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USGS1986

Formation of methane and carbon dioxide from dimethylselenide in anoxic sediments and by a methanogenic bacterium

Anaerobic San Francisco Bay salt marsh sediments rapidly metabolized [ 14 C]dimethylselenide (DMSe) to 14 CH 4 and 14 CO 2 . Addition of selective inhibitors (2-bromoethanesulfonic acid or molybdate) to these sediments indicated that both methanogenic and sulfate-respiring bacteria could degrade DMSe to gaseous products. However, sediments taken from the selenium-contaminated Kesterson Wildlife Refuge produced only 14 CO 2 from [ 14 C]DMSe, implying that methanogens were not important in the Kesterson samples. A pure culture of a dimethylsulfide (DMS)-grown methylotrophic methanogen converted [ 14 C]DMSe to 14 CH 4 and 14 CO 2 . However, the organism could not grow on DMSe. Addition of DMS to either sediments or the pure culture retarded the metabolism of DMSe. This effect appeared to be caused by competitive inhibition, thereby indicating a common enzyme system for DMS and DMSe metabolism. DMSe appears to be degraded as part of the DMS pool present in anoxic environments. These results suggest that methylotrophic methanogens may demethylate methylated forms of other metals and metalloids found in nature.

California↗

USGS1986

Metabolism of reduced methylated sulfur compounds in anaerobic sediments and by a pure culture of an estuarine methanogen

Addition of dimethylsulfide (DMS), dimethyldisulfide (DMDS), or methane thiol (MSH) to a diversity of anoxic aquatic sediments (e.g., fresh water, estuarine, alkaline/hypersaline) stimulated methane production. The yield of methane recovered from DMS was often 52 to 63%, although high concentrations of DMS (as well as MSH and DMDS) inhibited methanogenesis in some types of sediments. Production of methane from these reduced methylated sulfur compounds was blocked by 2-bromoethanesulfonic acid. Sulfate did not influence the metabolism of millimolar levels of DMS, DMDS, or MSH added to sediments. However, when DMS was added at &sim;2-μM levels as [ 14 C]DMS, metabolism by sediments resulted in a 14 CH 4 / 14 CO 2 ratio of only 0.06. Addition of molybdate increased the ratio to 1.8, while 2-bromoethanesulfonic acid decreased it to 0, but did not block 14 CO 2 production. These results indicate the methanogens and sulfate reducers compete for DMS when it is present at low concentrations; however, at high concentrations, DMS is a “noncompetitive” substrate for methanogens. Metabolism of DMS by sediments resulted in the appearance of MSH as a transient intermediate. A pure culture of an obligately methylotrophic estuarine methanogen was isolated which was capable of growth on DMS. Metabolism of DMS by the culture also resulted in the transient appearance of MSH, but the organism could grow on neither MSH nor DMDS. The culture metabolized [ 14 C]-DMS to yield a 14 CH 4 / 14 CO 2 ratio of &sim;2.8. Reduced methylated sulfur compounds represent a new class of substrates for methanogens and may be potential precursors of methane in a variety of aquatic habitats.

California↗

USGS1987

Growth determinations for unattached bacteria in a contaminated aquifer

Growth rates of unattached bacteria in groundwater contaminated with treated sewage and collected at various distances from the source of contamination were estimated by using frequency of dividing cells and tritiated-thymidine uptake and compared with growth rates obtained with unsupplemented, closed-bottle incubations. Estimates of bacterial generation times [(In 2)/ μ ] along a 3-km-long transect in oxygen-depleted (0.1 to 0.7 mg of dissolved oxygen liter -1 ) groundwater ranged from 16 h at 0.26 km downgradient from an on-land, treated-sewage outfall to 139 h at 1.6 km and correlated with bacterial abundance ( r 2 = 0.88 at P < 0.001). Partitioning of assimilated thymidine into nucleic acid generally decreased with distance from the contaminant source, and one population in heavily contaminated groundwater assimilated little thymidine during a 20-h incubation. Several assumptions commonly made when frequency of dividing cells and tritiated-thymidine uptake are used were not applicable to the groundwater samples.

