Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. he respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on ea...

Four Magnetospirillum strains degrading toluene, phenol, benzoate, and other aromatic compounds under anaerobic conditions were isolated from denitrifying enrichment cultures. One of the isolates, toluene-degrading strain TS-6, contained genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria. Transcription of the genes was confirmed. It was controlled by growth substrates and oxygen conditions, but bcr genes were unexpectedly expressed in aerobic cells grown on benzoate. It was confirmed that the genus Magnetospirillum represents the third genus of denitrifying bacteria capable of degrading aromatic compounds under anaerobic conditions, besides the genera Thauera and Azoarcus.   

The anaerobic degradation of tetradecylamine and other long-chain alkylamines by a newly isolated denitrifying bacterium was studied. Strain ZN6 was isolated from a mixture of soil and active sludge and was identified as representing Pseudomonas stutzeri, based on partial 16S rRNA gene sequence analysis. Strain ZN6 was a mesophilic, motile, Gram-negative rod-shaped bacterium and was able to grow on a variety of compounds including even-numbered primary fatty amines with alkyl chains ranging from C(4) to C(18) coupled to nitrate reduction. Alkylamines were used as sole carbon, energy and nitrogen source and were completely mineralized. Nitrate was dissimilated by ZN6 to nitrite. When strain ZN6 was grown under nitrate limitation, nitrite was slowly dissimilated further. When cocultivated with the complete denitrifier Castellaniella defragens ZN3, anaerobic degradation under denitrifying of alkylamines by strain ZN6 was slightly faster. Strain ZN3 is a complete denitrifier, unable to convert tetradecylamine, and was copurified from the same enrichment culture as strain ZN6. The proposed pathway for the degradation of alkylamines in strain ZN6 starts with C-N cleavages to alkanals and further oxidation to the corresponding fatty acids. PMID:18721145

The aerobic and anaerobic degradation of trimethylamine by a newly isolated denitrifying bacterium from an enrichment culture with trimethylamine inoculated with activated sludge was studied. Based on 16S rDNA analysis, this strain was identified as a Paracoccus sp. The isolate, strain T231, aerobically degraded trimethylamine, dimethylamine and methylamine and released a stoichiometric amount of ammonium ion into the culture fluid as a metabolic product, indicating that these methylated amines were completely degraded to formaldehyde and ammonia. The strain degraded trimethylamine also under denitrifying conditions and consumed a stoichiometric amount of nitrate, demonstrating that complete degradation of trimethylamine was coupled with nitrate reduction. Cell-free extract prepared from cells grown aerobically on trimethylamine exhibited activities of trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase, dimethylamine mono-oxygenase, and methylamine mono-oxygenase. Cell-free extract from cells grown anaerobically on trimethylamine and nitrate exhibited activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase. These results indicate that strain T231 had two different pathways for aerobic and anaerobic degradation of trimethylamine. This is a new feature for trimethylamine metabolism in denitrifying bacteria. PMID:11685371

Triclosan (2, 4, 4???-trichloro-2???-hydroxyl diphenyl ether) is a broad-spectrum antimicrobial agent present in a number of house hold consumables. Aerobic and anaerobic enrichment cultures tolerating triclosan were developed and 77 bacterial strains tolerating triclosan at different levels were isolated from different inoculum sources. Biodegradation of triclosan under aerobic, anoxic (denitrifying and sulphate reducing conditions), and anaerobic conditions was studied in batch cultures with isolated pure strains and enrichment consortium developed. Under aerobic conditions, the isolated strains tolerated triclosan up to 1 g/L and degraded the compound in inorganic-mineral-broth and agar media. At 10 mg/L level triclosan, 95???±???1.2% was degraded in 5 days, producing phenol, catech...

In recent sediments, microbial biodegradation provides a control on the long-term preservation of organic matter, through the preferential loss of certain biomolecules and the alteration and concentration of other more recalcitrant molecules. Biodegradation of hydrocarbons derived from membrane lipids, has been demonstrated by both aerobic and strictly anaerobic culturing experiments. The isoprenoid pristane, once considered stable under anaerobic conditions, is in fact degraded by a denitrifying microcosm (BREGNARD et al., 1997) and a methanogenic, sulphate-reducing enrichment culture (GROSSI, 2000). We recently demonstrated pristane biodegradation and accompanying loss of nitrate by an activated sludge isolate. The measured nitrate consumption accounts for a 7.1 +/- 0.4 mg loss of pristane, 4.74% of the initial substrate, in 181 days, assuming pristane conversion to CO2. We have characterized the microorganisms active in the biodegradation process, through the creation of a 16S rDNA clone library, as well as fluorescence in situ hybridization (FISH). Experiments are in progress to enrich cultures of sulfate reducing bacteria that utilize pristane as a sole carbon source and to characterize reaction mechanisms in pristane-oxidizing pathways.

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 ?M of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form. PMID:22678127

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dime...

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species...

We compared the development of microalgal and bacterial-denitrifier communities within biofilms over 28 days in a restored-prairie stream (RP) and a stream receiving treated wastewater effluent (DER). Inorganic nutrient concentrations were an order of magnitude greater in DER, and stream waters differed in the quality of dissolved organics (characterized via pyrolysis-GC/MS). Biofilm biomass and the densities of algae and bacteria increased over time in both systems; however, algal and denitrifier community composition and the patterns of development differed between systems. Specifically, algal and denitrifier taxonomic composition stabilized more quickly in DER than RP, whereas the rates of algal and denitrifier succession were more closely coupled in RP than DER. We hypothesize that, under unenriched conditions, successional changes in algal assemblages influence bacterial denitrifiers due to their dependence on algal exudates, while under enriched conditions, this relationship is decoupled. Between-system differences in organic signatures supported this, as RP biofilms contained more labile, aliphatic compounds than DER. In addition, potential denitrification rates (DNP) were negatively correlated with the percentage of aromatic compounds within the biofilm organic signatures, suggesting a significant relationship between algal exudate composition and denitrification. These results are significant because anthropogenic factors that affect biofilm community composition may alter their capacity to perform critical ecosystem services. PMID:21585403

In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition   Michael V.W. Kofoed, Peter Stief, Morten Poulsen, and Andreas Schramm Department of Biological Sciences, Microbiology, University of Aarhus, Denmark Denitrification, the sequential reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying (active) denitrifiers. The goal of this study was therefore to develop a protocol for the in situ detection of denitrifying bacterial cells by targeting the mRNA of denitrification genes, hereby linking denitrification activity directly to the single-cell level. Target genes were narG (encoding nitrate reductase) and nosZ (encoding nitrous oxide reductase), to detect nitrate-reducing and completely denitrifying bacteria, respectively. Enzyme-labelled oligonucleotide probes and digoxygenin-labelled polynucleotide probes were evaluated for in situ hybridization in combination with immunochemical detection and catalyzed fluorescent reporter deposition (CARD-FISH). The general feasibility of the approach was first tested with pure cultures of Pseudomonas stutzeri and various denitrifying and nitrate-reducing isolates. Detailed studies of probe specificity and hybridization conditions using Clone-FISH of narG and nosZ libraries prepared from freshwater sediment revealed a sequence similarity threshold of about 80% for detectable hybridization with polynucleotide probes. Consequently, polynucleotide probes need to be designed based on habitat-specific sequence information. In contrast, oligonucleotide probes can be designed to target a broader range of denitrifying bacteria; however, they require two-pass CARD-FISH, which may result in (too) high background fluorescence. In a first application example, habitat-specific polynucleotide probes were used to quantify bacteria expressing narG and nosZ in freshwater sediments and the guts of benthic invertebrates.    

We examined the influence of elevated CO2 concentration on denitrifier enzyme activity in wheat rhizoplanes by using controlled environments and solution culture techniques. Potential denitrification activity was from 3 to 24 times higher on roots that were grown under an elevated CO2 concentration of 1,000 micromoles of CO2 mol-1 than on roots grown under ambient levels of CO2. Nitrogen loss, as determined by a nitrogen mass balance, increased with elevated CO2 levels in the shoot environment and with a high NO3- concentration in the rooting zone. These results indicated that aerial CO2 concentration can play a role in rhizosphere denitrifier activity.

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23...

The authors investigated the effect of the partial pressure of oxygen (pO/sub 2/) on the production of NO and N/sub 2/O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO/sub 2/ in the range tested (0.5 to 10%), whereas N/sub 2/O production was inversely proportional to pO/sub 2/. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N/sub 2/O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of No and N/sub 2/O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N/sub 2/O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sprayed with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N/sub 2/O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.

As part of a highly interdisciplinary study of in situ reductive immobilization of Cr at DOE's Hanford 100H site, we are developing a systems biology approach (employing metagenomic and meta-transcriptomic data) to identify highly expressed genes in the native microbial community under conditions of interest, without requiring any a priori sequence information or assumptions about what processes might be occurring. A key scientific goal is to determine if there are diagnostic biomolecular signatures indicative of important aquifer biogeochemical processes that can be used to (a) help discriminate between direct (enzymatic) and indirect (abiotic) oxidation-reduction processes relevant to bioremediation and (b) to inform and constrain reactive transport models even when geochemical field measurements do not reveal all relevant processes. We are in the process of collecting metagenomic and meta-transcriptomic sequence information from various experimental systems under conditions relevant to in situ chromate reduction at Hanford 100H. This poster focuses on Hanford microcosm studies. To characterize functional changes in an aquifer-derived, chromate-reducing microbial community as it transitions successively through electron-accepting conditions relevant to the Hanford subsurface, we inoculated anaerobic microcosms with groundwater from the Cr-contaminated Hanford 100H site and supplemented them with lactate and electron acceptors present at the site [e.g., nitrate, sulfate, and Fe(III)]. Metagenomic and meta-transcriptomic "snapshots" were taken during denitrification, sulfate and Fe(III) reduction, and nitrate-dependent oxidation of Fe(II) and sulfide. We conducted Illumina paired-end sequencing, assembled with ABySS-pe, and initially annotated using MG-RAST and CAMERA. cDNA samples for meta-transcriptome sequencing represented mRNA enriched using a new subtractive hybridization method resulting in 61-78% of reads mapping to their corresponding metagenomes. Observations from the analyses to date include the following: (1) consistent phylogenetic community transitions were documented by 16S rRNA pyrotag and metagenome sequence data as Hanford microcosms passed successively through denitrifying conditions (dominated initially by beta-Proteobacteria) to fermentative and sulfate- and iron-reducing conditions (dominated by Firmicutes); (2) the greatest diversity of denitrification genes occurred during initial denitrifying phase; (3) high expression of nitrate reductase (nar) and S oxidation (soxWXYZABCD) genes occurred after nitrate was added to cultures following sulfate-reducing phase, even though S oxidation was not detectable based on sulfate measurements; and (4) highly expressed genes in Hanford microcosms and groundwater included "hypothetical proteins", which supports the monitoring approach that we are pursuing, namely, to focus on highly expressed genes specific to Hanford rather than genes chosen a priori.

Abstract in english Ammonium oxidation was thought to be an exclusively aerobic process; however, as recently described in the literature, it is also possible under anaerobic conditions and this process was named ANAMMOX. This work describes the operation of a system consisting of a denitrifying reactor coupled to a nitrifying reactor used for removal of nitrogen from slaughterhouse wastewater. During operation of the denitrifying reactor an average nitrogen ammonium removal rate of 50 mg/Ld (more) was observed. This biomass was used to seed a second reactor, operated in repeated fed batch mode, fed with synthetic medium specific to the growth of bacteria responsible for the ANAMMOX process. The nitrogen loading rate varied between 33 and 67 mgN/Ld and average nitrogen removal was 95% and 40%, respectively. Results of fluorescence in situ hybridization (FISH) confirmed the presence of anammox-like microorganisms in the enriched biomass.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.   

Anaerobic fermentative degradation of resorcinol and resorcylates was studied in enrichment cultures inoculated with marine or freshwater sediments or digested sludge, e-Resorcylate (3,5-dihydroxybenzoate) was degraded very rapidly to acetate and methane by enrichment cultures inoculated with freshw...

By adopting two sequencing batch reactors (SBRs) A and B, nitrate as the substrate, and the intermittent aeration mode, activated sludge was domesticated to enrich aerobic denitrifiers. The pHs of reactor A were approximately 6.3 at DOs 2.2–6.1 mg/l for a carbon source of 720 mg/l COD; the pHs of reactor B were 6.8–7.8 at DOs 2.2–3.0 mg/l for a carbon source of 1500 mg/l COD. Both reactors maintained an influent nitrate concentration of 80 mg/l NO3–-N. When the total inorganic nitrogen (TIN) removal efficiency of both reactors reached 60%, aerobic denitrifier accumulation was regarded completed. By bromthymol blue (BTB) medium, 20 bacteria were isolated from the two SBRs and DNA samples of 8 of these 20 strains were amplified by PCR and processed for 16SrRNA sequencing. The obtained results were analysed by a Blast similarity search of the GenBank database, and constructing a phylogenetic tree for identification by comparison. The 8 bacteria were found to belong to the genera Pseudomonas, Delftia, Herbaspirillum and Comamonas. At present, no Delftia has been reported to be an aerobic denitrifier.   

Considerable information has been accumulated on the toxicity of hydrazine to soil bacterial cultures and on the degradation of hydrazne by soil bacterial cultures. The activities of the autotrophic nitrifiers Nitrosomonas and Nitrobacter and of denitrifying bacteria, and the growth of Enterobacter cloacae, were all inhibited by hydrazine. An enzyme system has been found in heterotrophic N/sub 2/-fixing bacteria capable of degrading hydrazine. Information concerning the effect of hydrazine on microbial activity in soils is not available, however. Accidental spills to soil can occur during transportation and storage. Therefore, this study was initiated to determine degradation rates of hydrazine in soils and its effect on soil microbial activity.

