Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Euryarchaeota, using the model organism Haloferax volcanii. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.

Recycling and metabolic flexibility dictate life in the lower oceanic crust.

The lithified lower oceanic crust is one of Earth's last biological frontiers as it is difficult to access. It is challenging for microbiota that live in marine subsurface sediments or igneous basement to obtain sufficient carbon resources and energy to support growth1-3 or to meet basal power requirements4 during periods of resource scarcity. Here we show how limited and unpredictable sources of carbon and energy dictate survival strategies used by low-biomass microbial communities that live 10-750 m below the seafloor at Atlantis Bank, Indian Ocean, where Earth's lower crust is exposed at the seafloor. Assays of enzyme activities, lipid biomarkers, marker genes and microscopy indicate heterogeneously distributed and viable biomass with ultralow cell densities (fewer than 2,000 cells per cm3). Expression of genes involved in unexpected heterotrophic processes includes those with a role in the degradation of polyaromatic hydrocarbons, use of polyhydroxyalkanoates as carbon-storage molecules and recycling of amino acids to produce compounds that can participate in redox reactions and energy production. Our study provides insights into how microorganisms in the plutonic crust are able to survive within fractures or porous substrates by coupling sources of energy to organic and inorganic carbon resources that are probably delivered through the circulation of subseafloor fluids or seawater.

Formation of ethane and propane via abiotic reductive conversion of acetic acid in hydrothermal sediments.

A mechanistic understanding of formation pathways of low-molecular-weight hydrocarbons is relevant for disciplines such as atmospheric chemistry, geology, and astrobiology. The patterns of stable carbon isotopic compositions (δ13C) of hydrocarbons are commonly used to distinguish biological, thermogenic, and abiotic sources. Here, we report unusual isotope patterns of nonmethane hydrocarbons in hydrothermally heated sediments of the Guaymas Basin; these nonmethane hydrocarbons are notably 13C-enriched relative to sedimentary organic matter and display an isotope pattern that is reversed relative to thermogenic hydrocarbons (i.e., δ13C ethane > δ13C propane > δ13C n-butane > δ13C n-pentane). We hypothesized that this pattern results from abiotic reductive conversion of volatile fatty acids, which were isotopically enriched due to prior equilibration of their carboxyl carbon with dissolved inorganic carbon. This hypothesis was tested by hydrous pyrolysis experiments with isotopically labeled substrates at 350 °C and 400 bar that demonstrated 1) the exchange of carboxyl carbon of C2 to C5 volatile fatty acids with 13C-bicarbonate and 2) the incorporation of 13C from 13C-2-acetic acid into ethane and propane. Collectively, our results reveal an abiotic formation pathway for nonmethane hydrocarbons, which may be sufficiently active in organic-rich, geothermally heated sediments and petroleum systems to affect isotopic compositions of nonmethane hydrocarbons.

Environmental micro-niche filtering shapes bacterial pioneer communities during primary colonization of a Himalayas' glacier forefield.

The pedogenesis from the mineral substrate released upon glacier melting has been explained with the succession of consortia of pioneer microorganisms, whose structure and functionality are determined by the environmental conditions developing in the moraine. However, the microbiome variability that can be expected in the environmentally heterogeneous niches occurring in a moraine at a given successional stage is poorly investigated. In a 50 m2 area in the forefield of the Lobuche glacier (Himalayas, 5050 m above sea level), we studied six sites of primary colonization presenting different topographical features (orientation, elevation and slope) and harbouring greyish/dark biological soil crusts (BSCs). The spatial vicinity of the sites opposed to their topographical differences, allowed us to examine the effect of environmental conditions independently from the time of deglaciation. The bacterial microbiome diversity and their co-occurrence network, the bacterial metabolisms predicted from 16S rRNA gene high-throughput sequencing, and the microbiome intact polar lipids were investigated in the BSCs and the underlying sediment deep layers (DLs). Different bacterial microbiomes inhabited the BSCs and the DLs, and their composition varied among sites, indicating a niche-specific role of the micro-environmental conditions in the bacterial communities' assembly. In the heterogeneous sediments of glacier moraines, physico-chemical and micro-climatic variations at the site-spatial scale are crucial in shaping the microbiome microvariability and structuring the pioneer bacterial communities during pedogenesis.

Natural Asphalt Seeps Are Potential Sources for Recalcitrant Oceanic Dissolved Organic Sulfur and Dissolved Black Carbon.

