origins were compared. The native PHA synthase of A. mimigardefordensis (phaCAm) was obviously the best choice to establish homopolythioester production in this strain. In addition, the cultivation conditions, including an appropriate provision of the carbon source, were further optimized to enhance PMP production. The engineered strain accumulated PMP up to approximately 25% (wt/wt) of the cell dry weight when cultivated in mineral salts medium containing glycerol as the carbon source in addition to DTDP as the sulfur-providing precursor. According to our knowledge, this is the first report of PMP homopolymer production by a metabolically engineered bacterium using DTDP, which is nontoxic, as the precursor substrate. PMID:22344658

  19. Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

    Directory of Open Access Journals (Sweden)

    Katherine J Robins

    Full Text Available Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI to insoluble and relatively non-toxic Cr(III, bacterial bioremediation of Cr(VI pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI remediation. To identify novel Cr(VI reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI reductase (k(cat/K(M= 1.1×10(5 M(-1 s(-1 with NADH as cofactor. Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI remediation.

  20. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328

    Energy Technology Data Exchange (ETDEWEB)

    Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-05-13

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.

  1. Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328

    International Nuclear Information System (INIS)

    Kirimura, Kohtaro; Watanabe, Shotaro; Kobayashi, Keiichi

    2016-01-01

    Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni"2"+-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.

  2. Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA.

    Science.gov (United States)

    Weichler, Maria-Teresa; Kurteva-Yaneva, Nadya; Przybylski, Denise; Schuster, Judith; Müller, Roland H; Harms, Hauke; Rohwerder, Thore

    2015-07-01

    The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of

  3. Investigations into polymer and carbon nanomaterial separations

    Science.gov (United States)

    Owens, Cherie Nicole

    The work of this thesis follows a common theme of research focused on innovative separation science. Polyhydroxyalkanoates are biodegradable polyesters produced by bacteria that can have a wide distribution in molecular weight and monomer composition. This large distribution often leads to unpredictable physical properties making commercial applications challenging. To improve polymer homogeneity and obtain samples with a clear set of physical characteristics, poly-3-hydroxyvalerate-co-3-hydroxybutyrate copolymers were fractionated using gradient polymer elution chromatography (GPEC) with carefully optimized gradients. The resulting fractions were analyzed using Size Exclusion Chromatography (SEC) and NMR. As the percentage of “good” solvent was increased in the mobile phase, the polymers eluted with decreasing percentage of 3-hydroxyvalerate and increasing molecular weight, which indicates the importance of precipitation/redissolution in the separation. As such, GPEC is an excellent choice to provide polyhydroxyalkanoate samples with a narrower distribution in composition than the original bulk copolymer. Additionally, the critical condition was found for 3-hydroxybutyrate to erase its effects on retention of the copolymer. Copolymer samples were then separated using Liquid Chromatography at the Critical Condition (LCCC) and it was determined that poly(3-hydroxvalerate-co-3-hydroxybutyrate) is a statistically random copolymer. The second project uses ultra-thin layer chromatography (UTLC) to study the performance and behavior of polyhydroxybutyrate (P3HB) as a chromatographic substrate. One specific polyhydroxyalkanoate, polyhydroxybutyrate, is a liquid crystalline polymer that can be electrospun. Electrospinning involves the formation of nanofibers though the application of an electric potential to a polymer solution. Precisely controlled optimization of electrospinning parameters was conducted to achieve the smallest diameter PHA nanofibers to date to

  4. Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

    Science.gov (United States)

    Riedel, Sebastian L; Lu, Jingnan; Stahl, Ulf; Brigham, Christopher J

    2014-02-01

    Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

  5. Recombinant protein blends: silk beyond natural design.

    Science.gov (United States)

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. Copyright © 2016. Published by Elsevier Ltd.

  6. Sustainable multistage process for enhanced productivity of bioplastics from waste remediation through aerobic dynamic feeding strategy: Process integration for up-scaling.

    Science.gov (United States)

    Amulya, K; Jukuri, Srinivas; Venkata Mohan, S

    2015-01-01

    Polyhydroxyalkanoates (PHA) production was evaluated in a multistage operation using food waste as a renewable feedstock. The first step involved the production of bio-hydrogen (bio-H2) via acidogenic fermentation. Volatile fatty acid (VFA) rich effluent from bio-H2 reactor was subsequently used for PHA production, which was carried out in two stages, Stage II (culture enrichment) and Stage III (PHA production). PHA-storing microorganisms were enriched in a sequencing batch reactor (SBR), operated at two different cycle lengths (CL-24; CL-12). Higher polymer recovery as well as VFA removal was achieved in CL-12 operation both in Stage II (16.3% dry cell weight (DCW); VFA removal, 84%) and Stage III (23.7% DCW; VFA removal, 88%). The PHA obtained was a co-polymer [P(3HB-co-3HV)] of PHB and PHV. The results obtained indicate that this integrated multistage process offers new opportunities to further leverage large scale PHA production with simultaneous waste remediation in the framework of biorefinery. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. The Opportunity for High-Performance Biomaterials from Methane.

    Science.gov (United States)

    Strong, Peter James; Laycock, Bronwyn; Mahamud, Syarifah Nuraqmar Syed; Jensen, Paul Douglas; Lant, Paul Andrew; Tyson, Gene; Pratt, Steven

    2016-02-03

    Polyhydroxyalkanoate (PHA) biopolymers are widely recognised as outstanding candidates to replace conventional petroleum-derived polymers. Their mechanical properties are good and can be tailored through copolymer composition, they are biodegradable, and unlike many alternatives, they do not rely on oil-based feedstocks. Further, they are the only commodity polymer that can be synthesised intracellularly, ensuring stereoregularity and high molecular weight. However, despite offering enormous potential for many years, they are still not making a significant impact. This is broadly because commercial uptake has been limited by variable performance (inconsistent polymer properties) and high production costs of the raw polymer. Additionally, the main type of PHA produced naturally is poly-3-hydroxybutyrate (PHB), which has limited scope due to its brittle nature and low thermal stability, as well as its tendency to embrittle over time. Production cost is strongly impacted by the type of the feedstock used. In this article we consider: the production of PHAs from methanotrophs using methane as a cost-effective substrate; the use of mixed cultures, as opposed to pure strains; and strategies to generate a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer (PHBV), which has more desirable qualities such as toughness and elasticity.

  8. Volatile fatty acids influence on the structure of microbial communities producing PHAs

    Directory of Open Access Journals (Sweden)

    Slawomir Ciesielski

    2014-06-01

    Full Text Available Polyhydroxyalkanoates (PHAs can be produced by microorganisms and are a biodegradable alternative to fossil-fuel based plastics. Currently, the focus is on reducing production costs by exploring alternative substrates for PHAs production, and on producing copolymers which are less brittle than monomers. Accordingly, this study used a substrate consisting of wastewater from waste-glycerol fermentation, supplemented with different amounts of acetic and propionic acids. These substrates were used to feed mixed microbial communities enriched from activated sludge in a sequencing batch reactor. A reactor supplemented with 2 mL of acetic acid produced 227.8 mg/L of a homopolymer of hydroxybutyrate (3HB; 4 mL of acetic acid produced 279.8 mg/L 3HB; whereas 4 mL of propionic acid produced 673.0 mg/L of a copolymer of 3HB and 3HV (hydroxyvalerate. Ribosomal Intergenic Spacer Analysis (RISA was used to show the differences between the communities created in the reactors. Thauera species predominated in biomass that produced 3HB; Paracoccus denitrificans in the biomass that produced 3HB-co-3HV. Because P. denitrificans produced the more desirable copolymer, it may be advantageous to promote its growth in PHAs-producing reactors by adding propionate.

  9. Poly(3-hydroxybutyrate production in an integrated electromicrobial setup: Investigation under stress-inducing conditions.

    Directory of Open Access Journals (Sweden)

    Israa Salem Al Rowaihi

    Full Text Available Poly(3-hydroxybutyrate (PHB, a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS.

  10. Molecular Characterization of the Bacterial Community in Biofilms for Degradation of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) Films in Seawater.

    Science.gov (United States)

    Morohoshi, Tomohiro; Ogata, Kento; Okura, Tetsuo; Sato, Shunsuke

    2018-03-29

    Microplastics are fragmented pieces of plastic in marine environments, and have become a serious environmental issue. However, the dynamics of the biodegradation of plastic in marine environments have not yet been elucidated in detail. Polyhydroxyalkanoates (PHAs) are biodegradable polymers that are synthesized by a wide range of microorganisms. One of the PHA derivatives, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has flexible material properties and a low melting temperature. After an incubation in seawater samples, a significant amount of biofilms were observed on the surfaces of PHBH films, and some PHBH films were mostly or partially degraded. In the biofilms that formed on the surfaces of unbroken PHBH films, the most dominant operational taxonomic units (OTUs) showed high similarity with the genus Glaciecola in the family Alteromonadaceae. On the other hand, the dominant OTUs in the biofilms that formed on the surfaces of broken PHBH films were assigned to the families Rhodobacteraceae, Rhodospirillaceae, and Oceanospirillaceae, and the genus Glaciecola mostly disappeared. The bacterial community in the biofilms on PHBH films was assumed to have dynamically changed according to the progression of degradation. Approximately 50 colonies were isolated from the biofilm samples that formed on the PHBH films and their PHBH-degrading activities were assessed. Two out of three PHBH-degrading isolates showed high similarities to Glaciecola lipolytica and Aestuariibacter halophilus in the family Alteromonadaceae. These results suggest that bacterial strains belonging to the family Alteromonadaceae function as the principal PHBH-degrading bacteria in these biofilms.

  11. Production of polyhydroxy alkanoates by Ralstonia eutropha from volatile fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Jung Hyun; Sawant, Shailesh S.; Kim, Beom Soo [Chungbuk National University, Cheongju (Korea, Republic of)

    2013-12-15

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible thermoplastics that can be synthesized in various microorganisms. Volatile fatty acids (VFAs) are produced by anaerobic treatment of organic wastes that can be utilized as inexpensive substrates for PHA synthesis. In this study, several Ralstonia eutropha strains were grown on the mixture of VFAs (acetic, propionic, and butyric acid) as its carbon and energy source for growth and PHA synthesis. R. eutropha KCTC 2658 accumulated PHAs up to 50% of dry cell weight from total 5 g/L of mixed VFAs (acetic acid : propionic acid : butyric acid=1 : 2 : 2). In batch culture of R. eutropha KCTC2658 in a 5 L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from 20 g/L glucose as a sole carbon source with dry cell weight of 8.4 g/L and PHA content of 30%. In fed-batch culture, two feeding strategies, pulse or pH-stat, were applied to add VFAs to the fermentor. When VFAs were fed using pH-stat feeding strategy after 40 h, a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was produced with dry cell weight of 8.1 g/L, PHA content of 50%, and 3HV fraction of 20 mol%.

  12. In silico design of PHA synthase and its validation by PHAs producing bacterial isolates

    Directory of Open Access Journals (Sweden)

    Susrita Sahoo

    2017-10-01

    Full Text Available Biopolymers are important alternatives to the petroleum-based plastics due to environment friendly manufacturing processes, biodegradability and biocompatibility. Therefore use of novel biopolymers such as polylactide, polysaccharides, aliphatic polyesters and polyhydroxyalkonoates (PHAs is of interest. PHAs are biodegradable polyesters of hydroxyalkanoates (HA produced from renewable resources by using microorganisms as intracellular carbon and energy storage compounds.  Even though PHAs are promising candidate for biodegradable polymers, however, the production cost limits their application on an industrial scale. Therefore an attempt was made to model different PHAs synthases which are the key enzyme in the biosynthesis of Polyhydroxyalkanoates as the structural information of this enzyme is in dark veil.Then molecular docking  of class I  PHA  Synthase from Ralstonia Eutrophia was done to study the PHA synthase activity. As there are lots of strain which needs to explore for the production of PHA. This investigation leads to find out the most industrial applicable microbes. Few bacterial isolates from soil sample were screened for production of PHA followed by the validation of the enzymatic activity and its product characterization to understand its structural properties.

  13. Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions

    Science.gov (United States)

    Al Rowaihi, Israa Salem; Paillier, Alexis; Rasul, Shahid; Karan, Ram; Grötzinger, Stefan Wolfgang; Eppinger, Jörg

    2018-01-01

    Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS). PMID:29698424

  14. Production and characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar Lake, India.

    Science.gov (United States)

    Kulkarni, S O; Kanekar, P P; Nilegaonkar, S S; Sarnaik, S S; Jog, J P

    2010-12-01

    Several microorganisms produce polyhydroxyalkanoates (PHA). They are accumulated intracellularly as energy storage compounds. The PHAs are of interest because of their potential in biomedical applications. Halophilic bacteria and archaea are known to produce polyhydroxybutyrate (PHB). This paper describes production of a biodegradable copolymer, PHB-co-PHV by a moderately haloalkalitolerant Halomonas campisalis, isolated from Lonar Lake, India. The production of PHA was in the range of 45-81% on dry cell weight basis when the organism was grown in a production medium containing 1% (w/v) maltose and 0.1% (w/v) yeast extract, at pH ranging from 6 to 9 with an inoculum density of 10(5)-10(7) cells/ml of medium, for incubation period of 15-30 h and at 37 degrees C. The polymer produced by the organism is a hydroxyester with molecular weight of 1.3014 x 10(6). Its melting temperature was 171 degrees C. The (1)H NMR analysis revealed that the polymer was a copolymer of PHB-co-PHV. This could be achieved by providing simple carbon source viz. maltose. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  15. The Opportunity for High-Performance Biomaterials from Methane

    Directory of Open Access Journals (Sweden)

    Peter James Strong

    2016-02-01

    Full Text Available Polyhydroxyalkanoate (PHA biopolymers are widely recognised as outstanding candidates to replace conventional petroleum-derived polymers. Their mechanical properties are good and can be tailored through copolymer composition, they are biodegradable, and unlike many alternatives, they do not rely on oil-based feedstocks. Further, they are the only commodity polymer that can be synthesised intracellularly, ensuring stereoregularity and high molecular weight. However, despite offering enormous potential for many years, they are still not making a significant impact. This is broadly because commercial uptake has been limited by variable performance (inconsistent polymer properties and high production costs of the raw polymer. Additionally, the main type of PHA produced naturally is poly-3-hydroxybutyrate (PHB, which has limited scope due to its brittle nature and low thermal stability, as well as its tendency to embrittle over time. Production cost is strongly impacted by the type of the feedstock used. In this article we consider: the production of PHAs from methanotrophs using methane as a cost-effective substrate; the use of mixed cultures, as opposed to pure strains; and strategies to generate a poly(3-hydroxybutyrate-co-3-hydroxyvalerate copolymer (PHBV, which has more desirable qualities such as toughness and elasticity.

  16. PHA production by mixed cultures: a way to valorize wastes from pulp industry.

    Science.gov (United States)

    Queirós, Diogo; Rossetti, Simona; Serafim, Luísa S

    2014-04-01

    In this work, hardwood spent sulfite liquor (HSSL), a complex feedstock originating from the pulp industry, was tested for the first time as a substrate for polyhydroxyalkanoate (PHA) production by a mixed microbial culture (MMC) under aerobic dynamic feeding (ADF) conditions. A sequencing batch reactor (SBR) fed with HSSL was operated for 67days and the selected MMC reached a maximum PHA content of 67.6%. The MMC demonstrated a differential utilization of the carbon sources present in HSSL. Acetic acid was fully consumed, while xylose and lignosulphonates were partially consumed (30% and 14%, respectively). The selected culture was characterized by Fluorescence in Situ Hybridization (FISH). Bacteria belonging to the three main classes were identified: Alpha- (72.7±4.0%), Beta- (11.1±0.37%) and Gammaproteobacteria (10.3±0.3%). Within Alphaproteobacteria, a small amount of Paracoccus (4.2±0.51%) and Defluvicoccus related to Tetrad Forming Organisms (9.0±0.28%) were detected. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Electro-Fermentation in Aid of Bioenergy and Biopolymers

    Directory of Open Access Journals (Sweden)

    Prasun Kumar

    2018-02-01

    Full Text Available The soaring levels of industrialization and rapid progress towards urbanization across the world have elevated the demand for energy besides generating a massive amount of waste. The latter is responsible for poisoning the ecosystem in an exponential manner, owing to the hazardous and toxic chemicals released by them. In the past few decades, there has been a paradigm shift from “waste to wealth”, keeping the value of high organic content available in the wastes of biological origin. The most practiced processes are that of anaerobic digestion, leading to the production of methane. However; such bioconversion has limited net energy yields. Industrial fermentation targeting value-added bioproducts such as—H2, butanediols; polyhydroxyalkanoates, citric acid, vitamins, enzymes, etc. from biowastes/lignocellulosic substrates have been planned to flourish in a multi-step process or as a “Biorefinery”. Electro-fermentation (EF is one such technology that has attracted much interest due to its ability to boost the microbial metabolism through extracellular electron transfer during fermentation. It has been studied on various acetogens and methanogens, where the enhancement in the biogas yield reached up to 2-fold. EF holds the potential to be used with complex organic materials, leading to the biosynthesis of value-added products at an industrial scale.

  18. Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters

    Directory of Open Access Journals (Sweden)

    Helga Stan-Lotter

    2013-02-01

    Full Text Available Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl, whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing hypotheses on prokaryotic evolution, such as the molecular clock concept, or the net-like history of genome evolution. A comparison of available taxonomic characteristics from strains of Hcc. salifodinae and other Halococcus species, most of them originating from surface waters, is presented. The cell wall polymer of Hcc. salifodinae was examined and found to be a heteropolysaccharide, similar to that of Hcc. morrhuae. Polyhydroxyalkanoate granules were present in Hcc. salifodinae, suggesting a possible lateral gene transfer before Permian times.

  19. Polymer blends made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and epoxidized natural rubber: Thermal and mechanical response

    Science.gov (United States)

    Salim, Yoga Sugama; Han, Chan Chin; Kammer, Hans-Werner; Kumar, Sudesh; Neon, Gan Seng

    2015-08-01

    The ever-increasing demand of biodegradable over conventional polymers places microbial polyhydroxyalkanoates (PHA) as an ideal choice of research material for specific applications. In this study, polymer blends made of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx) and epoxidized natural rubber (ENR) were prepared using solution casting technique. The influence of ENR on thermal, morphological and mechanical properties of P(3HB-co-3HHx) was investigated. There are two glass transition (Tg) temperatures observed using differential scanning calorimeter. This indicates that P(3HB-co-3HHx) and ENR are immiscible at macroscopic level. Although the Tg of P(3HB-co-3HHx) is seen to shift toward ENR in the least manner, infrared analysis suggests that the crystal structure of P(3HB-co-3HHx) retains its conformational structure. In terms of morphology, ENR exists as droplets in P(3HB-co-3HHx)-rich phase, e.g. at ENR weight fraction (wENR) of 0.3. In dynamic mechanical analysis, all blend compositions exhibit solid-like behavior, with storage moduli larger than loss moduli, across the frequency sweep at room temperature.

  20. A new biological recovery approach for PHA using mealworm, Tenebrio molitor.

    Science.gov (United States)

    Murugan, Paramasivam; Han, Lizhu; Gan, Chee-Yuen; Maurer, Frans H J; Sudesh, Kumar

    2016-12-10

    Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Application of polymer nanocomposites in the nanomedicine landscape: envisaging strategies to combat implant associated infections.

    Science.gov (United States)

    Dwivedi, Poushpi; Narvi, Shahid S; Tewari, Ravi P

    2013-12-16

    This review article presents an overview of the potential biomedical application of polymer nanocomposites arising from different chemistries, compositions, and constructions. The interaction between the chosen matrix and the filler is of critical importance. The existing polymer used in the biomedical arena includes aliphatic polyesters such as polylactide (PLA), poly(ε-caprolactone) (PCL), poly(p-dioxanone) (PPDO), poly(butylenes succinate) (PBS), poly(hydroxyalkanoate)s, and natural biopolymers such as starch, cellulose, chitin, chitosan, lignin, and proteins. The nanosized fillers utilized to fabricate the nanocomposites are inorganic, organic, and metal particles such as clays, magnetites, hydroxyapatite, nanotubes chitin whiskers, lignin, cellulose, Au, Ag, Cu, etc. These nanomaterials are taking root in a variety of diverse healthcare applications in the sector of nanomedicine including the domain of medical implants and devices. Despite sterilization and aseptic procedures the use of these biomedical devices and prosthesis to improve the patient's 'quality of life' is facing a major impediment because of bacterial colonization causing nosocomial infection, together with the multi-drug-resistant 'super-bugs' posing a serious threat to its utility. This paper discusses the current efforts and key research challenges in the development of self-sterilizing nanocomposite biomaterials for potential application in this area.