Continental margin sediments constitute only about 10% of the total sediment surface area in the world’s oceans, nevertheless they are the dominant sites of nitrogen (N) cycling. Recent studies suggest that the oceanic nitrogen budget is unbalanced, primarily due to a higher nitrogen removal rate in contrast to the fixation rate, and it has been suggested that denitrification activity contributes significantly to this imbalance. Although denitrification in marine environments has been studied intensively at the process level, little is known about the species abundance, composition, distribution, and functional differences of the denitrifying population. Understanding the diversity of microbial populations in marine environments, their responses to various environmental factors such as NO3-, and how this impact the rate of denitrification is critical to predict global N dynamics. Environmental Microbiology has the prompt to study the influence of each microbial population on a biogeochemical process within a given ecosystem. Culture-dependent and –independent techniques using nucleic acid probes can access the identity and activity of cultured and uncultured microorganisms. Nucleic acid probes can target distintict genes which set phylogenetic relationships, such as rDNA 16S, DNA gyrase (gyrB) and RNA polymerase sigma 70 factor (rpoD). In the other hand, the genetic capabilities and their expression could be tracked using probes that target several functional genes, such as nirS, nirK, nosZ, and nifH, which are genes involved in denitrification. Selective detection of cells actively expressing functional genes within a community using In Situ Reverse Transcription-PCR (ISRT-PCR) could become a powerful culture-independent technique in microbial ecology. Here we describe an approach to study the expression of nirS genes in denitrifying bacteria. Pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans, as well as co-cultures with non-denitrifying populations were used to optimize the ISRT-PCR protocol. Cells grown on nitrate broth were harvested and fixed at both logarithmic (24-48 h) and stationary phase (7 days). Fixed and RNA protectedTMcc cells were spotted on microscope slides to optimize cell wall permeabilization conditions with lyzozyme and proteinase K. Subsequently, ISRT-PCR was performed with NirS 1F and NirS 6R primers using the QIAGEN® OneStep RT-PCR Kit. Amplification products within the cell were detected by Fluorescent In Situ Hybridization (FISH) at 40ºC overnight using a Cy3 labeled internal probe, specifically designed to detect the nirS gene. After hybridization, the cells were counterstained with DAPI and examined by confocal fluorescence microscopy. P. stutzeri cells treated with RNase and Pseudomonas G179 (a nirK denitrifying strain) were used as negative controls. Optimal cell permeabilization was achieved using 1 mg ml-1 lyzozyme for 30 min and 2 µg ml-1 Proteinase K. RNase treated cells did not fluoresce after FISH, but were detectable by DAPI. Only nirS-type denitrifying cells in log phase (80-95% of total direct cell counts) were detected by this approach while fewer cells (5-10%) were detectable after 7 days in stationary phase. Co-cultures of P. denitrificans with a non-denitrifying isolate resulted in selective identification of target cells, thus supporting the potential use of this approach for gene expression analysis at the community level.

A denitrifying consortium capable of transforming carbon tetrachloride (CCl[sub 4]) was cultured from aquifer sediment from the US Department of Energy's Hanford Site in southeastern Washington State. To understand the kinetics of the biological destruction of CCl[sub 4] by these microbes, a set of experiments, the conditions of which were chosen according to a fractional factorial experimental design, were completed. This article reports on the experimental design along with the results for CCl[sub 4], biomass, acetate, nitrate, and nitrite concentrations. These data indicate that growth is inhibited by high nitrite concentrations, whereas CCl[sub 4] degradation is slowed by the presence of nitrate and/or nitrite.

We have been successful in isolating and characterizing sulfate reducing bacteria and denitrifying Thiobacillus sp. from oil field waters which can use volatile fatty acids and dissolve carbonates found in these waters and formations. We have shown that these cultures can feed each other in sequential cultures and survive in mixed culture as long as sterile conditions are maintained. When nonsterile conditions are present both organism types are only successful to a limited degree, and, instead, an additional microflora develop belonging to the heterotrophic denitrifying bacteria. These microorganisms appear to be potentially useful in new MEOR processes through the production of pressure forming gases, viscosifying agents and the removal and prevention of de novo synthesis of hydrogen sulfide and are currently being pursued. They are capable of producing large amounts of gas, predominantly nitrogen and carbon dioxide and also form viscosifying agents under conditions which we believe we can easily maintain in an oil reservoir. In this report we show that they are also potent agents for the control of SRB growth, development and production of sulfide.

Widely present in the environment, the highly-toxic compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been found to resist microbial biodegradation. To develop an anaerobic biodegradation approach for soils and sediments contaminated with TCDD, methanogenic and denitrifying cultures were established on a variety of chloroaromatic substrates, including 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,3-dichlorophenol, 3,4-dichlorophenol, 4,5-dichlorocatechol and catechol, using an inoculum from Newtown Creek (New York, NY). Dehalogenation was observed, with monochlorophenols producing phenol and dichlorophenols producing monochlorinated phenols and phenol. Based on gas production, the chlorinated catechol did not appear to undergo biodegradation under any condition, while catechol was degraded under methanogenic conditions. Select cultures amended with a mixture of chloroaromatics and n-butanol, a solubilizing agent, exhibited depressed gas production under both anaerobic conditions. Biodegradation of TCDD adsorbed onto particles of gallium oxide is under investigation with an amalgamation of the active single-substrate methanogenic cultures. 29 refs., 10 figs., 2 tabs.

Abstract The hydrocarbon-degrading bacterium Dietzia maris WR-3 was isolated from a consortium comprising ammonia-oxidizing and denitrifying bacteria derived from marine sediments. Here, we examined biosurfactant production by strain WR-3 when cultured using several different carbon (D-glucose, n -decane, n -hexadecane, motor oil, olive oil, and rapeseed oil) and nitrogen (NH4)2SO4, NaNO3, yeast extract, and polypeptone) sources as growth substrates. Strain WR-3 was able to grow and reduce the surface tension of culture broth to 311.0 mN m-1 when cultured using n -hexadecane and nitrate ions. The surface-active compounds produced by strain WR-3 were extracted and analyzed by thin layer chromatography. Moreover, the main components in the extract were further purified and subjected to gas c...

Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C6-C10) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic ...

The ability of diverse microbial species to concentrate uranium, cesium, and radium was examined. Saccharomyces cerevisiae, Pseudomonas aeruginosa, and a mixed culture of denitrifying bacteria accumulated uranium to 10 to 15% of the dry cell weight. Only a fraction of the cells in a given population had visible uranium deposits in electron micrographs. While metabolism was not required for uranium uptake, mechanistic differences in the metal uptake process were indicated. Uranium accumulated slowly (hours) on the surface of S. cerevisiae and was subject to environmental factors (i.e., temperature, pH, interfering cations and anions). In contrast, P. aeruginosa and the mixed culture of denitrifying bacteria accumulated uranium rapidly (minutes) as dense, apparently random, intracellular deposits. This very rapid accumulation has prevented us from determining whether the uptake rate during the transient between the initial and equilibrium distribution of uranium is affected by environmental conditions. However, the final equilibrium distributions are not affected by those conditions which affect uptake by S. cerevisiae. Cesium and radium were concentrated to a considerably lesser extent than uranium by the several microbial species tested. The potential utility of microorganisms for the removal and concentration of these metals from nuclear processing wastes and several bioreactor designs for contacting microorganisms with contaminated waste streams will be discussed.

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24h incubation in half-Fraser broth, 4h i...

A mixed culture was enriched from surface soil obtained from an eastern United States site highly contaminated with chromate. Growth of the culture was inhibited by a chromium concentration of 12 mg/L. Another mixed culture was enriched from subsurface soil obtained from the Hanford reservation, at the fringe of a chromate plume. The enrichment medium was minimal salts solution augmented with acetate as the carbon source, nitrate as the terminal electron acceptor, and various levels of chromate. This mixed culture exhibited chromate tolerance, but not chromate reduction capability, when growing anaerobically on this medium. However, this culture did exhibit chromate reduction capability when growing anaerobically on TSB. Growth of this culture was not inhibited by a chromium concentration of 12 mg/L. Mixed cultures exhibited decreasing diversity with increasing levels of chromate in the enrichment medium. An in situ bioremediation strategy is suggested for chromate contaminated soil and groundwater. 16 refs., 5 figs., 1 tab.

Soils emit variable amounts of NO and N2O, with environmental consequences (atmosphere chemistry and global warming). Nitrification was for some time considered the main source of NO emission, but several investigations have indicated that denitrification may be a potent source as well. However, strong emission of NO from denitrifying organisms is in some conflict with common understanding of the role of NO in the regulation of denitrification, as based on paradigm model strains. NO appears to be an important signal molecule for denitrifying organisms by exerting a positive feedback on the expression of the genes coding for denitrification. On the other hand, a careful control of the NO concentrations at nanomolar concentrations has long been considered an essential fitness character for denitrifying organisms, since micromolar concentrations of NO is toxic to many organisms. For the same reason, organisms lacking genes encoding NO reductase (NOR) have been considered unfit for denitrification. This view is challenged by isolation of organisms whose primary product of denitrification is NO, either because they lack the genes for NO reductase, or because their synthesis of the denitrification proteome is extremely unbalanced, resulting in transient NO accumulation to micromolar concentrations when grown in pure culture. Such paralyzing NO concentrations are probably never reached in natural environments, however, due to diffusion and NO-absorption by adjacent organisms, be it by NOR or other NO scavenging enzymes. Hypothetically, the production of NO by denitrifying organisms may be an advantage by fending off nearby competitors. We have embarked on a comparative study of denitrification phenotypes regarding their denitrification gene expression and control of NO and N2O concentrations in response to anoxic spells. This includes model strains (Paracoccus denitrificans and Agrobacterium tumefaciens) and recently isolated strains within several genera. Some are found to robustly control NO at nanomolar levels, others reach micromolar concentrations of NO. These phenotypes appear to be congruent with phylogeny: closely related strains have very similar phenotypes regarding NO concentrations. This contrasts N2O production, which appears not to be related to phylogeny. We hypothesize that community DNA or transcriptome analyses may have predictive capacity for a soil’s propensity to produce NO but not N2O. More data on the relationship between phylogeny and phenotypes is clearly needed, however. Biogeochemical models of denitrification and its NO and N2O emission are based on a ”community phenotype”. In theory, the characteristics of this community phenotype could be predicted by analyses of community DNA or transcriptomes, but to reach this goal we need more information about the genotype/phenotype relationship.

In this study, a real-time reverse transcriptase PCR (real-time RT-PCR) assay for the detection of Salmonella was designed and developed. The real-time RT-PCR assay targeted the multi-copy RNA, tmRNA, had an inclusivity and exclusivity of 100% and a sensitivity of @?1 cell equivalent. The assay was combined with culture enrichment and an initial validation was performed in accordance with ISO 16140: 2003. Culture enrichment required 18h primary enrichment in buffered peptone water (BPW) and 6h selective enrichment in Rappaport-Vassiliadis Soya Peptone Broth (RVS). The combined culture enrichment/real-time RT-PCR method had a relative specificity of 100% when compared to the traditional culture method. When compared to an assay targeting the ssrA gene, which encodes for tmRNA, an approximat...

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-as...

Alcaligenes faecalis no. 4 has heterotrophic nitrification and aerobic denitrification abilities. By taking the nitrogen balance under different culture conditions, 40–50% of removed NH4+-N was denitrified and about one-half of removed NH4+-N was converted to intracellular nitrogen. The maximum ammonium removal rate of no. 4 (28.9 mg-N/l/h) and its denitrification rate at high-strength NH4+-N of about 1200 ppm in aerated batch experiments at a C/N ratio of 10 were 5–40 times higher than those of other bacteria with the same ability. Only a few percent of the removed ammonium was converted to nitrite, and the main denitrification process was speculated to be via hydroxylamine which was produced by ammonium oxidation.   

To clarify the genetic basis to the diversity of denitrifying ability in soybean bradyrhizobia, we compared the end-products of denitrification (N2, N2O and NO2-) with the existence of denitrifying genes (napA, nirK, norCB and nosZ) of sixty phylogenetically diverse strains of Bradyrhizobium japonicum and B. elkanii. The results indicate that the existence of denitrifying genes directly determines phenotype (end-products) in most strains of B. japonicum. The denitrifying capability and gene set were reflected by phylogenetic position based on repeated sequences (RS)-fingerprints and 16S rRNA gene sequences. However, the denitrifying genes in HRS (highly repeated sequence-possessing) strains of B. japonicum, which were identified based on RS-fingerprints as having heavy hybridization, resulted in an inconsistent correlation probably because of genomic rearrangements. The evolutionary and ecological implications of the denitrifying genes and capability in soybean bradyrhizobia are discussed.   

