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 (hexafluroethane) tracers and was confirmed by the appearance of 13C-enriched carbon dioxide in experiments in which 13C-enriched methane was used as the tracer. A Vmax 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 adevection-dispersion transport model to include a Michaelis-Menten-based consumption term for methane oxidation. The Km values for methane oxidation that gave the best match for the breakthrough curve peaks were 6.0 and 9.0 microM 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.     

Distribution and Rate of Methane Oxidation in Sediments of the Florida Everglades

Rates of methane emission from intact cores were measured during anoxic dark and oxic light and dark incubations. Rates of methane oxidation were calculated on the basis of oxic incubations by using the anoxic emissions as an estimate of the maximum potential flux. This technique indicated that methane oxidation consumed up to 91% of the maximum potential flux in peat sediments but that oxidation was negligible in marl sediments. Oxygen microprofiles determined for intact cores were comparable to profiles measured in situ. Thus, the laboratory incubations appeared to provide a reasonable approximation of in situ activities. This was further supported by the agreement between measured methane fluxes and fluxes predicted on the basis of methane profiles determined by in situ sampling of pore water. Methane emissions from peat sediments, oxygen concentrations and penetration depths, and methane concentration profiles were all sensitive to light-dark shifts as determined by a combination of field and laboratory analyses. Methane emissions were lower and oxygen concentrations and penetration depths were higher under illuminated than under dark conditions; the profiles of methane concentration changed in correspondence to the changes in oxygen profiles, but the estimated flux of methane into the oxic zone changed negligibly. Sediment-free, root-associated methane oxidation showed a pattern similar to that for methane oxidation in the core analyses: no oxidation was detected for roots growing in marl sediment, even for roots of Cladium jamaicense, which had the highest activity for samples from peat sediments. The magnitude of the root-associated oxidation rates indicated that belowground plant surfaces may not markedly increase the total capacity for methane consumption. However, the data collectively support the notion that the distribution and activity of methane oxidation have a major impact on the magnitude of atmospheric fluxes from the Everglades.     

Cooccurrence of aerobic and anaerobic methane oxidation in the water column of Lake Plusssee

Dissolved methane was investigated in the water column of eutrophic Lake Plusssee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and delta-13C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.     

Methane production and methane consumption: a review of processes underlying wetland methane fluxes

Potential rates of both methane production and methane consumptionvary over three orders of magnitude and their distribution is skew.These rates are weakly correlated with ecosystem type, incubationtemperature, in situ aeration, latitude, depth and distanceto oxic/anoxic interface. Anaerobic carbon mineralisation is amajor control of methane production. The large range in anaerobicCH₄:CO₂ production rates indicate that a largepart of the anaerobically mineralised carbon is used for reduction ofelectron acceptors, and, hence, is not available for methanogenesis.Consequently, cycling of electron acceptors needs to be studied tounderstand methane production. Methane and oxygen half saturationconstants for methane oxidation vary about one order of magnitude.Potential methane oxidation seems to be correlated withmethanotrophic biomass. Therefore, variation in potential methaneoxidation could be related to site characteristics with a model ofmethanotrophic biomass.     

Methane oxidation and methylotroph population dynamics in groundwater mesocosms

Extraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand-packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty-five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of ¹³C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty-nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.     

Methane oxidation and its coupled electron-sink reactions in ruminal fluid

AIMS: This study was conducted to investigate the occurrence of methane oxidation in the rumen, and to identify the electron-sink reaction coupled to the oxidation if it occurred. METHODS AND RESULTS: Mixed ruminal microbes taken from sheep were incubated with 13CH4. Oxidation of methane, estimated from the flux of 13C to CO2 and microbial cells, occurred, but represented only 0.2-0.5% of the methane produced. Methane oxidation was suppressed by the presence of oxygen, and was also inhibited by 2-bromoethane-sulphonate, and molybdate, but not by tungstate. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Methane could be oxidized anaerobically in the rumen by reverse methanogenesis in consort with sulphate reduction.     

Monitoring bacterial transport by stable isotope enrichment of cells

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (delta(13)C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of (13)C-enriched bacteria resulted in increased delta(13)C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly (13)C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.     

Cooccurrence of Aerobic and Anaerobic Methane Oxidation in the Water Column of Lake Plußsee

Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and δ-¹³C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.     

Spatial Distribution and Inhibition by Ammonium of Methane Oxidation in Intertidal Freshwater Marshes

In two intertidal marshes, the vertical distribution in the sediment and inhibition by ammonium of methane oxidation were investigated by slurry incubation experiments. The two sites differ in their dominant vegetation type, i.e., reed and bulrush, and in their heights above sea level. The reed site was elevated with respect to the bulrush site, resulting in a lower frequency and duration of flooding and, consequently, a higher potential for methane oxidation. Methane oxidation decreased with depth in the bulrush and reed slurries, although methane oxidation associated with root material from the bulrush plants increased with depth. Reed root material had a limited capacity for methane oxidation and showed no significant increase with depth. Inhibition of methane oxidation by ammonium was observed in all samples and depended on methane and ammonium concentrations. Increasing ammonium concentrations resulted in greater inhibition, and increasing methane concentrations resulted in less. Ammonium concentrations had to exceed methane concentrations by at least 30-fold to become effective for inhibition. This ratio was found only in the surface layer of the sediment. Hence, the ecological relevance for ammonium inhibition of methane oxidation in intertidal marshes is rather limited and is restricted to the surface layer. Nitrate production was restricted to the 0- to 5-cm-depth slurries.     

