Sulfate-reducing Bacterial Enrichment Research Articles

Removal of azo dye in innovative constructed wetlands: Influence of iron scrap and sulfate reducing bacterial enrichment

The research work reported here is about an innovative way of designing constructed wetlands (CW) for high removal rate of pollutants and to make them less land intensive. Influence of iron scrap (IS) and sulfate reducing bacterial enrichment (SRB) was studied in Canna indica planted CW microcosms. This is the first ever report which deals with such interaction for enhancing the performance of CW. Four sets of experiments were performed: first with normal-CW microcosm; second with SRB enriched CW microcosm; third with SRB and IS enriched, CW microcosm; and fourth with SRB and copper doped IS enriched CW microcosm. The microcosms were tested for color removal of methyl orange dye from synthetic wastewater. The SRB enriched CW achieved highest removal (99.5, 98.53, 97.05, 94.62, and 96.52%) and the normal-CW achieved lowest removal (93, 89.18, 48.72, 50.14, and 52.34%) from wastewater containing 500, 1000, 1500, 2000 and 2500mgl−1 dye respectively. Higher removal was achieved in SRB and IS enriched CW in comparison to other CW microcosms. At higher concentrations, enhancement in performance of SRB and SRB–IS enriched CW over normal CW was clearly achieved which was not observed in lower concentrations. During treatment of 2500mgl−1 dye containing wastewater, it was observed that the dye removal percentage was 37.02 higher in SRB-CW over normal-CW and 19.48% higher in SRB–IS-CW over SRB-CW in 24h of contact time. These results clearly revealed that higher removal rate of color of dye can be achieved in innovative constructed wetlands.

Preliminary investigation on the role of microorganisms in the production of phosphine

The relationships between the phosphine content and various microbial populations, activities of different enzymes were investigated firstly. The results indicated that the phosphine content of samples from various environments was positively related to total anaerobic microorganisms, organic phosphate compound-dissolving bacteria, denitrifying bacteria, and the activities of alkaline phosphatase and dehydrogenase, with correlation coefficients (R2) up to 0.93, 0.90, 0.69, 0.79, and 0.82, respectively. Results also showed that the phosphine content was not related to total aerobic microorganisms, inorganic phosphate compound-dissolving bacteria, sulfate-reducing bacteria, and the acidic phosphatase activity. Nutrients such as yeast extract and glucose were added, at a time and individually, to normal or autoclaved soil samples. The soil samples were inoculated with sulfate-reducing bacterial (SRB) enrichments and/or denitrifying bacterial (DNB) enrichment. After incubation for one month at 30 degrees C, the phosphane content of these samples was analyzed. The results indicated that the addition of glucose or yeast extract could greatly increase the phosphane content. Moreover, it was revealed that inoculation with SRB or DNB could also promote the formation of phosphine. The DNB, however, was more efficient in this regard. The highest phosphine content, about 5 times that of the control, was detected in the sample that was added with both glucose and yeast extract and inoculated with SRB and DNB simultaneously. SRB and DNB were enriched for several generations and the phosphane content of different generations was analyzed. Furthermore, SRB and DNB enrichments were inoculated into different media, in the beginning of enriching, the phosphane content was about the same for different enrichments, and differed more significantly as the enrichment process was carried further. In fourth generation, the phosphane content of DNB enrichment was about 3 times of that of SRB enrichment, indicating that the inoculation of different enrichments into different media caused the variations of the phosphine content. The highest phosphine content was detected in the sample which was inoculated with DNB enrichment into the denitrifying medium without nitrate. It is inferred from these results that microorganisms play an important role in the production of phosphine in different environments.

Anaerobic phenanthrene mineralization by a carboxylating sulfate-reducing bacterial enrichment

Information on the susceptibility of higher molecular weight polynuclear aromatic hydrocarbons to anaerobic biodegradation is relatively rare. We obtained a sulfate-reducing bacterial enrichment capable of phenanthrene metabolism from a hydrocarbon-contaminated marine sediment. Phenanthrene degradation was in stoichiometric agreement with the theoretically expected amount of sulfate reduction and inhibited by molybdate. Mineralization of (14)C-phenanthrene by the enrichment was confirmed by the recovery of the expected amount of (14)CO(2). Stable isotope studies with protonated or deuterated phenanthrene resulted in the detection of the correspondingly labeled phenanthrene carboxylic acid by gas chromatography-mass spectrometry. Comparison of the metabolite profile with a synthesized standard confirmed that the parent molecule was carboxylated at the C-2 position. Incorporation of (13)C-bicarbonate into the carboxyl group implicated a direct carboxylation of phenanthrene as a likely key initial reaction. Denaturing gradient gel electrophoresis analysis of the enrichment showed only two major bands and 16S rRNA sequences obtained by cloning clustered with known hydrocarbon-degrading sulfate-reducing delta-proteobacteria, indicating their possible involvement in the anaerobic oxidation of phenanthrene via carboxylation as the initial activation reaction.