The purpose of this study was to compare a conventional culture broth method (Bolton enrichment), a newly developed proprietary broth method (TECRA Campylobacter enrichment), and direct plating for recovery of Campylobacter spp. from chicken carcass rinsates. Whole carcass rinses were taken from 140 carcasses at rehang (immediately after defeathering but before evisceration) and from 140 carcasses at postchill from eight different processing plants in the United States. The rinsate samples were packed in ice and shipped overnight to the laboratory. Aliquots of the rinsate were transferred into Bolton and TECRA enrichment broths and were direct plated. Standard laboratory procedures with Campy-cefex plates were followed for recovery of Campylobacter spp. For rehang carcasses, 94% were positive for Campylobacter spp. with the TECRA enrichment broth and 74% were positive with the Bolton enrichment broth. For postchill carcasses, 74% were positive for Campylobacter spp. with the TECRA enrichment broth and 71% were positive with the Bolton enrichment broth. Compared with the Bolton enrichment broth, TECRA enrichment broth significantly suppressed non-Campylobacter microflora (P < 0.05). Overall, TECRA enrichment broth yielded an 11% higher total number of Campylobacter-positive samples compared with the Bolton enrichment broth. Campylobacter spp. detection in postchill samples was significantly greater (P < 0.05) by enrichment (84%) than by direct plating (19%). The high number of Campylobacter-positive samples obtained with all procedures indicated that 99% of the carcass rinsates obtained at rehang and 84% obtained at postchill contained Campylobacter spp. PMID:19517723

The ability of malachite green/magnesium chloride broth (Rappaport's medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were ...

Enrichment cultures were obtained, after prolonged incubation on a shale oil as the sole source of nitrogen, that selectively degraded nitriles. Capillary gas chromatographic analyses showed that the mixed microbial populations in the enrichments degraded the homologous series of aliphatic nitriles ...

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A ...

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence th...

lysine) in contrast to the casein enriched diet. These data suggest that ... isolated from the culture media and tested for their characteristic biochemical re- actions. The bacterial ...... 10: 957-980, 1.933. "Thyroid function in experimental strep- ...

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was establis...

A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chlori...

Chlordecone, known under the tradename Kepone, is a highly chlorinated cyclodiene pesticide that is resistant to microbial degradation. Six month chlordecone enrichment cultures yielded several Gram-negative microorganisms that were resistant to and/or degraded chlordecone. Three...

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene gl...

Aims: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. Methods and Results: A total of 286 samples (faecal, meat, neckskin and environmental samples) were collected over a period of 4 months at a turkey slaughterhouse and meat-cutting plant in Denmark. Faecal and environmental samples were tested by the conventional culture method and by the two EIAs, whereas meat and neckskin samples were tested by the two EIAs only. Two enrichment broths were used, Campylobacter Enrichment Broth (CEB) and Preston Broth (PB). Verification of positive test results was carried out by conventional culture on selective solid media. The specificities of all methods were high. The sensitivities of the EIAs were higher than that of the conventional culture technique but varied depending on the type of sample and enrichment broth. For neckskin samples, the Minividas had a significantly higher sensitivity than the EiaFoss and using PB instead of CEB as the enrichment broth significantly improved the sensitivity for both EIAs. Conclusions: Both EIAs provided more accurate results than the conventional culture technique. Furthermore, neckskin samples enriched in PB resulted in more positive test results and Campylobacter growth than samples enriched in CEB. Significance and Impact of the Study: The Eiafoss and Minividas proved to be reliable methods for detecting Campylobacter spp. in various samples. However, the results emphasize the need for the development of specific enrichment protocols for specific samples.

In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and ?-Proteobacteria but the heterotrophic community comprised many Dokdonella-related ?-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to th...

Abstract in english Isolation and identification of etiological agents found in body fluids can be of critical importance for the recovery of patients suffering from potentially-severe infections, which are often followed by serious sequels. Eighty-two samples of different body fluids were analyzed using two different methods: (1) the conventional culture method (agar plating) and (2) the enrichment culture technique, using the Bact/Alert® blood culture bottle. The number of positive cultur (more) es increased on average from 9.7% to 23.1% with the enrichment culture technique. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were the most frequently isolated bacteria. The enrichment method could provide a more accurate means the identifying etiological agents.

The use of mixed microbial cultures enriched for biological mercury removal is explored in this paper, focusing on the ecological shifts occurring throughout acclimatization to mercury and on the long-term stability of four microbial enrichments. The 16S rRNA genetic profiles obtained by denaturing gradient gel electrophoresis (DGGE) revealed that the glucose and ethanol cultures had similar profiles, whereas the acetate cultures diverged into a totally dissimilar cluster. Quantification of the merA gene copies in each enrichment showed higher values for the glucose culture, followed by the ethanol and then the acetate cultures, which was consistent with the mercury removal performance throughout the study. Isolates were obtained from the four cultures and analyzed with respect to their genetic (16S rRNA) and functional (merA) phylogenies in order to identify mercury-resistant species enriched with different carbon sources. All mercury-resistant isolates obtained from the glucose and ethanol cultures belonged to the Gammaproteobacteria, whereas acetate cultures also contained members of other phyla, with differences in merA sequences. Higher phylogenetic than functional diversity of the isolates, together with increasing merA copies even after culture stabilisation, highlight the role of horizontal gene transfer in the acclimatization process.   

In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to enrich hydrogen-producing bacteria. By varying the influent substrate concentrations and hydraulic retention times (HRT), the volumetric loading rate (VLR) of 55.64kg-COD/m3/day seemed to be optimum for this enriched culture for fermentative hydrogen production from lactate and acetate. The results of batch experiments confirmed that the enriched culture tended to fulfill the e- equiv requirement for cell growth at a lower VLR condition (21.77kg-COD/m3/day), while it could largely distribute the e- equiv for hydrogen production at a higher VLR condition. However, a maximum lactate/acetate concentration allowed for enriching this culture existed, especially at a lower HRT condition in w...

The red seaweed Hypnea spinella (Gigartinales, Rhodophyta), was cultured at laboratory scale under three different CO2 conditions, non-enriched air (360 ppm CO2) and CO2-enriched air at two final concentrations (750 and 1,600 ppm CO2), in order to evaluate the influence of increased CO2 concentrations on growth, photosynthetic capacity, nitrogen removal efficiency, and chemical cellular composition. Average specific growth rates of H. spinella treated with 750 and 1,600 ppm CO2-enriched air increased by 85.6% and 63.2% compared with non-enriched air cultures. CO2 reduction percentages close to 12% were measured at 750 ppm CO2 with respect to 5% and 7% for cultures treated with air and 1,600 ppm CO2, respectively. Maximum photosynthetic rates were enhanced significantly for high CO2 treatme...

Abstract Aims: The aim of this work was to enrich stable mixed cultures from atrazine-contaminated soil. The cultures were examined for their atrazine biodegradation efficiencies in comparison with J14a, a known atrazine-degrading strain of Agrobacterium radiobacter. The cultures were also characterized to identify community structure and bacterial species present. Methods and Results: The cultures were enriched and then stabilized in bacterial media. The stable mixed cultures and J14a were tested in a medium containing 100 mg l-1 of atrazine. For all cultures, atrazine was removed 33-51% within 7 days and the cell optical density increased from 005 to between 050 and 070. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence ...

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth foll...

A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier. When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers faile...

The hydrocarbon-degrading bacterium Dietzia maris WR-3 was isolated from a consortium comprising ammonia-oxidizing and denitrifying bacteria derived from marine sediments. Here, we examined biosurfactant production by strain WR-3 when cultured using several different carbon (D-glucose, n -decane, n -hexadecane, motor oil, olive oil, and rapeseed oil) and nitrogen (NH(4) )(2) SO(4) , NaNO(3) , yeast extract, and polypeptone) sources as growth substrates. Strain WR-3 was able to grow and reduce the surface tension of culture broth to 31±1.0 mN m(-1) when cultured using n -hexadecane and nitrate ions. The surface-active compounds produced by strain WR-3 were extracted and analyzed by thin layer chromatography. Moreover, the main components in the extract were further purified and subjected to gas chromatography/mass spectrometry (GC/MS). From the analysis, the surface-active compounds were tentatively identified as wax ester-like compounds, which were synthesized from the degradation process of n -alkane. The production of surface-active compounds by strain WR-3 promoted attachment of cells to hydrocarbon droplets via increased cell hydrophobicity, thus allowing enhanced degradation of water immiscible substrates. As Dietzia spp. can grow and produce wax esters from the degradation process of hydrocarbons, these marine bacteria are potentially useful for the bioremediation of hydrocarbon-contaminated environments. PMID:21656811

Enrichment cultures with enantiomeric 2-(4-sulfophenyl)butyrate (SPB) as the sole added source(s) of carbon and energy for growth yielded a pure culture of a degradative bacterium, which was identified as Delftia acidovorans SPB1. The organism utilized the enantiomers sequentially. R-SPB was utilize...

An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype w...

Culture methods were developed for concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, fecal, and hide samples. Several enrichment conditions were tested based on overall growth of pure cultures; tryptic soy broth for 2 h at 25 deg C, then for 6 h at 42 deg C, was sel...

Enrichment cultures of phototrophic purple bacteria rapidly oxidized up to 10 mM dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO was qualitatively identified by proton nuclear magnetic resonance. By using a biological assay, DMSO was always quantitatively recovered from the culture media. ...

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55??C for 528?days with a mixture of long-chain n-alkanes (C15?C20). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicu...

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm-forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so-far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm-enrichment approach was exemplified by the successful isolation of a not-yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media. PMID:22970793

Nutrient characteristics of four water masses in the light of their thermohaline properties are examined in the eastern Equatorial Indian Ocean during winter, spring and summer monsoon. The presence of low salinity water mass with "Surface enrichments" of inorganic nutrients was observed relative to 20 m in the mixed layer. Lowest oxygen levels of 19 microM at 3 degrees N in the euphotic zone indicate mixing of low oxygen high salinity Arabian Sea waters with the equatorial Indian Ocean. The seasonal variability of nutrients was regulated by seasonally varying physical processes like thermocline elevation, meridional and zonal transport, the equatorial undercurrent and biological processes of uptake and remineralization. Circulation of Arabian Sea high salinity waters with nitrate deficit could also be seen from low N/P ratio with a minimum of 8.9 in spring and a maximum of 13.6 in winter. This large deviation from Redfield N/P ratio indicates the presence of denitrified high salinity waters with a seasonal nitrate deficit ranging from -4.85 to 1.52 in the Eastern Equatorial Indian Ocean. PMID:20547419

Abstract in english Group B Streptococcus (GBS) is still not routinely screened during pregnancy in Brazil, being prophylaxis and empirical treatment based on identification of risk groups. This study aimed to investigate GBS prevalence in Brazilian pregnant women by culture or polymerase chain reaction (PCR) associated to the enrichment culture, and to determine the antimicrobial susceptibility patterns of isolated bacteria, so as to support public health policies and empirical prophylaxis. (more) After an epidemiological survey, vaginal and anorectal specimens were collected from 221 consenting laboring women. Each sample was submitted to enrichment culture and sheep blood agar was used to isolate suggestive GBS. Alternatively, specific PCR was performed from enrichment cultures. Antimicrobial susceptibility patterns were determined for isolated bacteria by agar diffusion method. No risk groups were identified. Considering the culture-based methodology, GBS was detected in 9.5% of the donors. Twenty five bacterial strains were isolated and identified. Through the culture-PCR methodology, GBS was detected in 32.6% specimens. Bacterial resistance was not detected against ampicillin, cephazolin, vancomycin and ciprofloxacin, whereas 22.7% were resistant to erythromycin and 50% were resistant to clindamycin. GBS detection may be improved by the association of PCR and enrichment culture. Considering that colony selection in agar plates may be laboring and technician-dependent, it may not reflect the real prevalence of streptococci. As in Brazil prevention strategies to reduce the GBS associated diseases have not been adopted, prospective studies are needed to anchor public health policies especially considering the regional GBS antimicrobial susceptibility patterns.

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism?s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air?liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a sig...

Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. In this study, hydrogen producing, cellulolytic cultures were enriched from compost material at 52, 60 and 70°C. Highest cellulose degradation and highest H(2) yield were 57% and 1.4 mol-H(2) mol-hexose(-1) (2.4 mol-H(2) mol-hexose-degraded(-1)), respectively, obtained at 52°C with the heat-treated (80°C for 20 min) enrichment culture. Heat-treatments as well as the sequential enrichments decreased the diversity of microbial communities. The enrichments contained mainly bacteria from families Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen production and bacteria closely related to Clostridium cellulosi and Clostridium stercorarium were responsible for cellulose degradation. PMID:21251819

The growth, biochemical properties, and chlorophyll fluorescence of cultures of the symbiotic Symbiodinium species from clade A, B, and F and the free-living dinoflagellate Prorocentrum minimum in response to ammonium enrichment were examined following transfer from ammonium-limited to ammonium-enriched conditions. Cultures were grown under a light:dark (L:D) cycle of 300mmol photons m^-^2s^-^1. Cell growth was initiated on day 1, reached saturation on approximately day 5, and decreased due to ammonium on day 10 (ammonium limitation experiments). Cells were removed from the ammonium limitation series on days 1, 5, and 10 and were enriched to 50mM ammonium followed by incubation for 4days (ammonium enrichment experiments). The cell density, cellular carbon, nitrogen, chlorophyll a contents,...