Natural oil seepages contribute about one-half of the annual petroleum input to marine systems. Yet, environmental implications and the persistence of water-soluble hydrocarbons from these seeps are vastly unknown. We investigated the release of oil-derived dissolved organic matter (DOM) from natural deep sea asphalt seeps using laboratory incubation experiments. Fresh asphalt samples collected at the Chapopote asphalt volcano in the Southern Gulf of Mexico were incubated aerobically in artificial seawater over 4 weeks. The compositional changes in the water-soluble fraction of asphalt-derived DOM were determined with ultrahigh-resolution mass spectrometry (Fourier-transform ion cyclotron resonance mass spectrometry, FT-ICR-MS) and by excitation-emission matrix spectroscopy to characterize fluorescent DOM (FDOM) applying parallel factor (PARAFAC) analysis. Highly reduced aliphatic asphalt-derived DOM was readily biodegraded, while aromatic and sulfur-enriched DOM appeared to be less bioavailable and accumulated in the aqueous phase. A quantitative molecular tracer approach revealed the abundance of highly condensed aromatic molecules of thermogenic origin. Our results indicate that natural asphalt and potentially other petroleum seepages can be sources of recalcitrant dissolved organic sulfur and dissolved black carbon to the ocean.

Meta-omics highlights the diversity, activity and adaptations of fungi in deep oceanic crust.

The lithified oceanic crust, lower crust gabbros in particular, has remained largely unexplored by microbiologists. Recently, evidence for heterogeneously distributed viable and transcriptionally active autotrophic and heterotrophic microbial populations within low-biomass communities was found down to 750 m below the seafloor at the Atlantis Bank Gabbro Massif, Indian Ocean. Here, we report on the diversity, activity and adaptations of fungal communities in the deep oceanic crust from ~10 to 780 mbsf by combining metabarcoding analyses with mid/high-throughput culturing approaches. Metabarcoding along with culturing indicate a low diversity of viable fungi, mostly affiliated to ubiquitous (terrestrial and aquatic environments) taxa. Ecophysiological analyses coupled with metatranscriptomics point to viable and transcriptionally active fungal populations engaged in cell division, translation, protein modifications and other vital cellular processes. Transcript data suggest possible adaptations for surviving in the nutrient-poor, lithified deep biosphere that include the recycling of organic matter. These active communities appear strongly influenced by the presence of cracks and veins in the rocks where fluids and resulting rock alteration create micro-niches.

Fluid mixing and the deep biosphere of a fossil Lost City-type hydrothermal system at the Iberia Margin.

Subseafloor mixing of reduced hydrothermal fluids with seawater is believed to provide the energy and substrates needed to support deep chemolithoautotrophic life in the hydrated oceanic mantle (i.e., serpentinite). However, geosphere-biosphere interactions in serpentinite-hosted subseafloor mixing zones remain poorly constrained. Here we examine fossil microbial communities and fluid mixing processes in the subseafloor of a Cretaceous Lost City-type hydrothermal system at the magma-poor passive Iberia Margin (Ocean Drilling Program Leg 149, Hole 897D). Brucite-calcite mineral assemblages precipitated from mixed fluids ca. 65 m below the Cretaceous paleo-seafloor at temperatures of 31.7 ± 4.3 °C within steep chemical gradients between weathered, carbonate-rich serpentinite breccia and serpentinite. Mixing of oxidized seawater and strongly reducing hydrothermal fluid at moderate temperatures created conditions capable of supporting microbial activity. Dense microbial colonies are fossilized in brucite-calcite veins that are strongly enriched in organic carbon (up to 0.5 wt.% of the total carbon) but depleted in (13)C (δ(13)C(TOC) = -19.4‰). We detected a combination of bacterial diether lipid biomarkers, archaeol, and archaeal tetraethers analogous to those found in carbonate chimneys at the active Lost City hydrothermal field. The exposure of mantle rocks to seawater during the breakup of Pangaea fueled chemolithoautotrophic microbial communities at the Iberia Margin, possibly before the onset of seafloor spreading. Lost City-type serpentinization systems have been discovered at midocean ridges, in forearc settings of subduction zones, and at continental margins. It appears that, wherever they occur, they can support microbial life, even in deep subseafloor environments.

OlsG (Sinac_1600) Is an Ornithine Lipid N-Methyltransferase from the Planctomycete Singulisphaera acidiphila.