  2. Eco-Efficiency Assessment of Bioplastics Production Systems and End-of-Life Options

    Directory of Open Access Journals (Sweden)

    Kunnika Changwichan

    2018-03-01

    Full Text Available Bioplastics demand has been increased globally due to concerns regarding environmentally friendly consumption and production. Polylactic acid (PLA, polyhydroxyalkanoates (PHAs, and polybutylene succinate (PBS are promising bioplastics with bio-based feedstocks and property of biodegradability. They are produced by bacterial fermentation of sugars from carbohydrate sources. With flexibility in their properties, PLA, PHAs, and PBS can potentially substitute conventional plastics such as polypropylene (PP, polyethylene terephthalate (PET, and polystyrene (PS. This study aims at evaluating the environmental and economic sustainability of bioplastics production together with end-of-life (EOL options. The combination of environmental and economic indicators, eco-efficiency (E/E, was selected to investigate the performance of PLA, PHAs, and PBS from sugarcane and cassava in comparison with PP. The environmental impacts were determined using life cycle assessment. The product cost was used to represent the economic value. The E/E results showed that the environmental and economic sustainability could be enhanced with 100% mechanical recycling of all kinds of studied plastics. It is also important to highlight that mechanical recycling showed a better performance in terms of E/E than composting of bioplastics.

  3. Oxidized Polyethylene Wax as a Potential Carbon Source for PHA Production

    Directory of Open Access Journals (Sweden)

    Iza Radecka

    2016-05-01

    Full Text Available We report on the ability of bacteria to produce biodegradable polyhydroxyalkanoates (PHA using oxidized polyethylene wax (O-PEW as a novel carbon source. The O-PEW was obtained in a process that used air or oxygen as an oxidizing agent. R. eutropha H16 was grown for 48 h in either tryptone soya broth (TSB or basal salts medium (BSM supplemented with O-PEW and monitored by viable counting. Study revealed that biomass and PHA production was higher in TSB supplemented with O-PEW compared with TSB only. The biopolymers obtained were preliminary characterized by nuclear magnetic resonance (NMR, gel permeation chromatography (GPC, differential scanning calorimetry (DSC, and thermogravimetric analysis (TGA. The detailed structural evaluation at the molecular level was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS. The study revealed that, when TSB was supplemented with O-PEW, bacteria produced PHA which contained 3-hydroxybutyrate and up to 3 mol % of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units. The ESI-MS/MS enabled the PHA characterization when the content of 3-hydroxybutyrate was high and the appearance of other PHA repeating units was very low.

  4. Isolation and Characterization of PHA-Producing Bacteria from Propylene Oxide Saponification Wastewater Residual Sludge.

    Science.gov (United States)

    Li, Ruirui; Gu, Pengfei; Fan, Xiangyu; Shen, Junyu; Wu, Yulian; Huang, Lixuan; Li, Qiang

    2018-03-21

    A polyhydroxyalkanoate (PHA)-producing strain was isolated from propylene oxide (PO) saponification wastewater activated sludge and was identified as Brevundimonas vesicularis UJN1 through 16S rDNA sequencing and Biolog microbiological identification. Single-factor and response surface methodology experiments were used to optimize the culture medium and conditions. The optimal C/N ratio was 100/1.04, and the optimal carbon and nitrogen sources were sucrose (10 g/L) and NH 4 Cl (0.104 g/L) respectively. The optimal culture conditions consisted of initial pH of 6.7 and an incubation temperature of 33.4 °C for 48 h, with 15% inoculum and 100 mL medium at an agitation rate of 180 rpm. The PHA concentration reached 34.1% of the cell dry weight and increased three times compared with that before optimization. The only report of PHA-producing bacteria by Brevundimonas vesicularis showed that the conversion rate of PHAs using glucose as the optimal carbon source was 1.67%. In our research, the conversion rate of PHAs with sucrose as the optimal carbon source was 3.05%, and PHA production using sucrose as the carbon source was much cheaper than that using glucose as the carbon source.

  5. Mathematical Modeling of Nitrous Oxide Production during Denitrifying Phosphorus Removal Process.

    Science.gov (United States)

    Liu, Yiwen; Peng, Lai; Chen, Xueming; Ni, Bing-Jie

    2015-07-21

    A denitrifying phosphorus removal process undergoes frequent alternating anaerobic/anoxic conditions to achieve phosphate release and uptake, during which microbial internal storage polymers (e.g., Polyhydroxyalkanoate (PHA)) could be produced and consumed dynamically. The PHA turnovers play important roles in nitrous oxide (N2O) accumulation during the denitrifying phosphorus removal process. In this work, a mathematical model is developed to describe N2O dynamics and the key role of PHA consumption on N2O accumulation during the denitrifying phosphorus removal process for the first time. In this model, the four-step anoxic storage of polyphosphate and four-step anoxic growth on PHA using nitrate, nitrite, nitric oxide (NO), and N2O consecutively by denitrifying polyphosphate accumulating organisms (DPAOs) are taken into account for describing all potential N2O accumulation steps in the denitrifying phosphorus removal process. The developed model is successfully applied to reproduce experimental data on N2O production obtained from four independent denitrifying phosphorus removal study reports with different experimental conditions. The model satisfactorily describes the N2O accumulation, nitrogen reduction, phosphate release and uptake, and PHA dynamics for all systems, suggesting the validity and applicability of the model. The results indicated a substantial role of PHA consumption in N2O accumulation due to the relatively low N2O reduction rate by using PHA during denitrifying phosphorus removal.

  6. PHA Productivity and Yield of Ralstonia eutropha When Intermittently or Continuously Fed a Mixture of Short Chain Fatty Acids

    Directory of Open Access Journals (Sweden)

    Panchali Chakraborty

    2012-01-01

    Full Text Available The research described in this present study was part of a larger effort focused on developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate (PHA. The focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs (simulated ARF and maximizing PHA production in a cost-effective way. Three different feeding strategies were examined in this study. The substrate evaluated in this study for the growth phase of R. eutropha was condensed corn solubles, a low-value byproduct of the dry-mill, corn ethanol industry. The culture was grown to high cell densities in nitrogen-supplemented condensed corn solubles media in 5 L bioreactors. The overall growth rate of R. eutropha was 0.2 h−1. The 20 mL ARF feeding every 3 h from 48 to 109 h strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L−1 h−1, PHA concentration (8.37 g L−1, and PHA content (39.52% were the highest when ARF was fed every 3 h for 61 h. This study proved that condensed corn solubles can be potentially used as a growth medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer production.

  7. PHA Productivity and Yield of Ralstonia eutropha When Intermittently or Continuously Fed a Mixture of Short Chain Fatty Acids

    Science.gov (United States)

    Chakraborty, Panchali; Muthukumarappan, Kasiviswanathan; Gibbons, William R.

    2012-01-01

    The research described in this present study was part of a larger effort focused on developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate (PHA). The focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs (simulated ARF) and maximizing PHA production in a cost-effective way. Three different feeding strategies were examined in this study. The substrate evaluated in this study for the growth phase of R. eutropha was condensed corn solubles, a low-value byproduct of the dry-mill, corn ethanol industry. The culture was grown to high cell densities in nitrogen-supplemented condensed corn solubles media in 5 L bioreactors. The overall growth rate of R. eutropha was 0.2 h−1. The 20 mL ARF feeding every 3 h from 48 to 109 h strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L−1 h−1), PHA concentration (8.37 g L−1), and PHA content (39.52%) were the highest when ARF was fed every 3 h for 61 h. This study proved that condensed corn solubles can be potentially used as a growth medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer production. PMID:23118512

  8. PhaC Synthases and PHA Depolymerases: The Enzymes that Produce and Degrade Plastic

    Directory of Open Access Journals (Sweden)

    Amro A. Amara

    2011-12-01

    Full Text Available PHAs are a group of intracellular biodegradable polymer produced by (most bacteria under unbalanced growth conditions. A series of enzymes are involved in different PHAs synthesis, however PhaC synthases are responsible for the polymerization step. PHAs are accumulated in bacterial cells from soluble to insoluble form as storage materials inside the inclusion bodies during unbalanced nutrition or to save organisms from reduces equivalents. PHAs are converted again to soluble components by another pathways and enzymes for the degradation process. PHAs depolymerases are the responsible enzymes. This review is designed to give the non-specialists a condense background about PHAs especially for researcher and students in medicinal and pharmaceutical filled. ABSTRAK: PHAs (polyhydroxyalkanoate merupakan sekumpulan polimer terbiodegradasikan intrasel yang dihasilkan oleh (kebanyakan bakteria di bawah keadaan tumbesaran tak seimbang. Satu rangkaian enzim terlibat dalam sistesis PHAs yang berbeza, namun sintesis PhaC bertanggungjawab dalam peringkat pempolimeran. PHAs dikumpulkan dalam sel bakteria dari bentuk larut dan tak larut sebagai bahan simpan di dalam jasad terangkum semasa nutrisi tak seimbang atau untuk menyelamatkan organisma daripada pengurangan tak keseimbangan. PHAs ditukarkan sekali lagi kepada komponen larut dengan cara lain dan enzim lain untuk proses degradasi. PHAs depoly-merases (enzim yang memangkin penguraian makro molekul kepada molekul yang lebih mudah merupakan enzim yang bertanggunjawab. Kajian semula ini direka untuk memberi mereka yang bukan pakar, satu ringkasan tentang PHAs terutamanya penyelidik dan penuntut dalam bidang peubatan dan farmaseutikal.

  9. Enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer and antimicrobial yellow pigmentation from Cupriavidus sp. USMAHM13 with antibiofilm capability.

    Science.gov (United States)

    Ismail, Iszatty; Gurusamy, Tana Poorani; Ramachandran, Hema; Al-Ashraf Amirul, Abdullah

    2017-04-21

    Antibiofilm polymers have the ability to inhibit bacterial biofilm formation, which is known to occur ubiquitously in the environment and pose risks of infection. In this study, production of P(3HB-co-4HB) copolymer and antimicrobial yellow pigment from Cupriavidus sp. USMAHM13 are enhanced through medium optimization. Before the improvement of yellow pigment production, screening for the best additional supplement was performed resulting in high-yield yellow pigmentation using yeast extract with optimum concentration of 2 g/L. Effects of different concentrations of 1,4-butanediol, ammonium acetate, and yeast extract were studied using central composite design. Under optimal conditions, 53 wt% of polyhydroxyalkanoate (PHA) content, 0.35 g/L of pigment concentration, and 5.87 g/L of residual biomass were achieved at 0.56 wt% C of 1,4-butanediol, 1.14 g/L of ammonium acetate, and 2 g/L of yeast extract. Antibiofilm tests revealed that the yellow pigment coated on P(3HB-co-4HB) copolymer had significant effect on the inhibition of bacteria proliferation and colonization from 6 hr onward reaching 100% inhibition by 12 hr, hence effectively inhibiting the biofilm formation.

  10. Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons.

    Science.gov (United States)

    Manilla-Pérez, Efraín; Lange, Alvin Brian; Hetzler, Stephan; Steinbüchel, Alexander

    2010-05-01

    Petroleum (or crude oil) is a complex mixture of hydrocarbons. Annually, millions of tons of crude petroleum oil enter the marine environment from either natural or anthropogenic sources. Hydrocarbon-degrading bacteria (HDB) are able to assimilate and metabolize hydrocarbons present in petroleum. Crude oil pollution constitutes a temporary condition of carbon excess coupled to a limited availability of nitrogen that prompts marine oil-degrading bacteria to accumulate storage compounds. Storage lipid compounds such as polyhydroxyalkanoates (PHAs), triacylglycerols (TAGs), or wax esters (WEs) constitute the main accumulated lipophilic substances by bacteria under such unbalanced growth conditions. The importance of these compounds as end-products or precursors to produce interesting biotechnologically relevant chemicals has already been recognized. In this review, we analyze the occurrence and accumulation of lipid storage in marine hydrocarbonoclastic bacteria. We further discuss briefly the production and export of lipophilic compounds by bacteria belonging to the Alcanivorax genus, which became a model strain of an unusual group of obligate hydrocarbonoclastic bacteria (OHCB) and discuss the possibility to produce neutral lipids using A. borkumensis SK2.

  11. Halophiles, coming stars for industrial biotechnology.

    Science.gov (United States)

    Yin, Jin; Chen, Jin-Chun; Wu, Qiong; Chen, Guo-Qiang

    2015-11-15

    Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Enzyme Engineering for In Situ Immobilization.

    Science.gov (United States)

    Rehm, Fabian B H; Chen, Shuxiong; Rehm, Bernd H A

    2016-10-14

    Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

  13. Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria

    Directory of Open Access Journals (Sweden)

    Asuncion eMartinez

    2013-11-01

    Full Text Available Aerobic degradation of methylphosphonate (MPn by marine bacterioplankton has been hypothesized to contribute significantly to the ocean’s methane supersaturation, yet little is known about MPn utilization by marine microbes. To identify the microbial taxa and metabolic functions associated with MPn-driven methane production we performed parallel metagenomic, metatranscriptomic, and functional screening of microcosm perturbation experiments using surface water collected in North Pacific Subtropical Gyre. In nutrient amended microcosms containing MPn, a substrate-driven microbial succession occurred. Initially, the addition of glucose and nitrate resulted in a bloom of Vibrionales and a transcriptional profile dominated by glucose-specific PTS transport and polyhydroxyalkanoate biosynthesis. Transcripts associated with phosphorus (P acquisition were also overrepresented and suggested that the addition of glucose and nitrate had driven the community to P depletion. At this point, a second community shift occurred characterized by the increase in C-P lyase containing microbes of the Vibrionales and Rhodobacterales orders. Transcripts associated with C-P lyase components were among the most highly expressed at the community level, and only C-P lyase clusters were recovered in a functional screen for MPn utilization, consistent with this pathway being responsible for the majority, if not all the methane accumulation we observed. Our results identify specific bacterioplankton taxa that can utilize MPn aerobically under conditions of P limitation using the C-P lyase pathway, and thereby elicit a significant increase in the dissolved methane concentration.

  14. Valorization of waste glycerol for the production of poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by Cupriavidus necator and extraction in a sustainable manner.

    Science.gov (United States)

    Gahlawat, Geeta; Soni, Sanjeev Kumar

    2017-11-01

    Glycerol is a by-product of many industrial processes and huge amounts of it are generated in the form of waste, thereby necessitating a search for the method of its disposal. An interesting solution is the valorization of crude glycerol into value added product such as polyhydroxyalkanoates (PHAs). The feasibility of producing PHAs by Cupriavidus necator was evaluated using crude glycerol (WG). Various cultivation strategies were designed for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by adding different organic acids as precursors at different concentrations levels. Batch cultivation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production showed accumulation of 6.76g/L biomass containing 4.84g/L copolymer on WG with a maximum 3-hydroxyvalerate content of 24.6mol%. PHAs extraction using a non-toxic and recyclable solvent, 1,2 propylene carbonate, showed the highest recovery yield (90%) and purity (93%) at 120°C temperature and 30min incubation. This is the first report on jatropha based glycerol valorization for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production coupled with extraction using non-toxic solvent. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. A comparative study on poly(xylitol sebacate) and poly(glycerol sebacate): mechanical properties, biodegradation and cytocompatibility

    International Nuclear Information System (INIS)

    Li, Yuan; Huang, Wenchao; Cook, Wayne D; Chen, Qizhi

    2013-01-01

    In order to develop degradable elastomers with a satisfactory combination of flexibility and enzyme-mediated degradation rate, the mechanical properties, enzymatic degradation kinetics and biocompatibility of poly(xylitol sebcate) (PXS) has been systematically investigated in comparison with poly(glycerol sebacate) (PGS). Under the same level of crosslinked density, the PXS elastomer networks have approximately twice the stretchability (elongation at break) of their PGS counterparts. This observation is attributable to the relatively longer and more orientable xylitol monomers, compared with glycerol molecules. Although xylitol monomers have two more hydroxyl groups, we, surprisingly, found that the hydrophilic side chains did not accelerate the water attack on the ester bonds of the PXS network, compared with their PGS counterpart. This observation was attributed to a steric hindrance effect, i.e. the large-sized hydroxyl groups can shield ester bonds from the attack of water molecules. In conclusion, the use of polyols of more than three –OH groups is an effective approach enhancing flexibility, whilst maintaining the degradation rate of polyester elastomers. Further development could be seen in the copolymerization of PPS with appropriate thermoplastic polyesters, such as poly(lactic acid) and polyhydroxyalkanoate. (paper)

  16. Elucidation of innovative antibiofilm materials.

    Science.gov (United States)

    Marcano, Aracelys; Ba, Ousmane; Thebault, Pascal; Crétois, Raphaël; Marais, Stéphane; Duncan, Anthony C

    2015-12-01

    It is known for roughly a decade that bacterial communities (called biofilms) are responsible for significant enhanced antibiotherapy resistance. Biofilms are involved in tissue persistent infection, causing direct or collateral damage leading to chronic wounds development and impairing natural wound healing. In this study, we are interested in the development of supported protein materials which consist of asymmetric membranes as reservoir supports for the incorporation and controlled release of biomolecules capable of dissolving biofilms (or preventing their formation) and their use as wound dressing for chronic wound treatment. In a first step, polyhydroxyalkanoates (PHAs) asymmetric membranes were prepared using wet phase inversion technique. Scanning microscopy (SEM) analysis has showed the influence of different processing parameters. In a second step, the porous side of the membranes were functionalized with a surface treatment and then loaded with the antibiofilm agent (dispersin B). In a third step, the properties and antibiofilm performance of the loaded-membranes were evaluated. Exposure of Staphylococcus epidermidis biofilms to such systems weakly inhibited biofilm formation (weak preventive effect) but caused their detachment and disaggregation (strong curative effect). These initial results are promising since they open the way to a new generation of effective tools in the struggle against persistent bacterial infections exhibiting enhanced antibiotherapy resistance, and in particular in the case of infected chronic wounds. Copyright © 2015. Published by Elsevier B.V.

  17. Processing and characterization of bio-based composites

    Science.gov (United States)

    Lu, Hong

    Much research has focused on bio-based composites as a potential material to replace petroleum-based plastics. Considering the high price of Polyhydroxyalkanoates (PHAs), PHA/ Distiller's Dried Grains with Solubles (DDGS) composite is a promising economical and high-performance biodegradable material. In this paper, we discuss the effect of DDGS on PHA composites in balancing cost with material performance. Poly (lactic acid) PLA/DDGS composite is another excellent biodegradable composite, although as a bio-based polymer its degradation time is relatively long. The goal of this research is therefore to accelerate the degradation process for this material. Both bio-based composites were extruded through a twin-screw microcompounder, and the two materials were uniformly mixed. The morphology of the samples was examined using a Scanning Electron Microscope (SEM); thermal stability was determined with a Thermal Gravimetric Analyzer (TGA); other thermal properties were studied using Differential Scanning Calorimetry (DSC) and a Dynamic Mechanical Analyzer (DMA). Viscoelastic properties were also evaluated using a Rheometer.

  18. Effect of saturated and unsaturated fatty acid supplementation on bio-plastic production under submerged fermentation.

    Science.gov (United States)

    Srivastava, S K; Tripathi, Abhishek Dutt

    2013-10-01

    Polyhydroxyalkanoates (PHAs) are intracellular reserve material stored by gram-negative bacteria under nutrient-limited condition. PHAs are utilized in biodegradable plastics (bio-plastics) synthesis due to their similarity with conventional synthetic plastic. In the present study, the effect of addition of saturated and unsaturated fatty acids (palmitic acid, stearic acid, oleic acid and linoleic acid) on the production of PHAs by the soil bacterium Alcaligenes sp. NCIM 5085 was studied. Fatty acid supplementation in basal media produced saturated and unsaturated PHAs of medium and short chain length. Gas chromatography analysis of palmitic acid-supplemented media showed the presence of short chain length (scl) PHAs which could potentially serve as precursors for bio-plastic production. The scl PHA was subsequently characterized as PHB by NMR and FTIR. On the other hand, oleic acid and linoleic acid addition showed both saturated and unsaturated PHAs of different chain lengths. Palmitic acid showed maximum PHB content of 70.8 % at concentration of 15 g l -1 under shake flask cultivation. When shake flask cultivation was scaled up in a 7.5-l bioreactor (working volume 3 l), 7.6 g l -1 PHA was produced with a PHB yield (Y P/X ) and productivity of 75.89 % and 0.14 g l -1  h, respectively.

  19. xylA and xylB overexpression as a successful strategy for improving xylose utilization and poly-3-hydroxybutyrate production in Burkholderia sacchari.

    Science.gov (United States)

    Guamán, Linda P; Oliveira-Filho, Edmar R; Barba-Ostria, Carlos; Gomez, José G C; Taciro, Marilda K; da Silva, Luiziana Ferreira

    2018-03-01

    Despite the versatility and many advantages of polyhydroxyalkanoates as petroleum-based plastic substitutes, their higher production cost compared to petroleum-based polymers has historically limited their large-scale production. One appealing approach to reducing production costs is to employ less expensive, renewable feedstocks. Xylose, for example is an abundant and inexpensive carbon source derived from hemicellulosic residues abundant in agro-industrial waste (sugarcane bagasse hemicellulosic hydrolysates). In this work, the production of poly-3-hydroxybutyrate P(3HB) from xylose was studied to develop technologies for conversion of agro-industrial waste into high-value chemicals and biopolymers. Specifically, this work elucidates the organization of the xylose assimilation operon of Burkholderia sacchari, a non-model bacterium with high capacity for P(3HB) accumulation. Overexpression of endogenous xylose isomerase and xylulokinase genes was successfully assessed, improving both specific growth rate and P(3HB) production. Compared to control strain (harboring pBBR1MCS-2), xylose utilization in the engineered strain was substantially improved with 25% increase in specific growth rate, 34% increase in P(3HB) production, and the highest P(3HB) yield from xylose reported to date for B. sacchari (Y P3HB/Xil  = 0.35 g/g). This study highlights that xylA and xylB overexpression is an effective strategy to improve xylose utilization and P(3HB) production in B. sacchari.

