Methanogenesis and sulfate reduction in timber and drainage water from a gold mine

Biogenesis of methane in the heartwood of diseased trees has been shown, but never in timber in service. Studies were undertaken to establish whether methan‐ogens and sulfate‐reducers were present in wooden pit props and drainage water from underground sites in a gold mine. The predominant methanogen in the mine ecosystem was tentatively identified as Methanobacterium bryantii. The sulfate‐reducers comprised Desulfovibrio desulfuricans and Desulfotomaculum antarcticum. Most probable numbers (MPN) of bacteria indicated that 3.5 × 10⁵ methanogenic and 7.9 × 10³ sulfate‐reducing bacteria were present per milliliter of stagnant drainage water. MPN values per gram of timber were lower for methanogens but comparable for sulfate‐reducers. Laboratory model systems predicted a maximum rate of methan‐ogenesis of 2.3 mL methane/g wood per day; however, rates would never attain this value because of nutrient limitations and environmental restrictions. Analysis of gas samples extracted from sealed areas of the gold mine verified the presence of methane.     

Methanogenic potential of tailings samples from oil sands extraction plants

Approximately 20% of Canada's oil supply now comes from the extraction of bitumen from the oil sands deposits in northeastern Alberta. The oil sands are strip-mined, and the bitumen is typically separated from sand and clays by an alkaline hot water extraction process. The rapidly expanding oil sands industry has millions of cubic metres of tailings for disposal and large areas of land to reclaim. There are estimates that the consolidation of the mature fine tails (MFT) in the settling ponds will take about 150 years. Some of the settling ponds are now evolving microbially produced methane, a greenhouse gas. To hasten consolidation, gypsum (CaSO4 x 2H2O) is added to MFT, yielding materials called consolidated or composite tailings (CT). Sulfate from the gypsum has the potential to stimulate sulfate-reducing bacteria (SRB) to out-compete methanogens, thereby stopping methanogenesis. This investigation examined three MFT and four CT samples from three oil sands extractions companies. Each was found to contain methanogens and SRB. Serum bottle microcosm studies showed sulfate in the CT samples stopped methane production. However, if the microcosms were amended with readily utilizable electron donors, the sulfate was consumed, and when it reached approximately 20 mg/L, methane production began. Some unamended microcosms were incubated for 372 days, with no methane production detected. This work showed that each MFT and CT sample has the potential to become methanogenic, but in the absence of exogenous electron donors, the added sulfate can inhibit methanogenesis for a long time.     

Methanogenesis in a saltern in the Bretagne (France)

Abstract Salterns in the Bretagne (France), exhibiting different stages of salinity of 5% to 33% evolved up to 0.7 mmol methane per m² per day. Methane concentrations of up to 0.4 mM were found. High methane evolution rates and increased methane concetration were restricted to basins of up to 7% and more than 15% salinity, and to the upper 10 cm of the sediments, where high sulfate concentrations (50 to 100 mM) occurred as well. Basins of 10–15% salinity exhibited only low methane evolution rates (less than 0.05 mmol methane per m² per day) and low methane concentrations (less than 0.02 mM). Gas bubbles arising during times of increased photosynthetic activity from the microbial mats covering the sediments of the saltern basins contained up to 2% methane. Addition of methylated amines and methanol, but not of H₂/CO₂, formate or acetate, to sediments slurries from basins of up to 12% salinity resulted in a rapid enrichment of methanogenic populations. Enriched methanogenic bacteria did not grow at salinities exceeding 15% or temperatures exceeding 45°C, and showed characteristics similar to those documented for Methanococcus halophilus and strain SF1 (DSM 3243). No enrichments were obtained from basins of more than 20% salinity in spite of methane being produced and evolved from those basins.     

Bioprocessing-Based Approach for Bitumen/Water/Fines Separation and Hydrocarbon Recovery from Oil Sands Tailings

Oil sands are a major source of oil, but their industrial processing generates tailings ponds that are an environmental hazard. The main concerns are mature fine tailings (MFT) composed of residual hydrocarbons, water, and fine clay. Tailings ponds include toxic contaminants such as heavy metals, and toxic organics including naphthenics. Naphthenic acids and polyaromatic hydrocarbons (PAHs) degrade very slowly and pose a long-term threat to surface and groundwater, as they can be transported in the MFT. Research into improved technologies that would enable densification and settling of the suspended particles is ongoing. In batch tests, BioTiger?, a microbial consortium that can metabolize PAHs, demonstrated improved oil sands tailings settling from a Canadian tailings pond. Results also showed, depending on the timing of the measurements, lower suspended solids and turbidity. Elevated total organic carbon was observed in the first 48 hours in the BioTiger?-treated columns and then decreased in overlying water. Oil sands tailings mixed with BioTiger? showed a two-fold reduction in suspended solids within 24 hours as compared to abiotic controls. The tailings treated with BioTiger? increased in microbial densities three orders of magnitude from 8.5 × 105 CFU/mL to 1.2 × 108 CFU/mL without any other carbon or energy source added, indicating metabolism of hydrocarbons and other available nutrients. Results demonstrated that bioaugmentation of BioTiger? increased separation of organic carbon from particles in oil sands and enhanced settling with tailings with improved water quality. Journal style is for Abstract to be less than 200 words, and contain no citations to other sources; please edit as needed     

