Geochemical Peculiarities of the Carbonate Constructions Formed during Microbial Oxidation of Methane under Anaerobic Conditions

The aragonite constructions of the Black Sea are formed in a stable anaerobic zone and are a perfect object to study the natural mechanism of anaerobic methane oxidation. The most probable pathway of methane oxidation is its methanogen-mediated reaction with bicarbonates, dissolved in seawater, with the formation of water and acetate, which is then consumed by other components of the anaerobic community. Comparison of the ¹³C values of carbonate minerals and organic matter once more demonstrated that the formation of the organic matter of biomass is accompanied by intense fractionation of carbon isotopes, as a result of which the total organic matter of biomass acquires an extremely light isotopic composition, characterized by ¹³C values as low as –83.8.     

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Sulfate-induced isotopic variation in biogenic methane from a tropical swamp without anaerobic methane oxidation

The oxidative consumption of methane (CH4) generally proceeds with a significant isotope fractionation, and isotopic variations in CH4 observed in sulfate-containing anaerobic sediments have often been interpreted as an indicator of anaerobic methane oxidation at the expense of sulfate. However, we found variations in δ13C value of CH4 depending on sulfate availability in tropical swamp sediments, in which no anaerobic CH4 oxidation was detected. In one sediment, the range of δ13C variation due to sulfate was as large as 20‰. The variations in δ13C of decomposed organic matter and CO2 failed to explain the variation in CH4 δ¹³C. We postulate a syntrophic linkage between sulfate-reducing and methanogenic bacteria via acetate as a mechanism of the observed δ'13C variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro study of lipid biosynthesis in an anaerobically methane-oxidizing microbial mat

The anaerobic oxidation of methane (AOM) is a key process in the global methane cycle, and the majority of methane formed in marine sediments is oxidized in this way. Here we present results of an in vitro 13CH4 labeling study (delta13CH4, approximately 5,400 per thousand) in which microorganisms that perform AOM in a microbial mat from the Black Sea were used. During 316 days of incubation, the 13C uptake into the mat biomass increased steadily, and there were remarkable differences for individual bacterial and archaeal lipid compounds. The greatest shifts were observed for bacterial fatty acids (e.g., hexadec-11-enoic acid [16:1Delta11]; difference between the delta13C at the start and the end of the experiment [Deltadelta13C(start-end)], approximately 160 per thousand). In contrast, bacterial glycerol diethers exhibited only slight changes in delta13C (Deltadelta13C(start-end), approximately 10 per thousand). Differences were also found for individual archaeal lipids. Relatively high uptake of methane-derived carbon was observed for archaeol (Deltadelta13C(start-end), approximately 25 per thousand), a monounsaturated archaeol, and biphytanes, whereas for sn-2-hydroxyarchaeol there was considerably less change in the delta13C (Deltadelta13C(start-end), approximately 2 per thousand). Moreover, an increase in the uptake of 13C for compounds with a higher number of double bonds within a suite of polyunsaturated 2,6,10,15,19-pentamethyleicosenes indicated that in methanotrophic archaea there is a biosynthetic pathway similar to that proposed for methanogenic archaea. The presence of group-specific biomarkers (for ANME-1 and ANME-2 associations) and the observation that there were differences in 13C uptake into specific lipid compounds confirmed that multiple phylogenetically distinct microorganisms participate to various extents in biomass formation linked to AOM. However, the greater 13C uptake into the lipids of the sulfate-reducing bacteria (SRB) than into the lipids of archaea supports the hypothesis that there is autotrophic growth of SRB on small methane-derived carbon compounds supplied by the methane oxidizers.     

Effect of microorganisms and seasonal factors on the isotope composition of organic carbon from Black Sea suspensions

The isotopic composition of particulate organic carbon (POC) from the Black Sea deep-water zone was studied during a Russian-Swiss expedition in May 1998. POC from the upper part of the hydrogen sulfide zone (the C-layer) was found to be considerably enriched with the 12C isotope, as compared to the POC of the oxycline and anaerobic zone. In the C-layer waters, the concurrent presence of dissolved oxygen and hydrogen sulfide and an increased rate of dark CO2 fixation were recorded, suggesting that the change in the POC isotopic composition occurs at the expense of newly formed isotopically light organic matter of the biomass of autotrophic bacteria involved in the sulfur cycle. In the anaerobic waters below the C-layer, the organic matter of the biomass of autotrophs is consumed by the community of heterotrophic microorganisms; this results in weighting of the POC isotopic composition. Analysis of the data obtained and data available in the literature allows an inference to be made about the considerable seasonable variability of the POC delta 13C value, which depends on the ratio of terrigenic and planktonogenic components in the particulate organic matter.     