  20. Viscoelastic, Spectroscopic, and Microscopic Characterization of Novel Bio-Based Plasticized Poly(vinyl chloride Compound

    Directory of Open Access Journals (Sweden)

    Mei Chan Sin

    2014-01-01

    Full Text Available Plasticized poly(vinyl chloride (PVC is one of the most widely consumed commodity plastics. Nevertheless, the commonly used plasticizers, particularly phthalates, are found to be detrimental to the environment and human health. This study aimed to investigate the ability of an alternative greener material, medium-chain-length polyhydroxyalkanoates (mcl-PHA, a kind of biopolyester and its thermally degraded oligoesters, to act as a compatible bioplasticizer for PVC. In this study, mcl-PHA were synthesized by Pseudomonas putida PGA1 in shake flask fermentation using saponified palm kernel oil (SPKO and subsequently moderately thermodegraded to low molecular weight oligoesters (degPHA. SEM, ATR-FTIR, 1H-NMR, and DMA were conducted to study the film morphology, microstructure, miscibility, and viscoelastic properties of the PVC-PHA and PVC/degPHA binary blends. Increased height and sharpness of tan δmax⁡ peak for all binary blends reveal an increase in chain mobility in the PVC matrix and high miscibility within the system. The PVC-PHA miscibility is possibly due to the presence of specific interactions between chlorines of PVC with the C=O group of PHA as evidenced by spectroscopic analyses. Dynamic viscoelastic measurements also showed that mcl-PHA and their oligoesters could reduce the Tg of PVC, imparting elasticity to the PVC compounds and decreasing the stiffness of PVC.

  1. Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    GarcIa-Fruitos, Elena; Seras-Franzoso, JoaquIn; Vazquez, Esther; Villaverde, Antonio [CIBER en BioingenierIa, Biomateriales y Nanomedicina, Bellaterra, 08193 Barcelona (Spain); Institut de Biotecnologia i de Biomedicina and Departament de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Cerdanyola del Valles), Barcelona (Spain)

    2010-05-21

    A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

  2. Modeling the competition between PHA-producing and non-PHA-producing bacteria in feast-famine SBR and staged CSTR systems.

    Science.gov (United States)

    Marang, Leonie; van Loosdrecht, Mark C M; Kleerebezem, Robbert

    2015-12-01

    Although the enrichment of specialized microbial cultures for the production of polyhydroxyalkanoates (PHA) is generally performed in sequencing batch reactors (SBRs), the required feast-famine conditions can also be established using two or more continuous stirred-tank reactors (CSTRs) in series with partial biomass recirculation. The use of CSTRs offers several advantages, but will result in distributed residence times and a less strict separation between feast and famine conditions. The aim of this study was to investigate the impact of the reactor configuration, and various process and biomass-specific parameters, on the enrichment of PHA-producing bacteria. A set of mathematical models was developed to predict the growth of Plasticicumulans acidivorans-as a model PHA producer-in competition with a non-storing heterotroph. A macroscopic model considering lumped biomass and an agent-based model considering individual cells were created to study the effect of residence time distribution and the resulting distributed bacterial states. The simulations showed that in the 2-stage CSTR system the selective pressure for PHA-producing bacteria is significantly lower than in the SBR, and strongly affected by the chosen feast-famine ratio. This is the result of substrate competition based on both the maximum specific substrate uptake rate and substrate affinity. Although the macroscopic model overestimates the selective pressure in the 2-stage CSTR system, it provides a quick and fairly good impression of the reactor performance and the impact of process and biomass-specific parameters. © 2015 Wiley Periodicals, Inc.

  3. Polyhydroxyalcanoates of strains of Azospirillum spp. isolated of roots of Lycopersicon esculentum Mill. “tomato” and Oryza sativa L. “rice” in Lambayeque

    Directory of Open Access Journals (Sweden)

    Katty Baca

    2010-12-01

    Full Text Available In this work was determined the concentration of polyhydroxyalkanoates (PHAs of Azospirillum strains isolated from roots of Lycopersicon esculentum Mill "tomato" and Oryza sativa L. "rice" as an alternative to accumulation of petroleum-based plastics. Previously disinfected root were plated in Nfb semisolid medium where nitrogen-fixing bacteria were recognized by a whitish film on the surface and turn from green to blue. The genus Azospirillum was identified in Congo red agar medium, obtained 96 isolates of A. lipoferum and A. brasilense on tomato and rice. Batch fermentation was performed with broth Azotobacter modified feeding a saturated solution of malic acid every 12 hours and were stained with Sudan Black B. Strains were selected with the greatest number of PHAs granules (in tomato, 18 of A. lipoferum and 2 of A. brasilense; in rice, 10 of A. lipoferum and 10 of A. brasilense and quantified the biomass and PHAs. PHAs concentration reached 0.661 gL-1 in A. lipoferum KM(T-73 and 0.738 gL-1 in A. brasilense KM(T-19, both isolated from tomato. Strains of A. lipoferum and A. brasilense isolated from tomato reached a higher concentration of biomass and PHAs against the strains of rice.

  4. Recent Advances in 3D Printing of Aliphatic Polyesters.

    Science.gov (United States)

    Chiulan, Ioana; Frone, Adriana Nicoleta; Brandabur, Călin; Panaitescu, Denis Mihaela

    2017-12-24

    3D printing represents a valuable alternative to traditional processing methods, clearly demonstrated by the promising results obtained in the manufacture of various products, such as scaffolds for regenerative medicine, artificial tissues and organs, electronics, components for the automotive industry, art objects and so on. This revolutionary technique showed unique capabilities for fabricating complex structures, with precisely controlled physical characteristics, facile tunable mechanical properties, biological functionality and easily customizable architecture. In this paper, we provide an overview of the main 3D-printing technologies currently employed in the case of poly (lactic acid) (PLA) and polyhydroxyalkanoates (PHA), two of the most important classes of thermoplastic aliphatic polyesters. Moreover, a short presentation of the main 3D-printing methods is briefly discussed. Both PLA and PHA, in the form of filaments or powder, proved to be suitable for the fabrication of artificial tissue or scaffolds for bone regeneration. The processability of PLA and PHB blends and composites fabricated through different 3D-printing techniques, their final characteristics and targeted applications in bioengineering are thoroughly reviewed.

  5. Functional redundancy ensures performance robustness in 3-stage PHA-producing mixed cultures under variable feed operation.

    Science.gov (United States)

    Carvalho, Gilda; Pedras, Inês; Karst, Soren M; Oliveira, Catarina S S; Duque, Anouk F; Nielsen, Per H; Reis, Maria A M

    2018-01-25

    Polyhydroxyalkanoates (PHA) are biopolymers that can be produced by mixed microbial cultures using wastes or industrial by-products, which represent an economical and environmental advantage over pure culture processes. The use of alternate feedstocks enables using seasonal by-products, providing that the process is resilient to transient conditions. The mixed microbial communities of a 3-stage PHA producing system fed initially with molasses and then cheese whey were investigated through amplicon sequencing of the 16S rRNA gene. The transition in feedstock resulted in an adaptation of the acidogenic community, where Actinobacteria dominated with sugarcane molasses (up to 93% of the operational taxonomic units) and Firmicutes, with cheese whey (up to 97%). The resulting fermentation products profile also changed, with a higher fraction of HV precursors obtained with molasses than cheese whey (7.1±0.5 and 1.7±0.7 gCOD/L, respectively). As for the PHA storing culture, the genera Azoarcus, Thauera and Paracoccus were enriched with fermented molasses (average 89% of Bacteria). Later, fermented cheese whey fostered a higher diversity, including some less characterised PHA-storers such as the genera Paenibacillus and Lysinibacillus. Although the microbial community structure was significantly affected by the feedstock shift, the acidogenic and PHA storing performance of the 3-stage system was very similar once a pseudo steady state was attained, showing that a reliable level of functional redundancy was attained in both mixed cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. A spent coffee grounds based biorefinery for the production of biofuels, biopolymers, antioxidants and biocomposites.

    Science.gov (United States)

    Karmee, Sanjib Kumar

    2018-02-01

    Spent coffee grounds are composed of lipid, carbohydrates, carbonaceous, and nitrogen containing compounds among others. Using n-hexane and n-hexane/isopropanol mixture highest oil yield was achived during soxhlet extraction of oil from spent coffee grounds. Alternatively, supercritical carbon dioxide can be employed as a green solvent for the extraction of oil. Using advanced chemical and biotechnological methods, spent coffee grounds are converted to various biofuels such as, biodiesel, renewable diesel, bioethanol, bioethers, bio-oil, biochar, and biogas. The in-situ transesterification of spent coffee grounds was carried out in a large scale (4 kg), which led to 80-83% biodiesel yield. In addition, a large number of value added and diversified products viz. polyhydroxyalkanoates, biosorbent, activated carbon, polyol, polyurethane foam, carotenoid, phenolic antioxidants, and green composite are obtained from spent coffee grounds. The principles of circular economy are applied to develop a sustanaible biorefinery based on valorisation of spent coffee grounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation

    Directory of Open Access Journals (Sweden)

    Stephanie Karmann

    2017-06-01

    Full Text Available Poly(hydroxyalkanoates (PHAs are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

  8. Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation.

    Science.gov (United States)

    Slaninova, Eva; Sedlacek, Petr; Mravec, Filip; Mullerova, Lucie; Samek, Ota; Koller, Martin; Hesko, Ondrej; Kucera, Dan; Marova, Ivana; Obruca, Stanislav

    2018-02-01

    Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

  9. Properties of PHA bi-, ter-, and quarter-polymers containing 4-hydroxybutyrate monomer units.

    Science.gov (United States)

    Zhila, Natalia; Shishatskaya, Ekaterina

    2018-05-01

    The present study investigates physicochemical, mechanical, and biological properties of polyhydroxyalkanoate (PHA) copolymers containing 4-hydroxybutyrate (4HB) synthesized in Cupriavidus eutrophus B10646 culture. In poly(3-hydroxybutyrate/4-hydroxybutyrate) [P(3HB/4HB)] bipolymers, 4HB varied between 10.4 and 75.0 mol%; in poly(3-hydroxybutyrate/3-hydroxyvalerate/4-hydroxybutyrate) terpolymers, 4HB constituted 28.7-55.6 mol%; and in poly(3-hydroxybutyrate/3-hydroxyvalerate/4-hydroxybutyrate/3-hydroxyhexanoate) quaterpolymers, 4HB varied between 9.3 and 13.3 mol%. The degree of crystallinity of P(3HB/4HB) copolymers decreased consistently with an increase in 4HB content, reaching 38%. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate into copolymers enhanced that effect. The effect of 4HB monomer units on temperature properties of copolymers was exhibited as lowering of the melting temperature and crystallization temperature, which improved the processing-related properties of the copolymers. All copolymers containing 4HB showed enhanced elongation at break compared to poly(3-hydroxybutyrate). Polymer films prepared from PHAs with different chemical composition had similar microstructure and porosity and had no toxic effect on mouse fibroblast NIH 3 T3 cells, proving their high biocompatibility. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Characterisation of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027), its biodegradability and potential application.

    Science.gov (United States)

    Kulkarni, Snehal O; Kanekar, Pradnya P; Jog, Jyoti P; Patil, Prashant A; Nilegaonkar, Smita S; Sarnaik, Seema S; Kshirsagar, Pranav R

    2011-06-01

    Characterisation of polyhydroxyalkanoate (PHA) film produced by haloalkalitolerant Halomonas campisalis (MCM B-1027) in 14L SS fermenter revealed it to have composition of monomer units, HB:HV as 96:4 as analysed by (1)H NMR indicating the PHA as a co-polymer of PHB-co-PHV, molecular weight by gel permeation chromatography as 2.08 × 10(6), melting temperature 166.51°C, tensile strength 18.8 MPa; two relaxations namely beta transition corresponding to the glass rubber transition and alpha transition corresponding to crystalline relaxation by Dynamic Mechanical Thermal analysis and only one relaxation corresponding to MWS interfacial polarisation with activation energy of 129 kJ/mol by broadband dielectric spectroscopy. Optical microscopic studies showed typical Maltese-cross pattern of spherulites. The PHA film was found to be biodegradable by standard ASTM method as well as by soil burial method. The leak proof polymer bags prepared from the film could be used as a packaging material. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

    International Nuclear Information System (INIS)

    GarcIa-Fruitos, Elena; Seras-Franzoso, JoaquIn; Vazquez, Esther; Villaverde, Antonio

    2010-01-01

    A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

  12. Post-processing optimization of electrospun submicron poly(3-hydroxybutyrate) fibers to obtain continuous films of interest in food packaging applications.

    Science.gov (United States)

    Cherpinski, Adriane; Torres-Giner, Sergio; Cabedo, Luis; Lagaron, Jose M

    2017-10-01

    Polyhydroxyalkanoates (PHAs) are one of the most researched family of biodegradable polymers based on renewable materials due to their thermoplastic nature and moisture resistance. The present study was targeted to investigate the preparation and characterization of poly(3-hydroxybutyrate) (PHB) films obtained through the electrospinning technique. To convert them into continuous films and then to increase their application interest in packaging, the electrospun fiber mats were subsequently post-processed by different physical treatments. Thus, the effect of annealing time and cooling method on morphology, molecular order, thermal, optical, mechanical, and barrier properties of the electrospun submicron PHB fibers was studied. Annealing at 160°C, well below the homopolyester melting point, was found to be the minimum temperature at which homogeneous transparent films were produced. The film samples that were cooled slowly after annealing showed the lowest permeability to oxygen, water vapor, and limonene. The optimally post-processed electrospun PHB fibers exhibited similar rigidity to conventional compression-molded PHA films, but with enhanced elongation at break and toughness. Films made by this electrospinning technique have many potential applications, such as in the design of barrier layers, adhesive interlayers, and coatings for fiber- and plastic-based food packaging materials.

  13. Recent Advances in 3D Printing of Aliphatic Polyesters

    Science.gov (United States)

    Frone, Adriana Nicoleta; Brandabur, Călin

    2017-01-01

    3D printing represents a valuable alternative to traditional processing methods, clearly demonstrated by the promising results obtained in the manufacture of various products, such as scaffolds for regenerative medicine, artificial tissues and organs, electronics, components for the automotive industry, art objects and so on. This revolutionary technique showed unique capabilities for fabricating complex structures, with precisely controlled physical characteristics, facile tunable mechanical properties, biological functionality and easily customizable architecture. In this paper, we provide an overview of the main 3D-printing technologies currently employed in the case of poly (lactic acid) (PLA) and polyhydroxyalkanoates (PHA), two of the most important classes of thermoplastic aliphatic polyesters. Moreover, a short presentation of the main 3D-printing methods is briefly discussed. Both PLA and PHA, in the form of filaments or powder, proved to be suitable for the fabrication of artificial tissue or scaffolds for bone regeneration. The processability of PLA and PHB blends and composites fabricated through different 3D-printing techniques, their final characteristics and targeted applications in bioengineering are thoroughly reviewed. PMID:29295559

  14. Solvent production by engineered Ralstonia eutropha: channeling carbon to biofuel.

    Science.gov (United States)

    Chakravarty, Jayashree; Brigham, Christopher J

    2018-06-01

    Microbial production of solvents like acetone and butanol was a couple of the first industrial fermentation processes to gain global importance. These solvents are important feedstocks for the chemical and biofuel industry. Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H 2 and CO 2 under aerobic conditions. This bacterium is a natural producer of polyhydroxyalkanoate biopolymers. Recently, with the advances in the development of genetic engineering tools, the range of metabolites R. eutropha can produce has enlarged. Its ability to utilize various carbon sources renders it an interesting candidate host for synthesis of renewable biofuel and solvent production. This review focuses on progress in metabolic engineering of R. eutropha for the production of alcohols, terpenes, methyl ketones, and alka(e)nes using various resources. Biological synthesis of solvents still presents the challenge of high production costs and competition from chemical synthesis. Better understanding of R. eutropha biology will support efforts to engineer and develop superior microbial strains for solvent production. Continued research on multiple fronts is required to engineer R. eutropha for truly sustainable and economical solvent production.

  15. The initial metabolic conversion of levulinic acid in Cupriavidus necator.

    Science.gov (United States)

    Jaremko, Matt; Yu, Jian

    2011-09-20

    Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The μ(max) and K(m) of LA alone were 0.26 h⁻¹ and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Genome-engineered Sinorhizobium meliloti for the production of poly(lactic-co-3-hydroxybutyric) acid copolymer.

    Science.gov (United States)

    Tran, Tam T; Charles, Trevor C

    2016-02-01

    Economically competitive commercial production of biodegradable bioplastics with desirable properties is an important goal. In this study, we demonstrate the use of chromosome engineering of an alternative bacterial host, Sinorhizobium meliloti, for production of the copolymer, poly(lactate-co-3-hydroxybutyrate). Codon-optimized genes for 2 previously engineered enzymes, Clostridium propionicum propionate CoA transferase (Pct532Cp) and Pseudomonas sp. strain MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1400Ps6-19), were introduced into S. meliloti Rm1021 by chromosome integration, replacing the native phbC gene. On the basis of phenotypic analysis and detection of polymer product by gas chromatography analysis, synthesis and accumulation of the copolymer was confirmed. The chromosome integrant strain, with the introduced genes under the control of the native phbC promoter, is able to produce over 15% cell dry mass of poly(lactate-co-3-hydroxybutyrate), containing 30 mol% lactate, from growth on mannitol. We were also able to purify the polymer from the culture and confirm the structure by NMR and GC-MS. To our knowledge, this is the first demonstration of production of this copolymer in the Alphaproteobacteria. Further optimization of this system may eventually yield strains that are able to produce economically viable commercial product.

  17. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

    Science.gov (United States)

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-03-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

  18. PHA bioplastics, biochemicals, and energy from crops.

    Science.gov (United States)

    Somleva, Maria N; Peoples, Oliver P; Snell, Kristi D

    2013-02-01

    Large scale production of polyhydroxyalkanoates (PHAs) in plants can provide a sustainable supply of bioplastics, biochemicals, and energy from sunlight and atmospheric CO(2). PHAs are a class of polymers with various chain lengths that are naturally produced by some microorganisms as storage materials. The properties of these polyesters make them functionally equivalent to many of the petroleum-based plastics that are currently in the market place. However, unlike most petroleum-derived plastics, PHAs can be produced from renewable feedstocks and easily degrade in most biologically active environments. This review highlights research efforts over the last 20 years to engineer the production of PHAs in plants with a focus on polyhydroxybutryrate (PHB) production in bioenergy crops with C(4) photosynthesis. PHB has the potential to be a high volume commercial product with uses not only in the plastics and materials markets, but also in renewable chemicals and feed. The major challenges of improving product yield and plant fitness in high biomass yielding C(4) crops are discussed in detail. Plant Biotechnology Journal © 2013 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  19. Conversion of food industrial wastes into bioplastics.

    Science.gov (United States)

    Yu, P H; Chua, H; Huang, A L; Lo, W; Chen, G Q

    1998-01-01

    The usage of plastics in packaging and disposable products, and the generation of plastic waste, have been increasing drastically. Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. In the authors' laboratories, various carbohydrates in the growth media, including sucrose, lactic acid, butyric acid, valeric acid, and various combinations of butyric and valeric acids, were utilized as the carbon (c) sources for the production of bioplastics by Alcaligenes eutrophus. As the first step in pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesize bioplastics, the authors investigated the usage of malt wastes from a beer brewery plant as the C sources for the production of bioplastics by microorganisms. Specific polymer production yield by A. Latus DSM 1124 increased to 70% polymer/cell (g/g) and 32 g/L cell dry wt, using malt wastes as the C source. The results of these experiments indicated that, with the use of different types of food wastes as the C source, different polyhydroxyalkanoate copolymers could be produced with distinct polymer properties.

  20. In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

    Science.gov (United States)

    Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

    2004-06-01

    A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

  1. RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Wang Jiangxin

    2012-12-01

    Full Text Available Abstract Background Fermentation production of biofuel ethanol consumes agricultural crops, which will compete directly with the food supply. As an alternative, photosynthetic cyanobacteria have been proposed as microbial factories to produce ethanol directly from solar energy and CO2. However, the ethanol productivity from photoautotrophic cyanobacteria is still very low, mostly due to the low tolerance of cyanobacterial systems to ethanol stress. Results To build a foundation necessary to engineer robust ethanol-producing cyanobacterial hosts, in this study we applied a quantitative transcriptomics approach with a next-generation sequencing technology, combined with quantitative reverse-transcript PCR (RT-PCR analysis, to reveal the global metabolic responses to ethanol in model cyanobacterial Synechocystis sp. PCC 6803. The results showed that ethanol exposure induced genes involved in common stress responses, transporting and cell envelope modification. In addition, the cells can also utilize enhanced polyhydroxyalkanoates (PHA accumulation and glyoxalase detoxication pathway as means against ethanol stress. The up-regulation of photosynthesis by ethanol was also further confirmed at transcriptional level. Finally, we used gene knockout strains to validate the potential target genes related to ethanol tolerance. Conclusion RNA-Seq based global transcriptomic analysis provided a comprehensive view of cellular response to ethanol exposure. The analysis provided a list of gene targets for engineering ethanol tolerance in cyanobacterium Synechocystis.