After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis. nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd1 nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that nirS, together with nirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of nirS lasting for about 30 min. The continued transcription of the nirS operon required the presence of nitrate or nitrite. This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes. PMID:9864326

A fuel cell was used to enrich a microbial consortium generating electricity, using organic wastewater as the fuel. Within 30 days of enrichment the maximum current of 0.2 mA was generated with a resistance of 1 kOhms. Current generation was coupled to a fall in chemical oxygen demand from over 1,700 mg l(-1) down to 50 mg l(-1). Denaturing gradient gel electrophoresis showed a different microbial population in the enriched electrode from that in the sludge used as the inoculum. Electron microscopic observation showed a biofilm on the electrode surface and microbial clumps. Nanobacteria-like particles were present on the biofilm surface. Metabolic inhibitors and electron acceptors inhibited the current generation. 16S ribosomal RNA gene analysis showed a diverse bacterial population in the enrichment culture. These findings demonstrate that an electricity-generating microbial consortium can be enriched using a fuel cell and that the electrochemical activity is a form of anaerobic electron transfer. PMID:12908088

Plantlets of Nicotiana tabacum (var. Samsun) were grown under CO/sub 2/ enriched air supplied by a Warburg buffer. Growth of all plant parts was enhanced. The maximum growth increase was found for roots (120%). Addition of 30 g.1/sup -1/ sucrose in the medium resulted in a three time faster growth. However, the effect of CO/sub 2/ enrichment was still positive in these conditions, although less pronounced than in autotrophic cultures.

In terrestrial subsurface environments where nitrate is a critical groundwater contaminant, few cultivated representatives are available with which to verify the metabolism of organisms that catalyze denitrification. In this study, five species of denitrifying bacteria from three phyla were isolated from subsurface sediments exposed to metal radionuclide and nitrate contamination as part of the U.S. Department of Energy s Oak Ridge Integrated Field Research Challenge (OR-IFRC). Isolates belonged to the genera Afipia and Hyphomicrobium (Alphaproteobacteria), Rhodanobacter (Gammaproteobacteria), Intrasporangium (Actinobacteria) and Bacillus (Firmicutes). Isolates from the phylum Proteobacteria were confirmed as complete denitrifiers, whereas the Gram-positive isolates reduced nitrate to nitrous oxide. Ribosomal RNA gene analyses reveal that bacteria from the genus Rhodanobacter comprise a diverse population of circumneutral to moderately acidophilic denitrifiers at the ORIFRC site, with a high relative abundance in areas of the acidic source zone. Rhodanobacter species do not contain a periplasmic nitrite reductase and have not been previously detected in functional gene surveys of denitrifying bacteria at the OR-IFRC site. Sequences of nitrite and nitrous oxide reductase genes were recovered from the isolates and from the terrestrial subsurface by designing primer sets mined from genomic and metagenomic data and from draft genomes of two of the isolates. We demonstrate that a combination of cultivation, genomic and metagenomic data are essential to the in situ characterization of denitrifiers and that current PCR-based approaches are not suitable for deep coverage of denitrifying microorganisms. Our results indicate that the diversity of denitrifiers is significantly underestimated in the terrestrial subsurface.

Abstract Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus`Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus`Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus`Brocadia fulgida' did not incorporate acetate d...

Hydrogen was produced vigorously by adding tuber mill of Dioscorea zingiberensis to enrich a culture of biogas sludge. Hydrogen-producig bacteria were able to be enriched in this way and twenty-four strains of hydrogen-producing bacteria were isolated. The amount of hydrogen produced varied with the species of bacteria and the media used. These bacteria were identified as Enterobacter cloacae, Escherichia coli, Serratia marcescens, Citrobacter freudii, Hafina alvei and Clostridium acetobutylicum. E. cloacae may be the major component. Its relative number was about 58.3% of the total number of bacteria isolated, and S. marcescens, about 16.7% and C. acetobutylicum, about 12.5%. The methane content in the biogas was greatly increased by adding a mixed culture of hydrogen-producing bacteria to an enriched culture of biogas sludge. The carbon dioxide content in it was obviously reduced. 8 references.

To understand the extent of natural adaptation to chemical exposure within a microbial consortium, we scrutinized the dynamic relationship between the microbial diversity and biodegradation capacity of octylphenol polyethoxylates (OPEOn), as representative alkylphenol polyethoxylates (APEOn), using enrichment cultures of various sediments from the Iwata River system. To address the potential of microbes in river sediments to transform a surfactant into endocrine-disrupting chemicals, the ability of the microbes to degrade OPEOn in six different sediment samples was assessed by enrichment culture. In addition, 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis was conducted to elucidate the microbial communities before and after enrichment of OPEOn-degrading microbes. Functional gene (adh1) copies were also enumerated to estimate the potential of OPEOn degradation in the six sediment samples. Moreover, microbial communities in three of the six sediment samples were characterized by 16S rRNA-based clone analysis. OPEOn-degrading activity was determined to be present in five of the six enrichment cultures; however, no predominant species could be found by clone analysis. DGGE analysis revealed that the genus Pseudomonas, which contributes to the degradation of OPEOn, was one of the major populations in the enrichment cultures. The adh1 gene enumeration was shown to be relatively high when river flow was relatively slow. These results suggest that potential OPEOn degraders are widely distributed in Iwata River with differences in their history of chemical exposure.   

Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants. PMID:22956759

Microbial methane from coal beds accounts for a significant and growing percentage of natural gas worldwide. Our knowledge of physical and geochemical factors regulating methanogenesis is still in its infancy. We hypothesized that in these closed systems, trace elements (as micronutrients) are a limiting factor for methanogenic growth and activity. Trace elements are essential components of enzymes or cofactors of metabolic pathways associated with methanogenesis. This study examined the effects of eight trace elements (iron, nickel, cobalt, molybdenum, zinc, manganese, boron, and copper) on methane production, on mcrA transcript levels, and on methanogenic community structure in enrichment cultures obtained from coal bed methane (CBM) well produced water samples from the Powder River Basin, Wyoming. Methane production was shown to be limited both by a lack of additional trace elements as well as by the addition of an overly concentrated trace element mixture. Addition of trace elements at concentrations optimized for standard media enhanced methane production by 37%. After 7?days of incubation, the levels of mcrA transcripts in enrichment cultures with trace element amendment were much higher than in cultures without amendment. Transcript levels of mcrA correlated positively with elevated rates of methane production in supplemented enrichments (R(2)?=?0.95). Metabolically active methanogens, identified by clone sequences of mcrA mRNA retrieved from enrichment cultures, were closely related to Methanobacterium subterraneum and Methanobacterium formicicum. Enrichment cultures were dominated by M. subterraneum and had slightly higher predicted methanogenic richness, but less diversity than enrichment cultures without amendments. These results suggest that varying concentrations of trace elements in produced water from different subsurface coal wells may cause changing levels of CBM production and alter the composition of the active methanogenic community. PMID:22590465

The presence of microcystin-LR -degrading bacteria in an active anthracite biofilter and in Lake Mead, Nevada was investigated. Four bacterial isolates from enrichment culture were identified using 16S rRNA analysis. Microcystin biodegradation tests were performed with both, the enrichment cultures and the respective isolates, using microcystin alone and acetate as carbon sources. A newly recognized microcystin-degrading bacterium, Morganella morganii, was isolated from the biofilter and from Lake Mead. The results of the biodegradation tests indicated that addition of a carbon source (acetate), significantly repressed the degradation of microcystin-LR. The findings of this study inform on the prevalence of microcystin-degrading bacteria in the environment indicating bioaugmentation may no...

Background We compared the LightCycler MRSA advanced test (Roche Diagnostics, Germany) with enrichment culture methods to evaluate the relative diagnostic performance of the LightCycler MRSA advanced test for active surveillance in a high-prevalence setting. Methods A total of 342 nasal swab specimens were obtained from patients in the intensive care unit at admission and on the seventh day for follow-up. The results of LightCycler MRSA advanced test were compared to those of the enrichment culture. For discrepant results, mecA gene PCR was performed. Results For the detection of methicillin-resistant Staphylococcus aureus (MRSA), the LightCycler MRSA advanced test showed 98.5% sensitivity and 78.6% specificity and had positive and negative predictive values of 75.0% and 98.8%, respectively. A total of 46 samples had discrepant results between the LightCycler MRSA advanced test and enrichment culture. Of the 44 specimens that were positive in the LightCycler MRSA advanced test but negative by enrichment culture, mecA genes were detected in 37 specimens. In addition, of the original 44 cases, 21 patients had a history of MRSA colonization or infection within the last month; of those 21 specimens, 20 were positive for mecA gene as shown by PCR. Seven mecA-negative discrepant specimens comprised 3 methicillin-sensitive S. aureus-culture positive and only 2 patients had MRSA infections. Conclusions Despite its low specificity and positive predictive value, the LightCycler MRSA advanced test could serve as a rapid test for patients colonized with MRSA.

The cometabolic biotransformation of 2,4-dinitrotoluene (DNT) under nitrogen reducing conditions by enrichment cultures using ethanol as the main source of carbon and energy was studied. Two microbial cultures were enriched from two different sources representing environments with and without previous exposure to DNT: the wastewater treatment system at the Radford Army Ammunition Plant in Radford, VA, and the municipal wastewater treatment plant in Urbana, IL. DNT was completely biotransformed. by both enrichment cultures. However, experiments with uniformly labeled [14C]-DNT demonstrated that no significant mineralization occurred. Losses by volatilization accounted for 9 and 7% of the original {sup 14}C-tracer in the unacclimated and acclimated cultures, respectively. The formation of insoluble materials was significant, with 41% of the tracer associated with this fraction in the unacclimated and 33% in the acclimated cultures. Most of the soluble metabolites were hydrophilic in the cultures from the unacclimated source. These metabolites were characterized according to their charge by HPLC and ion pairing chromatography. In the acclimated culture, 60% of the soluble metabolites were classified as hydrophobic materials. GC-MS analyses of this fraction revealed the presence of 2- and 4- mononitrotoluenes, 6-nitroindazole, acetylaminotoluene, and 4-acetylamino-2- nitrotoluene. These results indicate the inadequacy of setting a discharge limit only for DNT, since a number of the metabolites formed may also pose a significant toxicological hazard.

Enrichment cultures of phototrophic purple bacteria rapidly oxidized up to 10 mM dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO was qualitatively identified by proton nuclear magnetic resonance. By using a biological assay, DMSO was always quantitatively recovered from the culture media. DMS oxidation was not detected in cultures incubated in the dark, and it was slow in cultures exposed to full daylight. Under optimal conditions, the second-order rate constant for DMS oxidation was 6 day/sup -1/ mg of protein/sup -1/ ml/sup -1/. The rate constant was reduced in the presence of high concentration of sulfide (>1 mM), but was not affected by the addition of acetate. DMS was also oxidized to DMSO by a pure strain (tentatively identified as a Thiocystis sp.) isolated from the enrichment cultures. DMS supported growth of the enrichment cultures and of the pure strain by serving as an electron source for photosynthesis. A determination of the amount of protein produced in the cultures and an estimation of the electron balance suggested that the two electrons liberated during the oxidation of DMS to DMSO were quantitatively used to reduce carbon dioxide to biomass. The oxidation of DMS by phototrophic purple bacteria may be an important source of DMSO detected in anaerobic ponds and marshes.

Sulfur-driven autotrophic denitrification refers to the chemolithotrophic process coupling denitrification with the oxidation of reduced inorganic sulfur compounds. Ever since 1904, when Thiobacillus denitrificans was isolated, autotrophic denitrifiers and their uncultured close relatives have been ...

The denitrifying betaproteobacterium “Aromatoleum aromaticum” strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the re...

  The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO2?) but not nitrate (NO3?) to form nitrous oxide (N2O). Here we found, however, that C. tonkinense can denitrify NO3? under certain conditions. Presence of ammonium (NH3+) in addition to NO3? and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity. Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO3? metabolism. These activities contained assimilatory type NADPH (or NADH)-dependent NO3? reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO3? reductase (dNar). The denitrification was inhibited by tungstate, an inhibitor of Nar. These results demonstrated occurrence of a novel type of denitrification in C. tonkinense, in which assimilatory type Nar is possibly involved.   

The goal of the research described herein was to examine the feasibility of biodegradation of mono and polycyclic aromatic hydrocarbons typically present in a manufactured gas processing (MGP) site groundwater and subsurface sediments under mixed oxygen/denitrifying conditions. ...

Detoxification of perchlorate by microbial communities under denitrifying conditions has been recently reported, although the identity of the mixed populations involved in perchlorate reduction is not well understood. In order to address this, the bacterial diversity of membrane ...

Continuous cultures were carried out for methane production from formate, methanol, and acetate by formate or acetate enrichment methanogenic cultures. Substrate-limited chemostat cultures could be established at different dilution rates with each substrate. The growth characteristics of enrichment cultures were analysed and compared. The maximum values of specific growth rates and specific methane production rates obtained with formate, acetate, and methanol were 1.3 day/sup -1/ and 0.5 mol CH/sub 4//g cell/day, 0.2 day/sup -1/ and 25 mmol CH/sub 4//g cell/day, and 1.0 day/sup -1/ and 0.20 mol CH/sub 4//g cell/day, respectively. Substrate carbons converted to carbons of methane, carbon dioxide and cells were 22.2, 70.2, and 5.6% in formate, 46, 46, and 11% in acetate, and 60, 20, and 13% in methanol, respectively. 14 references, 4 figures.

We have previously demonstrated that the chemoautotroph and facultative anaerobe Thiobacillus denitrificans may be readily cultured aerobically or anoxically in batch and continuous reactors on hydrogen sulfide under sulfide-limiting conditions. A sulfide-tolerant strain of T. denitrificans (strain F) was isolated by enrichment and recently used in a successful field test of a microbial process for the treatment of sour water coproduced with petroleum at an Amoco Production Co. site in Wyoming. Prior to the initiation of this field test, it was determined that the sour water at this site contained low concentrations of indigenous autotrophs, which could grow on thiosulfate as an energy source. Samples of this sour water have now been used to produce an enrichment culture for sulfide oxidizers. This enrichment has been characterized with respect to hydrogen sulfide oxidation, response to oxygen, pH and temperature optima, and sulfide tolerance. The enrichment was shown to be strictly aerobic and to grow on sulfide as an energy source with complete oxidation of sulfide to sulfate. The enrichment has a tolerance of sulfide comparable to that of T. denitrificans strain F. However, the enrichment has a higher optimum temperature (35{degrees}C) than strain F and was shown to oxidize sulfides over a much broader range of pH values.