The Late Eocene ‘Whiskey Creek’ methane-seep deposit (western Washington State)

The Late Eocene ‘Whiskey Creek’ deposit (Pysht Formation, Olympic Penisula, Washington State) formed at a methane-seep. Early diagenetic micrites and aragonite cement have δ¹³C values as low as −36‰ indicating that the seepage fluids contained methane. With respect to micrite samples, low δ¹³C values correlate with relatively high δ¹³O values andvice versa. Ongoing micrite formation after the cessation of the seepage during increased burial might have altered the isotopic composition of the microcrystalline carbonates toward lower δ¹³O values and higher δ¹³C values. Alternatively, the trend in isotope values may reflect a change in the composition of seepage fluids. The principal difference between these scenarios is the duration of seepage with respect to micrite formation. Two petrographically similar varieties of blocky calcite spar are related to different carbonate sources. The δ¹³C values range from −32 to −29‰ for one type of blocky spar and are either the result of methane oxidation or indicate thermal decarboxylation of organic matter. Low δ¹⁸O values are in favour of the latter. For the other type of spar, δ¹³C values as high as +6‰ indicate carbonate formation within the zone of methanogensis. The ‘Whiskey Creek’ limestone exhibits a chaotic fabric produced by a variety of processes, including bioturbation, concretionary carbonate formation, earlyin situ brecciation, carbonate corrosion, and late fracturing of the rock. Two varieties of micrite aggregates are responsible for the nodular fabric of the limestone. Smoothly-shaped pyritiferous micrite nodules are of diagenetic origin and formed in a manner similar to that which produces carbonate concretions. Apart from being induced by anaerobic oxidation of methane, their formation is proposed to be linked to iron reduction and sulphide formation. The second, dominant variety is represented by irregularly-shaped, nodular to angular micrite aggregates surrounded by massive rims of pyrite, resulting from carbonate corrosion. A pure, fluorescent seam-micrite, constructive in origin, lines cavities or surrounds micritic aggregates.     

The biogeochemical controls of N2O production and emission in landfill cover soils: the role of methanotrophs in the nitrogen cycle

Emissions of N2O from cover soils of both abandoned (> 30 years) and active landfills greatly exceed the maximum fluxes previously reported for tropical soils, suggesting high microbial activities for N2O production. Low soil matrix potentials (<-0.7 MPa) indicate that nitrification was the most likely mechanism of N2O formation during most of the time of sampling. Soil moisture had a strong influence on N2O emissions. The production of N2O was stimulated by as much as 20 times during laboratory incubations, when moisture was increased from -2.0 MPa to -0.6 MPa. Additional evidence from incubation experiments and delta13C analyses of fatty acids (18:1) diagnostic of methanotrophs suggests that N2O is formed in these soils by nitrification via methanotrophic bacteria. In a NH3(g)-amended landfill soil, the rate of N2O production was significantly increased when incubated with 100 ppmv methane compared with 1.8 ppmv (atmospheric) methane. Preincubation of a landfill soil with 1% CH4 for 2 weeks resulted in higher rates of N2O production when subsequently amended with NH3(g) relative to a control soil preincubated without CH4. At one location, at the soil depth (9-16 cm) of maximum methane consumption and N2O production, we observe elevated concentrations of organic carbon and nitrogen and distinct minima in delta15N (+1.0%) and delta13C (-33.8%) values for organic nitrogen and organic carbon respectively. A delta13C value of -39.3% was measured for 18:1 carbon fatty acids in this soil, diagnostic of type II methanotrophs. The low delta15N value for organic nitrogen is consistent with N2 fixation by type II methanotrophs. These observations all point to a methanotrophic origin for the organic matter at this depth. The results of this study corroborate previous reports of methanotrophic nitrification and N2O formation in aqueous and soil environments and suggest a predominance of type II rather than type I or type X methanotrophs in this landfill soil.     