The performance of a phase separated granular bed bioreactor treating brewery wastewater

This study presents the performance characteristics of a plug flow phase separated anaerobic granular bed baffled reactor (GRABBR) fed with brewery wastewater at various operating conditions. The reactor achieved chemical oxygen demand (COD) removal of 93-96% with high methane production when operated at organic loading rates (OLRs) of 2.16-13.38kg COD m(-3)d(-1). The reactor configuration and microbial environment encouraged the acidogenic dominant zone to produce intermediate products suitable for degradation in the predominantly methanogenic zone. Noticeable phase separation between acidogenesis and methanogenesis mainly occurred at high OLR, involving a greater number of compartments to contribute to wastewater treatment. The highly active nature and good settling characteristics of methanogenic granular sludge offered high biomass retention and enhanced methanogenic activities within the system. The granular structure in the acidogenic dominant zone of the GRABBR was susceptible to disintegration and flotation. Methanogenic granular sludge was a multi-layered structure with Methanosaeta-like organisms dominant in the core.     

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.     

Microbiological investigations of high-temperature horizons of the Kongdian petroleum reservoir in connection with field trial of a biotechnology for enhancement of oil recovery

The physicochemical conditions and microbiological characteristics of the formation waters of the Kongdian bed of the Dagang oil field (China) were studied. It was demonstrated that this bed is a high-temperature ecosystem with formation waters characterized by low mineralization. The concentrations of nitrogen and phosphorus compounds, as well as of electron acceptors, are low. Oil and oil gas are the main organic matter sources. The bed is exploited with water-flooding. The oil stratum was inhabited mostly by anaerobic thermophilic microorganisms, including fermentative (10(2)-10(5) cells/ml), sulfate-reducing (0-10(2) cells/ml), and methanogenic (0-10(3) cells/ml) microorganisms. Aerobic bacteria were detected mainly in the near-bottom zone of injection wells. The rate of sulfate reduction varied from 0.002 to 18.940 microg S(2-) l(-1) day(-1) and the rate of methanogenesis from 0.012 to 16.235 microg CH4 l(-1) day(-1). Microorganisms with great biotechnological potential inhabited the bed. Aerobic thermophilic bacteria were capable of oxidizing oil with the formation of biomass, the products of partial oxidation of oil (volatile acids), and surfactants. During growth on the culture liquid of oiloxidizing bacteria, methanogenic communities produced methane and carbon dioxide, which also had oil-releasing capabilities. Using various labeled tracers, the primary filtration flows of injected solutions at the testing site were studied. Our comprehensive investigations allowed us to conclude that the tested method for microbial enhancement of oil recovery based on the activation of the stratal microflora can be applied in the Kongdian bed horizons.     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明, 属于高温生态环境, 地层水矿化度较低, 氮、磷浓度低, 而且缺乏电子受体, 主要的有机物来源是油气。油田采用经过除油处理的油藏产出水回注方式开发, 油层中存在的微生物类型主要是厌氧嗜热菌, 包括发酵菌(102个/mL~105个/mL), 产甲烷菌(103个/mL); 好氧菌主要存在于注水井周围。硫酸盐还原菌(SRB)还原速率0.002 mg S2-/(L·d) ~18.9 mg S2-/(L·d), 产甲烷菌产甲烷速率0.012 mgCH4/(L·d)~16.2 mgCH4/(L·d)。好氧菌能够氧化油形成生物质, 部分氧化产物为挥发性脂肪酸和表面活性剂。产甲烷菌在油氧化菌液体培养基中产生CH4, CO2为好氧微生物和厌氧微生物的共同代谢产物。这些产物具有提高原油流动性的作用。用示踪剂研究了注入水渗流方向。通过综合分析, 油藏微生物具有较大的潜力, 基于激活油层菌的提高采收率方法在该油田是可行的。     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明,属于高温生态环境,地层水矿化度较低,氮、磷浓度低,而且缺乏电子受体,主要的有机物来源是油气.油田采用经过除油处理的油藏产出水回注方式开发,油层中存在的微生物类型主要是厌氧嗜热菌,包括发酵菌(102个/mL～105个/mL),产甲烷菌(103个/mL);好氧菌主要存在于注水井周围.硫酸盐还原菌(SRB)还原速率0.002 μg S2-/(L·d)～18.9 μg S2-/(L·d),产甲烷菌产甲烷速率0.012 μgCH4/(L·d)～16.2 μgCH4/(L·d).好氧菌能够氧化油形成生物质,部分氧化产物为挥发性脂肪酸和表面活性荆.产甲烷菌在油氧化菌液体培养基中产生CH4,CO2为好氧微生物和厌氧微生物的共同代谢产物.这些产物具有提高原油流动性的作用.用示踪剂研究了注入水渗流方向.通过综合分析,油藏微生物具有较大的潜力,基于激活油层茵的提高采收率方法在该油田是可行的.     