This topical report on ``Sulfur Control`` presents the results of work conducted by the Institute of Gas Technology (IGT), the Illinois Institute of Technology (IIT), and the Ohio State University (OSU) to develop three novel approaches for desulfurization that have shown good potential with coal and could be cost-effective for oil shales. These are (1) In-Bed Sulfur Capture using different sorbents (IGT), (2) Electrostatic Desulfurization (IIT), and (3) Microbial Desulfurization and Denitrification (OSU and IGT). The objective of the task on In-Bed Sulfur Capture was to determine the effectiveness of different sorbents (that is, limestone, calcined limestone, dolomite, and siderite) for capturing sulfur (as H{sub 2}S) in the reactor during hydroretorting. The objective of the task on Electrostatic Desulfurization was to determine the operating conditions necessary to achieve a high degree of sulfur removal and kerogen recovery in IIT`s electrostatic separator. The objectives of the task on Microbial Desulfurization and Denitrification were to (1) isolate microbial cultures and evaluate their ability to desulfurize and denitrify shale, (2) conduct laboratory-scale batch and continuous tests to improve and enhance microbial removal of these components, and (3) determine the effects of processing parameters, such as shale slurry concentration, solids settling characteristics, agitation rate, and pH on the process.

The removal of high explosives (HIE) from ordnance is being accomplished via washout steamout procedures. Because large volumes of waste water are generated by these processes, safe and efficient methods must be developed for their treatment. Activated carbon can be used to efficiently remove HE from aqueous waste streams, but carbon that is laden with HE constitutes a hazardous solid waste. Although conventional treatment methods (i.e., incineration, open burning) are available, they may not be in compliance with existing or future environmental regulations. New and cost-effective methods are therefore required for the elimination of this solid waste. We are developing and demonstrating coupled chemical and biological systems for the safe and economical treatment of HE-laden activated carbon. We have developed a completely engineered treatment system to accomplish this objective and have been operating a pilot treatment system at the Pantex Plant in Amarillo, TX. In this system, HE- contaminated waste water is treated first by activated-carbon adsorption columns. The HE sorbed to carbon is subsequently recovered via heated solvent elution or by base hydrolysis. The HE- or hydrolysate-laden fluid is then treated using a denitrifying culture of microorganisms, which converts the HE or hydrolysate byproducts to less hazardous endproducts. With these methods, the treated carbon can either be re-used or disposed as a nonhazardous waste. This strategy, which has been shown to be effective for the regeneration of carbon and the degradation of RDX and HMX, will be applicable to other energetic chemicals sorbed to activated carbon.

The effect of carbon source and COD/NO(3)(-)-N ratio on denitrification and methanogenesis in mixed methanogenic matrix was investigated in this study. Industrial wastewater, anaerobic treated cassava stillage (CS) and glucose synthetic wastewater were used as carbon sources respectively for comparison. Experimental results showed that denitrification was the main nitrate reduction pathway for all COD/NO(3)(-)-N ratios tested in two substrates. Simultaneous denitrification and methanogenesis occurred at COD/NO(3)(-)-N higher than 7 regardless of carbon sources. Incomplete denitrification was observed at COD/NO(3)(-)-N ratio below 7 in both the anaerobic effluent of CS and glucose-fed cultures due to the insufficient available organic carbon. The nature of carbon sources was observed to play a key role in the nitrate and organic carbon utilization rates. COD/NO(3)(-)-N ratio had a strong effect on the organic matter utilization pathways. Methanization consumed more organic matter than denitrification with further increase of COD/NO(3)(-)-N ratio above 7 in two substrates. Results of VFA variation suggested that propionate and butyrate were preferably utilized by the denitrifiers than acetate. PMID:22293676

Denitrifying bacteria convert nitrate (NO(3) (-) ) to dinitrogen (N(2) ) gas through an anaerobic respiratory process in which the potent greenhouse gas nitrous oxide (N(2) O) is a free intermediate. These bacteria can be grouped into classes that synthesize a nitrite (NO(2) (-) ) reductase (Nir) that is solely dependent on haem-iron as a cofactor (e.g. Paracoccus denitrificans) or a Nir that is solely dependent on copper (Cu) as a cofactor (e.g. Achromobacter xylosoxidans). Regardless of which form of Nir these groups synthesize, they are both dependent on a Cu-containing nitrous oxide reductase (NosZ) for the conversion of N(2) O to N(2) . Agriculture makes a major contribution to N(2) O release and it is recognized that a number of agricultural lands are becoming Cu-limited but are N-rich because of fertilizer addition. Here we utilize continuous cultures to explore the denitrification phenotypes of P.?denitrificans and A.?xylosoxidans at a range of extracellular NO(3) (-) , organic carbon and Cu concentrations. Quite distinct phenotypes are observed between the two species. Notably, P.?denitrificans emits approximately 40% of NO(3) (-) consumed as N(2) O under NO(3) (-) -rich Cu-deficient conditions, while under the same conditions A.?xylosoxidans releases approximately 40% of the NO(3) (-) consumed as NO(2) (-) . However, the denitrification phenotypes are very similar under NO(3) (-) -limited conditions where denitrification intermediates do not accumulate significantly. The results have potential implications for understanding denitrification flux in a range of agricultural environments. PMID:22642644

The geochemistry of deposition of the Meade Peak Member of the Phosphoria Formation (MPM) in southeast Idaho, USA, a world-class sedimentary phosphate deposit of Permian age that extends over 300,000 km2, is ascertained from its rare earth element (REE) composition. Ratios of REE:Al2O3 suggest two sources-seawater and terrigenous debris. The seawater-derived marine fraction identifies bottom water in the Phosphoria Sea as O2-depleted, denitrifying (suboxic) most of the time, and seldom sulfate-reducing (anoxic). This interpretation is supported by earlier research that showed progressively greater ratios in the marine sediment fraction of Cr:Ni>V:Ni???Mo:Ni, relative to their ratios in seawater; for which marine Cr, V, and Mo can have a dominantly O2-depleted bottom-water source and Ni a photic-zone, largely algal, source. The water chemistry was maintained by a balance between bacterial oxidation of organic matter settling through the water column, determined largely by primary productivity in the photic zone, and the flux of oxidants into the bottom water via advection of seawater from the open ocean. Samples strongly enriched in carbonate fluorapatite, the dominant REE host mineral, have variable Er/Sm, Tm/Sm, and Yb/Sm ratios. Their distribution may represent greater advection of seawater between the Phosphoria Sea and open ocean during deposition of two ore zones than a center waste and greater upwelling of nutrient-enriched water into the photic zone. However, the mean rate of deposition of marine Ni, a trace nutrient of algae, and PO43-, a limiting nutrient, indicate that primary productivity was probably high throughout the depositional history. An alternative interpretation of the variable enrichments of Er, Tm, and Yb, relative to Sm, is that they may reflect temporally variable carbonate alkalinity of open-ocean seawater in Permian time. A more strongly negative Ce anomaly for all phosphatic units than the Ce anomaly of modern pelletal phosphate is further indicative of an elevated O2 concentration in the Permo-Carboniferous open ocean, as proposed by others, in contrast to the depletion of O2 in the bottom water of the Phosphoria Sea itself. The oceanographic conditions under which the deposit accumulated were likely similar to conditions under which many sedimentary phosphate deposits have accumulated and to conditions under which many black shales that are commonly phosphate poor have accumulated. A shortcoming of several earlier studies of these deposits has resulted from a failure to examine the marine fraction of elements separate from the terrigenous fraction. ?? 2007 Elsevier Ltd. All rights reserved.

An enriched mixed culture was successfully grown on model alkyl and aryl carbonates. These compounds were degraded by microorganisms at different rates. P-Chlorophenyl-2-octyl carbonate and p-nitrobenzyl-2-octyl carbonate were metabolized through the formation of p-chlorophenol and p-nitrobenzyl alcohol respectively. A strain of Acinetobacter calcoaceticus isolated from the mixed culture utilized phenyl-2-octyl carbonate by an intracellular hydrolase to phenol and 2-octanol which were further metabolized. PMID:1366389

An Alcanivorax dieselolei strain, termed strain N1203, was isolated from the consortia of ammonia-oxidizing bacteria (AOB) combined with denitrifying bacteria from our previous study and was shown to have ability to reduce nitrate to nitrite to either nitrous oxide or molecular nitrogen. Analysis of 16S rRNA gene sequences established strain N1203 as a member of the species Alcanivorax dieselolei. In addition, the ability of strain N1203 to utilize various organic substrates as the sole carbon source, supplemented with carbohydrates, amino acids, and n-alkane compounds, was investigated, and this strain was found to have a narrow substrate range of growth such as grycerol, succinate, ethanol and n-alkane hydrocarbon. Furthermore, N1203's stepwise denitrifying activity, utilizing succinate and hexadecane as sole carbon sources, was measured. Gene fragments of nirK and qnorB genes, which are involved in denitrifying activities, were obtained, cloned and sequenced. Phylogenetic analysis for these two genes showed that both the nirK and qnorB sequences, although found in separate branches within clusters, formed subclusters branching from uncultured environmental clones. This demonstrated the typical uniqueness of these genes from any cultivated denitrifiers. Thus, strain N1203 is novel type of denitrifying bacteria that demonstrated denitrifying activities when cultivated using succinate as the sole carbon source.   

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine ecosystems. PMID:18462401

To understand the potential of cultivating tropical marine diatom Thalassiosira sp. to produce biofuel, biodiesel product properties and growth characteristics of Thalassiosira sp. in three different media were investigated. After medium evaluation, significant Thalassiosira sp. cell growth was observed in both Walne and enriched seawater media, but not in plain seawater medium. The microalgae grew well in alkaline condition (pH range of 8.0-8.8). The average biomass density cultured in Walne and enriched seawater media on the 6th day was 4.36 and 2.50gL-1, respectively. Based on ESI-IT-MS spectra, the TAGs of algal oil were identified as POP, POO, and SOO, and the FAMEs as oleic acid methyl ester. The oil productivity of Thalassiosira sp. cultured in Walne and enriched seawater media were...

A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42 degrees C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35 degrees C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42OC for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98% (88/90) and 100% (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87% (78/90) and 83% (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 1-2 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products. PMID:17955973

Twenty-three nasal swab samples that tested positive for methicillin-resistant Staphylococcus aureus (MRSA) on initial testing by the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA) were culture positive only for methicillin-susceptible S. aureus (MSSA) from an enrichment broth. The 2...

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper,...

The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel e...

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals. PMID:14982773

Dibenzothiophenes (DBTs) bearing alkyl substitutions adjacent to the sulfur atom, such as 4,6-diethyldibenzothiophene (4,6-DEDBT), are referred to as sterically hindered with regard to access to the sulfur moiety. By using enrichment cultures with 4,6-DEDBT as the sole sulfur source, bacterial isola...

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the or...

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide co...

1. Functional foods is a relatively new concept covering food products enriched with various kinds of (natural) substances (eg vitamins, minerals or probiotic cultures) or modified so as to provide consumers with an additional physiological benefit presumed to prevent disease or promote health, with...

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55°C). This isolate exhibited a temperature optimum of 55 to 60°C and a maximum near 70°C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth nea...

The protein PCM-1 localizes to cytoplasmic granules known as “centriolar satellites” that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific de...

Conventional Salmonella isolation involves multiple sample transfers to culture media performed by an experienced microbiologist. The Reaction (RX) Plate method, a modification of the RX tube designed by Gailey et al. (2004), consolidates pre-enrichment (buffered peptone water or GN Hajna), enrichm...

The amiloride-binding Na+ channel protein of high electrical resistance epithelia was solubilized and purified from cultured A6 toad kidney cells and bovine renal papilla. Purification was assessed by enrichment in [3H]methylbromoamiloride specific binding. Chromatography of 3-[(3-cholamidopropyl)di...

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidati...

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube- substrate contact (Bloch, R.J., and B. Geiger, 1980, Cell, 21:25-35). We have used two chole...

  We isolated aromatics-degrading bacteria from the gut of a lower termite, Coptotermes formosanus, using a mineral salt medium containing various aromatic compounds as the sole carbon source. Two species, Burkholderia sp. strain VE22 and Citrobacter sp. strain VA53, were isolated by aerobic enrichment culture with veratraldehyde and vanillin, respectively. Strain VA53 could also grow and metabolize vanillin anaerobically.   

This study aimed to evaluate the usefulness of a novel differential culture medium, chromID VRE agar, for the isolation of VRE in a clinical laboratory. It was shown that ChromID VRE agar may be useful for rapid and selective isolation of VRE especially after inclusion of broth enrichment step. PMID:19167435

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid ...

Bombesin, a polypeptide derived from frog skin, has been shown to stimulate gastrin release from the gastric antrum in vivo and in vitro. To elucidate the mechanisms of this effect, we developed a method to culture isolated and enriched G cells from canine stomach. After digestion of antral mucosa w...

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogeneti...