Methane oxidation potential in the water column of two diverse coastal marine sites

Methane oxidation in the water column was investigated at two nearshore marine environments with relatively high concentrations of dissolved methane. In the northern Gulf of Mexico, high methane oxidation rates were observed at the pycnocline, with the highest oxidation rate corresponding to the most negative bacterial ¹³C values. These low isotopic values occurred during the winter when overall bacterial productivity was low, suggesting that at this time of the year, methanotrophs in the Gulf could make up a significant portion of the overall bacterial assemblage. Although methane oxidation also occurred during more productive times (i.e., summer), the isotopic signal of methane oxidation was not observed in the bacterial biomass because of the higher overall bacterial productivity. The other site, Cape Lookout Bight, NC, is a small marine embayment where methane is produced in the organic-rich sediments. No measurable rates of methane oxidation in the water column occurred, and no anomalously low ¹³C values of the bacterioplankton were measured. In both environments, methane production and oxidation appear to be spatially coupled, occurring at/near the pycnocline in the northern Gulf of Mexico and at the sediment-water interface at Cape Lookout Bight, NC.     

Biogeochemical processes of methane cycle in the soils, swamps and lakes of Western Siberia

The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and of raised bogs and lake bottom sediments nearby the Tarkosalinsk gas field in western Siberia. Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm3, respectively). In bogs and lake bottom sediments, methane concentration was as high as 11 microliters/cm3. Acetate was the major precursor of the newly formed methane. The rate of aceticlastic methanogenesis reached 55 ng C/(cm3 day), whereas that of autotrophic methanogenesis was an order of magnitude lower. The most active methane production and oxidation were observed in bogs and lake sediments where the delta 13C values of CO2 were inversely related to the intensity of bacterial methane oxidation. Methane diffusing from bogs and lake bottom sediments showed delta 13C values ranging from -78 to -47@1000, whereas the delta 13C value of carbon dioxide ranged from -18 to -6@1000. In these ecosystems, methane emission comprised from 3 to 206 mg CH4/(m2 day). Conversely, the dry and water-logged soils of tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH4/(m2 day). Methane consumption in soils was of biological rather than of adsorptive nature. This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the delta 13C value changed from -51 to -41@1000).     

Consumption of methane and CO2 by methanotrophic microbial mats from gas seeps of the anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Biomarker Evidence for Widespread Anaerobic Methane Oxidation in Mediterranean Sediments by a Consortium of Methanogenic Archaea and Bacteria

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in ¹³C (δ¹³C values are as low as −95‰). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of ¹³C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.     

Dissolved methane oxidation and competition for oxygen in down-flow hanging sponge reactor for post-treatment of anaerobic wastewater treatment

Post-treatment of anaerobic wastewater was undertaken to biologically oxidize dissolved methane, with the aim of preventing methane emission. The performance of dissolved methane oxidation and competition for oxygen among methane, ammonium, organic matter, and sulfide oxidizing bacteria were investigated using a lab-scale closed-type down-flow hanging sponge (DHS) reactor. Under the oxygen abundant condition of a hydraulic retention time of 2h and volumetric air supply rate of 12.95m(3)-airm(-3)day(-1), greater than 90% oxidation of dissolved methane, ammonium, sulfide, and organic matter was achieved. With reduction in the air supply rate, ammonium oxidation first ceased, after which methane oxidation deteriorated. Sulfide oxidation was disrupted in the final step, indicating that COD and sulfide oxidation occurred prior to methane oxidation. A microbial community analysis revealed that peculiar methanotrophic communities dominating the Methylocaldum species were formed in the DHS reactor operation.     