Massachusetts↗

USGS1987

Reduction of selenate to selenide by sulfate-respiring bacteria: Experiments with cell suspensions and estuarine sediments

Washed cell suspensions of Desulfovibrio desulfuricans subsp. aestuarii were capable of reducing nanomolar levels of selenate to selenide as well as sulfate to sulfide. Reduction of these species was inhibited by 1 mM selenate or tungstate. The addition of 1 mM sulfate decreased the reduction of selenate and enhanced the reduction of sulfate. Increasing concentrations of sulfate inhibited rates of selenate reduction but enhanced sulfate reduction rates. Cell suspensions kept in 1 mM selenate were incapable of reducing either selenate or sulfate when the selenate/sulfate ratio was ≥0.02, indicating that irreversible inhibition occurs at high selenate concentrations. Anoxic estuarine sediments having an active flora of sulfate-respiring bacteria were capable of a small amount of selenate reduction when ambient sulfate concentrations were low (<4 mM). These results indicate that sulfate is an inhibitor of the reduction of trace quantities of selenate. Therefore, direct reduction of traces of selenate to selenide by sulfate-respiring bacteria in natural environments is constrained by the ambient concentration of sulfate ions. The significance of this observation with regard to the role sediments play in sequestering selenium is discussed.

Applied and Environmental Microbiology↗

USGS1987

Rapid assay for microbially reducible ferric iron in aquatic sediments

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.

Potomac River estuary↗

USGS1988

Microbial hydroxylation of quinoline in contaminated groundwater: evidence for incorporation of the oxygen atom of water.

Studies conducted in an aquifer contaminated by creosote suggest that quinoline is converted to 2(1H)quinolinone by an indigenous consortium of microorganisms. Laboratory microbial experiments using H 2 18 O indicate that water is the source of the oxygen atom for this hydroxylation reaction under aerobic and anaerobic conditions.

Applied and Environmental Microbiology↗

USGS1988

Denitrification in a sand and gravel aquifer

Denitrification was assayed by the acetylene blockage technique in slurried core material obtained from a freshwater sand and gravel aquifer. The aquifer, which has been contaminated with treated sewage for more than 50 years, had a contaminant plume greater than 3.5-km long. Near the contaminant source, groundwater nitrate concentrations were greater than 1 mM, whereas 0.25 km downgradient the central portion of the contaminant plume was anoxic and contained no detectable nitrate. Samples were obtained along the longitudinal axis of the plume (0 to 0.25 km) at several depths from four sites. Denitrification was evident at in situ nitrate concentrations at all sites tested; rates ranged from 2.3 to 260 pmol of N 2 O produced (g of wet sediment) −1 h −1 . Rates were highest nearest the contaminant source and decreased with increasing distance downgradient. Denitrification was the predominant nitrate-reducing activity; no evidence was found for nitrate reduction to ammonium at any site. Denitrifying activity was carbon limited and not nitrate limited, except when the ambient nitrate level was less than the detection limit, in which case, even when amended with high concentrations of glucose and nitrate, the capacity to denitrify on a short-term basis was lacking. These results demonstrate that denitrification can occur in groundwater systems and, thereby, serve as a mechanism for nitrate removal from groundwater.

Massachusetts↗

USGS1989

Requirement for a microbial consortium to completely oxidize glucose in Fe(III)- reducing sediments

In various sediments in which Fe(III) reduction was the terminal electron-accepting process, [ 14 C]glucose was fermented to 14 C-fatty acids in a manner similar to that observed in methanogenic sediments. These results are consistent with the hypothesis that, in Fe(III)-reducing sediments, fermentable substrates are oxidized to carbon dioxide by the combined activity of fermentative bacteria and fatty acid-oxidizing, Fe(III)-reducing bacteria.

Maryland, Minnesota↗

USGS1989

Hydrogen and formate oxidation coupled to dissimilatory reduction of iron or manganese by Alteromonas putrefaciens

The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.

Applied and Environmental Microbiology↗

USGS1989

Description of an estuarine methylotrophic methanogen, which grows on dimethyl sulfite