The anaerobic biodegradability and toxicity of fourteen aromatic compounds were evaluated over a wide range of concentrations using a serum bottle technique. Benzene, toluene, and all three isomers of xylene were not significantly degraded to methane in a phenol-enriched culture. Complete degradation of 1000 mg/L phenol, 800 mg/L catechol, 100 mg/L 2-NP, 100 mg/L 3-NP, and 100 mg/L 4-NP was observed within two months while depletion of 100 mg/L resorcinol and 1000 mg/L hydroquinone required more than six and eight months incubation, respectively. None of the three isomers of chlorophenol were degraded in the phenol-enriched culture. Batch toxicity assay revealed that the phenol-enriched culture was more susceptible to inhibition caused by substituted phenols than the acetate-enriched culture. In general, the inhibitory effects on both phenol degradation and acetate utilization did not vary significantly with the isomer but rather with the substituted group. The degree of inhibition was in the order of nitrophenols chlorophenols hydroxyphenols. The Haldane inhibition model was used to fit experimental data from phenol and catechol. The inhibition of phenol degradation by chlorophenols, resorcinol, and hydroquineone was described rather well by a Monod-type noncompetitive model.

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A...

Two bacterial strains were dominant in anaerobic enrichment cultures with betaine (N,N,N-trimethylglycine) as a substrate and intertidal mud as an inoculum. One was a coccoid bacterium which was a trimethylamine (TMA)-fermenting methanogen similar to Methanococcoides methylutens. The other strain, a...

An anaerobic enrichment culture inoculated with a sample of sediments from soda lakes of the Kulunda Steppe with elemental sulfur as electron acceptor and formate as electron donor at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in ...

A microbial fuel cell (MFC) was inoculated with a random transposon insertion mutant library of Shewanella oneidensis MR-1 and operated with lactate as the sole fuel to select for mutants that preferentially grew in it. Agar plate cultivation of the resultant MFC enrichment culture detected an incre...

A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converte...

Anaerobic ammonium-oxidizing (anammox) bacteria are key players in the global nitrogen cycle and responsible for significant global nitrogen loss. Moreover, the anammox process is widely implemented for nitrogen removal from wastewaters as a cost-effective and environment-friendly alternative to conventional nitrification-denitrification systems. Currently, five genera of anammox bacteria have been identified, together forming a deep-branching order in the Planctomycetes-Verrucomicrobium-Chlamydiae superphylum. Members of all genera have been detected in wastewater treatment plants and have been enriched in lab-scale bioreactors, but genome information is not yet available for all genera. Here we report the metagenomic analysis of a granular sludge anammox reactor dominated (?50%) by “Candidatus Jettenia asiatica.” The metagenome was sequenced using both Illumina and 454 pyrosequencing. After de novo assembly 37,432 contigs with an average length of 571?nt were obtained. The contigs were then analyzed by BLASTx searches against the protein sequences of “Candidatus Kuenenia stuttgartiensis” and a set of 25 genes essential in anammox metabolism were detected. Additionally all reads were mapped to the genome of an anammox strain KSU-1 and de novo assembly was performed again using the reads that could be mapped on KSU-1. Using this approach, a gene encoding copper-containing nitrite reductase NirK was identified in the genome, instead of cytochrome cd1-type nitrite reductase (NirS, present in “Ca. Kuenenia stuttgartiensis” and “Ca. Scalindua profunda”). Finally, the community composition was investigated through MetaCluster analysis, 16S rRNA gene analysis and read mapping, which showed the presence of other important community members such as aerobic ammonia-oxidizing bacteria, methanogens, and the denitrifying methanotroph “Ca. Methylomirabilis oxyfera”, indicating a possible active methane and nitrogen cycle in the bioreactor under the prevailing operational conditions. PMID:21040513

The demand for ethanol as an oxygenate and octane booster in automobile fuel is growing. A number of processes are being investigated for conversion of biomass to ethanol. The Hydrolysis-Fermentation-Combustion (HFC) process for fuel ethanol production developed at the University of California Forest Products Laboratory, Richmond, California is at the stage of technology transfer following over two decades of research and development. This study addresses the technology to be used in treatment of spent streams to be discharged from this process. The treatment design combines a single stage denitrification and anaerobic digestion (SSDAD) for the biological treatment of a representative stream from this process. A typical spent stream contained a wide range of soluble organic materials including: unfermented sugars, components of the feedstocks solubilized in the hydrolysis, acid degradation products of carbohydrates, cleavage products of lignin, water-soluble extractives and phenolics, terpenes and other unfermented organic material, and nitrate ion from the nitric acid used as a catalyst in the hydrolysis reaction. Three sets of experiments were conducted in laboratory scale anaerobic digesters. Commonly available anaerobic sludge from local sewage treatment plants was used as a starter seed and was successfully acclimated to the high nitrate substrate leading to enrichment of denitrifiers. Necessary nutrients and trace elements were identified and supplied to satisfy the obligatory requirements of different groups of bacterial groups present. A major finding was the unique role of ammonium hydroxide in controlling pH leading to steady-state operation of the digester. At steady state operation the reduction in COD was 65%, the nitrate reduction was 88% and the nitrite reduction was 100%. Nitrate was reduced to safe nitrogen gas without buildup of any intermediate products. Organic material was converted to useful methane gas and carbon dioxide. The SSDAD system was shown to be effective in treating spent streams having high COD and nitrate concentrations.

Biodegradation is a potentially important loss process for haloacetic acids (HAAs), a class of chlorination byproducts, in water treatment and distribution systems, but little is known about the organisms involved (i.e., identity, substrate range, biodegradation kinetics). In this research, 10 biomass samples (i.e., tap water, distribution system biofilms, and prechlorinated granular activated carbon filters) from nine drinking water systems were used to inoculate a total of thirty enrichment cultures fed monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), or trichloroacetic (TCAA) as sole carbon and energy source. HAA degraders were successfully enriched from the biofilm samples (GAC and distribution system) but rarely from tap water. Half of the MCAA and DCAA enrichment cultures were positive, whereas only one TCAA culture was positive (two were inconclusive). Eight unique HAA-degrading isolates were obtained including several Afipia spp. and a Methylobacterium sp.; all isolates were members of the phylum Proteobacteria. MCAA, monobromoacetic acid (MBAA), and monoiodoacetic acid (MIAA) were rapidly degraded by all isolates, and DCAA and tribromoacetic (TBAA) were also relatively labile. TCAA and dibromoacetic acid (DBAA)were degraded by only three isolates and degradation lagged behind the other HAAs. Detailed DCAA biodegradation kinetics were obtained for two selected isolates and two enrichment cultures. The maximum biomass-normalized degradation rates (Vm) were 0.27 and 0.97 microg DCAA/ microg protein/h for Methylobacterium fujisawaense strain PAWDI and Afipia felis strain EMD2, respectively, which were comparable to the values obtained for the enrichment cultures from which those organisms were isolated (0.39 and 1.37 microg DCAN/microg protein/h, respectively). The half-saturation constant (Km) values ranged from 4.38 to 77.91 microg DCAA/L and the cell yields ranged from 14.4 to 36.1 mg protein/g DCAA. PMID:19534130

Growth parameters and biochemical composition of the green microalga Chlorella vulgaris cultivated under different mixotrophic conditions were determined and compared to those obtained from a photoautotrophic control culture. Mixotrophic microalgae showed higher specific growth rate, final biomass concentration and productivities of lipids, starch and proteins than microalgae cultivated under photoautotrophic conditions. Moreover, supplementation of the inorganic culture medium with hydrolyzed cheese whey powder solution led to a significant improvement in microalgal biomass production and carbohydrate utilization when compared with the culture enriched with a mixture of pure glucose and galactose, due to the presence of growth promoting nutrients in cheese whey. Mixotrophic cultivation of...

In this study ground pine (Pinus sylvestris) sawdust was used as the source of lignocellulose. Two pretreatment methods, acid and alkali hydrolysis, were examined and the variables of concentration, temperature and time optimized. Biomethanation of the two pretreatment products, hydrolysates and residual solids, in closed cultures by isolated microbial associations, resulted in 8.5-fold increases in total methane generation compared with untreated controls. Closed culture methods were used to enrich and isolate microbial associations from freshwater sediment capable of catabolizing selected monomers under anoxic conditions. In all, 20 associations were isolated of which two, a veratric acid-catabolizing and a syringic acid-catabolizing culture, were chosen for further study.

In response to general shortages of energy, examination of the anaerboic digestion process as a potential source of a combustible, methane-rich fuel has intensified in recent years. It has been suggested that orgaic intermediates (such as fatty acids), produced during digestion, might also be recovered for use as chemical feedstocks. This investigation has been concerned with combining ultrafiltration separation techniques with anaerobic digestion for the development of a process in which the total production of acetic acid (the most valuable intermediate in anaerobic digestion) and methane are optimized. Enrichment cultures, able to utilize glucose as a sole carbon source, were adapted from sewage digesting cultures using conventional techniques. An ultrafiltration system was constructed and coupled to an anaerobic digester culture vessel which contained the glucose enrichment. The membrane controlled anaerobic digester appears to show promise as a means of producing high rates of both methane gas and acetic acid.

Stem cells of the side population (SP) phenotype are found in many self-renewing tissues and can be identified by their unique ability to effectively exclude the dye Hoechst 33342. We previously established a method for expanding spermatogonial stem cells (SSCs) in vitro, but the frequency of SSCs is only about 1 to 2%, limiting detailed SSC analyses. In this study, we sought to isolate SSCs from in vitro cultures by exploiting their ability to exclude Hoechst 33342. In contrast to the findings of previous in vivo studies, we found that SP cells developed in a stochastic manner in vitro. Moreover, SP cells in culture were not enriched in SSCs, but they were interconvertible with non-SP cells. Although SP cells were consistently found in testes after transplantation of cultured cells, they were not enriched in SSCs. These results show that SSCs have an unstable SP phenotype and provide evidence that SSCs change their phenotype characteristics in response to their microenvironment.   

An efficient enrichment method using immobilized metal affinity chromatography (IMAC) was developed for selective extraction of bioactive sphingoid base-1-phosphates (SB1Ps) from adventitious roots of Hypericum perforatum cultured in bioreactor. The phosphate-selective IMAC enrichment coupled with LC-MS/MS enabled sensitive analysis of low-abundance SB1Ps present in the root biomass, which would not be feasible otherwise due to severe interferences from complex biological matrices. The time-dependent growth rate and production of SB1Ps from adventitious roots were investigated. The level of phytosphingosine-1-phosphate, which was found to be the major SB1Ps, reached a maximum amount of 635.6pmolpergram of dry weight after 3weeks of culture and decreased between 3 and 5weeks of culture subs...

The conventional culture methods of hatching eggs using shell and/or egg contents for detection of salmonella organisms give mostly unsatisfactory results. The aim of the present study is to evaluate selection of other samples and techniques of culturing hatching eggs and freshly hatched chicks. This current study provides the best evidence of Salmonella enteritidis in artificially contaminated eggs (Layer type) by using enrichment broth in empty egg shell samples in comparison to culturing samples from yolk, albumen or from shell above the air cell (with the outer shell membrane). The isolation rates could be enhanced if empty egg shell was initially filled with Buffered Pepton Water as pre-enrichment broth. Examination of organs from freshly hatched chicks revealed that crop samples give mostly higher reisolation rates. PMID:1289043

We developed istotropically etched silicon chip micro-arrays for co-culture of metastatic human breast cancer (MDA-MB-231) and non-tumorigenic human breast (MCF10A) cells. The micro-arrays were fabricated using a single-mask, single-etch step process. Each chip contained a 16x16 array of cavities 140mm wide by 60 mm deep separated by planar silicon surfaces. Cells occupied 97-100% of the etched cavities. The cavities were enriched three-fold in MDA-MB-231 cells relative to the seeding ratio of, MDA-MB-231(1): MCF10A(10) cells. Micro co-cultures comprised of both MCF10A and MDA-MB-231 cells formed in 26% of cavities and contained 2-10 cells per cavity. Heterotropic cell interactions were seen in co-culture, and sites of these interactions were enriched with vinculin spikes. A selective morp...