Ornithine lipids (OLs) are phosphorus-free membrane lipids widespread in bacteria but absent from archaea and eukaryotes. In addition to the unmodified OLs, a variety of OL derivatives hydroxylated in different structural positions has been reported. Recently, methylated derivatives of OLs were described in several planctomycetes isolated from a peat bog in Northern Russia, although the gene/enzyme responsible for the N-methylation of OL remained obscure. Here we identify and characterize the OL N-methyltransferase OlsG (Sinac_1600) from the planctomycete Singulisphaera acidiphila. When OlsG is co-expressed with the OL synthase OlsF in Escherichia coli, methylated OL derivatives are formed. An in vitro characterization shows that OlsG is responsible for the 3-fold methylation of the terminal δ-nitrogen of OL. Methylation is dependent on the presence of the detergent Triton X-100 and the methyldonor S-adenosylmethionine.

Inactive hydrothermal vent microbial communities are important contributors to deep ocean primary productivity.

Active hydrothermal vents are oases for productivity in the deep ocean, but the flow of dissolved substrates that fuel such abundant life ultimately ceases, leaving behind inactive mineral deposits. The rates of microbial activity on these deposits are largely unconstrained. Here we show primary production occurs on inactive hydrothermal deposits and quantify its contribution to new organic carbon production in the deep ocean. Measured incorporation of 14C-bicarbonate shows that microbial communities on inactive deposits fix inorganic carbon at rates comparable to those on actively venting deposits. Single-cell uptake experiments and nanoscale secondary ion mass spectrometry showed chemoautotrophs comprise a large fraction (>30%) of the active microbial cells. Metagenomic and lipidomic surveys of inactive deposits further revealed that the microbial communities are dominated by Alphaproteobacteria and Gammaproteobacteria using the Calvin-Benson-Bassham pathway for carbon fixation. These findings establish inactive vent deposits as important sites for microbial activity and organic carbon production on the seafloor.

Quantifying the effects of hydrogen on carbon assimilation in a seafloor microbial community associated with ultramafic rocks.

Thermodynamic models predict that H2 is energetically favorable for seafloor microbial life, but how H2 affects anabolic processes in seafloor-associated communities is poorly understood. Here, we used quantitative 13C DNA stable isotope probing (qSIP) to quantify the effect of H2 on carbon assimilation by microbial taxa synthesizing 13C-labeled DNA that are associated with partially serpentinized peridotite rocks from the equatorial Mid-Atlantic Ridge. The rock-hosted seafloor community was an order of magnitude more diverse compared to the seawater community directly above the rocks. With added H2, peridotite-associated taxa increased assimilation of 13C-bicarbonate and 13C-acetate into 16S rRNA genes of operational taxonomic units by 146% (±29%) and 55% (±34%), respectively, which correlated with enrichment of H2-oxidizing NiFe-hydrogenases encoded in peridotite-associated metagenomes. The effect of H2 on anabolism was phylogenetically organized, with taxa affiliated with Atribacteria, Nitrospira, and Thaumarchaeota exhibiting the most significant increases in 13C-substrate assimilation in the presence of H2. In SIP incubations with added H2, an order of magnitude higher number of peridotite rock-associated taxa assimilated 13C-bicarbonate, 13C-acetate, and 13C-formate compared to taxa that were not associated with peridotites. Collectively, these findings indicate that the unique geochemical nature of the peridotite-hosted ecosystem has selected for H2-metabolizing, rock-associated taxa that can increase anabolism under high H2 concentrations. Because ultramafic rocks are widespread in slow-, and ultraslow-spreading oceanic lithosphere, continental margins, and subduction zones where H2 is formed in copious amounts, the link between H2 and carbon assimilation demonstrated here may be widespread within these geological settings.

Systematic fragmentation patterns of archaeal intact polar lipids by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry.

Archaea are ubiquitous and abundant microorganisms on Earth that mediate key global biogeochemical cycles. The headgroup attached to the sn-1 position of the glycerol backbone and the ether-linked isoprenoid lipids are among the diagnostic traits that distinguish Archaea from Bacteria and Eukarya. Over the last 30 years, numerous archaeal lipids have been purified and described in pure cultures. Coupled high-performance liquid chromatography (HPLC) ion-trap mass spectrometry (ITMS) now enables the detection and rapid identification of intact polar lipids in relatively small and complex samples, revealing a wide range of archaeal lipids in natural environments. Although major structural groups have been identified, the lack of a systematic evaluation of MS/MS fragmentation patterns has hindered the characterization of several atypical components that are therefore considered as unknowns. Here, we examined mass spectra resulting from lipid analysis of natural microbial communities using HPLC/electrospray ionization (ESI)-ITMS(n), and depicted the systematics in MS(2) fragmentation of intact archaeal lipids. This report will be particularly useful for environmental scientists interested in a rapid and straightforward characterization of intact archaeal membrane lipids.