Inhibition Experiments on Anaerobic Methane Oxidation

Anaerobic methane oxidation is a general process important in controlling fluxes of methane from anoxic marine sediments. The responsible organism has not been isolated, and little is known about the electron acceptors and substrates involved in the process. Laboratory evidence indicates that sulfate reducers and methanogens are able to oxidize small quantities of methane. Field evidence suggests anaerobic methane oxidation may be linked to sulfate reduction. Experiments with specific inhibitors for sulfate reduction (molybdate), methanogenesis (2-bromoethanesulfonic acid), and acetate utilization (fluoroacetate) were performed on marine sediments from the zone of methane oxidation to determine whether sulfate-reducing bacteria or methanogenic bacteria are responsible for methane oxidation. The inhibition experiment results suggest that methane oxidation in anoxic marine sediments is not directly mediated by sulfate-reducing bacteria or methanogenic bacteria. Our results are consistent with two possibilities: anaerobic methane oxidation may be mediated by an unknown organism or a consortium involving an unknown methane oxidizer and sulfate-reducing bacteria.     

Activity and metabolic regulation of methane production in deep peat profiles of boreal bogs

The potential activity of methane production was determined in the vertical profiles of the peat deposits of three bogs in Tver oblast, which were representative of the boreal zone. In the minerotrophic fen, the rates of methane production measured throughout the profile did not change significantly with depth and comprised 3-6 ng CH4-C g(-1) h(-1). In ombrotrophic peat bogs, the rate did not exceed 5 ng CH4-C g(-1) h(-1) in the upper layer of the profile (up to 1.5 m) and increased to 15-30 ng CH4-C g(-1) h(-1) in the deep layers of the peat deposits. The distribution of fermentative microorganisms and methanogens in the profiles of peat deposits was uniform in all the studied bogs. In bog water samples, the presence of butyrate (up to 14.1 mg l(-1)) and acetate (up to 2.4 mg l(-1)) was revealed throughout the whole profile; in the upper 0.5-m layer of the ombrotrophic bogs, formate (up to 8.9 mg l(-1)) and propionate (up to 0.3 mg l(-1)) were detected as well. The arrangement of local maxima of the fatty acid content and methanogenic activity in the peat deposits, as well as the decrease in the acetate concentrations during summer, support the hypothesis that the initial substrates for methanogenesis come from the upper peat layers. It was established that the addition of sulfate and nitrate inhibits methane production in peat samples: the changes in the concentrations, recorded in situ, may also influence the methane content in peat layers.     

Comparative anaerobic treatment of wastewater from pharmaceutical, brewery, paper and amino acid producing industries

This study concerned the anaerobic treatment of five different industrial wastewaters with a diverse and complex chemical composition. The kinetics of biotransformation of this wastewater at different chemical oxygen demand (COD) were studied in a batch reactor. Wastewater from an amino acid producing industry (Fermex) and from a tank that received several types of wastewaters (collector) contained 0.83 g l(-1) and 0.085 g l(-1) sulfate, respectively. During the study period of 20 days, methane formation was observed in all types of wastewaters. Studies on COD biodegradation showed the reaction velocity was higher for Fermex wastewater and lower for collector wastewater, with values of 0.0022 h(-1) and 0.0011 h(-1), respectively. A lower methanogenic activity of 0.163 g CH4 day(-1) g(-1) volatile suspended solids (VSS) and 0.20 g CH4 day(-1) g(-1) VSS, respectively, was observed for paper producing and brewery wastewater. Adapted granular sludge showed the best biodegradation of COD during the 20-day period. The sulfate-reducing activity in pharmaceutical and collector wastewater was studied. A positive effect of sulfate-reducing activity on methanogenic activity was noted for both types of wastewaters, both of which contained sulfate ions. All reactions of methane generation for the tested industrial wastewaters were first-order. The results of this study suggest that the tested wastewaters are amenable to anaerobic treatment.     