The aim of the present study was to evaluate a new one-step enrichment protocol, consisting of a combined preenrichment and enrichment broth (Cronobacter Enrichment Broth [CEB]) used in conjunction with selective-differential agar ChromID Sakazakii, to facilitate a shortened 2-day cultural method for detection of Cronobacter spp. (Enterobacter sakazakii) in powdered infant formula (PIF). The CEB was evaluated using samples artificially inoculated with low concentrations of 10 lyophilized strains, representative of the genus Cronobacter. The detection of strains was compared in parallel with the enrichment medium from ISO/TS 22964 and a recently proposed differential screening broth for the detection of Cronobacter. All of the Cronobacter strains were recovered using the CEB, and a significantly higher final bacterial concentration was obtained with the CEB than with the other enrichment broths (P < 0.01). There was no significant difference between the cell concentrations for cultures grown in CEB at 37 degrees C and those grown at 41.5 degrees C. Cronobacter was recovered from both 1/10 (50 g:450 ml) and 1/5.5 (100 g:450 ml) sample-to-broth ratios, with no significant difference observed between the final bacterial concentrations obtained from the two ratios. Further studies on a wider range of PIFs, including naturally contaminated samples, are warranted to determine if the use of this protocol may facilitate the rapid release (within 40 to 48 h) of PIF. PMID:19681272

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential, previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage, culture milieu, and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA, 39% and LS, 28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion, IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore, temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation. PMID:20060001

Microbial metabolism of residual hydrocarbons, primarily short-chain n-alkanes and certain monoaromatic hydrocarbons, in oil sands tailings ponds produces large volumes of CH(4) in situ. We characterized the microbial communities involved in methanogenic biodegradation of whole naphtha (a bitumen extraction solvent) and its short-chain n-alkane (C(6)-C(10)) and BTEX (benzene, toluene, ethylbenzene, and xylenes) components using primary enrichment cultures derived from oil sands tailings. Clone libraries of bacterial 16S rRNA genes amplified from these enrichments showed increased proportions of two orders of Bacteria: Clostridiales and Syntrophobacterales, with Desulfotomaculum and Syntrophus/Smithella as the closest named relatives, respectively. In parallel archaeal clone libraries, sequences affiliated with cultivated acetoclastic methanogens (Methanosaetaceae) were enriched in cultures amended with n-alkanes, whereas hydrogenotrophic methanogens (Methanomicrobiales) were enriched with BTEX. Naphtha-amended cultures harbored a blend of these two archaeal communities. The results imply syntrophic oxidation of hydrocarbons in oil sands tailings, with the activities of different carbon flow pathways to CH(4) being influenced by the primary hydrocarbon substrate. These results have implications for predicting greenhouse gas emissions from oil sands tailings repositories. PMID:22894132

Enrichment cultures of microorganisms separated from soil contaminated with pentachlorophenol and creosote were able to grow on and degrade phenol (300 mg/l), 2-chlorophenol (100 mg/l), or 4-chlorophenol (100 mg/l) when added as the sole carbon source, but were unable to degrade 3-chlorophenol (100 mg/l) even after more than 127 days of incubation. Phenol biodegradation by enrichment cultures was completely inhibited by temperatures at or above 37 C or phenol concentrations greater than 1200 mg/l Phenol degradation rates were reduced in the absence of an inorganic nitrogen source. No isolates were found that degraded any of the chlorinated compounds. Phenol biodegradation by the yeast was completely inhibited by substrate concentrations greater than 1000 mg/l; it was partly inhibited by low dissolved-oxygen concentrations, substrate concentrations greater than 500 mg/l, and the presence of alternative carbon sources such as acetate or glucose. Acetate also inhibited yeast growth in the presence of phenol, while glucose stimulated it. The addition of yeast extract or thiamine stimulated yeast growth and phenol degradation by the yeast. In enrichment cultures, growth factors were provided to yeast by other microorganisms. The rapid rates of growth and phenol degradation by isolates and enrichments suggest that biodegradation of phenol in the subsurface should not be substrate limited. Rather the transport of dissolved oxygen by advection/dispersion or vertical diffusion should limit phenol degradation by aerobic metabolic pathways in groundwater.

The biodegradation of estradiol (E2), estrone (E1), and ethinylestradiol (EE2) was investigated using mixed bacterial cultures enriched from activated sludge. Enrichments were carried out on E2 or EE2 in batch conditions with acetonitrile as additional carbon source. Degradation experiments were performed both using hormones as sole carbon source or with an additional source. The hormones were completely degraded by these cultures. Estradiol was rapidly converted to E1 within 24 h. Thereafter, E1 degradation began, displaying a lag phase ranging from 3 to 4 days. Estrone depletion took from 48 h to more than 6 days, depending on the culture conditions. For EE2 degradation, when it was the sole carbon source, the lag phase and the time required for its complete removal (7 and 15 days, respectively) were shorter that in cultures with a supplementary carbon source. The specific degradation rates observed for E2 both with and without an additional carbon source were similar. By contrast, the specific degradation rates for E1 and EE2 were, respectively, seven and 20 times faster when these hormones were supplied as the sole carbon source. The bacterial community structure of each culture was characterized by molecular and cultural methods. The mixed cultures were made up of species belonging to Alcaligenes faecalis, Pusillimonas sp., Denitrobacter sp., and Brevundimonas diminuta or related to uncultured Bacteroidetes. The isolated strain B. diminuta achieved the conversion of E2 to E1. PMID:19685239

In situ reductive immobilization, whereby highly soluble Cr(VI) species are reduced to poorly soluble Cr(III) species, is a favored approach for remediating Cr-contaminated groundwater. How microbial populations respond phylogenetically and functionally to the injection of an organic electron donor to stimulate Cr(VI) reduction is unclear, as are the relative contributions of direct enzymatic Cr(VI) reduction versus indirect (e.g. sulfide-mediated) reduction. In this study, we inoculated anaerobic microcosms with groundwater from the Cr-contaminated Hanford 100H site (WA) and supplemented them with lactate and the electron acceptors nitrate, sulfate, and amorphous ferric oxyhydroxide. The microcosms progressed successively through nitrate-reducing, sulfate-reducing, and Fe(III)-reducing conditions, and after a second nitrate amendment, nitrate-dependent Fe(II)-oxidizing conditions. Cr(VI) reduction occurred during both the denitrification and the sulfate/iron reduction phases. DNA and RNA were harvested during each major biogeochemical phase and were subjected to PhyloChip analysis, qPCR, and transcript sequencing. Bacterial community succession followed a trajectory related to the sequential use of electron acceptors. During denitrification, bacterial communities were enriched in known denitrifiers within the Beta- and Gamma-proteobacteria and became phylogenetically clustered. Fermenters became enriched following nitrate reduction, preceding both iron and sulfate reduction. Iron reduction was stoichiometrically related to the formation of hydrogen sulfide and, although iron reducers were detected during this phase, their iron-reducing activity was not confirmed. Following the depletion of lactate and sulfate, iron reduction rates decreased and acetate and propionate concentrations stabilized, indicating a marginal contribution of acetate-coupled iron reduction. Rapid Fe(II) oxidation occurred following the nitrate amendment with a concomitant reduction of nitrate to nitrite and an increased abundance of Beta-proteobacterial species related to known anaerobic Fe(II)-oxidizing bacteria. To uncover the microbial mechanisms contributing to the biogeochemical complexity encountered, even under controlled laboratory incubations, requires alternatives to standard phylogenetic analyses. Our ongoing efforts in analyzing the community transcriptomes (mRNA) should provide valuable insight into the relative rates of direct versus indirect mechanisms of Cr(VI) immobilization in contaminated aquifers.

Brain and primary neuron fractions enriched in synaptic terminals are important tools for neuroscientists in biochemical, neuroanatomical and physiological studies. We describe an annotated updated micro-method for preparing synaptoneurosomes (SNs) enriched in presynaptic and postsynaptic elements. An easy to follow, step-by-step, protocol is provided for making SNs from small amounts of mammalian brain tissue. This includes novel applications for material obtained from human neurosurgical procedures and primary rat neuronal cultures. Our updated method for preparing SNs using smaller amounts of tissue provides a valuable new tool and expands the capabilities of neuroscientists.

Nutrient enrichment from shrimp aquaculture poses an increasing environmental threat due to the industry's projected rapid growth and unsustainable management practices. Traditional methods to monitor impacts emphasize water quality sampling; however, there are many advantages to bioindicators, especially in developing countries. We investigated the usefulness of three bioindicators-growth, tissue nitrogen content and nitrogen stable isotope signature (d15N)-in the tropical red macroalga Acanthophora spicifera. Algae were collected, cultured, and deployed in a spatial array around the outflow from a shrimp farm in Moorea, French Polynesia, to detect nitrogenous wastes. All three parameters were highest adjacent to the shrimp farm indicating nutrient enrichment, and d15N values confirmed th...

Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. PRACTICAL APPLICATION: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices. PMID:22860597

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders...

This paper addresses the need to inform all students of the importance of multicultural issues and the impact that cultural diversity will have on global society and offers a model that examines the problematic issue of incorporating a multicultural curriculum into what has been termed "traditional curriculum." The author develops the criteria needed for cultural totality in curricula and examines various social determinants that significantly impact curriculum development as it relates to the historical racism as a social deterrent to a culturally enriched curriculum. In addition, the paper highlights specific techniques and strategies for infusing culturally diverse materials into curricula that are currently being used to assist all students to become better-informed citizens and to understand the rich history and culture of various diverse groups in the United States and globally. (Contains 16 references.) (GLR)

Studies have been conducted to investigate the biological potential of methane generation from two types of poultry waste: broiler chicken litter and laying hen manure. Through the systematic study, thermophilic bacterial cultures were initiated and established to produce methane at their highest rates. It was found that different kinds of waste with different chemical compositions required different operational conditions to reach the individual maximal potential. The microbiology of the methane-producing bacteria in the poultry waste-based anaerobic digester was studied. An enriched thermophilic methane-producing culture was isolated. The methanogenic culture can use acetate, ethanol, methylamine, propionate, and H/sub 2/-CO/sub 2/, but not formate and methanol, for growth and methanogenesis. The methanogenic culture was found to be a mixed culture from which a thermophilic Methanococcus sp. and an unidentified rod-shaped microorganism were isolated. The two organisms produced methane symbiotically in the acetate medium.

Climate warming in temperate regions may lead to decreased soil temperatures over winter as a result of reduced snow cover. We examined the effects of temperatures near the freezing point on N(2) O emissions, denitrification, and on the abundance and structure of soil nitrifiers and denitrifiers. Soil microcosms supplemented with NO3 - and/or NO3 - plus red clover residues were incubated for 120 days at -4 °C, -1 °C, +2 °C or +5 °C. Among microcosms amended with residues, N(2) O emission and/or denitrification increased with increasing temperature on Days 2 and 14. Interestingly, N(2) O emission and/or denitrification after Day 14 were the greatest at -1 °C. Substantial N(2) O emissions were only observed on Day 2 at +2 °C and +5 °C, while at -1 °C, N(2) O emissions were consistently detected over the duration of the experiment. Abundances of ammonia oxidizing bacteria (AOB) and archaea (AOA), Nitrospira-like bacteria and nirK denitrifiers were the lowest in soils at -4 °C, while abundances of Nitrobacter-like bacteria and nirS denitrifiers did not vary among temperatures. Community structures of nirK and nirS denitrifiers and Nitrobacter-like bacteria shifted between below-zero and above-zero temperatures. Structure of AOA and AOB communities also changed but not systematically among frozen and unfrozen temperatures. Results indicated shifts in some nitrifier and denitrifier communities with freezing and a surprising stimulation of N(2) O emissions at -1 °C when NO3 - and C are present. PMID:22882277

'The objectives of the research within this grant are: (1) Isolation and characterization of chlororespiring organisms responsible for the complete dehalogenation of chlorinated ethenes and propanes. (2) Development of conditions that yield high cell densities and induce dechlorinating activity. (3) Development of assay systems to detect the dechlorinating activity in cell-free extracts. (4) Purification and characterization of the dehalogenating enzymes. Anaerobic microcosms were obtained from a variety of geographically different sediment samples. In several microcosms complete dechlorination of tetrachloroethene (PCE) to ethene (ETH), and 1,2-dichloropropane ( 1,2-D) and/or 1,2,3-trichloropropane to propene was observed. Upon subsequent transfers to anaerobic medium, sediment-free, methanogenic enrichment cultures were obtained that dechlorinated PCE to ETH, and 1,2-D to propene, respectively. 2-Bromoethanesulfonate (BES), a well known inhibitor of methanogens, did not inhibit the dechlorination of 1,2-D to propene and the dechlorination of PCE to cis-dichloroethene (cis-DCE). However,-the complete dechlorination of PCE to vinyl chloride (VC) and ETH was severely inhibited. The authors could show that BES inhibited the dechlorination of chloroethenes in cultures not containing methanogens. Previous to this study, BES was believed to be aspecific inhibitor of methanogens and the inhibitory effect of BES on declorination was explained by the involvement of methanogens in the dechlorination process. The non-methanogenic cultures obtained after the BES treatment were subsequently transferred to medium riot containing BES and complete dechlorination of PCE to ETH was observed as was in the original microcosms. Subcultures were further enriched with PCE, cis-DCE, VC, or 1,2-D as the only available electron acceptor and acetate, or acetate plus hydrogen as the only available electron donor(s). To date these cultures have undergone up to 45 transfers. Interestingly, two cultures that originally dechlorinated PCE to ETH, but were then enriched with cis-DCE or VC, lost their ability to-dechlorinate PCE or TCE. This finding indicates that different populations are involved in the complete dechlorination of PCE to ETH in these cultures. In contrast, one culture that was enriched with cis-DCE and VC, respectively, maintained its ability to dechlorinate PCE. Using molecular tools ({sup 16}S rDNA targeted PCR,TA cloning of {sup 1}6S rDNA genes, ARDRA analysis and sequencing) they showed that this culture consisted of three distinct organisms. Two of them could be isolated in pure Culture but neither of them showed any dechlorinating activity, indicating that one organism was responsible for the complete dechlorination of PCE to ETH in the mixed culture. The authors are currently focusing on the isolation and phylogenetic characterization of this organism.'

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species. PMID:20382819

A series of experiments were conducted with greenhouse cucumber and pepper plants to determine the effects of oxygen enrichment of the irrigation water on yield and fruit shelf-life. The experiments were carried out in soilless culture in research greenhouses. Depending on the experiment, treatments included sub-ambient (2mgL^-^1), ambient (5-6mgL^-^1), medium (16mgL^-^1) and high (30-40mgL^-^1) levels of oxygen in the supply tank. Cucumber plants were grown in yellow cedar sawdust and pepper plants in either sawdust or perlite. Oxygen enrichment resulted in a promotion of cucumber yield in only one experiment; in two other experiments, none of the oxygen treatments, including those at sub-ambient levels, had an effect. There were no effects of oxygen enrichment on pepper yield. However, i...