  2. Recent Advances in 3D Printing of Aliphatic Polyesters

    Directory of Open Access Journals (Sweden)

    Ioana Chiulan

    2017-12-01

    Full Text Available 3D printing represents a valuable alternative to traditional processing methods, clearly demonstrated by the promising results obtained in the manufacture of various products, such as scaffolds for regenerative medicine, artificial tissues and organs, electronics, components for the automotive industry, art objects and so on. This revolutionary technique showed unique capabilities for fabricating complex structures, with precisely controlled physical characteristics, facile tunable mechanical properties, biological functionality and easily customizable architecture. In this paper, we provide an overview of the main 3D-printing technologies currently employed in the case of poly (lactic acid (PLA and polyhydroxyalkanoates (PHA, two of the most important classes of thermoplastic aliphatic polyesters. Moreover, a short presentation of the main 3D-printing methods is briefly discussed. Both PLA and PHA, in the form of filaments or powder, proved to be suitable for the fabrication of artificial tissue or scaffolds for bone regeneration. The processability of PLA and PHB blends and composites fabricated through different 3D-printing techniques, their final characteristics and targeted applications in bioengineering are thoroughly reviewed.

  3. PHB Biosynthesis Counteracts Redox Stress in Herbaspirillum seropedicae

    Directory of Open Access Journals (Sweden)

    Marcelo B. Batista

    2018-03-01

    Full Text Available The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate (PHB enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a ΔphaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae. The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c-branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the ΔphaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.

  4. PHB Biosynthesis Counteracts Redox Stress in Herbaspirillum seropedicae.

    Science.gov (United States)

    Batista, Marcelo B; Teixeira, Cícero S; Sfeir, Michelle Z T; Alves, Luis P S; Valdameri, Glaucio; Pedrosa, Fabio de Oliveira; Sassaki, Guilherme L; Steffens, Maria B R; de Souza, Emanuel M; Dixon, Ray; Müller-Santos, Marcelo

    2018-01-01

    The ability of bacteria to produce polyhydroxyalkanoates such as poly(3-hydroxybutyrate) (PHB) enables provision of a carbon storage molecule that can be mobilized under demanding physiological conditions. However, the precise function of PHB in cellular metabolism has not been clearly defined. In order to determine the impact of PHB production on global physiology, we have characterized the properties of a Δ phaC1 mutant strain of the diazotrophic bacterium Herbaspirillum seropedicae . The absence of PHB in the mutant strain not only perturbs redox balance and increases oxidative stress, but also influences the activity of the redox-sensing Fnr transcription regulators, resulting in significant changes in expression of the cytochrome c -branch of the electron transport chain. The synthesis of PHB is itself dependent on the Fnr1 and Fnr3 proteins resulting in a cyclic dependency that couples synthesis of PHB with redox regulation. Transcriptional profiling of the Δ phaC1 mutant reveals that the loss of PHB synthesis affects the expression of many genes, including approximately 30% of the Fnr regulon.

  5. Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

    Science.gov (United States)

    Oliveira, Lilian C.G.; Ramos, Patricia Locosque; Marem, Alyne; Kondo, Marcia Y.; Rocha, Rafael C.S.; Bertolini, Thiago; Silveira, Marghuel A.V.; da Cruz, João Batista; de Vasconcellos, Suzan Pantaroto; Juliano, Luiz; Okamoto, Debora N.

    2015-01-01

    Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications. PMID:26273248

  6. Polyhydroyalkanoates: from Basic Research and Molecular Biology to Application

    Directory of Open Access Journals (Sweden)

    Amro Abd alFattah Amara

    2010-09-01

    Full Text Available This review describes the Polyhydroxyalkanoate (PHA, an intracellular biodegradable microbial polymer. PHAs is formed from different types of three hydroxyalkanoic acids monomers, each unit forms an ester bond with the hydroxyl group of the other one and the hydroxyl substituted carbon has R configuration. The C-3 atom in β position is branched with at least one carbon atom in the form of methyl group (C1 to thirteen carbons in the form of tridecyl (C13. This alkyl side chain is not necessarily saturated. PHAs are biosynthesized through regulated pathways by specific enzymes. PHAs are accumulated in bacterial cells from soluble to insoluble form as storage materials inside the inclusion bodies during unbalanced nutrition or to save organisms from reducing equivalents. PHAs are converted again to soluble components by PHAs depolymerases and the degraded materials enter various metabolic pathways. Until now, four classes of enzymes responsible for PHAs polymerization are known. PHAs were well studied regarding their promising applications, physical, chemical and biological properties. PHAs are biodegradable, biocompatible, have good material properties, renewable and can be used in many applications. The most limiting factor in PHAs commercialization is their high cost compared to the petroleum plastics. This review highlights the new knowledge and that established by the pioneers in this field as well as the factors, which affect PHAs commercialization.

  7. Anaerobic acidogenic digestion of olive mill wastewaters in biofilm reactors packed with ceramic filters or granular activated carbon.

    Science.gov (United States)

    Bertin, Lorenzo; Lampis, Silvia; Todaro, Daniela; Scoma, Alberto; Vallini, Giovanni; Marchetti, Leonardo; Majone, Mauro; Fava, Fabio

    2010-08-01

    Four identically configured anaerobic packed bed biofilm reactors were developed and employed in the continuous acidogenic digestion of olive mill wastewaters to produce volatile fatty acids (VFAs), which can be exploited in the biotechnological production of polyhydroxyalkanoates. Ceramic porous cubes or granular activated carbon were used as biofilm supports. Aside packing material, the role of temperature and organic loading rate (OLR) on VFA production yield and mixture composition were also studied. The process was monitored through a chemical, microbiological and molecular biology integrated procedure. The highest wastewater acidification yield was achieved with the ceramic-based technology at 25 degrees C, with an inlet COD and an OLR of about 17 g/L and 13 g/L/day, respectively. Under these conditions, about the 66% of the influent COD (not including its VFA content) was converted into VFAs, whose final amount represented more than 82% of the influent COD. In particular, acetic, propionic and butyric acids were the main VFAs by composing the 55.7, 21.5 and 14.4%, respectively, of the whole VFA mixture. Importantly, the relative concentrations of acetate and propionate were affected by the OLR parameter. The nature of the packing material remarkable influenced the process performances, by greatly affecting the biofilm bacterial community structure. In particular, ceramic cubes favoured the immobilization of Firmicutes of the genera Bacillus, Paenibacillus and Clostridium, which were probably involved in the VFA producing process. (c) 2010 Elsevier Ltd. All rights reserved.

  8. Microbial degradation of polyurethane, polyester polyurethanes and polyether polyurethanes.

    Science.gov (United States)

    Nakajima-Kambe, T; Shigeno-Akutsu, Y; Nomura, N; Onuma, F; Nakahara, T

    1999-02-01

    Polyurethane (PUR) is a polymer derived from the condensation of polyisocyanate and polyol and it is widely used as a base material in various industries. PUR, in particular, polyester PUR, is known to be vulnerable to microbial attack. Recently, environmental pollution by plastic wastes has become a serious issue and polyester PUR had attracted attention because of its biodegradability. There are many reports on the degradation of polyester PUR by microorganisms, especially by fungi. Microbial degradation of polyester PUR is thought to be mainly due to the hydrolysis of ester bonds by esterases. Recently, polyester-PUR-degrading enzymes have been purified and their characteristics reported. Among them, a solid-polyester-PUR-degrading enzyme (PUR esterase) derived from Comamonas acidovorans TB-35 had unique characteristics. This enzyme has a hydrophobic PUR-surface-binding domain and a catalytic domain, and the surface-binding domain was considered as being essential for PUR degradation. This hydrophobic surface-binding domain is also observed in other solid-polyester-degrading enzymes such as poly(hydroxyalkanoate) (PHA) depolymerases. There was no significant homology between the amino acid sequence of PUR esterase and that of PHA depolymerases, except in the hydrophobic surface-binding region. Thus, PUR esterase and PHA depolymerase are probably different in terms of their evolutionary origin and it is possible that PUR esterases come to be classified as a new solid-polyester-degrading enzyme family.

  9. Short-chain fatty acids and poly-beta-hydroxyalkanoates: (New) Biocontrol agents for a sustainable animal production.

    Science.gov (United States)

    Defoirdt, Tom; Boon, Nico; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter

    2009-01-01

    Because of the risk of antibiotic resistance development, there is a growing awareness that antibiotics should be used more carefully in animal production. However, a decreased use of antibiotics could result in a higher frequency of pathogenic bacteria, which in its turn could lead to a higher incidence of infections. Short-chain fatty acids (SCFAs) have long been known to exhibit bacteriostatic activity. These compounds also specifically downregulate virulence factor expression and positively influence the gastrointestinal health of the host. As a consequence, there is currently considerable interest in SCFAs as biocontrol agents in animal production. Polyhydroxyalkanoates (PHAs) are polymers of beta-hydroxy short-chain fatty acids. Currently, PHAs are applied as replacements for synthetic polymers. These biopolymers can be depolymerised by many different microorganisms that produce extracellular PHA depolymerases. Interestingly, different studies provided some evidence that PHAs can also be degraded upon passage through the gastrointestinal tract of animals and consequently, adding these compounds to the feed might result in biocontrol effects similar to those described for SCFAs.

  10. Footprint area analysis of binary imaged Cupriavidus necator cells to study PHB production at balanced, transient, and limited growth conditions in a cascade process.

    Science.gov (United States)

    Vadlja, Denis; Koller, Martin; Novak, Mario; Braunegg, Gerhart; Horvat, Predrag

    2016-12-01

    Statistical distribution of cell and poly[3-(R)-hydroxybutyrate] (PHB) granule size and number of granules per cell are investigated for PHB production in a five-stage cascade (5CSTR). Electron microscopic pictures of cells from individual cascade stages (R1-R5) were converted to binary pictures to visualize footprint areas for polyhydroxyalkanoate (PHA) and non-PHA biomass. Results for each stage were correlated to the corresponding experimentally determined kinetics (specific growth rate μ and specific productivity π). Log-normal distribution describes PHA granule size dissimilarity, whereas for R1 and R4, gamma distribution best reflects the situation. R1, devoted to balanced biomass synthesis, predominately contains cells with rather small granules, whereas with increasing residence time τ, maximum and average granule sizes by trend increase, approaching an upper limit determined by the cell's geometry. Generally, an increase of intracellular PHA content and ratio of granule to cell area slow down along the cascade. Further, the number of granules per cell decreases with increasing τ. Data for μ and π obtained by binary picture analysis correlate well with the experimental results. The work describes long-term continuous PHA production under balanced, transient, and nutrient-deficient conditions, as well as their reflection on the granules size, granule number, and cell structure on the microscopic level.

  11. Bioconversion of fish solid waste into PHB using Bacillus subtilis based submerged fermentation process.

    Science.gov (United States)

    Mohapatra, S; Sarkar, B; Samantaray, D P; Daware, A; Maity, S; Pattnaik, S; Bhattacharjee, S

    2017-12-01

    Currently, one of the major problem affecting the world is solid waste management, predominantly petroleum-based plastic and fish solid waste (FSW). However, it is very difficult to reduce the consumption of plastic as well as fish products, but it is promising to convert FSW to biopolymer to reduce eco-pollution. On account of that, the bioconversion of FSW extract to polyhydroxybutyrate (PHB) was undertaken by using Bacillus subtilis (KP172548). Under optimized conditions, 1.62 g/L of PHB has been produced by the bacterium. The purified compound was further characterized by advanced analytical technologies to elucidate its chemical structure. Results indicated that the biopolymer was found to be PHB, the most common homopolymer of polyhydroxyalkanoates (PHAs). This is the first report demonstrating the efficacy of B. subtilis to utilize FSW extract to produce biopolymer. The biocompatibility of the PHB against murine macrophage cell line RAW264.7 demonstrated that, it was comparatively less toxic, favourable for surface attachment and proliferation in comparison with poly-lactic acid (PLA) and commercially available PHB. Thus, further exploration is highly indispensable to use FSW extract as a substrate for production of PHB at pilot scale.

  12. Model-Based Nutrient Feeding Strategies for the Increased Production of Polyhydroxybutyrate (PHB) by Alcaligenes latus.

    Science.gov (United States)

    Gahlawat, Geeta; Srivastava, Ashok K

    2017-10-01

    Polyhydroxyalkanoates (PHAs) are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, the widespread use of these polymers is still hampered due to their higher cost of production as compared to plastics. The production cost could be overcome by obtaining high yields and productivity. The goal of the present research was to enhance the yield of polyhydroxybutyrate (PHB) with the help of two simple fed-batch cultivation strategies. In the present study, average batch kinetic and substrate limitation/inhibition study data of Alcaligenes latus was used for the development of PHB model which was then adopted for designing various off-line nutrient feeding strategies to enhance PHB accumulation. The predictive ability of the model was validated by experimental implementation of two fed-batch strategies. One such dynamic strategy of fed-batch cultivation under pseudo-steady state with respect to nitrogen and simultaneous carbon feeding strategy resulted in significantly high biomass and PHB concentration of 39.17 g/L and 29.64 g/L, respectively. This feeding strategy demonstrated a high PHB productivity and PHB content of 0.6 g/L h and 75%, respectively, which were remarkably high in comparison to batch cultivation. The mathematical model can also be employed for designing various other nutrient feeding strategies.

  13. Packaging related properties of commercially available biopolymers – An overview of the status quo

    Directory of Open Access Journals (Sweden)

    V. Jost

    2018-05-01

    Full Text Available Several commercially available thermoplastic biopolymers were processed in a continuous extrusion line. The molecular weight, crystallinity, and mechanical and permeation properties of the cast films were determined in order to evaluate the status quo of biopolymers currently commercially available. The biopolymers that were evaluated were polylactic acid (PLA, several polyhydroxyalkanoates (PHAs (Poly(3-hydroxybutyrate (PHB, poly(3-hydroxybutyrate-co-4-hydroxybutyrate (PHBHB, poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV, thermoplastic starch (TPS, polybutylene adipate terephthalate (PBAT, polybutylene succinate (PBS, polycaprolactone (PCL and biobased polyethylene (BioPE. Due to its potential for biobased production, thermoplastic polyurethane elastomer (TPU was also analysed. Mechanical analysis showed the PLA and PHA films had high strength and extremely low elongation at break. These were also the materials with the highest molecular weights. Films made of TPU, PCL, TPS, PBAT and BioPE had a significantly lower Young’s modulus and significantly higher elongation at break; these films had comparatively low molecular weights. Permeation measurements showed that PHA films, and particularly PHBV, had the lowest oxygen and water vapour permeability of the biopolymers that were analysed. The biopolymers BioPE, TPS, PCL, TPU and PBAT were highly permeable to oxygen, and had comparatively low molecular weight. The biopolymers TPU, PBS, PBAT, PCL and TPS were highly permeable to water vapour.

  14. Direct Production of Propene from the Thermolysis of Poly(..beta..-hydroxybutyrate)

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Ashutosh; Pilath, Heidi M.; Johnson, David K.

    2015-03-22

    To transform biomass components into hydrocarbon fuels it is clear that there are two main transformations that need to occur, i.e., deoxygenation and carbon chain extension. The potential routes for decreasing the oxygen content of biomass intermediates include dehydration, hydrodeoxygenation and decarboxylation. One route that is examined here is the conversion of polyhydroxyalkanoates (PHA) to alkenes that would be intermediates to hydrocarbon fuels.Thermal breakdown of PHA proceeds via an intermediate carboxylic acid, which can then be decarboxylated to an alkene. Oligomerization of alkenes by well-known commercial technologies would permit production of a range of hydrocarbon fuels from a carbohydrate derived intermediate. Moreover, polyhydroxybutyrate (PHB) can be produced in Cupriavidus necator (formerly known as Ralstonia eutropha) and Alcaligenes eutrophus on a variety of carbon sources including glucose, fructose and glycerol with PHB accumulation reaching 75 percent of dry cell mass. We conducted thermal conversion of PHB and pure crotonic acid (CA), the intermediate carboxylic acid produced by thermal depolymerization of PHB, in a flow-through reactor. The results of initial experiments on the thermal conversion of CA showed that up to 75 mole percent yields of propene could be achieved by optimizing the residence time and temperature of the reactor. Further experiments are being investigated to optimize the reactor parameters and enhance propene yields via thermal conversion of PHB.

  15. Nanocomposite microcapsules from powders of polyhydroxybutyrate (PHB) and smectite clays

    International Nuclear Information System (INIS)

    Silva-Valenzuela, Maria das Gracas da; Wang, Shu Hui; Wiebeck, Helio; Valenzuela-Diaz, Francisco R.

    2009-01-01

    Drug delivery systems involving microcapsules provide an attractive way to improve the performance of many chemical and biological substances. These systems may be used for several industrial segments, especially medical, pharmaceuticals and cosmetics. PHB is a polyhydroxyalkanoate available in powder form, biocompatible, biodegradable and inert towards animal tissues. The obtained PHB/smectite clay nanocomposite improved the physical-chemical properties of PHB, including its biodegradability. In this work, we describe the preparation of microcapsules from two nanocomposites systems: a) PHB and Cloisite 20A organoclay (PHB1) and b) PHB and natural Brazilian green polycationic clay (PHB2). When analyzed by XRD, the films and microcapsules did not show a d (001) peak, demonstrating an exfoliated structure for the nanocomposites. The films have shown by SEM an homogeneous distribution with the clay mineral particles spread homogeneously by the PHB film. The new microcapsules/nanocomposites showed an 'hydrangea' morphology. The diameter of the microcapsules was variable between 0.5-15 μm. (author)

  16. Characterization of poly-3-hydroxybutyrate (PHB) produced from Ralstonia eutropha using an alkali-pretreated biomass feedstock.

    Science.gov (United States)

    Saratale, Ganesh D; Oh, Min-Kyu

    2015-09-01

    Alkaline pretreatment using NaOH, KOH, or NaOCl has been applied to various types of waste biomass to enhance enzymatic digestibility. Pretreatment (2% NaOH, 121 °C, 30 min) of rice paddy straw (PS) resulted in a maximum yield of 703 mg of reducing sugar per gram of PS with 84.19% hydrolysis yield after a two-step enzymatic hydrolysis process. Ralstonia eutropha ATCC 17699 was tested for its ability to synthesize poly-3-hydroxybutyrate (PHB) using PS hydrolysates as its sole carbon source. It is noteworthy that dry cell weight, polyhydroxyalkanoate (PHA) accumulation and PHB yield with the use of laboratory-grade sugars were similar to those achieved with PS-derived sugars. Under optimized conditions, we observed maximal PHA accumulation (75.45%) and PHB production (11.42 g/L) within 48 h of fermentation. After PHB recovery, the physicochemical properties of PHB were determined by various analytical techniques, showed the results were consistent with the characteristics of a standard polymer of PHB. Thus, the PS hydrolysate proved to be an excellent cheap carbon substrate for PHB production. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Production of polyhydroxy alkanoates by Ralstonia eutropha from volatile fatty acids

    International Nuclear Information System (INIS)

    Yun, Jung Hyun; Sawant, Shailesh S.; Kim, Beom Soo

    2013-01-01

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible thermoplastics that can be synthesized in various microorganisms. Volatile fatty acids (VFAs) are produced by anaerobic treatment of organic wastes that can be utilized as inexpensive substrates for PHA synthesis. In this study, several Ralstonia eutropha strains were grown on the mixture of VFAs (acetic, propionic, and butyric acid) as its carbon and energy source for growth and PHA synthesis. R. eutropha KCTC 2658 accumulated PHAs up to 50% of dry cell weight from total 5 g/L of mixed VFAs (acetic acid : propionic acid : butyric acid=1 : 2 : 2). In batch culture of R. eutropha KCTC2658 in a 5 L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from 20 g/L glucose as a sole carbon source with dry cell weight of 8.4 g/L and PHA content of 30%. In fed-batch culture, two feeding strategies, pulse or pH-stat, were applied to add VFAs to the fermentor. When VFAs were fed using pH-stat feeding strategy after 40 h, a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was produced with dry cell weight of 8.1 g/L, PHA content of 50%, and 3HV fraction of 20 mol%

  18. Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: Investigation under stress-inducing conditions

    KAUST Repository

    Al Rowaihi, Israa Salem

    2018-04-26

    Poly(3-hydroxybutyrate) (PHB), a biodegradable polymer, can be produced by different microorganisms. The PHB belongs to the family of polyhydroxyalkanoate (PHA) that mostly accumulates as a granule in the cytoplasm of microorganisms to store carbon and energy. In this study, we established an integrated one-pot electromicrobial setup in which carbon dioxide is reduced to formate electrochemically, followed by sequential microbial conversion into PHB, using the two model strains, Methylobacterium extorquens AM1 and Cupriavidus necator H16. This setup allows to investigate the influence of different stress conditions, such as coexisting electrolysis, relatively high salinity, nutrient limitation, and starvation, on the production of PHB. The overall PHB production efficiency was analyzed in reasonably short reaction cycles typically as short as 8 h. As a result, the PHB formation was detected with C. necator H16 as a biocatalyst only when the electrolysis was operated in the same solution. The specificity of the source of PHB production is discussed, such as salinity, electricity, concurrent hydrogen production, and the possible involvement of reactive oxygen species (ROS).