The treatment of textile wastewater including chromium(VI) and reactive dye by sulfate-reducing bacterial enrichment

Sulfate-reducing bacteria (SRB) that could grow on modified Postgate C medium (PC) containing chromium(VI) were isolated from industrial wastewaters and their chromium(VI) reduction capacities were investigated as a function of changes in the initial pH values, chromium, sulfate, NaCl and reactive dye concentrations. The optimum pH value at 50 mg l −1 initial chromium(VI) concentration was determined to be 8. Chromium(VI) reduction by SRB was investigated at 22.7–98.4 mg l −1 initial chromium(VI) concentrations. At the end of the experiments, the mixed cultures of SRB were found to reduce within 2–6 days more than 99% of the initial chromium(VI) levels, which ranged from 22.7 to 74.9 mg l −1. The effects of the initial 0–9.0 g l −1 concentrations of disodium sulfate and 0–6% (w/v) concentrations of NaCI to chromium reduction showed that the lowest concentrations of sulfate and NaCI were the best for chromium reduction in the PC medium including 50 mg l −1 chromium(VI). Chromium(VI) reduction in 50 mg l −1 and 25–100 mg l −1 Remazol Blue, Reactive Black B or Reactive Red RB containing media were also investigated. In the experiments, 25–30% of the initial dye concentrations and 95% of the chromium(VI) was removed from the medium at the end of 72-h and 24-h incubation periods, respectively.

Stable Isotopic Studies of n -Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.

Biodegradation of an alicyclic hydrocarbon by a sulfate-reducing enrichment from a gas condensate-contaminated aquifer.

We used ethylcyclopentane (ECP) as a model alicyclic hydrocarbon and investigated its metabolism by a sulfate-reducing bacterial enrichment obtained from a gas condensate-contaminated aquifer. The enrichment coupled the consumption of ECP with the stoichiometrically expected amount of sulfate reduced. During ECP biodegradation, we observed the transient accumulation of metabolite peaks by gas chromatography-mass spectrometry, three of which had identical mass spectrometry profiles. Mass-spectral similarities to analogous authentic standards allowed us to identify these metabolites as ethylcyclopentylsuccinic acids, ethylcyclopentylpropionic acid, ethylcyclopentylcarboxylic acid, and ethylsuccinic acid. Based on these findings, we propose a pathway for the degradation of this alicyclic hydrocarbon. Furthermore, a putative metabolite similar to ethylcyclopentylsuccinic acid was also found in samples of contaminated groundwater from the aquifer. However, no such finding was evident for samples collected from wells located upgradient of the gas condensate spill. Microbial community analysis of the ECP-degrading enrichment by denaturing gradient gel electrophoresis revealed the presence of at least three different organisms using universal eubacterial primers targeting 550 bp of the 16S rRNA gene. Based on sequence analysis, these organisms are phylogenetically related to the genera Syntrophobacter and Desulfotomaculum as well as a member of the Cytophaga-Flexibacter-Bacteroides group. The evidence suggests that alicyclic hydrocarbons such as ECP can be anaerobically activated by the addition to the double bond of fumarate to form alkylsuccinate derivatives under sulfate-reducing conditions and that the reaction occurs in the laboratory and in hydrocarbon-impacted environments.

Anaerobic oxidation of n-dodecane by an addition reaction in a sulfate-reducing bacterial enrichment culture.

We identified trace metabolites produced during the anaerobic biodegradation of H(26)- and D(26)-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C---H or C---D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H(26)- and D(26)-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H(26)-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.

Detection of Phenol and Benzoate as Intermediates of Anaerobic Benzene Biodegradation under Different Terminal Electron-Accepting Conditions

A sulfate-reducing bacterial enrichment that anaerobically metabolized benzene was obtained from a petroleum-contaminated aquifer. During biodegradation, we observed the transient accumulation of p...

Carboxylation and mineralization ofm-cresol by a sulfate-reducing bacterial enrichment

An enrichment culture that anaerobically degradedm-cresol under sulfate-reducing conditions was obtained from an anoxic aquifer.m-Cresol removal by the culture was greatest when sulfate or thiosulfate served as electron acceptors; sulfite, nitrate, and CO2 were poor substitutes for sulfate. A14C-labeled carboxylated intermediate was detected when the culture was given14C-labeled bicarbonate and nonlabeledm-cresol or nonlabeled bicarbonate and14C-labeledm-cresol. Metabolism of the carboxylated intermediate yielded14C-acetate, which was eventually converted to14CO2. Trace quantities of methylbenzoic acid were also detected as a putativem-cresol intermediate. The importance of this dehydroxylated intermediate in the anaerobic degradation ofm-cresol is unclear, since an amendment of 2-methylbenzoic acid was not degraded by the culture. The stoichiometry of electron acceptor consumption and carbon mass balances confirm thatm-cresol was mineralized by the culture.

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.

The anaerobic biodegradation ofo-, m- andp-cresol by sulfate-reducing bacterial enrichment cultures obtained from a shallow anoxic aquifer

Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize eithero-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition ofp-cresol proceeds by the initial oxidation of the aryl methyl group to formp-hydroxybenzoic acid. This intermediate was then converted to benzoic acid. Benzoic acid and a hydroxybenzaldehyde were also found in spent culture fluids from ano-cresol-degrading enrichment culture. This result, in addition to others, suggested thato-cresol may also be anaerobically degraded by the oxidation of the methyl substituent. An alternate pathway for anaerobicm-cresol decomposition might exist. Enrichment cultures obtained with eitherp- oro-cresol degraded both of these substrates but notm-cresol. In contrast, am-cresol enrichment culture did not metabolize theortho orpara isomers. Anaerobic biodegradation in all enrichment cultures was inhibited by molybdate and oxygen, and was dependent on the presence of sulfate as a terminal electron acceptor. The stoichiometry of sulfate-reduction and substrate depletion by the various enrichment cultures indicated that the parent cresol isomers were completely mineralized. This result was confirmed by the conversion of14C-labeledp-cresol to14CO2. These results help clarify the fate of alkylated aromatic chemicals in anoxic aquifers.