Laboratory studies, using bacterial enrichments from geographically diverse aquifers, show that certain transition metal catalysts can effect partial dechlorination of chlorinated methanes when these catalysts are added to aquifer enrichments. The mechanism for this activity appears to involve direct catalysis by the transition metal complexes. Catalytic enhancement involves electron transfer reactions between the extracellular cytochrome c{sub 3} of sulfate-reducing bacteria and the exogenously added transition metal complex. This reaction is shown to occur with purified proteins, with pure cultures of sulfate-reducers, and in crude sulfate-reducing aquifer enrichments. The results suggest a defined process for aquifer remediation involving: (1) generation of sulfate-reducing populations within the aquifer that do not necessarily need to possess dechlorination activity, and (2) addition of a transition metal complex that accepts electrons from the extracellular cytochrome of the sulfate-reducer and catalyzes the reductive dehalogenation.

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibiotic-resistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pate. For any...

Sequence analysis of the 16S ribosomal RNA gene was used to study bacterial diversity of a pristine forest soil and of two cultures of the same soil enriched with cellulolytic bacteria. Our analysis revealed high bacterial diversity in the native soil sample, evidencing at least 10 phyla, in which Actinobacteria, Proteobacteria and Acidobacteria accounted for more than 76% of all sequences. In both enriched samples, members of Proteobacteria were the most frequently represented. The majority of bacterial genera in both enriched samples were identified as Brevundimonas and Caulobacter, but members of Devosia, Sphingomonas, Variovorax, Acidovorax, Pseudomonas, Xanthomonas, Stenotrophomonas, Achromobacter and Delftia were also found. In addition, it was possible to identify cellulolytic taxa such as Acidothermus, Micromonospora, Streptomyces, Paenibacillus and Pseudomonas, which indicates that this ecosystem could be an attractive source for study of novel enzymes for cellulose degradation. PMID:22202170

Abstract BACKGROUND: Denitrification is a microbial process that has received considerable attention during the past decade since it can result in losses of added nitrogen fertilisers from agricultural soils. Paddy soil has been known to have strong denitrifying activity, but the denitrifying microorganisms responsible for fertilisers in paddy soil are not well known. The objective of this study was to explore the impacts of 17-year application of inorganic and organic fertiliser (rice straw) on the abundance and composition of a nosZ-denitrifier community in paddy soil. Soil samples were collected from CK plots (no fertiliser), N (nitrogen fertiliser), NPK (nitrogen, phosphorus and potassium fertilisers) and NPK + OM (NPK plus organic matter). The nitrous oxide reductase gene (nosZ) commu...

Nucleic acid methods based on sequencing of clone libraries provide sequence and the phylogenetic information of an individual clone. In the present study, ammonia monooxygenase (amoA), nitrite-oxido-reductase(norB), nitrite reductase (nirS) and nitrous oxide reductase (nosZ) genes are chosen to detect nitrifiers and denitrifiers in coastal aquaculture using gene-specific primers. The abundance of these functional genes revealed the presence of nitrifying and denitrifying organisms in coastal aquaculture. Nitrifying and denitrifying communities were analyzed by parallel DNA extractions and clone library construction for amoA, norB, nirS and nosZ obtained from coastal aquaculture soil. Amino acids in parts of the amoA, norB, nirS and nosZ encoded proteins were aligned to know conserved amin...

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by (/sup 3/H)thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.

An organ culture of slices of livers from adult rats was used to study effect of vitamin A (all-trans retinol) on lysosome stability. Lysosomes were purified by centrifugation in Percoll gradients. Preparations were monitored by electron microscopy and evaluated by morphometry and assays of marker enzymes. Enrichments relative to homogenates and crude pellets were estimated from latent (triton X-100) acid p-nitrophenylphosphatase specific activities. Lysosomes prepared from unincubated slices were enriched 50-fold in latent acid phosphatase relative to homogenates. In contrast, lysosomes prepared from slices incubated for 30 min in PBS alone were enriched only 20-fold. When 25 ..mu..g/ml retinol was included in the incubation medium, enrichments of 40-fold were obtained. The integrity of the slices was monitored by electron microscopy and their viability was confirmed by a sustained uptake and incorporation of (/sup 3/H)leucine into protein (up to 2 h in culture). The loss of lysosomes from homogenates of slices incubated in the absence of retinol was accompanied by a loss of acid phosphatase from the lysosomal pellet to the supernatant during purification. Addition of retinol to slices just prior to homogenization was without effect. The results demonstrate a stabilizing influence of vitamin A on lysosomes during incubation of licer slices. The findings contrast earlier reports of retinol-induced lysosome fragility in other in vitro systems.

Two hundred and fifty tracheal aspirates were subjected to direct antimicrobial susceptibility testing by disk diffusion, Etest and inoculation on antibiotic-enriched MacConkey agar plates. Results were compared with those obtained using an automated system on microorganisms recovered from standard quantitative culture. A total of 255 microorganisms were isolated from 194 positive samples by the standard quantitative procedure. A total of 85.1%, 82.5% and 72.5% agreement between direct disk diffusion, Etest and antibiotic-enriched MacConkey agar plates, respectively, and the standard procedure was observed in 64 microorganisms obtained from monomicrobial cultures that corresponded to 240 individual microorganism-antimicrobial agent combinations. Three (1.3%) and four (1.7%) very major erro...

Sulfur is almost insoluble in water at ambient temperatures, and therefore polysulfide (Sn^2^-) has been considered as a possible intermediate that is used directly by bacteria in sulfur respiration. Sulfur-reducing reductases have been purified and characterized from a few sulfur reducers. However, polysulfide reduction has only been confirmed in Wolinella succinogenes. In our previous study, the direct production of hydrogen sulfide from polysulfide was confirmed by an enrichment culture obtained from natural samples under sulfate-reducing conditions. The present study attempted to isolate and identify polysulfide-reducing bacteria from the enrichment cultures. Almost all the isolated strains were classified into the genus Clostridium, based on 16S rRNA gene sequence analysis. The isolat...

Abstract An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the ?top six? non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin?producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 105 colony-forming units (CFU)/g before enrichment and 102 CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samp...

Many waste streams that are suitable substrates for mixed culture bioplastic (polyhydroxyalkanoate, PHA) production are nutrient limited and may need to be supplemented to allow sufficient growth of PHA accumulating bacteria. The scope of this study was to investigate the necessity of nutrient supplementation for the enrichment of an efficient PHA producing mixed culture. We studied the influence of different degrees of carbon and nitrogen limitation on the performance of an acetate-fed feast-famine sequencing batch reactor (SBR) employed to enrich PHA storing bacteria. The microbial reaction rates in the SBR showed a shift with a change in the limiting substrate: high acetate uptake rates were found in carbon-limited SBRs (medium C/N ratios 6-13.2 Cmol/Nmol), while nitrogen-limited SBRs (...

Abstract in english Comparative growth studies of Pediococcus pentosaceus were done in MRS (control) culture medium as well as in culture medium of 5% sugar cane blackstrap molasses (Broth 1) enriched with yeast extract (Broth 2) and enriched with beef extract (Broth 30. The experiment was carried out in a shaker (96 rpm) in 250 mL Erlenmeyers flasks with working volume of 150 mL, initial inoculum of about 10³ - 10(4) FCU/mL, at 35 ± 1ºC temperature and 28-hour fermentation. The biomass p (more) ruductivity was higher in MRS broth (0.0375 g/L.h), followed by Broth 2 (0.0275), Broth 3 (0.0189) and Broth 1 (0.0151). The higher viable cells number occured in MRS (13.96 log10 FCU/mL), followed by Broth 2 (12.09), Broth 3 (10.12) and Broth 1 (8.42).

A possibility of dissimilatory MnO2 reduction at extremely high salt and pH was studied in sediments from hypersaline alkaline lakes in Kulunda Steppe (Altai, Russia). Experiments with anaerobic sediment slurries demonstrated a relatively rapid reduction of colloidal MnO2 in the presence of acetate and formate as electron donor at in situ conditions (i.e., pH 10 and a salt content from 0.6 to 4?M total Na+). All reduced Mn at these conditions remained in the solid phase. A single, stable enrichment culture was obtained from the slurries consistently reducing MnO2 at pH 10 and 0.6?M total Na+ with formate. A pure culture of a haloalkaliphilic Mn-reducing bacterium obtained from the positive enrichment was phylogenetically closely related to the anaerobic haloalkaliphilic Bacillus arsenicise...

Hydrogen production from desugared molasses (DM) was investigated in both batch and continuous reactors using thermophilic mixed cultures enriched from digested manure by load shock (loading with DM concentration of 50.1 g-sugar/L) to suppress methanogens. H"2 gas, free of methane, was produced during batch cultivations, at different (DM) concentrations ranging from 1.5 g-sugars/L to 50.1 g-sugars/L. The highest yield of 237 ml-H"2/g-sugar was achieved during the DM batch fermentation at concentration of 2.1 g-sugars/L, whereafter the yield decreased with increasing DM concentration. The enriched hydrogen producing mixed culture achieved from the 16.7 g-sugars/L DM batch cultivation was immobilized on heat treated anaerobic sludge granules in an up-flow anaerobic sludge blanket (UASB) reac...

Subcultures that reductively dechlorinate cis-dichloroethene (cis-DCE) or vinyl chloride (VC) were derived from three independent enrichments that completely dechlorinated tetrachloroethene (PCE) to ethene in order to study the reductive dechlorination of the lesser chlorinated ethenes. These subcultures completely dechlorinated cis-DCE and VC and could be transferred indefinitely in basal salts minimal medium with H{sub 2} as the electron donor. After 10 transfers (1% V/V) the cis-DCE and VC-dechlorinating subcultures from two of the PCE enrichments failed to dechlorinate PCE, but the subcultures from the third PCE enrichment maintained the ability to dechlorinate PCE. Analysis of the 16S rRNA genes from these enrichments by terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) demonstrated shifts in the community composition of the subcultures that had lost the PCE-dechlorinating activity but not in the subcultures that maintained the PCE-dechlorinating activity. Analysis of the changes in community composition of the different enrichments suggested that at least two populations were responsible for the sequential dechlorination of PCE to ethene in these cultures and that consortia can cooperate in the complete dechlorination of PCE.

Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobactereium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.

Impacts of divergent arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Gigaspora margarita, on denitrifying and diazotrophic bacterial communities of Plantago lanceolata in nutrient-limited dune soil were assessed. We hypothesized AM species-related modifications that were confirmed in respective bacterial nirK and nifH sequence polymorphism -based community clustering and community variance allocation. The denitrifying community appeared more responsive to AM fungi than the nitrogen-fixing community. Nevertheless, the main explanatory variable, in both cases, was plant age. We conclude that AM fungi can modify N-cycling microbial rhizosphere communities and future work should aim to verify the functional significance and mechanistic basis.

Denitrification is applied in the tertiary treatment of wastewater to reduce nitrogen pollution. Fluorescence in situ hybridization (FISH), catalyzed reporter deposition (CARD)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in chemolithotrophic denitrifying bioreactors fed with nitrate and thiosulfate. FISH oligonucleotide probes for the chemolitoautotrophic denitrifiers Thiobacillus denitrificans and Thiomicrospira denitrificans were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity in the biofilms changed during the reactor operation. Chemoorganotrophic fermentative Gram-positive strains in the phyla, Actinobacteria and Firmicutes, were dom...

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.   

Prokaryotic diversities of 12 geothermal hot springs located in Northern, Central and Southern Tunisia were investigated by culture-based and molecular approaches. Enrichment cultures for both aerobic and anaerobic microorganisms were successfully obtained at temperatures ranging from 50 to 75?C. Fourteen strains including four novel species were cultivated and assigned to the phyla Firmicutes (9), Thermotogae (2), Betaproteobacteria (1), Synergistetes (1) and Bacteroidetes (1). Archaeal or universal oligonucleotide primer sets were used to generate 16S rRNA gene libraries. Representative groups included Proteobacteria, Firmicutes, Deinococcus-Thermus, Thermotogae, Synergistetes, Bacteroidetes, Aquificae, Chloroflexi, candidate division OP9 in addition to other yet unclassified strains. Th...

We evaluated the MVPlex assay (Geneco Biomedical Products), which uses target-enriched multiplex PCR amplification followed by liquid array identification, for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from 307 dual-swab specimens. By using a combination of culture (Trypticase soy agar-5% sheep blood agar and Columbia CNA agar-5% sheep blood) and an FDA-approved MRSA PCR assay as the "gold standard," the MVPlex MRSA assay and culture were found to have sensitivities of 97.8% and 84.4% (P = 0.002) and specificities of 95.8% and 98.6% (P < 0.05), respectively. PMID:18614646

An enrichment culture obtained from anaerobic granular sludge of a bench-scale anarobic reactor degraded methanol at 65 C via sulfate reduction and acetogenesis. Sulfate reduction was the dominant process (S{sup 2-}/acetate=2.5). No methane formation was observed. Approximately 30% of the methanol was converted by acetogenic bacteria to acetate, while the remainder was degraded by these bacteria to H{sub 2} and CO{sub 2} in syntrophy with hydrogen-consuming sulfate-reducing bacteria. Pure cultures of sulfate-reducing and acetogenic bacteria were isolated and characterized. (orig.)

A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of 14CO2 from [U-ring-14C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.

Abstract in spanish Este artículo analiza algunos de los paradigmas más importantes de la cultura política, con la finalidad de intentar una síntesis fenomenológica desde las ciencias antropológicas. Al mismo tiempo, a partir de la noción de cultura política, se propone la construcción de una ruta nueva de investigación para enriquecer los estudios regionales. Abstract in english This article examines some of the most important paradigms of political culture in order to attempt a phenomenological synthesis founded on the anthropological sciences. At the same time, based on the notion of political culture, we build a new research path aimed at enriching regional studies.