  19. Multi-scale individual-based model of microbial and bioconversion dynamics in aerobic granular sludge.

    Science.gov (United States)

    Xavier, Joao B; De Kreuk, Merle K; Picioreanu, Cristian; Van Loosdrecht, Mark C M

    2007-09-15

    Aerobic granular sludge is a novel compact biological wastewater treatment technology for integrated removal of COD (chemical oxygen demand), nitrogen, and phosphate charges. We present here a multiscale model of aerobic granular sludge sequencing batch reactors (GSBR) describing the complex dynamics of populations and nutrient removal. The macro scale describes bulk concentrations and effluent composition in six solutes (oxygen, acetate, ammonium, nitrite, nitrate, and phosphate). A finer scale, the scale of one granule (1.1 mm of diameter), describes the two-dimensional spatial arrangement of four bacterial groups--heterotrophs, ammonium oxidizers, nitrite oxidizers, and phosphate accumulating organisms (PAO)--using individual based modeling (IbM) with species-specific kinetic models. The model for PAO includes three internal storage compounds: polyhydroxyalkanoates (PHA), poly phosphate, and glycogen. Simulations of long-term reactor operation show how the microbial population and activity depends on the operating conditions. Short-term dynamics of solute bulk concentrations are also generated with results comparable to experimental data from lab scale reactors. Our results suggest that N-removal in GSBR occurs mostly via alternating nitrification/denitrification rather than simultaneous nitrification/denitrification, supporting an alternative strategy to improve N-removal in this promising wastewater treatment process.

  20. Novel poly(hydroxyalkanoates)-based composites containing Bioglass® and calcium sulfate for bone tissue engineering

    International Nuclear Information System (INIS)

    García-García, J M; Boccaccini, A R; Garrido, L; Quijada-Garrido, I; Kaschta, J; Schubert, D W

    2012-01-01

    Three different poly(hydroxyalkanoates) (PHAs), copolymers of poly(3-hydroxybutyrate) (P3HB), have been used to make composites using two different fillers, bioactive glass (type 45S5 Bioglass®) and calcium sulfate dihydrate. The PHAs used were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBHV] and two copolymers of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [PHBHHx]. The aim of the study was the fabrication and characterization of the new composites and the assessment of the influence of the particular filler combination on the physical properties and bioactivity of the films. The thermal behaviour was studied using differential scanning calorimetry while mechanical properties were evaluated using dynamic mechanic thermal analysis and tensile strength test. The mechanical and thermal properties were affected by particles addition. The distribution of the particles in the polymer matrix, observed by scanning electron microscopy, was directly related to the mechanical properties. The surface characteristics were investigated by contact angle measurements and Raman spectroscopy. The extent of formation of hydroxyapatite (HA) upon immersion in simulated body fluid (SBF) depended on the polymer used, the amount of fillers employed and the time of immersion in SBF. Bioactivity was enhanced in the composites with a rise of hydrophilicity. The HA formation was controllable with time in the case of PHBHHx composites. (paper)

  1. Impact of phosphate limitation on PHA production in a feast-famine process.

    Science.gov (United States)

    Korkakaki, Emmanouela; van Loosdrecht, Mark C M; Kleerebezem, Robbert

    2017-12-01

    Double-limitation systems have shown to induce polyhydroxyalkanoates (PHA) production in chemostat studies limited in e.g. carbon and phosphate. In this work the impact of double substrate limitation on the enrichment of a PHA producing community was studied in a sequencing batch process. Enrichments at different C/P concentration ratios in the influent were established and the effect on the PHA production capacity and the enrichment community structure was investigated. Experimental results demonstrated that when a double substrate limitation is imposed at a C/P ratio in the influent in a range of 150 (C-mol/mol), the P-content of the biomass and the specific substrate uptake rates decreased. Nonetheless, the PHA storage capacity remained high (with a maximum of 84 wt%). At a C/P ratio of 300, competition in the microbial community is based on phosphate uptake, and the PHA production capacity is lost. Biomass specific substrate uptake rates are a linear function of the cellular P-content, offering advantages for scaling-up the PHA production process due to lower oxygen requirements. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Biological Effects of Spirulina (Arthrospira Biopolymers and Biomass in the Development of Nanostructured Scaffolds

    Directory of Open Access Journals (Sweden)

    Michele Greque de Morais

    2014-01-01

    Full Text Available Spirulina is produced from pure cultures of the photosynthetic prokaryotic cyanobacteria Arthrospira. For many years research centers throughout the world have studied its application in various scientific fields, especially in foods and medicine. The biomass produced from Spirulina cultivation contains a variety of biocompounds, including biopeptides, biopolymers, carbohydrates, essential fatty acids, minerals, oligoelements, and sterols. Some of these compounds are bioactive and have anti-inflammatory, antibacterial, antioxidant, and antifungal properties. These compounds can be used in tissue engineering, the interdisciplinary field that combines techniques from cell science, engineering, and materials science and which has grown in importance over the past few decades. Spirulina biomass can be used to produce polyhydroxyalkanoates (PHAs, biopolymers that can substitute synthetic polymers in the construction of engineered extracellular matrices (scaffolds for use in tissue cultures or bioactive molecule construction. This review describes the development of nanostructured scaffolds based on biopolymers extracted from microalgae and biomass from Spirulina production. These scaffolds have the potential to encourage cell growth while reducing the risk of organ or tissue rejection.

  3. Strategies for Automated Control of the Bioproduction of Mcl-PHA Biopolymers

    Directory of Open Access Journals (Sweden)

    P. Hrnčiřík

    2017-10-01

    Full Text Available Medium-chain-length polyhydroxyalkanoates (mcl-PHAs are polyesters synthesized by numerous bacteria as storage material. Despite being promising candidates for biodegradable materials of industrial interest and environmental value, their usage is still rather limited because of high production costs. One of the areas with considerable potential for further improvements is control of the production process. This paper deals with the experimental work related to the design of control strategies for mcl-PHA biopolymer production process (Pseudomonas putida KT2442 fed-batch cultivations. For this bioprocess, a set of five control strategies (two main and three auxiliary strategies have been proposed, together with the proper sequence of their switching during the fed-batch part of the production process. The application of these strategies with octanoic acid as a sole carbon source resulted in intracellular PHA content (max. mass fraction 65 % of mcl-PHA in cell dry mass (g g–1 and PHA productivity (max. 0.89 g L–1 h–1 comparable to the best results reported in the literature for this type of strain and carbon substrate.

  4. Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

    Science.gov (United States)

    Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

    2016-05-01

    Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-07-01

    Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

  6. Identificación de bacterias productoras de Polihidroxialcanoatos (PHAs en suelos contaminados con desechos de fique

    Directory of Open Access Journals (Sweden)

    Silvia Alexandra Sánchez Moreno

    2012-07-01

    Full Text Available Título en ingles: Identification of polyhydroxyalkanoate-producing bacteria in soils contaminated with fique wastes Resumen: Los Polihidroxialcanoatos (PHAs son biopolímeros con características similares a los plásticos sintéticos, pero rápidamente biodegradables dado su origen microbiano. En esta investigación se aislaron 248 colonias bacteriales de suelos contaminados con residuos del beneficio de fique en Guarne (Antioquia, evaluándose su capacidad como productoras de PHAs. Se realizaron tinciones con rojo y azul de Nilo y detección por PCR del gen PhaC. Las bacterias positivas a dichas pruebas, fueron identificadas utilizando análisis filogenético de secuencias de 16S del ADNr y pruebas bioquímicas. Finalmente, se evaluó, mediante cromatografía de gases con detector selectivo de masas GC-MS/SIM, la naturaleza química del biopolímero, a partir de la biomasa generada en un ensayo de fermentación en cultivo sumergido, con medio mínimo de sales suplementado con glucosa como fuente de carbono. Cuatro cepas de los morfotipos bacteriales encontrados, presentaron potencial para producir PHAs, de los cuales dos fueron identificados como miembros de la especie Bacillus megaterium, uno como B. mycoides y el otro como Gordonia sp. El gen PhaC se detectó en los dos aislamientos de B. megaterium. El análisis cromatográfico permitió detectar al Polihidroxibutirato (PHB como el principal componente de los PHAs presentes en B. megaterium, cuantificándose entre 63.8 mg/g y 95.3 mg/g de PHB en los ensayos de fermentación. Las bacterias aisladas tienen potencial en la producción de PHAs a partir de residuos agroindustriales, incluyendo el jugo de fique, lo que contribuiría a la reducción de su condición contaminante. Palabras clave: ADNr 16S; Bacillus; biopolímeros; Furcraea bedinghausii; PhaC. Abstract: Polyhydroxyalkanoates (PHAs are biodegradable biopolymers of bacterial origin with properties similar to conventional plastics. In

  7. PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris.

    Science.gov (United States)

    Jang, Seong Han; Jang, Ho Am; Lee, Junbeom; Kim, Jong Uk; Lee, Seung Ah; Park, Kyoung-Eun; Kim, Byung Hyun; Jo, Yong Hun; Lee, Bok Luel

    2017-06-01

    Five genes encoding PhaP family proteins and one phaR gene have been identified in the genome of Burkholderia symbiont strain RPE75. PhaP proteins function as the surface proteins of polyhydroxyalkanoate (PHA) granules, and the PhaR protein acts as a negative regulator of PhaP biosynthesis. Recently, we characterized one phaP gene to understand the molecular cross talk between Riptortus insects and Burkholderia gut symbionts. In this study, we constructed four other phaP gene-depleted mutants (Δ phaP1 , Δ phaP2 , Δ phaP3 , and Δ phaP4 mutants), one phaR gene-depleted mutant, and a phaR -complemented mutant (Δ phaR/phaR mutant). To address the biological roles of four phaP family genes and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second-instar nymphs, and colonization ability and fitness parameters were examined. In vitro , the Δ phaP3 and Δ phaR mutants cannot make a PHA granule normally in a stressful environment. Furthermore, the Δ phaR mutation decreased the colonization ability in the host midgut and negatively affected the host insect's fitness compared with wild-type Burkholderia -infected insects. However, other phaP family gene-depleted mutants colonized well in the midgut of the fifth-instar nymph insects. However, in the case of females, the colonization rate of the Δ phaP3 mutant was decreased and the host's fitness parameters were decreased compared with the wild-type-infected host, suggesting that the environment of the female midgut may be more hostile than that of the male midgut. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and that it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects' midgut. IMPORTANCE Bacterial polyhydroxyalkanoate (PHA) biosynthesis is a complex process requiring several enzymes. The biological roles of PHA granule synthesis enzymes and the surface proteins of PHA

  8. Microbial fixation of CO2 in water bodies and in drylands to combat climate change, soil loss and desertification.

    Science.gov (United States)

    Rossi, Federico; Olguín, Eugenia J; Diels, Ludo; De Philippis, Roberto

    2015-01-25

    The growing concern for the increase of the global warming effects due to anthropogenic activities raises the challenge of finding novel technological approaches to stabilize CO2 emissions in the atmosphere and counteract impinging interconnected issues such as desertification and loss of biodiversity. Biological-CO2 mitigation, triggered through biological fixation, is considered a promising and eco-sustainable method, mostly owing to its downstream benefits that can be exploited. Microorganisms such as cyanobacteria, green algae and some autotrophic bacteria could potentially fix CO2 more efficiently than higher plants, due to their faster growth. Some examples of the potential of biological-CO2 mitigation are reported and discussed in this paper. In arid and semiarid environments, soil carbon sequestration (CO2 fixation) by cyanobacteria and biological soil crusts is considered an eco-friendly and natural process to increase soil C content and a viable pathway to soil restoration after one disturbance event. Another way for biological-CO2 mitigation intensively studied in the last few years is related to the possibility to perform carbon dioxide sequestration using microalgae, obtaining at the same time bioproducts of industrial interest. Another possibility under study is the exploitation of specific chemotrophic bacteria, such as Ralstonia eutropha (or picketii) and related organisms, for CO2 fixation coupled with the production chemicals such as polyhydroxyalkanoates (PHAs). In spite of the potential of these processes, multiple factors still have to be optimized for maximum rate of CO2 fixation by these microorganisms. The optimization of culture conditions, including the optimal concentration of CO2 in the provided gas, the use of metabolic engineering and of dual purpose systems for the treatment of wastewater and production of biofuels and high value products within a biorefinery concept, the design of photobioreactors in the case of phototrophs are some

  9. Biocompatible Synthetic and Semi-synthetic Polymers - A Patent Analysis.

    Science.gov (United States)

    Ranganathan, Balu; Miller, Charles; Sinskey, Anthony

    2018-01-01

    Bioengineering has come of ages by setting up spare parts manufacturing units to be used in human body such as invasive implants and interventional controlled drug delivery in vivo systems. As a matter of fact patients on basis of their fiscal strength have the option to undergo prophylactic tactical manoeuvre for longer life spans. In this sphere of invasive implants, biocompatible polymer implants are a state of the art cutting edge technology with outstanding innovations leading to number of very successful start-up companies with a plethora of patent portfolios. From 2000 onwards, patent filings and grants for biocompatible polymers are expanding. Currently definition of biocompatibility is quite ambiguous with respect to the use of FDA approved polymeric materials. This article analysed patent portfolios for the trend patterns of prolific biocompatible polymers for capitalization and commercialization in the forthcoming years. Pair Bulk Data (PBD) portal was used to mine patent portfolios. In this patent preliminary analysis report, patents from 2000 to 2015 were evaluated using 317(c) filings, grants and classifications data for poly(vinyl alcohol) (PVA), poly(glycolic acid) (PGA), poly(hydroxyalkanoates) (PHAs) and poly(lactic acid) (PLA). This patent portfolio preliminary analysis embarks into patent analysis for New Product Development (NPD) for corporate R&D investment managerial decisions and on government advocacy for federal funding which is decisive for developmental advances. An in-depth patent portfolio investigation with return of investment (RoI) is in the pipeline. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Biodegradability of carbon nanotube/polymer nanocomposites under aerobic mixed culture conditions.

    Science.gov (United States)

    Phan, Duc C; Goodwin, David G; Frank, Benjamin P; Bouwer, Edward J; Fairbrother, D Howard

    2018-10-15

    The properties and commercial viability of biodegradable polymers can be significantly enhanced by the incorporation of carbon nanotubes (CNTs). The environmental impact and persistence of these carbon nanotube/polymer nanocomposites (CNT/PNCs) after disposal will be strongly influenced by their microbial interactions, including their biodegradation rates. At the end of consumer use, CNT/PNCs will encounter diverse communities of microorganisms in landfills, surface waters, and wastewater treatment plants. To explore CNT/PNC biodegradation under realistic environmental conditions, the effect of multi-wall CNT (MWCNT) incorporation on the biodegradation of polyhydroxyalkanoates (PHA) was investigated using a mixed culture of microorganisms from wastewater. Relative to unfilled PHA (0% w/w), the MWCNT loading (0.5-10% w/w) had no statistically significant effect on the rate of PHA matrix biodegradation. Independent of the MWCNT loading, the extent of CNT/PNC mass remaining closely corresponded to the initial mass of CNTs in the matrix suggesting a lack of CNT release. CNT/PNC biodegradation was complete in approximately 20 days and resulted in the formation of a compressed CNT mat that retained the shape of the initial CNT/PNC. This study suggests that although CNTs have been shown to be cytotoxic towards a range of different microorganisms, this does not necessarily impact the biodegradation of the surrounding polymer matrix in mixed culture, particularly in situations where the polymer type and/or microbial population favor rapid polymer biodegradation. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Enhanced Polyhydroxybutyrate Production for Long-Term Spaceflight Applications

    Science.gov (United States)

    Putman, Ryan J.; Rahman, Asif; Miller, Charles D.; Hadi, Masood Z.

    2015-01-01

    Synthetic biology holds the promise of advancing long term space fight by the production of medicine, food, materials, and energy. One such application of synthetic biology is the production of biomaterials, specifically polyhydroxyalkanoates (PHAs), using purposed organisms such as Escherichia coli. PHAs are a group of biodegradable bioplastics that are produced by a wide variety of naturally occurring microorganisms, mainly as an energy storage intermediate. PHAs have similar melting point to polypropylene and a Youngs modulus close to polystyrene. Due to limited resources and cost of transportation, large-scale extraction of biologically produced products in situ is extremely cumbersome during space flight. To that end, we are developing a secretion systems for exporting PHA from the cell in order to reduce unit operations. PHAs granules deposited inside bacteria are typically associated with proteins bound to the granule surface. Phasin, a granule bound protein, was targeted for type I secretion by fusion with HlyA signal peptide for indirect secretion of PHAs. In order to validate our secretion strategy, a green fluorescent protein (GFP) was tagged to the PHA polymerase enzyme (phaC), this three part gene cassette consists of phaA and phaB and are required for PHA production. Producing PHAs in situ during space flight or planet colonization will enable mission success by providing a valuable source of biomaterials that can have many potential applications thereby reducing resupply requirements. Biologically produced PHAs can be used in additive manufacturing such as three dimensional (3D) printing to create products that can be made on demand during space flight. After exceeding their lifetime, the PHAs could be melted and recycled back to 3D print other products. We will discuss some of our long term goals of this approach.

  12. Biodegradable and Biocompatible Biomaterial, Polyhydroxybutyrate, Produced by an Indigenous Vibrio sp. BM-1 Isolated from Marine Environment

    Directory of Open Access Journals (Sweden)

    Ho-Shing Wu

    2011-04-01

    Full Text Available Polyhydroxybutyrate (PHB is one of the polyhydroxyalkanoates (PHAs which has biodegradable and biocompatible properties. They are adopted in the biomedical field, in, for example, medical implants and drug delivery carriers. This study seeks to promote the production of PHB by Vibrio sp. BM-1, isolated from a marine environment by improving constituents of medium and implementing an appropriate fermentation strategy. This study successfully developed a glycerol-yeast extract-tryptone (GYT medium that can facilitate the growth of Vibrio sp. BM-1 and lead to the production of 1.4 g/L PHB at 20 h cultivation. This study also shows that 1.57 g/L PHB concentration and 16% PHB content were achieved, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium (mineral salts-supplemented GYT medium for 12 h. Both cell dry weight (CDW and residual CDW remained constant at around 8.2 g/L and 8.0 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. The results suggest that the secretion of PHB depolymerase that might be caused by the addition of mineral salts reduced PHB after 12 h of cultivation. However, work will be done to explain the effect of adding mineral salts on the production of PHB by Vibrio sp. BM-1 in the near future.

  13. Biological nitrate removal from water and wastewater by solid-phase denitrification process.

    Science.gov (United States)

    Wang, Jianlong; Chu, Libing

    2016-11-01

    Nitrate pollution in receiving waters has become a serious issue worldwide. Solid-phase denitrification process is an emerging technology, which has received increasing attention in recent years. It uses biodegradable polymers as both the carbon source and biofilm carrier for denitrifying microorganisms. A vast array of natural and synthetic biopolymers, including woodchips, sawdust, straw, cotton, maize cobs, seaweed, bark, polyhydroxyalkanoate (PHA), polycaprolactone (PCL), polybutylene succinate (PBS) and polylactic acid (PLA), have been widely used for denitrification due to their good performance, low cost and large available quantities. This paper presents an overview on the application of solid-phase denitrification in nitrate removal from drinking water, groundwater, aquaculture wastewater, the secondary effluent and wastewater with low C/N ratio. The types of solid carbon source, the influencing factors, the microbial community of biofilm attached on the biodegradable carriers, the potential adverse effect, and the cost of denitrification process are introduced and evaluated. Woodchips and polycaprolactone are the popular and competitive natural plant-like and synthetic biodegradable polymers used for denitrification, respectively. Most of the denitrifiers reported in solid-phase denitrification affiliated to the family Comamonadaceae in the class Betaproteobacteria. The members of genera Diaphorobacter, Acidovorax and Simplicispira were mostly reported. In future study, more attention should be paid to the simultaneous removal of nitrate and toxic organic contaminants such as pesticide and PPCPs by solid-phase denitrification, to the elucidation of the metabolic and regulatory relationship between decomposition of solid carbon source and denitrification, and to the post-treatment of the municipal secondary effluent. Solid-phase denitrification process is a promising technology for the removal of nitrate from water and wastewater. Copyright © 2016

  14. Biodegradable and biocompatible biomaterial, polyhydroxybutyrate, produced by an indigenous Vibrio sp. BM-1 isolated from marine environment.

    Science.gov (United States)

    Wei, Yu-Hong; Chen, Wei-Chuan; Wu, Ho-Shing; Janarthanan, Om-Murugan

    2011-01-01

    Polyhydroxybutyrate (PHB) is one of the polyhydroxyalkanoates (PHAs) which has biodegradable and biocompatible properties. They are adopted in the biomedical field, in, for example, medical implants and drug delivery carriers. This study seeks to promote the production of PHB by Vibrio sp. BM-1, isolated from a marine environment by improving constituents of medium and implementing an appropriate fermentation strategy. This study successfully developed a glycerol-yeast extract-tryptone (GYT) medium that can facilitate the growth of Vibrio sp. BM-1 and lead to the production of 1.4 g/L PHB at 20 h cultivation. This study also shows that 1.57 g/L PHB concentration and 16% PHB content were achieved, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium (mineral salts-supplemented GYT medium) for 12 h. Both cell dry weight (CDW) and residual CDW remained constant at around 8.2 g/L and 8.0 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. The results suggest that the secretion of PHB depolymerase that might be caused by the addition of mineral salts reduced PHB after 12 h of cultivation. However, work will be done to explain the effect of adding mineral salts on the production of PHB by Vibrio sp. BM-1 in the near future.

