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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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

Inactivation of infectious hematopoietic necrosis virus by low levels of iodine

The fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) was rapidly inactivated by extremely low concentrations of iodine in water. A 99.9% virus reduction was obtained in 7.5 s when virus (10 5 PFU/ml) and iodine (0.1 mg/liter, final concentration) were combined in distilled-deionized or hatchery water. Iodine efficacy decreased at pHs greater than 7.5 or when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Sodium thiosulfate effectively neutralized free iodine. Powder, iodophor, and crystalline iodine solutions inactivated IHNV equally. Iodine rapidly inactivated IHNV isolates representing each of the five electropherotypes. Under the conditions used in this study, inactivation was not affected by temperature, salinity, or water hardness. When Dworshak National Fish Hatchery water was continuously treated to provide a free iodine concentration of 0.14 mg/liter, a 7.5-s exposure to iodine was sufficient to inactivate 99.9% of the IHNV. Iodine added to water that contained IHNV prevented infection of rainbow trout ( Oncorhynchus mykiss ) fry. These results suggest that the waterborne route of IHNV transmission can be blocked by adding low iodine concentrations to the water supplies of hatcheries.

Applied and Environmental Microbiology↗

In situ measurement of methane oxidation in groundwater by using natural-gradient tracer tests

Methane oxidation was measured in an unconfined sand and gravel aquifer (Cape Cod, Mass.) by using in situ natural-gradient tracer tests at both a pristine, oxygenated site and an anoxic, sewage-contaminated site. The tracer sites were equipped with multilevel sampling devices to create target grids of sampling points; the injectate was prepared with groundwater from the tracer site to maintain the same geochemical conditions. Methane oxidation was calculated from breakthrough curves of methane relative to halide and inert gas (hexafluoroethane) tracers and was confirmed by the appearance of 13 C-enriched carbon dioxide in experiments in which 13 C-enriched methane was used as the tracer. A V max for methane oxidation could be calculated when the methane concentration was sufficiently high to result in zero-order kinetics throughout the entire transport interval. Methane breakthrough curves could be simulated by modifying a one-dimensional advection-dispersion transport model to include a Michaelis-Menten-based consumption term for methane oxidation. The K m values for methane oxidation that gave the best match for the breakthrough curve peaks were 6.0 and 9.0 μ M for the uncontaminated and contaminated sites, respectively. Natural-gradient tracer tests are a promising approach for assessing microbial processes and for testing in situ bioremediation potential in groundwater systems.

Massachusetts↗

Nitrate is a preferred electron acceptor for growth of freshwater selenate-respiring bacteria

An anaerobic, freshwater enrichment grew with either nitrate or selenate as an electron acceptor. With both ions present, nitrate reduction preceded selenate reduction. An isolate from the enrichment grew on either ion, but the presence of nitrate precluded the reduction of selenate. Stock cultures of denitrifiers grew anaerobically on nitrate but not on selenate.

Applied and Environmental Microbiology↗

A hydrogen-oxidizing, Fe(III)-reducing microorganism from the Great Bay estuary, New Hampshire

A dissimilatory Fe(III)- and Mn(IV)-reducing bacterium was isolated from bottom sediments of the Great Bay estuary, New Hampshire. The isolate was a facultatively anaerobic gram-negative rod which did not appear to fit into any previously described genus. It was temporarily designated strain BrY. BrY grew anaerobically in a defined medium with hydrogen or lactate as the electron donor and Fe(III) as the electron acceptor. BrY required citrate, fumarate, or malate as a carbon source for growth on H 2 and Fe(III). With Fe(III) as the sole electron acceptor, BrY metabolized hydrogen to a minimum threshold at least 60-fold lower than the threshold reported for pure cultures of sulfate reducers. This finding supports the hypothesis that when Fe(III) is available, Fe(III) reducers can outcompete sulfate reducers for electron donors. Lactate was incompletely oxidized to acetate and carbon dioxide with Fe(III) as the electron acceptor. Lactate oxidation was also coupled to the reduction of Mn(IV), U(VI), fumarate, thiosulfate, or trimethylamine n -oxide under anaerobic conditions. BrY provides a model for how enzymatic metal reduction by respiratory metal-reducing microorganisms has the potential to contribute to the mobilization of iron and trace metals and to the immobilization of uranium in sediments of Great Bay Estuary.

New Hampshire↗

Reduction of uranium by Desulfovibrio desulfuricans

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organisms for recovering uranium from contaminated waters and waste streams.

Applied and Environmental Microbiology↗

Inhibition of existing denitrification enzyme activity by chloramphenicol

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

Applied and Environmental Microbiology↗

Evaluation of methyl fluoride and dimethyl ether as inhibitors of aerobic methane oxidation

Methyl fluoride (MF) and dimethyl ether (DME) were effective inhibitors of aerobic methanotrophy in a variety of soils. MF and DME blocked consumption of CH 4 as well as the oxidation of 14 CH 4 to 14 CO 2 , but neither MF nor DME affected the oxidation of [ 14 C]methanol or [ 14 C]formate to 14 CO 2 . Cooxidation of ethane and propane by methane-oxidizing soils was also inhibited by MF. Nitrification (ammonia oxidation) in soils was inhibited by both MF and DME. Production of N 2 O via nitrification was inhibited by MF; however, MF did not affect N 2 O production associated with denitrification. Methanogenesis was partially inhibited by MF but not by DME. Methane oxidation was ∼100-fold more sensitive to MF than was methanogenesis, indicating that an optimum concentration could be employed to selectively block methanotrophy. MF inhibited methane oxidation by cell suspensions of Methylococcus capsulatus ; however, DME was a much less effective inhibitor.

California↗

Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H 2 -dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c 3 . Addition of cytochrome c 3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c 3 was oxidized by U(VI), as was a c -type cytochrome(s) in whole-cell suspensions. When cytochrome c 3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H 2 . Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c 3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c 3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c 3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium- contaminated waters and waste streams.

Applied and Environmental Microbiology↗

Aerobic and anaerobic degradation of a range of alkyl sulfides by a denitrifying marine bacterium

A pure culture of a bacterium was obtained from a marine microbial mat by using an anoxic medium containing dimethyl sulfide (DMS) and nitrate. The isolate grew aerobically or anaerobically as a denitrifier on alkyl sulfides, including DMS, dimethyl disulfide, diethyl sulfide (DES), ethyl methyl sulfide, dipropyl sulfide, dibutyl sulfide, and dibutyl disulfide. Cells grown on an alkyl sulfide or disulfide also oxidized the corresponding thiols, namely, methanethiol, ethanethiol, propanethiol, or butanethiol. Alkyl sulfides were metabolized by induced or derepressed cells with oxygen, nitrate, or nitrite as electron acceptor. Cells grown on DMS immediately metabolized DMS, but there was a lag before DES was consumed; with DES-grown cells, DES was immediately used but DMS was used only after a lag. Chloramphenicol prevented the eventual use of DES by DMS-grown cells and DMS use by DES-grown cells, respectively, indicating separate enzymes for the metabolism of methyl and ethyl groups. Growth was rapid on formate, acetate, propionate, and butyrate but slow on methanol. The organism also grew chemolithotrophically on thiosulfate with a decrease in pH; growth required carbonate in the medium. Growth on sulfide was also carbonate dependent but slow. The isolate was identified as a Thiobacillus sp. and designated strain ASN-1. It may have utility for removing alkyl sulfides, and also nitrate, nitrite, and sulfide, from wastewaters.

Georgia↗