Characteristics of an obligately methylotrophic coccoid methanogen (strain GS-16) previously isolated from estuarine sediment are described. Growth was demonstrated on dimethyl sulfide (DMS) or trimethylamine (TMA), but not on methane thiol, methane thiol plus hydrogen, dimethyl disulfide, or methionine. DMS-grown cells were able to metabolize DMS and TMA simultaneously when inoculated into media containing substrate levels of these compounds. However, TMA-grown cells could not metabolize [ 14 C]DMS to 14 CH 4 , although they could convert [ 14 C]methanol to 14 CH 4 . These results suggest that metabolism of DMS proceeds along a somewhat different route than that of TMA and perhaps also that of methanol. The organism exhibited doubling times of 23 and 32 h for growth (25°C) in mineral media on TMA and DMS, respectively. Doubling times were more rapid (∼6 h) when the organisms were grown on TMA in complex broth. In mineral media, the fastest growth on DMS occurred between pH levels of 7.0 and 8.7, at 29°C, and with 0.2 to 0.4 M Na + and 0.04 M Mg 2+ . Somewhat different results occurred for growth on TMA in complex broth. Cells had a moles percent G+C value of 44.5% for their DNA. Growth on DMS, TMA, and methanol yielded stable carbon isotope fractionation factors of 1.044, 1.037, and 1.063, respectively. Fractionation factors for hydrogen were 1.203 (DMS) and 1.183 (TMA).

Applied and Environmental Microbiology↗

USGS1989

Selenate reduction to elemental selenium by anaerobic bacteria in sediments and culture: Biogeochemical significance of a novel, sulfate-independent respiration

Interstitial water profiles of SeO 4 2− , SeO 3 2− , SO 4 2− , and Cl − in anoxic sediments indicated removal of the seleno-oxyanions by a near-surface process unrelated to sulfate reduction. In sediment slurry experiments, a complete reductive removal of SeO 4 2− occurred under anaerobic conditions, was more rapid with H 2 or acetate, and was inhibited by O 2 , NO 3 − , MnO 2 , or autoclaving but not by SO 4 2− or FeOOH. Oxidation of acetate in sediments could be coupled to selenate but not to molybdate. Reduction of selenate to elemental selenium was determined to be the mechanism for loss from solution. Selenate reduction was inhibited by tungstate and chromate but not by molybdate. A small quantity of the elemental selenium precipitated into sediments from solution could be resolublized by oxidation with either nitrate or FeOOH, but not with MnO 2 . A bacterium isolated from estuarine sediments demonstrated selenate-dependent growth on acetate, forming elemental selenium and carbon dioxide as respiratory end products. These results indicate that dissimilatory selenate reduction to elemental selenium is the major sink for selenium oxyanions in anoxic sediments. In addition, they suggest application as a treatment process for removing selenium oxyanions from wastewaters and also offer an explanation for the presence of selenite in oxic waters.

California↗

USGS1990

Dissimilatory selenate reduction potentials in a diversity of sediment types

We measured potential rates of bacterial dissimilatory reduction of 75 SeO 4 2− to 75 Se 0 in a diversity of sediment types, with salinities ranging from freshwater (salinity = 1 g/liter) to hypersaline (salinity = 320 g/liter and with pH values ranging from 7.1 to 9.8. Significant biological selenate reduction occurred in all samples with salinities from 1 to 250 g/liter but not in samples with a salinity of 320 g/liter. Potential selenate reduction rates (25 nmol of SeO 4 2− per ml of sediment added with isotope) ranged from 0.07 to 22 μmol of SeO 4 2− reduced liter −1 h −1 . Activity followed Michaelis-Menten kinetics in relation to SeO 4 2− concentration ( K m of selenate = 7.9 to 720 μM). There was no linear correlation between potential rates of SeO 4 2− reduction and salinity, pH, concentrations of total Se, porosity, or organic carbon in the sediments. However, potential selenate reduction was correlated with apparent K m for selenate and with potential rates of denitrification ( r = 0.92 and 0.81, respectively). NO 3 − , NO 2 − , MoO 4 2− , and WO 4 2− inhibited selenate reduction activity to different extents in sediments from both Hunter Drain and Massie Slough, Nev. Sulfate partially inhibited activity in sediment from freshwater (salinity = 1 g/liter) Massie Slough samples but not from the saline (salinity = 60 g/liter) Hunter Drain samples. We conclude that dissimilatory selenate reduction in sediments is widespread in nature. In addition, in situ selenate reduction is a first-order reaction, because the ambient concentrations of selenium oxyanions in the sediments were orders of magnitude less than their K m s.