  15. Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442.

    Science.gov (United States)

    Tripathi, Lakshmi; Wu, Lin-Ping; Chen, Jinchun; Chen, Guo-Qiang

    2012-04-05

    Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and -16.4°C, one melting temperature (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.

  16. Genetic Resources for Advanced Biofuel Production Described with the Gene Ontology

    Directory of Open Access Journals (Sweden)

    Trudy eTorto-Alalibo

    2014-10-01

    Full Text Available Dramatic increases in research in the area of microbial biofuel production coupled with high-throughput data generation on bioenergy-related microbes has led to a deluge of information in the scientific literature and in databases. Consolidating this information and making it easily accessible requires a unified vocabulary. The Gene Ontology (GO fulfills that requirement, as it is a well-developed structured vocabulary that describes the activities and locations of gene products in a consistent manner across all kingdoms of life. The Microbial Energy Gene Ontology (MENGO: http://www.mengo.biochem.vt.edu project is extending the GO to include new terms to describe microbial processes of interest to bioenergy production. Our effort has added over 600 bioenergy related terms to the Gene Ontology. These terms will aid in the comprehensive annotation of gene products from diverse energy-related microbial genomes. An area of microbial energy research that has received a lot of attention is microbial production of advanced biofuels. These include alcohols such as butanol, isopropanol, isobutanol, and fuels derived from fatty acids, isoprenoids, and polyhydroxyalkanoates. These fuels are superior to first generation biofuels (ethanol and biodiesel esterified from vegetable oil or animal fat, can be generated from non-food feedstock sources, can be used as supplements or substitutes for gasoline, diesel and jet fuels, and can be stored and distributed using existing infrastructure. Here we review the roles of genes associated with synthesis of advanced biofuels, and at the same time introduce the use of the GO to describe the functions of these genes in a standardized way.

  17. Screening and Evaluation of Polyhydroxybutyrate-Producing Strains from Indigenous Isolate Cupriavidus taiwanensis Strains

    Directory of Open Access Journals (Sweden)

    Om-Murugan Janarthanan

    2011-01-01

    Full Text Available Polyhydroxyalkanoate (PHA is a biodegradable material with many potential biomedical applications, including medical implants and drug delivery. This study developed a system for screening production strains in order to optimize PHA production in Cupriavidus taiwanensis 184, 185, 186, 187, 204, 208, 209 and Pseudomona oleovorans ATCC 29347. In this study, Sudan black B staining, Infrared (IR and Gas Chromatography (GC analysis indicated that the best strain for PHA synthesis is C. taiwanensis 184, which obtains polyhydroxybutyrate (PHB. Cultivation of C. taiwanensis 184 under a pH of 7.0, at 30 °C, and at an agitation rate of 200 rpm, obtained a PHB content of 10% and PHB production of 0.14 g/L. The carbon and nitrogen types selected for analysis of PHB production by C. taiwanensis 184 were gluconic acid and NH4Cl, respectively. Optimal carbon/nitrogen ratio for PHB production was also determined. This study demonstrated a PHB content of 58.81% and a PHB production of 2.44 g/L when the carbon/nitrogen ratio of 8/1 was selected for C. taiwanensis 184. A two‑stage fermentation strategy significantly enhanced PHB content and PHB production. Under a two-stage fermentation strategy with nutrient‑limited conditions, C. taiwanensis 184 obtained a PHB content of 72% and a PHB concentration of 7 g/L. Finally, experimental results confirmed that optimizing the growth medium and fermentation conditions for cultivating the indigenous C. taiwanensis 184 strain substantially elevated PHB content from 10% to 72% and PHB production from 0.14 g/L to 7 g/L, respectively.

  18. Producción de polihidroxialcanoatos por bacterias halófilas nativas utilizando almidón de cáscaras de Solanum tuberosum L.

    Directory of Open Access Journals (Sweden)

    César Guzmán

    2017-01-01

    Full Text Available The aim of this study was to determine the concentration of starch Solanum tuberosum L. “potato” peels for the production of polyhydroxyalkanoates, PHA, from native halophilic bacteria as an alternative to reduce production costs of these biopolymers, possible replacements for petrochemical plastics. The bacteria were isolated of water samples of eight saline Lamba yeque region and were enriched in HM 1 broth at 30 °C with 5, 10, 15, 20, 25 and 30 g 100 mL - 1 NaCl. Dilutions were performed subsequently, aliquots were taken and plated on HM 1 agar, 203 isolates of halophilic bacteria were obtained, they were grown in HM 2 broth with 10 g.L - 1 glucose as carbon source and the 38.92% of the isolates showed PHA granules stained by Sudan Black B. Twenty bacteria with PHA granules in 65 – 75% over carried to fermentation, reaching between 0.174 to 0.889 g . g - 1 of yield Y (p / x. Three isolates were selected with the highest values among which Halomonas sp M4C1 grew and synthesized PHA in HM 2 broth with 5, 10, 15 and 20 g . L - 1 of starch as carbon source, reaching 0.019; 0.016; 0.007 y 0.006 g . L - 1 of PHA, with 0.177; 0.111; 0.056 an d 0.066 g . L - 1 of biomass after 20, 40, 24 and 16 hours respectively. The highest yield of 0.144 g . g - 1 corresponded to 10 g . L - 1 of starch demonstrating that this concentration is feasible PHA production by native halophilic bacteria.

  19. Cloud-point extraction of green-polymers from Cupriavidus necator lysate using thermoseparating-based aqueous two-phase extraction.

    Science.gov (United States)

    Leong, Yoong Kit; Lan, John Chi-Wei; Loh, Hwei-San; Ling, Tau Chuan; Ooi, Chien Wei; Show, Pau Loke

    2017-03-01

    Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Integration of poly-3-(hydroxybutyrate-co-hydroxyvalerate) production by Haloferax mediterranei through utilization of stillage from rice-based ethanol manufacture in India and its techno-economic analysis.

    Science.gov (United States)

    Bhattacharyya, Anirban; Jana, Kuntal; Haldar, Saubhik; Bhowmic, Asit; Mukhopadhyay, Ujjal Kumar; De, Sudipta; Mukherjee, Joydeep

    2015-05-01

    Haloferax mediterranei has potential for economical industrial-scale production of polyhydroxyalkanoate (PHA) as it can utilize cheap carbon sources, has capacity for nonsterile cultivation and allows simple product recovery. Molasses-based Indian distilleries are converting themselves to cereal-based distilleries. Waste stillage (14 l) of rice-based ethanol industry was used for the production of PHA by H. mediterranei in the simple plug-flow reactor configuration of the activated sludge process. Cells utilized stillage and accumulated 63 ± 3 % PHA of dry cell weight and produced 13.12 ± 0.05 g PHA/l. The product yield coefficient was 0.27 while 0.14 g/l h volumetric productivity was reached. Simultaneous lowering of 5-day biochemical oxygen demand and chemical oxygen demand values of stillage by 82 % was attained. The biopolymer was characterized as poly-3-(hydroxybutyrate-co-17.9 mol%-hydroxyvalerate) (PHBV). Directional properties of decanoic acid jointly with temperature-dependent water solubility in decanoic acid were employed for two-step desalination of the spent stillage medium in a cylindrical baffled-tank with an immersed heater and a stirrer holding axial and radial impellers. 99.3 % of the medium salts were recovered and re-used for PHA production. The cost of PHBV was estimated as US$2.05/kg when the annual production was simulated as 1890 tons. Desalination contributed maximally to the overall cost. Technology and cost-analysis demonstrate that PHA production integrated with ethanol manufacture is feasible in India. This study could be the basis for construction of a pilot plant.

  1. Study on the Effect of Levulinic Acid on Whey-Based Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate by Hydrogenophaga pseudoflava

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2017-04-01

    Full Text Available Background and Objective: Production of polyhydroxyalkanoate copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate units was for the first time studied using the production strain Hydrogenophaga pseudoflava based on sustainable raw materials. This strategy provides for increased cost efficiency in PHA production and in enhanced material quality.Material and Methods: As a particularity, production of these poly(3-hydroxybutyrate-co-3- hydroxyvalerate copolyesters was based on a novel substrate/co-substrate combination: whey permeate from dairy industry, on the one hand, acted as substrate for biomass and 3HB biosynthesis; on the other hand, levulinic acid, accessible from various renewable resources, was used as 3HV-related precursor compound. The experiments were carried out on shaking flask scale using defined nutrient media.Results and Conclusion: Applied during nutritionally balanced growth of H. pseudoflava, levulinicacid displays drastic growth inhibition at rather low concentrations of 0.2 g l-1 (growth inhibition constant Ki = 0.032, which suggests the careful supply of this compound in the first phase of cultivation. Under nitrogen-free cultivation conditions, inhibition of the strain´s metabolism by levulinic acid was less pronounced. Here, poly(3-hydroxybutyrate-co- 3-hydroxyvalerate concentrations up to 4.2 g l-1 and volumetric poly(3-hydroxybutyrate-co-3- hydroxyvalerate productivities up to 0.06 g l-1 h -1 were achieved in dependence on the precursor supply. Investigating poly(3-hydroxybutyrate-co-3-hydroxyvalerate composition in setups supplied with differently composed whey/levulinic acid mixtures revealed 3- hydroxyvalerate fractions in the polymer between 0 and 0.6 mol mol-1 . This study successfully demonstrates the feasibility of combined utilization of different waste- and by-products from food industry and agriculture for generation of value-added 2nd generation biopolymers. Conflict of interest: The authors

  2. A dual-species co-cultivation system to study the interactions between Roseobacters and Dinoflagellates

    Directory of Open Access Journals (Sweden)

    Hui eWang

    2014-06-01

    Full Text Available Some microalgae in nature live in symbiosis with microorganisms that can enhance or inhibit growth, thus influencing the dynamics of phytoplankton blooms. In spite of the great ecological importance of these interactions, very few defined laboratory systems are available to study them in detail. Here we present a co-cultivation system consisting of the toxic phototrophic dinoflagellate Prorocentrum minimum and the photoheterotrophic alphaproteobacterium Dinoroseobacter shibae. In a mineral medium lacking a carbon source, vitamins for the bacterium and the essential vitamin B12 for the dinoflagellate, growth dynamics reproducibly went from a mutualistic phase, where both algae and bacteria grow, to a pathogenic phase, where the algae are killed by the bacteria. The data show a Jekyll and Hyde lifestyle that had been proposed but not previously demonstrated. We used RNAseq and microarray analysis to determine which genes of D. shibae are transcribed and differentially expressed in a light dependent way at an early time-point of the co-culture when the bacterium grows very slowly. Enrichment of bacterial mRNA for transcriptome analysis was optimized, but none of the available methods proved capable of removing dinoflagellate ribosomal RNA completely. RNAseq showed that a phasin encoding gene (phaP1 which is part of the polyhydroxyalkanoate (PHA metabolism operon represented approximately 10 % of all transcripts. Five genes for aerobic anoxygenic photosynthesis were down-regulated in the light, indicating that the photosynthesis apparatus was functional. A betaine-choline-carnitine-transporter (BCCT that may be used for dimethylsulfoniopropionate (DMSP uptake was the highest up-regulated gene in the light. The data suggest that at this early mutualistic phase of the symbiosis, PHA degradation might be the main carbon and energy source of D. shibae, supplemented in the light by degradation of DMSP and aerobic anoxygenic photosynthesis.

  3. Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization.

    Science.gov (United States)

    Franden, Mary Ann; Jayakody, Lahiru N; Li, Wing-Jin; Wagner, Neil J; Cleveland, Nicholas S; Michener, William E; Hauer, Bernhard; Blank, Lars M; Wierckx, Nick; Klebensberger, Janosch; Beckham, Gregg T

    2018-06-07

    Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (~124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and

  4. Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

    Science.gov (United States)

    Berlanga, Mercedes; Montero, M T; Fernández-Borrell, Jordi; Guerrero, Ricardo

    2006-06-01

    Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

  5. Reexamining intra and extracellular metabolites produced by Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Maria Shuja

    2016-02-01

    Full Text Available Objective: To isolate, screen and analyze bacteria from different areas of Pakistan for the production of antimicrobial compounds, zinc solubilization and bioplastic production. Methods: Isolation and purification was proceeding with streak plate method. Antagonistic assay was completed with well diffusion and thin-layer chromatography. In vivo analysis of bioplastic was analyzed with Nile blue fluorescence under UV and Sudan staining. Results: A total of 18 bacterial strains purified from soil samples while 148 strains form stock cultures were used. Out of 166 only 94 showed antimicrobial activity against each of Grampositive and Gram-negative; cocci and rods. In case of heavy metal (ZnO and Zn3(PO42.4H2O solubilization, 54 strains solubilized ZnO and 23 strains solubilized Zn3(PO42.4H2O, while 127 strains grown on polyhydroxyalkanoate detection meedia supplemented with Nile blue medium showed bioplastic production by producing fluorescence under UV light. Four bacterial strains (coded as 100, 101, 104 and 111 were selected for further characterization. Induction time assay showed that strains 101, 104, and 111 showed inhibitory activity after 4 h of incubation while strain 100 showed after 8 h. All four strains were tolerable to the maximum concentration of ZnO. Amplified products of both 16S rRNA and PhaC gene fragments of strain 111 were sequenced and submitted to GenBank as accession numbers EU781525 and EU781526. Conclusions: Bacterial strain Pseudomonas aeruginosa-111 has potential to utilize as biofertilize and bioplastic producer.

  6. Investigation of the phase morphology of bacterial PHA inclusion bodies by contrast variation SANS

    International Nuclear Information System (INIS)

    Russell, R.A.; Holden, P.J.; Garvey, C.J.; Wilde, K.L.; Hammerton, K.M.; Foster, L.J.

    2006-01-01

    Under growth-limiting conditions, many bacteria are able to metabolise excess organic acids into polyhydroxyalkanoates (PHA) and store these polymers as intracellular inclusions until the return of favourable conditions. Various models have been proposed for the macromolecular organisation of the boundary layer surrounding the polymer, and contrast-variation small-angle neutron scattering (SANS) was used to study its organisation. Inclusions formed by Pseudomonas oleovorans under hydrogenating conditions showed lowest scattering intensity at ca. 20% D 2 O. The inclusions consist of protein and membrane lipids in the boundary layer and polyhydroxyoctanoate (lipid) in the inclusion body. At 20% D 2 O the contributions of lipids were contrast matched with the solvent, indicating that lipids contributed the bulk of the scattering intensity observed at other D 2 O/H 2 O ratios. These results are inconsistent with a model of the boundary layer which proposed outer and inner layers of crystalline protein lattice sandwiching a membrane lipid membrane layer [E.S. Stuart, R.W. Lenz, R.C. Fuller, Can J Microbiol 41(Suppl 1) (1995) 84-93], and is more consistent with a model consisting of a lipid monolayer containing embedded proteins [U. Pieper-furst, M.H. Madkour, F. Mayer, A. Steinbuchel, J. Bacteriol. 176 (1994) 4328-4337.] By altering the H/D content of the precursors, we were able to collect SANS data from preparations of both deuterated and H/D copolymer inclusions, where initial PHA produced was hydrogenated followed by deuteration. Deuterated inclusions showed minimum intensity above 90% D 2 O/H 2 O whereas the sequentially produced copolymer (assumed to be in a core/shell arrangement) displayed minimum scattering some 20% lower, which is consistent with the increased hydrogenation of the boundary layer expected from its synthesis during supply of hydrogenated followed by deuterated precursors

  7. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    International Nuclear Information System (INIS)

    Kamnev, Alexander A.; Tugarova, Anna V.; Antonyuk, Lyudmila P.; Tarantilis, Petros A.; Kulikov, Leonid A.; Perfiliev, Yurii D.; Polissiou, Moschos G.; Gardiner, Philip H.E.

    2006-01-01

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with 57 Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour

  8. Biomass Biorefinery for the production of Polymers and Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Oliver P. Peoples

    2008-05-05

    The conversion of biomass crops to fuel is receiving considerable attention as a means to reduce our dependence on foreign oil imports and to meet future energy needs. Besides their use for fuel, biomass crops are an attractive vehicle for producing value added products such as biopolymers. Metabolix, Inc. of Cambridge proposes to develop methods for producing biodegradable polymers polyhydroxyalkanoates (PHAs) in green tissue plants as well as utilizating residual plant biomass after polymer extraction for fuel generation to offset the energy required for polymer extraction. The primary plant target is switchgrass, and backup targets are alfalfa and tobacco. The combined polymer and fuel production from the transgenic biomass crops establishes a biorefinery that has the potential to reduce the nation’s dependence on foreign oil imports for both the feedstocks and energy needed for plastic production. Concerns about the widespread use of transgenic crops and the grower’s ability to prevent the contamination of the surrounding environment with foreign genes will be addressed by incorporating and expanding on some of the latest plant biotechnology developed by the project partners of this proposal. This proposal also addresses extraction of PHAs from biomass, modification of PHAs so that they have suitable properties for large volume polymer applications, processing of the PHAs using conversion processes now practiced at large scale (e.g., to film, fiber, and molded parts), conversion of PHA polymers to chemical building blocks, and demonstration of the usefulness of PHAs in large volume applications. The biodegradability of PHAs can also help to reduce solid waste in our landfills. If successful, this program will reduce U.S. dependence on imported oil, as well as contribute jobs and revenue to the agricultural economy and reduce the overall emissions of carbon to the atmosphere.

  9. Lignin valorization through integrated biological funneling and chemical catalysis

    Science.gov (United States)

    Linger, Jeffrey G.; Vardon, Derek R.; Guarnieri, Michael T.; Karp, Eric M.; Hunsinger, Glendon B.; Franden, Mary Ann; Johnson, Christopher W.; Chupka, Gina; Strathmann, Timothy J.; Pienkos, Philip T.; Beckham, Gregg T.

    2014-01-01

    Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a “biological funnel” to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the β-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydrate-derived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials. PMID:25092344

  10. Expression of the entire polyhydroxybutyrate operon of Ralstonia eutropha in plants.

    Science.gov (United States)

    Mozes-Koch, Rita; Tanne, Edna; Brodezki, Alexandra; Yehuda, Ran; Gover, Ofer; Rabinowitch, Haim D; Sela, Ilan

    2017-01-01

    Previously we demonstrated that an entire bacterial operon (the PRN operon) is expressible in plants when driven by the Tomato -yellow-leaf-curl-virus (TYLCV) -derived universal vector IL-60.Petroleum-derived plastics are not degradable, and are therefore harmful to the environment. Fermentation of bacteria carrying operons for polyhydroxyalkanoates (PHAs) produces degradable bioplastics which are environmentally friendly. However, bacterial production of bioplastics is not cost-effective, and attention is turning to their production in plants. Such "green" plastics would be less expensive and environmentally friendly. Hence, attempts are being made to substitute petroleum-derived plastics with "green" plastics. However, transformation of plants with genes of operons producing bioplastics has deleterious effects. Transformation of plastids does not cause deleterious effects, however it is a complicated procedures. We have developed another TYLCV-based vector (SE100) and show that yet another bacterial operon (the phaCAB operon) when driven by SE100 is also expressed in plants. We employed the combination of SE100 and the phaCAB operon to drive the operon to the plastids and produce in plants a biodegradable plastic [polyhydroxybutyrate (PHB)].Here we indicate that the bacterial operon (phaCAB), when driven by the newly developed universal plant vector SE100 is directed to chloroplasts and produces in plants PHB, a leading PHA. The PHB-producing plants circumvent the need for complicated technical procedures. The viral vector system SE100 facilitated the production of the bio-plastic poly-3-hydroxybutyrate. This was achieved by using the full pha-CAB operon indicating that TYLCV based system can transcribe and translate genes from bacterial operons controlled by a single cis element. Our data hints to the participation of the chloroplasts in these processes.

  11. Novel bio-based and biodegradable polymer blends

    Science.gov (United States)

    Yang, Shengzhe

    Most plastic materials, including high performance thermoplastics and thermosets are produced entirely from petroleum-based products. The volatility of the natural oil markets and the increasing cost of petroleum have led to a push to reduce the dependence on petroleum products. Together with an increase in environmental awareness, this has promoted the use of alternative, biorenewable, environmentally-friendly products, such as biomass. The growing interest in replacing petroleum-based products by inexpensive, renewable, natural materials is important for sustainable development into the future and will have a significant impact on the polymer industry and the environment. This thesis involved characterization and development of two series of novel bio-based polymer blends, namely polyhydroxyalkanoate (PHA)/polyamide (PA) and poly(lactic acid) (PLA)/soy protein. Blends with different concentrations and compatible microstructures were prepared using twin-screw extruder. For PHA/PA blends, the poor mechanical properties of PHA improved significantly with an excellent combination of strength, stiffness and toughness by adding PA. Furthermore, the effect of blending on the viscoelastic properties has been investigated using small-amplitude oscillatory shear flow experiments as a function of blend composition and angular frequency. The elastic shear modulus (G‧) and complex viscosity of the blends increased significantly with increasing the concentration of PHA. Blending PLA with soy protein aims at reducing production cost, as well as accelerating the biodegradation rate in soil medium. In this work, the mechanical, thermal and morphological properties of the blends were investigated using dynamic mechanical analysis (DMA), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and tensile tests.