Regulation of geochemical activity of microorganisms in a petroleum reservoir by injection of H2O2 or water-air mixture

In the course of pilot trials of biotechnologies for the enhancement of oil recovery in the Gangxi bed of the Dagang oil field (China), microbiological processes were investigated. The biotechnologies were based on injection into the petroleum reservoir of different oxygen sources (H2O2 solution or a water-air mixture) with nitrogen and phosphorus salts. The injection of water-air mixture with nitrogen and phosphorus salts resulted in an increase in the number of aerobic and anaerobic organotrophic bacteria, rates of sulfate reduction and methanogenesis in formation water and also the content of CO2 (from 4.8-12 to 15-23.2%) and methane (from 86-88 to 91.8%) in the gas. The preferential consumption of isotopically light bicarbonate by methanogens resulted in a higher content of the light 12C in methane; the delta13C/CH4 value changed from -45.1...-48.3 to -50.7...-59.3 per thousand). At the same time, mineral carbonates of the formation water became isotopically heavier; the delta13C/Sigmacarbonates value increased from 3.4...4.0 to 5.4...9.6 per thousand. Growth of hydrocarbon-oxidizing bacteria was accompanied by production of biosurfactants and decreased interfacial tension of formation water. Injection of H2O2 solution resulted in the activation of aerobic processes and in suppression of both sulfate reduction and methanogenesis. Methane content in the gas decreased from 86-88 to 75.4-79.8%, probably due to its consumption by methanotrophs. Due to consumption of isotopically light methane, the residual methane carbon became heavier, with the delta13C/CH4 values from -39.0 to -44.3 per thousand. At the same time, mineral carbonates of the formation water became isotopically considerably lighter; the delta13C/Sigmacarbonates value decreased from 5.4... 9.6 to -1.4...2.7 per thousand). The additional amount of oil recovered during the trial of both variants of biotechnological treatment was 3819 t.     

Anaerobic filters for the treatment of coal gasification wastewater

A process train consisting of the following sequence of unit processes, a berl-saddle-packed anaerobic filter, an expanded bed, granular activated carbon anaerobic filter, and an activated sludge nitrification system was evaluated for the treatment of a synthetically prepared coal gasification wastewater. The first-stage anaerobic filter resulted in very little removal of organic matter and no methane production. Excellent reduction in organic matter occurred in the granular activated carbon anaerobic filter. The removal mechanism was initially adsorptive and near the end of the study, removal of organic matter was primarily through conversion to methane gas. It is felt that the success of the activated carbon anaerobic filter was due to the ability of the activated carbon to sequester some components of the wastewater that were toxic to the mixed culture of anaerobic microorganisms. The activated sludge nitrification system resulted in complete ammonia oxidation and was very efficient in final effluent polishing.     

Microbial transformation of chitin in soil under anaerobic conditions

Anaerobic chitinolytic complex was studied in three soil types: chernozem, gray forest soil, and chestnut soil. The abundance and biomass of anaerobic chitinolytic microbial complex of fungi, bacteria, and actinomycetes were evaluated by luminescent microscopy. The dynamics of methane emission from soil during chitinolytic succession was studied by gas chromatography. All three studied microbial groups proved to participate in chitin transformation in soil under anaerobic conditions. The highest biomass growth was observed among prokaryotes, particularly actinomycetes, whose biomass doubled. The increase in methane emission during chitinolytic succession was most pronounced in soils with low organic matter content.     

Microbial Utilization of Estuarine Dissolved Organic Carbon: a Stable Isotope Tracer Approach Tested by Mass Balance

The natural stable isotope values of different plants have been used to trace the fate of organic carbon that enters estuarine ecosystems. Experiments were designed to determine the magnitude of (delta) (sup13)C changes of dissolved organic carbon (DOC) derived from tidal marsh vegetation that occurred during bacterial decomposition. Bacteria were grown on DOC leached from estuarine Spartina alterniflora and Typhus angustifolia plants. In four experiments, 25 to 80% of the initial carbon (2.6 to 9.1 mM organic C) was converted to bacterial biomass and CO(inf2). Mass balance calculations showed good recovery of total C and (sup13)C at the end of these experiments (100% (plusmn) 14% total C; (plusmn) 1(permil) (delta) (sup13)C). The (delta) (sup13)C values of DOC, bacterial biomass, and respired CO(inf2) changed only slightly in the four experiments by average values of -0.6, +1.4, and +0.5(permil), respectively. These changes are small relative to the range of (delta) (sup13)C values represented by different organic carbon sources to estuaries. Thus, microbial (delta) (sup13)C values determined in the field helped to identify the source of the carbon assimilated by bacteria.     

Combination of biomolecular and stable isotope techniques to determine the origin of organic matter used by bacterial communities: application to sediment.