Monitoring Bacterial Transport by Stable Isotope Enrichment of Cells

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (δ¹³C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of ¹³C-enriched bacteria resulted in increased δ¹³C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly ¹³C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.     

Methane emissions from tropical freshwater wetlands located in different climatic zones of Costa Rica

Wetlands are the largest natural source of the greenhouse gas methane to the atmosphere. Despite the fact that a large percentage of wetlands occur in tropical latitudes, methane emissions from natural tropical wetlands have not been extensively studied. The objective this research was to compare methane emissions from three natural tropical wetlands located in different climatic and ecological areas of Costa Rica. Each wetland was within a distinct ecosystem: (1) a humid flow‐through wetland slough with high mean annual temperatures (25.9 °C) and precipitation (3700 mm yr^?1); (2) a stagnant rainforest wetland with high mean annual temperatures (24.9 °C) and precipitation (4400 mm yr^?1); or (3) a seasonally wet riverine wetland with very high mean annual temperatures (28.2 °C) and lower mean annual precipitation (1800 mm yr^?1). Methane emission rates were measured from sequential gas samples using nonsteady state plastic chambers during six sampling periods over a 29‐month period from 2006 to 2009. Methane emissions were higher than most rates previously reported for tropical wetlands with means (medians) of 91 (52), 601 (79), and 719 (257) mg CH₄‐C m^?2 day^?1 for the three sites, with highest rates seen at the seasonally flooded wetland site. Methane emissions were statistically higher at the seasonally wet site than at the humid sites (P<0.001). Highest methane emissions occurred when surface water levels were between 30 and 50 cm. The interaction of soil temperature, water depth, and seasonal flooding most likely affected methanogenesis in these tropical sites. We estimate that Costa Rican wetlands produce about 0.80 Tg yr^?1 of methane, or approximately 0.6% of global tropical wetland emissions. Elevated methane emissions at the seasonally wet/warmer wetland site suggest that some current humid tropical freshwater wetlands of Central America could emit more methane if temperatures increase and precipitation becomes more seasonal with climate change.     

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.     

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (¹³C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.     

Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers

Landfill sites are responsible for 6-12% of global methane emission. Methanotrophs play a very important role in decreasing landfill site methane emissions. We investigated the methane oxidation capacity and methanotroph diversity in lysimeters simulating landfill sites with different plant vegetations. Methane oxidation rates were 35 g methane m-2 day-1 or higher for planted lysimeters and 18 g methane m-2 day-1 or less for bare soil controls. Best methane oxidation, as displayed by gas depth profiles, was found under a vegetation of grass and alfalfa. Methanotroph communities were analysed at high throughput and resolution using a microbial diagnostic microarray targeting the particulate methane monooxygenase (pmoA) gene of methanotrophs and functionally related bacteria. Members of the genera Methylocystis and Methylocaldum were found to be the dominant members in landfill site simulating lysimeters. Soil bacterial communities in biogas free control lysimeters, which were less abundant in methanotrophs, were dominated by Methylocaldum. Type Ia methanotrophs were found only in the top layers of bare soil lysimeters with relatively high oxygen and low methane concentrations. A competetive advantage of type II methanotrophs over type Ia methanotrophs was indicated under all plant covers investigated. Analysis of average and individual results from parallel samples was used to identify general trends and variations in methanotroph community structures in relation to depth, methane supply and plant cover. The applicability of the technology for the detection of environmental perturbations was proven by an erroneous result, where an unexpected community composition detected with the microarray indicated a potential gas leakage in the lysimeter being investigated.     

Regulation of Root-Associated Methanotrophy by Oxygen Availability in the Rhizosphere of Two Aquatic Macrophytes

The relative importance of oxygen for root-associated methanotrophy was examined by using sediment-free, intact freshwater marsh plants (Pontederia cordata and Sparganium eurycarpum) incubated in split chambers. The root medium contained approximately 100 (mu)M methane. Methane oxidation was calculated from the difference between methane loss from chambers in the presence and absence of 1 mM 1-allyl-2-thiourea, a methanotrophic inhibitor. When the root medium was oxic, methane oxidation accounted for 88 and 63% of the total methane depletion for S. eurycarpum and P. cordata, respectively; the remainder represented diffusional loss to the atmosphere via roots, stems, and leaves. Under suboxic conditions, methane oxidation was not detectable for S. eurycarpum but accounted for 68% of total methane depletion for P. cordata. The introduction of a biological oxygen sink, Pseudomonas aeruginosa, resulted in complete loss of methane oxidation in S. eurycarpum chambers under oxic conditions, while methane consumption continued (51.6% of total methane depletion) in P. cordata chambers. The differences between plant species were consistent with their relative ability to oxygenate their rhizospheres: during a suboxic incubation, dissolved oxygen decreased by 19% in S. eurycarpum chambers but increased by 232% for P. cordata. An in situ comparison also revealed greater methanotrophic activity for P. cordata than S. eurycarpum.     