Rock weathering creates oases of life in a high Arctic desert.

During primary colonization of rock substrates by plants, mineral weathering is strongly accelerated under plant roots, but little is known on how it affects soil ecosystem development before plant establishment. Here we show that rock mineral weathering mediated by chemolithoautotrophic bacteria is associated to plant community formation in sites recently released by permanent glacier ice cover in the Midtre Lovénbreen glacier moraine (78 degrees 53'N), Svalbard. Increased soil fertility fosters growth of prokaryotes and plants at the boundary between sites of intense bacterial mediated chemolithotrophic iron-sulfur oxidation and pH decrease, and the common moraine substrate where carbon and nitrogen are fixed by cyanobacteria. Microbial iron oxidizing activity determines acidity and corresponding fertility gradients, where water retention, cation exchange capacity and nutrient availability are increased. This fertilization is enabled by abundant mineral nutrients and reduced forms of iron and sulfur in pyrite minerals within a conglomerate type of moraine rock. Such an interaction between microorganisms and moraine minerals determines a peculiar, not yet described model for soil genesis and plant ecosystem formation with potential past and present analogues in other harsh environments with similar geochemical settings.

Carbon Oxidation State in Microbial Polar Lipids Suggests Adaptation to Hot Spring Temperature and Redox Gradients.

The influence of oxidation-reduction (redox) potential on the expression of biomolecules is a topic of ongoing exploration in geobiology. In this study, we investigate the novel possibility that structures and compositions of lipids produced by microbial communities are sensitive to environmental redox conditions. We extracted lipids from microbial biomass collected along the thermal and redox gradients of four alkaline hot springs in Yellowstone National Park (YNP) and investigated patterns in the average oxidation state of carbon (ZC), a metric calculated from the chemical formulae of lipid structures. Carbon in intact polar lipids (IPLs) and their alkyl chains becomes more oxidized (higher ZC) with increasing distance from each of the four hot spring sources. This coincides with decreased water temperature and increased concentrations of oxidized inorganic solutes, such as dissolved oxygen, sulfate, and nitrate. Carbon in IPLs is most reduced (lowest ZC) in the hot, reduced conditions upstream, with abundance-weighted ZC values between -1.68 and -1.56. These values increase gradually downstream to around -1.36 to -1.33 in microbial communities living between 29.0 and 38.1°C. This near-linear increase in ZC can be attributed to a shift from ether-linked to ester-linked alkyl chains, a decrease in average aliphatic carbons per chain (nC), an increase in average degree of unsaturation per chain (nUnsat), and increased cyclization in tetraether lipids. The ZC of lipid headgroups and backbones did not change significantly downstream. Expression of lipids with relatively reduced carbon under reduced conditions and oxidized lipids under oxidized conditions may indicate microbial adaptation across environmental gradients in temperature and electron donor/acceptor supply.

A micrometer-scale snapshot on phototroph spatial distributions: mass spectrometry imaging of microbial mats in Octopus Spring, Yellowstone National Park.

Microbial mats from alkaline hot springs in the Yellowstone National Park are ideal natural laboratories to study photosynthetic life under extreme conditions, as well as the nuanced interactions of oxygenic and anoxygenic phototrophs. They represent distinctive examples of chlorophototroph (i.e., chlorophyll or bacteriochlorophyll-based phototroph) diversity, and several novel phototrophs have been first described in these systems, all confined in space, coexisting and competing for niches defined by parameters such as light, oxygen, or temperature. In a novel approach, we employed mass spectrometry imaging of chloropigments, quinones, and intact polar lipids (IPLs) to describe the spatial distribution of different groups of chlorophototrophs along the ~ 1 cm thick microbial mat at 75 µm resolution and in the top ~ 1.5 mm green part of the mat at 25 µm resolution. We observed a fine-tuned sequence of oxygenic and anoxygenic chlorophototrophs with distinctive biomarker signatures populating the microbial mat. The transition of oxic to anoxic conditions is characterized by an accumulation of biomarkers indicative of anoxygenic phototrophy. It is also identified as a clear boundary for different species and ecotypes, which adjust their biomarker inventory, particularly the interplay of quinones and chloropigments, to prevailing conditions. Colocalization of the different biomarker groups led to the identification of characteristic IPL signatures and indicates that glycosidic diether glycerolipids are diagnostic for anoxygenic phototrophs in this mat system. The zoom-in into the upper green part further reveals how oxygenic and anoxygenic phototrophs share this microenvironment and informs on subtle, microscale adjustments in lipid composition of Synechococcus spp.