California, Nevada↗

USGS1990

Nitrogen fixation dynamics of two diazotrophic communities in Mono Lake, California

Two types of diazotrophic microbial communities were found in the littoral zone of alkaline hypersaline Mono Lake, California. One consisted of anaerobic bacteria inhabiting the flocculent surface layers of sediments. Nitrogen fixation (acetylene reduction) by flocculent surface layers occurred under anaerobic conditions, was not stimulated by light or by additions of organic substrates, and was inhibited by O 2 , nitrate, and ammonia. The second community consisted of a ball-shaped association of a filamentous chlorophyte ( Ctenocladus circinnatus ) with diazotrophic, nonheterocystous cyanobacteria, as well as anaerobic bacteria ( Ctenocladus balls). Nitrogen fixation by Ctenocladus balls was usually, but not always, stimulated by light. Rates of anaerobic dark fixation equaled those in the light under air. Fixation in the light was stimulated by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and by propanil [ N -(3,4-dichlorophenyl)propanamide]. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea-elicited nitrogenase activity was inhibited by ammonia (96%) and nitrate (65%). Fixation was greatest when Ctenocladus balls were incubated anaerobically in the light with sulfide. Dark anaerobic fixation was not stimulated by organic substrates in short-term (4-h) incubations, but was in long-term (67-h) ones. Areal estimates of benthic N 2 fixation were measured seasonally, using chambers. Highest rates (∼29.3 μmol of C 2 H 4 m −2 h −1 ) occurred under normal diel regimens of light and dark. These estimates indicate that benthic N 2 fixation has the potential to be a significant nitrogen source in Mono Lake.

California↗

USGS1990

Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GS-15

The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p -cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ ring - 14 C]toluene was oxidized to 14 CO 2 , and the stoichiometry of 14 CO 2 production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p -cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p -Hydroxybenzoate was a transitory extracellular intermediate of phenol and p -cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p -cresol ( p -hydroxybenzylalcohol and p -hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.

Applied and Environmental Microbiology↗

USGS1991

Methylmercury decomposition in sediments and bacterial cultures: Involvement of methanogens and sulfate reducers in oxidative demethylation

Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with 14 CH 3 HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly 14 CO 2 as well as lesser amounts of 14 CH 4 . Acetogenic activity resulted in fixation of some 14 CO 2 produced from 14 CH 3 HgI into acetate. Aerobic demethylation in estuarine sediments produced only 14 CH 4 , while aerobic demethylation in freshwater sediments produced small amounts of both 14 CH 4 and 14 CO 2 . Two species of Desulfovibrio produced only traces of 14 CH 4 from 14 CH 3 HgI, while a culture of a methylotrophic methanogen formed traces of 14 CO 2 and 14 CH 4 when grown on trimethylamine in the presence of the 14 CH 3 HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.

California↗

USGS1991

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14 C-labeled organic compounds, and the evolution of 14 CO 2 was measured over time. Gas chromatographic analyses were used to monitor CO 2 production and O 2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14 CO 2 was measured from [ 14 C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [ 14 C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14 C label. [ 14 C]benzene and [ 14 C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO 3 , CO 2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 μ mol g -1 (dry weight) day -1 while oxygen concentration decreased at a rate of 0.124 μ mol g -1 (dry weight) day -1 . With no added nitrate, CO 2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n -alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific. Also, the community is strongly nitrogen limited, and nitrogen additions may be required to significantly enhance hydrocarbon biodegradation.

South Carolina↗

USGS1991

In situ bacterial selenate reduction in the agricultural drainage systems of western Nevada

Dissimilatory in situ selenate reduction to elemental selenium in sediments from irrigated agricultural drainage regions of western Nevada was measured at ambient Se oxyanion concentrations. Selenate reduction was rapid, with turnover rate constants ranging from 0.04 to 1.8 h -1 at total Se concentrations in pore water of 13 to 455 nM. Estimates of removal rates of selenium oxyanions were 14, 38, and 155 μ mol m -2 day -1 for South Lead Lake, Massie Slough, and Hunter Drain, respectively.

Nevada↗

USGS1991

Electron transport in the dissimilatory iron reducer, GS-15

Mechanisms for electron transport to Fe(III) were investigated in GS-15, a novel anaerobic microorganism which can obtain energy for growth hy coupling the complete oxidation of organic acids or aromatic compounds to the reduction of Fe(III) to Fe(II). The results indicate that Fe(III) reduction proceeds through a type b cytochrome and a membrane-bound Fe(III) reductase which is distinct from the nitrate reductase.

Applied and Environmental Microbiology↗