Effect of ammonia on the methanogenic activity of methylaminotrophic methane producing Archaea enriched biofilm

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH(3)-N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37 degrees C. The results showed that at 848.8 mg NH(3)-N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337+/-0.213 g COD methane/g VSS *day when 48.8 mg NH(3)-N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH(3)-N/L, producing under 1.658+/-0.185 g COD methane/g VSS *day. The lowest methanogenic activity reached was 0.639+/-0.162 g COD methane/g VSS *day at the system added with 848.8 mg NH(3)-N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH(3)-N/L.     

Mass transfer limitation of sulfate in methanogenic aggregates

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady-state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m(-3)) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 x 10(-5) kg SO(4) (2-) kg(-1) VSS S(-1) or biomass concentrations (40 KG VSS m(-3)), and in large aggregates (radius of 7.5 10(-4) m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. (c) 1994 John Wiley & Sons, Inc.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Bioreactor studies predict whole microbial population dynamics in oil sands tailings ponds

Microorganisms in oil sands fluid fine tailings (FFT) are critical to biogeochemical elemental cycling as well as to the degradation of residual hydrocarbon constituents and subsequent methane and CO₂ production. Microbial activity enhances particulate matter sedimentation rates and the dewatering of FFT materials, allowing water to be recycled back into bitumen extraction. A bulk of this evidence comes from bioreactor studies and has implications for engineering and environmental management of the FFT ponds. Yet, it is largely uncertain whether such laboratory populations are representative of whole field scale microbial communities. By using population ecology tools, we compared whole microbial communities present in FFT bioreactors to reference populations existing in Syncrude's West In Pit (WIP) tailings pond. Bacteria were found to be persistent in a sulfidic zone in both the oxic and anoxic bioreactors at all occasions tested. In contrast to the WIP, archaea only became predominant in bioreactors after 300 days, at which point analysis of similarity (global R statistic p?<?0.5) revealed no significant dissimilarities between the populations present in either system. A whole community succession pattern from bacterial dominated prevalence to a new assemblage predominated by archaea was suggested. These results have implications for the stepwise development of microbial model systems for predictive management of field scale FFT basins.     

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate–methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.     

Methanogenic bacteria in mangrove sediments

The occurrence of methanogenic bacteria in the Kodiakkarai (10° 18 N; 79° 52 E) mangrove sediments, whereAvicennia spp are predominant, was studied. Trimethylamine under N₂:CO₂ (80:20% v/v) was used as the substrate. Most Probable Number (MPN) of methanogenic bacteria was determined for a period of one year from July 1987 to June 1988 with monthly sampling. The methanogenic bacterial populations were found to be at the maximum of 1.1 × 10⁵ MPN gm^–1 of wet sediment during August 1987 and from February to June 1988. The bacterial numbers were found to decrease during October to December 1987 with a minimal value of 3.6 × 10² MPN gm^–1 during December 1987. Environmental factors were correlated with the methanogenic bacterial population.     

Microbiological investigations of high-temperature horizons of the Kongdian petroleum reservoir in connection with field trial of a biotechnology for enhancement of oil recovery

The physicochemical conditions and microbiological characteristics of the formation waters of the Kongdian oilfield of the Dagang oilfield (China) were studied. It was demonstrated that this oilfield is a high-temperature ecosystem with formation waters characterized by low mineralization. The concentrations of nitrogen and phosphorus compounds, as well as of electron acceptors, are low. Oil and oil gas are the main organic matter sources. The oilfield is exploited with water-flooding. The oil stratum was inhabited mostly by anaerobic thermophilic microorganisms, including fermentative (10²–10⁵ cells/ml), sulfate-reducing (0–10² cells/ml), and methanogenic (0–10³ cells/ml) microorganisms. Aerobic bacteria were detected mainly in the near-bottom zone of injection wells. The rate of sulfate reduction varied from 0.002 to 18.940 μg S^2? l^?1 day^?1 and the rate of methanogenesis from 0.012 to 16.235 μg CH₄ l^?1 day^?1. Microorganisms with great biotechnological potential inhabited the oilfield. Aerobic thermophilic bacteria were capable of oxidizing oil with formation of biomass, the products of partial oxidation of oil (volatile acids), and surfactants. During growth on the culture liquid of oil-oxidizing bacteria, methanogenic communities produced methane and carbon dioxide, which also had oil-releasing capabilities. Using various labeled tracers, the primary filtration flows of injected solutions at the test site were studied. Our comprehensive investigations allowed us to conclude that the method for microbial enhancement of oil recovery based on the activation of the stratal microflora can be applied in the Kongdian oilfield.     

Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates.

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.