Diel methane emission patterns from Scirpus lacustris and Phragmites australis

In mature Phragmites australis and Scirpus lacustris vegetated sediment methane was emitted almost exclusively by plant-mediated transport, whereas in unvegetated, but otherwise identical sediment, methane was emitted almost exclusively by ebullition. Diel variations in methane emission, with highest emission rates at daytime and emission peaks following sunrise, were demonstrated for Phragmites and Scirpus. The diel difference and magnitude of the emission peaks were much smaller for Scirpus than for Phragmites. In contrast to Phragmites, methane concentrations within Scirpus stems did not change significantly over the diel period. These patterns are consistent with a two-way transport mechanism for Phragmites (convective at daytime and diffusive at night-time) and an all day diffusive mechanism for Scirpus. The patterns could not be accounted for by diel variation in air and sediment temperature, plant transpiration, or photosynthetically coupled methane production. Comparison of the experimentally derived ratio of methane emission in helium and nitrogen under light and dark conditions with the theoretical derived ratio (calculated according to the kinetic theory of gases) confirmed the exploitation of the different transport mechanism for Phragmites and Scirpus. Methane emission from Phragmites correlated significantly with incident light, which probably drove the pressure differential associated with thermally induced convection. Decrease of the radial resistance of Scirpus stems for methane transport under light compared to dark conditions, in combination with morphological characteristics of the plant species, suggested that stomatal aperture, regulated by light, controls methane emission from Scirpus. Diel variation in bubble emission from the non-vegetated sediment coincided with sediment temperature changes. The results have important implications for sampling and scaling strategies for estimating methane emission from wetlands.     

Spatial Variability in Biodegradation Rates as Evidenced by Methane Production from an Aquifer

Accurate predictions of carbon and energy cycling rates in the environment depend on sampling frequencies and on the spatial variability associated with biological activities. We examined the variability associated with anaerobic biodegradation rates at two sites in an alluvial sand aquifer polluted by municipal landfill leachate. In situ rates of methane production were measured for almost a year, using anaerobic wells installed at two sites. Methane production ranged from 0 to 560 μmol · m^-2 · day^-1 at one site (A), while a range of 0 to 120,000 μmol · m^-2 · day^-1 was measured at site B. The mean and standard deviations associated with methane production at site A were 17 and 57 μmol · m^-2 · day^-1, respectively. The comparable summary statistics for site B were 2,000 and 9,900 μmol · m^-2 · day^-1. The coefficients of variation at sites A and B were 340 and 490%, respectively. Despite these differences, the two sites had similar seasonal trends, with the maximal rate of methane production occurring in summer. However, the relative variability associated with the seasonal rates changed very little. Our results suggest that (i) two spatially distinct sites exist in the aquifer, (ii) methanogenesis is a highly variable process, (iii) the coefficient of variation varied little with the rate of methane production, and (iv) in situ anaerobic biodegradation rates are lognormally distributed.     

Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase).

Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities.     

Distribution and Metabolic Diversity of Microorganisms in Deep Igneous Rock Aquifers of Finland

Groundwater samples from 200- to 950-m depths in four igneous rock sites in Finland were investigated for different metabolic groups of microorganisms, and the data were compared with the available geochemical record. Samples were collected with a pressurized groundwater sampling system developed for gas and microbiological sampling. Two of the sites had groundwater that was fresh, with &lt; 0.2 g/l dissolved solids, whereas that at the two other sites was much more saline, reaching a maximum of 24 g/l dissolved solids. The groundwater contained gases, 33 to 340 ml/l, with nitrogen or methane dominating. Total cell numbers were 10 5 to 10 6 cells/ml, which is typical for deep igneous rock aquifers. Growth media were designed to mimic the actual groundwater chemistry at each sampling point and used for most probable number enumeration of methanogens, acetogens, sulfate-reducing bacteria (SRB), and iron-reducing bacteria (IRB). SRB predominated in sites where iron sulfide fracture-filling minerals are common. IRB were the main population in one site where iron sulfide fracture minerals are not present, but iron hydroxide fracture minerals predominate. Fracture-filling minerals were a better indicator of microbial populations than was groundwater chemistry. Low numbers of autotrophic methanogens were cultured. One of several possible interpretations of stable isotope data suggested that most of the detected methane is thermogenic, which would correlate with few active methanogens. However, we concluded other interpretations were also possible.