  12. Life cycle risks for human health: a comparison of petroleum versus bio-based production of five bulk organic chemicals.

    Science.gov (United States)

    Roes, Alexander L; Patel, Martin K

    2007-10-01

    This article describes the development and application of a generic approach to the comparative assessment of risks related to the production of organic chemicals by petrochemical processes versus white biotechnology. White biotechnology, also referred to as industrial biotechnology, typically uses bio-based feedstocks instead of the fossil raw materials used in the petrochemical sector. The purpose of this study was to investigate whether the production of chemicals by means of white biotechnology has lower conventional risks than their production by petrochemical processes. Conventional risks are the risks of well-established processes, and not those related to genetically modified microorganisms and plants. Our approach combines classical risk assessment methods (largely based on toxicology), as developed by the life cycle assessment (LCA) community, with statistics on technological disasters, accidents, and work-related illnesses. Moreover, it covers the total process chain for both petrochemical and bio-based products from cradle to grave. The approach was applied to five products: the plastics polytrimethylene terephthalate (PTT), polyhydroxyalkanoates (PHA), polyethylene terephthalate (PET), polyethylene (PE), and ethanol. Our results show that the conventional risks related to the white biotechnology products studied are lower than those of the petrochemical products. However, considering the uncertainties with respect to the ranges of input data, the (incomplete) coverage of emissions by the environmental priority strategies (EPS) 2000 method, and the uncertainties of the assumptions made in this study (i.e., large to very large), the differences in results between bio-based and petrochemical products fall into the uncertainty range. Because of this, future research is necessary to decrease the uncertainties before we can conclude that the conventional risks of biotechnologically produced chemicals are lower than those of fossil-fuel-derived chemicals.

  13. The second green revolution? Production of plant-based biodegradable plastics.

    Science.gov (United States)

    Mooney, Brian P

    2009-03-01

    Biodegradable plastics are those that can be completely degraded in landfills, composters or sewage treatment plants by the action of naturally occurring micro-organisms. Truly biodegradable plastics leave no toxic, visible or distinguishable residues following degradation. Their biodegradability contrasts sharply with most petroleum-based plastics, which are essentially indestructible in a biological context. Because of the ubiquitous use of petroleum-based plastics, their persistence in the environment and their fossil-fuel derivation, alternatives to these traditional plastics are being explored. Issues surrounding waste management of traditional and biodegradable polymers are discussed in the context of reducing environmental pressures and carbon footprints. The main thrust of the present review addresses the development of plant-based biodegradable polymers. Plants naturally produce numerous polymers, including rubber, starch, cellulose and storage proteins, all of which have been exploited for biodegradable plastic production. Bacterial bioreactors fed with renewable resources from plants--so-called 'white biotechnology'--have also been successful in producing biodegradable polymers. In addition to these methods of exploiting plant materials for biodegradable polymer production, the present review also addresses the advances in synthesizing novel polymers within transgenic plants, especially those in the polyhydroxyalkanoate class. Although there is a stigma associated with transgenic plants, especially food crops, plant-based biodegradable polymers, produced as value-added co-products, or, from marginal land (non-food), crops such as switchgrass (Panicum virgatum L.), have the potential to become viable alternatives to petroleum-based plastics and an environmentally benign and carbon-neutral source of polymers.

  14. Microbial diversity and activity in the Nematostella vectensis holobiont: insights from 16S rRNA gene sequencing, isolate genomes, and a pilot-scale survey of gene expression

    Directory of Open Access Journals (Sweden)

    Jia Yi Har

    2015-09-01

    Full Text Available We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing 99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e. Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P, polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins. We hypothesize that such activities may mediate acclimation and persistence of bacteria in N. vectensis.

  15. Microbial diversity and activity in the Nematostella vectensis holobiont: insights from 16S rRNA gene sequencing, isolate genomes, and a pilot-scale survey of gene expression.

    Science.gov (United States)

    Har, Jia Y; Helbig, Tim; Lim, Ju H; Fernando, Samodha C; Reitzel, Adam M; Penn, Kevin; Thompson, Janelle R

    2015-01-01

    We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing 99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and

  16. Improved productivity of poly (3-hydroxybutyrate) (PHB) in thermophilic Chelatococcus daeguensis TAD1 using glycerol as the growth substrate in a fed-batch culture.

    Science.gov (United States)

    Cui, Bin; Huang, Shaobin; Xu, Fuqian; Zhang, Ruijian; Zhang, Yongqing

    2015-07-01

    A particularly successful polyhydroxyalkanoate (PHA) in industrial applications is poly (3-hydroxybutyrate) (PHB). However, one of the major obstacles for wider application of PHB is the cost of its production and purification. Therefore, it is desirable to discover a method for producing PHB in large quantities at a competitive price. Glycerol is a cheap and widely used carbon source that can be applied in PHB production process. There are numerous advantages to operating fermentation at elevated temperatures; only several thermophilic bacteria are able to accumulate PHB when glycerol is the growth substrate. Here, we report on the possibility of increasing PHB production at low cost using thermophilic Chelatococcus daeguensis TAD1 when glycerol is the growth substrate in a fed-batch culture. We found that (1) excess glycerol inhibited PHB accumulation and (2) organic nitrogen sources, such as tryptone and yeast extract, promoted the growth of C. daeguensis TAD1. In the batch fermentation experiments, we found that using glycerol at low concentrations as the sole carbon source, along with the addition of mixed nitrate (NH4Cl, tryptone, and yeast extract), stimulated PHB accumulation in C. daeguensis TAD1. The results showed that the PHB productivity decreased in the following order: two-stage fed-batch fermentation > fed-batch fermentation > batch fermentation. In optimized culture conditions, a PHB amount of 17.4 g l(-1) was obtained using a two-stage feeding regimen, leading to a productivity rate of 0.434 g l(-1) h(-1), which is the highest productivity rate reported for PHB to date. This high PHB biosynthetic productivity could decrease the total production cost, allowing for further development of industrial applications of PHB.

  17. Direct Production of Propene from the Thermolysis of Poly(β-hydroxybutyrate) (PHB). An Experimental and DFT Investigation.

    Science.gov (United States)

    Clark, Jared M; Pilath, Heidi M; Mittal, Ashutosh; Michener, William E; Robichaud, David J; Johnson, David K

    2016-01-28

    We demonstrate a synthetic route toward the production of propene directly from poly(β-hydroxybutyrate) (PHB), the most common of a wide range of high-molecular-mass microbial polyhydroxyalkanoates. Propene, a major commercial hydrocarbon, was obtained from the depolymerization of PHB and subsequent decarboxylation of the crotonic acid monomer in good yields (up to 75 mol %). The energetics of PHB depolymerization and the gas-phase decarboxylation of crotonic acid were also studied using density functional theory (DFT). The average activation energy for the cleavage of the R'C(O)O-R linkage is calculated to be 163.9 ± 7.0 kJ mol(-1). Intramolecular, autoacceleration effects regarding the depolymerization of PHB, as suggested in some literature accounts, arising from the formation of crotonyl and carboxyl functional groups in the products could not be confirmed by the results of DFT and microkinetic modeling. DFT results, however, suggest that intermolecular catalysis involving terminal carboxyl groups may accelerate PHB depolymerization. Activation energies for this process were estimated to be about 20 kJ mol(-1) lower than that for the noncatalyzed ester cleavage, 144.3 ± 6.4 kJ mol(-1). DFT calculations predict the decarboxylation of crotonic acid to follow second-order kinetics with an activation energy of 147.5 ± 6.3 kJ mol(-1), consistent with that measured experimentally, 146.9 kJ mol(-1). Microkinetic modeling of the PHB to propene overall reaction predicts decarboxylation of crotonic acid to be the rate-limiting step, consistent with experimental observations. The results also indicate that improvements made to enhance the isomerization of crotonic acid to vinylacetic acid will improve the direct conversion of PHB to propene.

  18. Controlling microbial PHB synthesis via CRISPRi.

    Science.gov (United States)

    Li, Dan; Lv, Li; Chen, Jin-Chun; Chen, Guo-Qiang

    2017-07-01

    Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47-75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

  19. Ralstonia eutropha's Poly(3-hydroxybutyrate)(PHB) polymerase PhaC1 and PHB depolymerase PhaZa1 are phosphorylated in vivo.

    Science.gov (United States)

    Jüngert, Janina R; Patterson, Cameron; Jendrossek, Dieter

    2018-04-20

    In this study, we screened PHB synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phase. Thr373 of PHB synthase PhaC1 was phosphorylated in the stationary growth phase but was not modified in the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in phosphorylated form both in the exponential and in the stationary growth phase. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modification of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for modulation of the activities of PHB synthase and PHB depolymerase. Importance Polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and other stress conditions. The simultaneous presence of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation Cupriavidus necator ) has been previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of identified residues. Furthermore, we conducted in vitro and in vivo analysis of PHB synthase activity and PHB contents. Copyright © 2018 American Society for Microbiology.

  20. Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems

    Directory of Open Access Journals (Sweden)

    Simon J. McIlroy

    2018-05-01

    Full Text Available Enhanced biological phosphorus removal (EBPR involves the cycling of biomass through carbon-rich (feast and carbon-deficient (famine conditions, promoting the activity of polyphosphate accumulating organisms (PAOs. However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs, which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal – as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the “Candidatus Accumulibacter” PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of

  1. Selective enrichment of a methanol-utilizing consortium using pulp & paper mill waste streams

    Energy Technology Data Exchange (ETDEWEB)

    Gregory R. Mockos; William A. Smith; Frank J. Loge; David N. Thompson

    2007-04-01

    Efficient utilization of carbon inputs is critical to the economic viability of the current forest products sector. Input carbon losses occur in various locations within a pulp mill, including losses as volatile organics and wastewater . Opportunities exist to capture this carbon in the form of value-added products such as biodegradable polymers. Waste activated sludge from a pulp mill wastewater facility was enriched for 80 days for a methanol-utilizing consortium with the goal of using this consortium to produce biopolymers from methanol-rich pulp mill waste streams. Five enrichment conditions were utilized: three high-methanol streams from the kraft mill foul condensate system, one methanol-amended stream from the mill wastewater plant, and one methanol-only enrichment. Enrichment reactors were operated aerobically in sequencing batch mode at neutral pH and 25°C with a hydraulic residence time and a solids retention time of four days. Non-enriched waste activated sludge did not consume methanol or reduce chemical oxygen demand. With enrichment, however, the chemical oxygen demand reduction over 24 hour feed/decant cycles ranged from 79 to 89 %, and methanol concentrations dropped below method detection limits. Neither the non-enriched waste activated sludge nor any of the enrichment cultures accumulated polyhydroxyalkanoates (PHAs) under conditions of nitrogen sufficiency. Similarly, the non-enriched waste activated sludge did not accumulate PHAs under nitrogen limited conditions. By contrast, enriched cultures accumulated PHAs to nearly 14% on a dry weight basis under nitrogen limited conditions. This indicates that selectively-enriched pulp mill waste activated sludge can serve as an inoculum for PHA production from methanol-rich pulp mill effluents.

  2. Thermochemical Wastewater Valorization via Enhanced Microbial Toxicity Tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Beckham, Gregg T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Thelhawadigedara, Lahiru Niroshan Jayakody [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Johnson, Christopher W [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Pleitner, Brenna P [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Cleveland, Nicholas S [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Michener, William E [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Olstad-Thompson, Jessica L [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Vardon, Derek R [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Whitham, Jason M. [Oak Ridge National Laboratory; Giannone, Richard J. [Oak Ridge National Laboratory; Klingeman, Dawn M. [Oak Ridge National Laboratory; Brown, Robert C. [Iowa State University; Brown, Steven D. [Oak Ridge National Laboratory; LanzaTech, Inc.; Hettich, Robert L. [Oak Ridge National Laboratory; Guss, Adam M. [Oak Ridge National Laboratory

    2018-04-17

    Thermochemical (TC) biomass conversion processes such as pyrolysis and liquefaction generate considerable amounts of wastewater, which often contains highly toxic compounds that are incredibly challenging to convert via standard wastewater treatment approaches such as anaerobic digestion. These streams represent a cost for TC biorefineries, and a potential valorization opportunity, if effective conversion methods are developed. The primary challenge hindering microbial conversion of TC wastewater is toxicity. In this study, we employ a robust bacterium, Pseudomonas putida, with TC wastewater streams to demonstrate that aldehydes are the most inhibitory compounds in these streams. Proteomics, transcriptomics, and fluorescence-based immunoassays of P. putida grown in a representative wastewater stream indicate that stress results from protein damage, which we hypothesize is a primary toxicity mechanism. Constitutive overexpression of the chaperone genes, groEL, groES, and clpB, in a genome-reduced P. putida strain improves the tolerance towards multiple TC wastewater samples up to 200-fold. Moreover, the concentration ranges of TC wastewater are industrially relevant for further bioprocess development for all wastewater streams examined here, representing different TC process configurations. Furthermore, we demonstrate proof-of-concept polyhydroxyalkanoate production from the usable carbon in an exemplary TC wastewater stream. Overall, this study demonstrates that protein quality control machinery and repair mechanisms can enable substantial gains in microbial tolerance to highly toxic substrates, including heterogeneous waste streams. When coupled to other metabolic engineering advances such as expanded substrate utilization and enhanced product accumulation, this study generally enables new strategies for biological conversion of highly-toxic, organic-rich wastewater via engineered aerobic monocultures or designer consortia.

  3. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kamnev, Alexander A. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation)]. E-mail: aakamnev@ibppm.sgu.ru; Tugarova, Anna V. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Antonyuk, Lyudmila P. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Tarantilis, Petros A. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Kulikov, Leonid A. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Perfiliev, Yurii D. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Polissiou, Moschos G. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Gardiner, Philip H.E. [Division of Chemistry, School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2006-07-28

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with {sup 57}Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

  4. The link of feast-phase dissolved oxygen (DO) with substrate competition and microbial selection in PHA production.

    Science.gov (United States)

    Wang, Xiaofei; Oehmen, Adrian; Freitas, Elisabete B; Carvalho, Gilda; Reis, Maria A M

    2017-04-01

    Polyhydroxyalkanoates (PHAs) are biobased and biodegradable polyesters with the potential to replace conventional plastics. Aeration requires large amounts of energy in PHA production by mixed microbial cultures (MMCs), particularly during the feast phase due to substrate uptake. The objective of this study was to investigate the impact of DO concentrations on microbial selection, substrate competition and PHA production performance by MMCs. This represents the first study investigating DO impact on PHA production while feeding the multiple volatile fatty acids (VFAs) typically encountered in real fermented feedstocks, as well as the substrate preferences at different DO levels. Efficient microbial cultures were enriched under both high (3.47 ± 1.12 mg/L) and low (0.86 ± 0.50 mg/L) DO conditions in the feast phase containing mostly the same populations but with different relative abundance. The most abundant microorganisms in the two MMCs were Plasticicumulans, Zoogloea, Paracoccus, and Flavobacterium. Butyrate and valerate were found to be the preferred substrates as compared to acetate and propionate regardless of DO concentrations. In the accumulation step, the PHA storage capacity and yield were less affected by the change of DO levels when applying the culture selected under low DO in the feast phase (PHA storage capacity >60% and yield > 0.9 Cmol PHA/Cmol VFA). A high DO level is required for maximal PHA accumulation rates with the four VFAs (acetate, propionate, butyrate and valerate) present, due to the lower specific uptake rates of acetate and propionate under low DO conditions. However, butyrate and valerate specific uptake rates were less impacted by DO levels and hence low DO for PHA accumulation may be effective when feed is composed of these substrates only. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts.

    Science.gov (United States)

    Bhattacharyya, Anirban; Saha, Jayeeta; Haldar, Saubhik; Bhowmic, Asit; Mukhopadhyay, Ujjal Kumar; Mukherjee, Joydeep

    2014-03-01

    Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2% (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD5 and COD values of stillage by 83% was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96% of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.

  6. Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4–55 PHA synthase and an insight into its catalytic mechanism

    Directory of Open Access Journals (Sweden)

    Ahmad Khairudin Nurul

    2006-11-01

    Full Text Available Abstract Background Polyhydroxyalkanoates (PHA, are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D model of the Type II Pseudomonas sp. USM 4–55 PHA synthase 1 (PhaC1P.sp USM 4–55. Results Sequence analysis demonstrated that PhaC1P.sp USM 4–55 lacked similarity with all known structures in databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the alpha/beta hydrolase fold family. Threading approach revealed that the most suitable template to use was the human gastric lipase (PDB ID: 1HLG. The superimposition of the predicted PhaC1P.sp USM 4–55 model with 1HLG covering 86.2% of the backbone atoms showed an RMSD of 1.15 Å. The catalytic residues comprising of Cys296, Asp451 and His479 were found to be conserved and located adjacent to each other. In addition to this, an extension to the catalytic mechanism was also proposed whereby two tetrahedral intermediates were believed to form during the PHA biosynthesis. These transition state intermediates were further postulated to be stabilized by the formation of oxyanion holes. Based on the sequence analysis and the deduced model, Ser297 was postulated to contribute to the formation of the oxyanion hole. Conclusion The 3D model of the core region of PhaC1P.sp USM 4–55 from residue 267 to residue 484 was developed using computational techniques and the locations of the catalytic residues were identified. Results from this study for the first time highlighted Ser297 potentially

  7. Film forming microbial biopolymers for commercial applications--a review.

    Science.gov (United States)

    Vijayendra, S V N; Shamala, T R

    2014-12-01

    Microorganisms synthesize intracellular, structural and extracellular polymers also referred to as biopolymers for their function and survival. These biopolymers play specific roles as energy reserve materials, protective agents, aid in cell functioning, the establishment of symbiosis, osmotic adaptation and support the microbial genera to function, adapt, multiply and survive efficiently under changing environmental conditions. Viscosifying, gelling and film forming properties of these have been exploited for specific significant applications in food and allied industries. Intensive research activities and recent achievements in relevant and important research fields of global interest regarding film forming microbial biopolymers is the subject of this review. Microbial polymers such as pullulan, kefiran, bacterial cellulose (BC), gellan and levan are placed under the category of exopolysaccharides (EPS) and have several other functional properties including film formation, which can be used for various applications in food and allied industries. In addition to EPS, innumerable bacterial genera are found to synthesis carbon energy reserves in their cells known as polyhydroxyalkanoates (PHAs), microbial polyesters, which can be extruded into films with excellent moisture and oxygen barrier properties. Blow moldable biopolymers like PHA along with polylactic acid (PLA) synthesized chemically in vitro using lactic acid (LA), which is produced by LA bacteria through fermentation, are projected as biodegradable polymers of the future for packaging applications. Designing and creating of new property based on requirements through controlled synthesis can lead to improvement in properties of existing polysaccharides and create novel biopolymers of great commercial interest and value for wider applications. Incorporation of antimicrobials such as bacteriocins or silver and copper nanoparticles can enhance the functionality of polymer films especially in food packaging

  8. A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli.

    Science.gov (United States)

    Mezzina, Mariela P; Álvarez, Daniela S; Egoburo, Diego E; Díaz Peña, Rocío; Nikel, Pablo I; Pettinari, M Julia

    2017-07-15

    The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals. IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E

  9. Characterization and modification of selected bioplastics

    Science.gov (United States)

    Wei, Liqing

    Bioplastics are becoming increasingly prominent mainly due to the growing environmental pollutions caused by non-biodegradable plastics. Polyhydroxybutyrate-co-hydroxyvalerate (PHBV), the major copolymer of polyhydroxyalkanoates (PHAs) family, was biosynthesized (by mixed microbial culture fed with fermented diary manure) and characterized. The monomeric composition (HV% ~ 40%) was determined by GC-MS and NMR. ESI-MSn and NMR analyses showed these PHBVs had random co-monomeric sequence distribution; meantime, they showed characteristic properties (crystallinity, single melting, and tensile properties) as studied by DSC and DMA. The homopolymer poly(3-hydroxybutyrate) (PHB), usually shows interesting properties such as high crystallinity and multiple melting behaviors. The effect of thermal history, such as crystallization (isothermal (temepratures = 80 to 140 °C) and nonisothermal (cooling rates = 2 to 50 °C/min)) and melting (heating rates = 5 to 50 °C/min), on the multiple melting behavior of PHB has been studied using conventional and temperature modulated DSC (TMDSC) techniques. Results showed PHB multiple melting was ascribed to the melting-recrystallization-remelting mechanism and its crystallization kinetics varied with crystallization temperatures and cooling rates. The brittleness and poor melt strength properties of the bioplastics PHB and poly L-lactic acid (PLLA) were imporved by two strategies: (i) to modify polymer structures by cross-linking, (ii) to introduce an external component by grafting, which were initiated by dicumyl peroxide (DCP) via reactive extrusion, have been developed. In method (i), rheological measurements showed cross-linked PHB and PLLA (0.25 to 1 wt% DCP) separately had higher melting strength than their linear polymers due to the formation of long chain branching. Their brittleness was reduced because smaller crystal sizes were observed by hot-stage polarized microscope (HS-POM), meanwhile the reduction of crystallinity was

  10. Microbial Population Dynamics and Ecosystem Functions of Anoxic/Aerobic Granular Sludge in Sequencing Batch Reactors Operated at Different Organic Loading Rates

    Directory of Open Access Journals (Sweden)

    Enikö Szabó

    2017-05-01

    Full Text Available The granular sludge process is an effective, low-footprint alternative to conventional activated sludge wastewater treatment. The architecture of the microbial granules allows the co-existence of different functional groups, e.g., nitrifying and denitrifying communities, which permits compact reactor design. However, little is known about the factors influencing community assembly in granular sludge, such as the effects of reactor operation strategies and influent wastewater composition. Here, we analyze the development of the microbiomes in parallel laboratory-scale anoxic/aerobic granular sludge reactors operated at low (0.9 kg m-3d-1, moderate (1.9 kg m-3d-1 and high (3.7 kg m-3d-1 organic loading rates (OLRs and the same ammonium loading rate (0.2 kg NH4-N m-3d-1 for 84 days. Complete removal of organic carbon and ammonium was achieved in all three reactors after start-up, while the nitrogen removal (denitrification efficiency increased with the OLR: 0% at low, 38% at moderate, and 66% at high loading rate. The bacterial communities at different loading rates diverged rapidly after start-up and showed less than 50% similarity after 6 days, and below 40% similarity after 84 days. The three reactor microbiomes were dominated by different genera (mainly Meganema, Thauera, Paracoccus, and Zoogloea, but these genera have similar ecosystem functions of EPS production, denitrification and polyhydroxyalkanoate (PHA storage. Many less abundant but persistent taxa were also detected within these functional groups. The bacterial communities were functionally redundant irrespective of the loading rate applied. At steady-state reactor operation, the identity of the core community members was rather stable, but their relative abundances changed considerably over time. Furthermore, nitrifying bacteria were low in relative abundance and diversity in all reactors, despite their large contribution to nitrogen turnover. The results suggest that the OLR has

  11. Final Report: Development of Renewable Microbial Polyesters for Cost Effective and Energy- Efficient Wood-Plastic Composites

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Emerick, Robert W.; England, Alfred B.; Flanders, James P.; Loge, Frank J.; Wiedeman, Katherine A.; Wolcott, Michael P.