Temperature limits to deep subseafloor life in the Nankai Trough subduction zone.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea.

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.

Anaerobic Degradation of Non-Methane Alkanes by "Candidatus Methanoliparia" in Hydrocarbon Seeps of the Gulf of Mexico.

Crude oil and gases in the seabed provide an important energy source for subsurface microorganisms. We investigated the role of archaea in the anaerobic degradation of non-methane alkanes in deep-sea oil seeps from the Gulf of Mexico. We identified microscopically the ethane and short-chain alkane oxidizers "Candidatus Argoarchaeum" and "Candidatus Syntrophoarchaeum" forming consortia with bacteria. Moreover, we found that the sediments contain large numbers of cells from the archaeal clade "Candidatus Methanoliparia," which was previously proposed to perform methanogenic alkane degradation. "Ca. Methanoliparia" occurred abundantly as single cells attached to oil droplets in sediments without apparent bacterial or archaeal partners. Metagenome-assembled genomes of "Ca. Methanoliparia" encode a complete methanogenesis pathway including a canonical methyl-coenzyme M reductase (MCR) but also a highly divergent MCR related to those of alkane-degrading archaea and pathways for the oxidation of long-chain alkyl units. Its metabolic genomic potential and its global detection in hydrocarbon reservoirs suggest that "Ca. Methanoliparia" is an important methanogenic alkane degrader in subsurface environments, producing methane by alkane disproportionation as a single organism.IMPORTANCE Oil-rich sediments from the Gulf of Mexico were found to contain diverse alkane-degrading groups of archaea. The symbiotic, consortium-forming "Candidatus Argoarchaeum" and "Candidatus Syntrophoarchaeum" are likely responsible for the degradation of ethane and short-chain alkanes, with the help of sulfate-reducing bacteria. "Ca. Methanoliparia" occurs as single cells associated with oil droplets. These archaea encode two phylogenetically different methyl-coenzyme M reductases that may allow this organism to thrive as a methanogen on a substrate of long-chain alkanes. Based on a library survey, we show that "Ca. Methanoliparia" is frequently detected in oil reservoirs and may be a key agent in the transformation of long-chain alkanes to methane. Our findings provide evidence for the important and diverse roles of archaea in alkane-rich marine habitats and support the notion of a significant functional versatility of the methyl coenzyme M reductase.

Cultivable microbial community in 2-km-deep, 20-million-year-old subseafloor coalbeds through ~1000 days anaerobic bioreactor cultivation.

Recent explorations of scientific ocean drilling have revealed the presence of microbial communities persisting in sediments down to ~2.5 km below the ocean floor. However, our knowledge of these microbial populations in the deep subseafloor sedimentary biosphere remains limited. Here, we present a cultivation experiment of 2-km-deep subseafloor microbial communities in 20-million-year-old lignite coalbeds using a continuous-flow bioreactor operating at 40 °C for 1029 days with lignite particles as the major energy source. Chemical monitoring of effluent samples via fluorescence emission-excitation matrices spectroscopy and stable isotope analyses traced the transformation of coalbed-derived organic matter in the dissolved phase. Hereby, the production of acetate and 13C-depleted methane together with the increase and transformation of high molecular weight humics point to an active lignite-degrading methanogenic community present within the bioreactor. Electron microscopy revealed abundant microbial cells growing on the surface of lignite particles. Small subunit rRNA gene sequence analysis revealed that diverse microorganisms grew in the bioreactor (e.g., phyla Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Spirochaetes, Tenericutes, Ignavibacteriae, and SBR1093). These results indicate that activation and adaptive growth of 2-km-deep microbes was successfully accomplished using a continuous-flow bioreactor, which lays the groundwork to explore networks of microbial communities of the deep biosphere and their physiologies.

Methane-producing microbial community in a coal bed of the Illinois basin.

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.