Natural isotopic composition is a good tool to trace organic matter in ecosystems. Recent studies used a combination of molecular and stable isotope techniques to determine the origin of the organic carbon used by bacteria in the water column. In our study, we show that this procedure can be used for analysis of sediment bacterial communities with few modifications. In the water column, bacterial recovery is done before DNA extraction. In the sediment, we tested qualitatively and quantitatively a direct and indirect extraction of DNA. The direct extraction was the most efficient. It recovered between 3.1 and 15.8 microg DNA g(-1) dry sediment and the contamination of field samples by eucaryotic DNA was less than 13%. In this preliminary study of the salt marsh ecosystem, the delta(13)C values of DNA (-26 to - 24%) recovered from the sediment were close to the delta(13)C values of halophytic plants (-26.4 and - 25.3%) showing a relationship between plants and microorganisms. Thus, this procedure can be used to trace the flow of carbon through the sediment microbial biomass and to understand the variation of bacterial activity according to the inputs of allocthonous and autochtonous organic matter.     

Factors affecting the isotopic composition of organic matter. (1) Carbon isotopic composition of terrestrial plant materials

The stable isotope composition of the light elements (i.e., H, C, N, O and S) of organic samples varies significantly and, for C, is also unique and distinct from that of inorganic carbon. This is the result of (1) the isotope composition of reactants, (2) the nature of the reactions leading to formation and post-formational modification of the samples, (3) the environmental conditions under which the reactions took place, and (4) the relative concentration of the reactants compared to that of the products (i.e., [products]/[reactants] ratio). This article will examine the carbon isotope composition of terrestrial plant materials and its relationship with the above factors. delta13C(PDB) values of terrestrial plants range approximately from -8 to -38%, inclusive of C3-plants (-22 to -38%), C4-plants (-8 to -15%) and CAM-plants (-13 to -30%). Thus, the delta13C(PDB) values largely reflect the photosynthesis pathways of a plant as well as the genetics (i.e., species difference), delta13C(PDB) values of source CO2, relevant humidity, CO2/O2 ratios, wind and light intensity etc. Significant variations in these values also exist among different tissues, different portions of a tissue and different compounds. This is mainly a consequence of metabolic reactions. Animals mainly inherit the delta13C(PDB) values of the foods they consume; therefore, their delta13C(PDB) values are similar. The delta13C(PDB) values of plant materials, thus, contain information regarding the inner workings of the plants, the environmental conditions under which they grow, the delta13C(PDB) values of CO2 sources etc., and are unique. Furthermore, this uniqueness is passed on to their derivative matter, such as animals, humus etc. Hence, they are very powerful tools in many areas of research, including the ecological and environmental sciences.     

Characterization of Specific Membrane Fatty Acids as Chemotaxonomic Markers for Sulfate-Reducing Bacteria Involved in Anaerobic Oxidation of Methane

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as m 103. Specific fatty acids released from bacterial membranes include C 16:1 y 5c , C 17:1 y 6c , and cyC 17:0 y 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest i 13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 y 5c and cyC 17:0 y 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.     

Microbiological and isotopic-geochemical investigations of meromictic lakes in Khakasia in winter

Microbiological and isotopic-geochemical investigations of the brackish meromictic lakes Shira and Shunet were performed in the steppe region of Khakasia in winter. Measurements made with a submersed sensor demonstrated that one-meter ice transmits light in a quantity sufficient for oxygenic and anoxygenic photosynthesis. As in the summer season, in the community of phototrophic bacteria found in Lake Shira, the purple sulfur bacteria Amoebobacter purpureus dominated, whereas, in Lake Shunet, the green sulfur bacteria Pelodictyon luteolum were predominant. Photosynthetic production, measured using the radioisotopic method, was several times lower than that in summer. The rates of sulfate reduction and production and oxidation of methane in the water column and bottom sediments were also lower than those recorded in summer. The process of anaerobic methane oxidation in the sediments was an exception, being more intense in winter than in summer. The data from radioisotopic measurements of the rates of microbial processes correlate well with the results of determination of the isotopic composition of organic and mineral carbon (delta13C) and hydrogen sulfide and sulfate (delta34S) and suggest considerable seasonal variations in the activity of the microbial community in the water bodies investigated.