    2010-03-31

    In this project, we proposed to produce wood fiber reinforced thermoplastic composites (WFRTCs) using microbial thermoplastic polyesters in place of petroleum-derived plastic. WFRTCs are a rapidly growing product area, averaging a 38% growth rate since 1997. Their production is dependent on substantial quantities of petroleum based thermoplastics, increasing their overall energy costs by over 230% when compared to traditional Engineered Wood Products (EWP). Utilizing bio-based thermoplastics for these materials can reduce our dependence on foreign petroleum. We have demonstrated that biopolymers (polyhydroxyalkanoates, PHA) can be successfully produced from wood pulping waste streams and that viable wood fiber reinforced thermoplastic composite products can be produced from these materials. The results show that microbial polyester (PHB in this study) can be extruded together with wastewater-derived cell mass and wood flour into deck products having performance properties comparable to existing commercial HDPE/WF composite products. This study has thus proven the underlying concept that the microbial polyesters produced from waste effluents can be used to make cost-effective and energy-efficient wood-plastic composites. The cost of purified microbial polyesters is about 5-20 times that of HDPE depending on the cost of crude oil, due to high purification (40%), carbon substrate (40%) and sterilized fermentation (20%) costs for the PHB. Hence, the ability to produce competitive and functional composites with unpurified PHA-biomass mixtures from waste carbon sources in unsterile systems—without cell debris removal—is a significant step forward in producing competitive value-added structural composites from forest products residuals using a biorefinery approach. As demonstrated in the energy and waste analysis for the project, significant energy savings and waste reductions can also be realized using this approach. We recommend that the next step for development of

  12. Waste cooking oil as substrate for biosynthesis of poly(3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate: Turning waste into a value-added product

    Directory of Open Access Journals (Sweden)

    Yang, T. A.

    2013-01-01

    Full Text Available Aims: Improper disposal of domestic wastes, such as waste cooking oil (WCO, contributes to the deterioration of the environment and may lead to health problems. In this study, we evaluated the potential of plant-based WCO as a carbon source for the commercial biosynthesis of the bio-plastics, poly(3-hydroxybutyrate and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate. The consumption of WCO for this purpose would mitigate their pollution of the environment at the same time.Methodology and Results: WCO collected from several cafeterias in USM was tested as the carbon source for polyhydroxyalkanoates (PHA production. A selection of suitable nitrogen source was first conducted in order to obtain an acceptable number of dry cell weight (DCW and PHA content. Urea was found to be a suitable nitrogen source for the two bacterial strains used in our study, Cupriavidus necator H16 and its transformed mutant, C. necator PHB¯4 harboring the PHA synthase gene of Aeromonas caviae (PHB¯4/pBBREE32d13. With WCO as the sole carbon source, C. necator H16 yielded a relatively good dry cell weight (DCW=25.4 g/L, with 71 wt% poly(3-hydroxybutyrate P(3HBcontent. In comparison, the DCW obtained with fresh cooking oil (FCO was 24.8 g/L. The production of poly(3 hydroxybutyrate-co-3- hydroxyhexanoate [P(3HB-co-3HHx] from WCO by the transformant C. necator PHB¯4 was comparable, yielding a DCW of 22.3 g/L and P(3HB-co-3HHx content of 85 wt%. Lipase activities for both bacterial strains reached a maximum after 72 h of cultivation when time profile was conducted. Conclusion, significance and impact of study: The use of WCO as a carbon source in the biosynthesis of the bioplastic, PHA, turns a polluting domestic waste into a value-added biodegradable product. This renewable source material can thus be exploited for the low cost production of PHA.

  13. Recovery of nitrogen and phosphorus from alkaline fermentation liquid of waste activated sludge and application of the fermentation liquid to promote biological municipal wastewater treatment.

    Science.gov (United States)

    Tong, Juan; Chen, Yinguang

    2009-07-01

    removal was mainly attributed to the increase of influent SCFA, or rather, the increase of intracellular polyhydroxyalkanoates (PHA) which served as the carbon and energy sources for denitrification and phosphorus uptake. The addition of alkaline fermentation liquid to municipal wastewater, however, increased the effluent COD, which was caused mainly by the increase of influent humic acid, not protein or carbohydrate.

  14. Poly(-β-hydroxybutyrate) (PHB) depolymerase PHAZ Pen from Penicillium expansum: purification, characterization and kinetic studies.

    Science.gov (United States)

    Gowda U S, Vaishnavi; Shivakumar, Srividya

    2015-12-01

    Very few studies have been dedicated to R-hydroxyacids (R-HA) production using extracellular polyhydroxyalkanoate depolymerases (ePhaZs). Penicillium expansum produced maximum extracellular polyhydroxybutyrate depolymerase (~6 U/mL) by 72 h when grown in mineral salt medium containing 0.2 % w/v PHB, pH 5.0, at 30 °C and 200 rpm shaking conditions. Partial purification of the extracellular poly(-β-hydroxybutyrate) depolymerase PHAZ Pen from P. expansum by two steps using ammonium sulphate (80 % saturation) and affinity chromatography using concanavalin A yielded 22.76-fold purity and 43.15 % recovery of protein. The enzyme composed of a single polypeptide chain of apparent molecular mass of 20 kDa, as determined by SDS-PAGE, stained positive for glycoprotein by periodic-schiff base (PAS) staining. Optimum enzyme activity was detected between pH 4.0 and 6.0 at 45-50 °C with pH 5.0 and 50 °C supporting maximum activity. The enzyme was stable between pH 4.0 and 6.0 at 55 °C for 1 h with a residual activity of almost 70-80 %. The enzyme was completely inhibited by 1 mM DTT/1 mM HgCl 2 and N-ethylmaleimide (10 mM) indicating the importance of essential disulphide bonds (cystine residues) and tyrosine for enzyme activity or probably for maintaining the native enzyme structure. Among the various divalent and trivalent metal ions, mercuric chloride, ferric citrate and ferrous sulphate inhibited enzyme activity. The enzyme showed substrate specificity towards only PHB and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and no other lipid or other p-nitrophenyl fatty acids or with polycaprolactone, showing that it was a true depolymerase and not any lipase or cutinase. Preliminary investigation revealed β-hydroxybutyrate as the end product of PHB hydrolysis by P. expansum, suggesting that the enzyme acted principally as an exo-type hydrolase. The above properties when compared with other fungal PHB depolymerases reported till date suggest the distinct nature

  15. Synthetic approaches to uniform polymers.

    Science.gov (United States)

    Ali, Monzur; Brocchini, Steve

    2006-12-30

    Uniform polymers are characterised by a narrow molecular weight distribution (MWD). Uniformity is also defined by chemical structure in respect of (1) monomer orientation, sequence and stereo-regularity, (2) polymer shape and morphology and (3) chemical functionality. The function of natural polymers such as polypeptides and polynucleotides is related to their conformational structure (e.g. folded tertiary structure). This is only possible because of their high degree of uniformity. While completely uniform synthetic polymers are rare, polymers with broad structure and MWD are widely used in medicine and the biomedical sciences. They are integral components in final dosage forms, drug delivery systems (DDS) and in implantable devices. Increasingly uniform polymers are being used to develop more complex medicines (e.g. delivery of biopharmaceuticals, enhanced formulations or DDS's for existing actives). In addition to the function imparted by any new polymer it will be required to meet stringent specifications in terms of cost containment, scalability, biocompatibility and performance. Synthetic polymers with therapeutic activity are also being developed to exploit their polyvalent properties, which is not possible with low molecular weight molecules. There is need to utilise uniform polymers for applications where the polymer may interact with the systemic circulation, tissues or cellular environment. There are also potential applications (e.g. stimuli responsive coatings) where uniform polymers may be used for their more defined property profile. While it is not yet practical to prepare synthetic polymers to the same high degree of uniformity as proteins, nature also effectively utilises many polymers with lower degrees of uniformity (e.g. polysaccharides, poly(amino acids), polyhydroxyalkanoates). In recent years it has become possible to prepare with practical experimental protocols sufficient quantities of polymers that display many aspects of uniformity. This

  16. Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

    Science.gov (United States)

    Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

    2017-10-15

    Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of

  17. Strategies for efficiently selecting PHA producing mixed microbial cultures using complex feedstocks: Feast and famine regime and uncoupled carbon and nitrogen availabilities.

    Science.gov (United States)

    Oliveira, Catarina S S; Silva, Carlos E; Carvalho, Gilda; Reis, Maria A

    2017-07-25

    Production of polyhydroxyalkanoates (PHAs) by open mixed microbial cultures (MMCs) has been attracting increasing interest as an alternative technology to PHA production by pure cultures, due to the potential for lower costs associated with the use of open systems (eliminating the requirement for sterile conditions) and the utilisation of cheap feedstock (industrial and agricultural wastes). Such technology relies on the efficient selection of an MMC enriched in PHA-accumulating organisms. Fermented cheese whey, a protein-rich complex feedstock, has been used previously to produce PHA using the feast and famine regime for selection of PHA accumulating cultures. While this selection strategy was found efficient when operated at relatively low organic loading rate (OLR, 2g-CODL -1 d -1 ), great instability and low selection efficiency of PHA accumulating organisms were observed when higher OLR (ca. 6g-CODL -1 d -1 ) was applied. High organic loading is desirable as a means to enhance PHA productivity. In the present study, a new selection strategy was tested with the aim of improving selection for high OLR. It was based on uncoupling carbon and nitrogen supply and was implemented and compared with the conventional feast and famine strategy. For this, two selection reactors were fed with fermented cheese whey applying an OLR of ca. 8.5g-CODL -1 (with 3.8g-CODL -1 resulting from organic acids and ethanol), and operated in parallel under similar conditions, except for the timing of nitrogen supplementation. Whereas in the conventional strategy nitrogen and carbon substrates were added simultaneously at the beginning of the cycle, in the uncoupled substrates strategy, nitrogen addition was delayed to the end of the feast phase (i.e. after exogenous carbon was exhausted). The two different strategies selected different PHA-storing microbial communities, dominated by Corynebacterium and a Xantomonadaceae, respectively with the conventional and the new approaches. The new

  18. Beyond feast and famine: Selecting a PHA accumulating photosynthetic mixed culture in a permanent feast regime.

    Science.gov (United States)

    Fradinho, J C; Reis, M A M; Oehmen, A

    2016-11-15

    Currently, the feast and famine (FF) regime is the most widely applied strategy to select for polyhydroxyalkanoate (PHA) accumulating organisms in PHA production systems with mixed microbial cultures. As an alternative to the FF regime, this work studied the possibility of utilizing a permanent feast regime as a new operational strategy to select for PHA accumulating photosynthetic mixed cultures (PMCs). The PMC was selected in an illuminated environment and acetate was constantly present in the mixed liquor to guarantee a feast regime. During steady-state operation, the culture presented low PHA accumulation levels, likely due to low light availability, which resulted in most of the acetate being used for biomass growth (Y x/s of 0.64 ± 0.18 Cmol X/Cmol Acet). To confirm the light limitation on the PMC, SBR tests were conducted with higher light availability, at similar levels as would be expectable from natural sunlight. In this case, the Y x/s reduced to 0.11 ± 0.01 Cmol X/Cmol Acet and the culture presented a PHB production yield on acetate of 0.67 ± 0.01 Cmol PHB/Cmol Acet, leading to a maximum PHB content of 60%. Unlike other studied PMCs, the PMC was capable of simultaneous growth and PHB accumulation continuously throughout the cycle. Thus far, 60% PHA content is the maximum value ever reported for a PMC, a result that prospects the utilization of feast regimes as an alternative strategy for the selection of PHA accumulating PMCs. Furthermore, the PMC also presented high phosphate removal rates, delivering an effluent that complies with phosphate discharge limits. The advantages of selecting PMCs under a permanent feast regime are that no aeration inputs are required; it allows higher PHA contents and phosphate removal rates in comparison to FF-operated PMC systems; and it represents a novel means of integrating wastewater treatment with resource recovery in the form of PHA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Análisis de las polihidroxialcanoato sintasas (PhaC1 y PhaC2 en una cepa de Pseudomonas fluorescens IBUN S1602, aislada en suelos colombianos

    Directory of Open Access Journals (Sweden)

    Julieth Serrano Riaño

    2011-07-01

    Full Text Available Título en ingles: Analysis of polyhydroxyalkanoate synthases (PhaC1 and PhaC2 in a strain of Pseudomonas fluorescens   IBUN S1602 isolated from Colombian soil. Resumen La cepa Pseudomonas fluorescens IBUN S1602 conforma el grupo de aislamientos provenientes de suelos colombianos de caña de azúcar, que acumula polihidrioxialcanoato (PHA, fue seleccionada como promisoria para escalamiento comercial por tener afinidad por sustratos alternativos y económicos como el glicerol, aceites usados, suero de leche, entre otros. Dada la importancia de la enzima sintasa en la síntesis de los PHAs, en el presente trabajo se realizó el análisis molecular de los genes phaC1 y phaC2 que codifican las enzimas sintasas tipo II (PhaC1 y PhaC2. Para la obtención de los amplímeros requeridos en la secuenciación, se utilizó la técnica de PCR bajo condiciones estandarizadas para iniciadores diseñados reportados en las bases de datos. Se identificaron dos fragmentos de 1680 pb y 1683 pb correspondientes a  phaC1 y phaC2. El análisis comparativo de las secuencias proteicas resultantes de estos genes demuestra que la sintasa  IBUN S1602 contiene la región α/β hidrolasa y 8 residuos de aminoácidos conservados, que son características de las sintasas examinadas a nivel mundial. Se analizó la estructura enzimática a nivel primario y se predijo la secundaria. Se concluyó que  las sintasas de la cepa Pseudomonas fluorescens IBUN S1602 presentan alta homología con las sintasas tipo II  que se reportan para Pseudomonas. Los resultados obtenidos contribuyen al entendimiento básico de la biosíntesis de PHA,  la cual permitirá, en un futuro, el aumento de la calidad de PHA debida a la modulación del nivel de sintasa que se exprese en un organismo recombinante, con el fin de variar el peso molecular del biopolímero, propiedad esencial en el estudio de aplicaciones industriales.   Palabras clave: polihidroxialcanoatos, PHA sintasa, bioinform

  20. Synthesis of bio-based nanocomposites for controlled release of antimicrobial agents in food packaging

    Science.gov (United States)

    DeGruson, Min Liu

    The utilization of bio-based polymers as packaging materials has attracted great attention in both scientific and industrial areas due to the non-renewable and nondegradable nature of synthetic plastic packaging. Polyhydroxyalkanoate (PHA) is a biobased polymer with excellent film-forming and coating properties, but exhibits brittleness, insufficient gas barrier properties, and poor thermal stability. The overall goal of the project was to develop the polyhydroxyalkanoate-based bio-nanocomposite films modified by antimicrobial agents with improved mechanical and gas barrier properties, along with a controlled release rate of antimicrobial agents for the inhibition of foodborne pathogens and fungi in food. The ability for antimicrobial agents to intercalate into layered double hydroxides depended on the nature of the antimicrobial agents, such as size, spatial structure, and polarity, etc. Benzoate and gallate anions were successfully intercalated into LDH in the present study and different amounts of benzoate anion were loaded into LDH under different reaction conditions. Incorporation of nanoparticles showed no significant effect on mechanical properties of polyhydroxybutyrate (PHB) films, however, significantly increased the tensile strength and elongation at break of polyhydroxybutyrate-co-valerate (PHBV) films. The effects of type and concentration of LDH nanoparticles (unmodified LDH and LDH modified by sodium benzoate and sodium gallate) on structure and properties of PHBV films were then studied. The arrangement of LDH in the bio-nanocomposite matrices ranged from exfoliated to phase-separated depending on the type and concentration of LDH nanoparticles. Intercalated or partially exfoliated structures were obtained using modified LDH, however, only phase-separated structures were formed using unmodified LDH. The mechanical (tensile strength and elongation at break) and thermo-mechanical (storage modulus) properties were significantly improved with low

  1. The rheology, degradation, processing, and characterization of renewable resource polymers

    Science.gov (United States)

    Conrad, Jason David

    Renewable resource polymers have become an increasingly popular alternative to conventional fossil fuel based polymers over the past couple decades. The push by the government as well as both industrial and consumer markets to go "green" has provided the drive for companies to research and develop new materials that are more environmentally friendly and which are derived from renewable materials. Two polymers that are currently being produced commercially are poly-lactic acid (PLA) and polyhydroxyalkanoate (PHA) copolymers, both of which can be derived from renewable feedstocks and have shown to exhibit similar properties to conventional materials such as polypropylene, polyethylene, polystyrene, and PET. PLA and PHA are being used in many applications including food packaging, disposable cups, grocery bags, and biomedical applications. In this work, we report on the rheological properties of blends of PLA and PHA copolymers. The specific materials used in the study include Natureworks RTM 7000D grade PLA and PHA copolymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Blends ranging from 10 to 50 percent PHA by weight are also examined. Shear and extensional experiments are performed to characterize the flow behavior of the materials in different flow fields. Transient experiments are performed to study the shear rheology over time in order to determine how the viscoelastic properties change under typical processing conditions and understand the thermal degradation behavior of the materials. For the blends, it is determined that increasing the PHA concentration in the blend results in a decrease in viscosity and increase in degradation. Models are fit to the viscosity of the blends using the pure material viscosities in order to be able to predict the behavior at a given blend composition. We also investigate the processability of these materials into films and examine the resultant properties of the cast films. The mechanical and thermal properties of the

  2. Characterization of a Bio-Based, Biodegradable Class of Copolymers, Poly[(R)-3-Hydroxybutyrate-Co-(R)-3- Hydroxyhexanoate], and Application Development

    Science.gov (United States)

    Sobieski, Brian

    As modern society begins to focus on sustainability and renewable resources there is a growing need for the polymer industry to develop more environmentally friendly materials and practices. Part of this movement can be seen in the use of recycled materials in new products and in the development of bio-based, biodegradable polymers. Bio-based, biodegradable polymers are produced from renewable carbon sources, such as vegetable oils, typically polymerized using fermentation reactions via bacteria, and are able to be consumed by bacteria in landfills to completely convert the polymers to water and CO2. One class of such polymers are poly(hydroxyalkanoate)'s (PHAs), which are chiral, aliphatic polyesters. Within this class of polyesters are poly(hydroxybutyrate) (PHB) and the copolymer poly[(R)-3-hydroxybutyrate- co-(R)-3-hydroxyhexanoate] (PHBHx), which have received extensive study due to their material properties as thermoplastics. Although the properties of PHB have been widely explored, much still remains to be understood about these promising biodegradable polymers. Specifically, PHB and its copolymers exhibit physical gelation in most solvents, yet the origin and mechanism of gelation and the properties of the resulting gel state are unknown. This research effort was primarily focused on investigating the physical gel state of PHBHx. Five goals were laid out and completed: determining the origin of gelation, the mechanism of gelation, the structure of the gel state, the properties of the gel state, and the effects of gelation on electrospun fibers of PHBHx. These goals were achieved through material characterization of the gel state utilizing infrared spectroscopy/two-dimensional correlation spectroscopy, differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, and many other analysis methods. Crystallization of the polymer in solution was found to cause gelation in PHBHx solutions, where the polymer crystals act as tie points forming