Comprehensive molecular-isotopic characterization of archaeal lipids in the Black Sea water column and underlying sediments.

The Black Sea is a permanently anoxic, marine basin serving as model system for the deposition of organic-rich sediments in a highly stratified ocean. In such systems, archaeal lipids are widely used as paleoceanographic and biogeochemical proxies; however, the diverse planktonic and benthic sources as well as their potentially distinct diagenetic fate may complicate their application. To track the flux of archaeal lipids and to constrain their sources and turnover, we quantitatively examined the distributions and stable carbon isotopic compositions (δ13 C) of intact polar lipids (IPLs) and core lipids (CLs) from the upper oxic water column into the underlying sediments, reaching deposits from the last glacial. The distribution of IPLs responded more sensitively to the geochemical zonation than the CLs, with the latter being governed by the deposition from the chemocline. The isotopic composition of archaeal lipids indicates CLs and IPLs in the deep anoxic water column have negligible influence on the sedimentary pool. Archaeol substitutes tetraether lipids as the most abundant IPL in the deep anoxic water column and the lacustrine methanic zone. Its elevated IPL/CL ratios and negative δ13 C values indicate active methane metabolism. Sedimentary CL- and IPL-crenarchaeol were exclusively derived from the water column, as indicated by non-variable δ13 C values that are identical to those in the chemocline and by the low BIT (branched isoprenoid tetraether index). By contrast, in situ production accounts on average for 22% of the sedimentary IPL-GDGT-0 (glycerol dibiphytanyl glycerol tetraether) based on isotopic mass balance using the fermentation product lactate as an endmember for the dissolved substrate pool. Despite the structural similarity, glycosidic crenarchaeol appears to be more recalcitrant in comparison to its non-cycloalkylated counterpart GDGT-0, as indicated by its consistently higher IPL/CL ratio in sediments. The higher TEX86 , CCaT, and GDGT-2/-3 values in glacial sediments could plausibly result from selective turnover of archaeal lipids and/or an archaeal ecology shift during the transition from the glacial lacustrine to the Holocene marine setting. Our in-depth molecular-isotopic examination of archaeal core and intact polar lipids provided new constraints on the sources and fate of archaeal lipids and their applicability in paleoceanographic and biogeochemical studies.

Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin.

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), ß-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A ß-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and ß-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and ß-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

Size-fractionated distribution of glycerol dialkyl glycerol tetraether core lipids in surface sediments of a large-river delta-front estuary.

Glycerol dialkyl glycerol tetraether core lipids (GDGTs) are microbial biomarkers ubiquitously distributed in terrestrial and marine environments. Dispersal and fate of GDGTs in an estuary largely depends on sediment grain size, however, their size distribution patterns remain poorly understood. Here, surface sediments collected from the Changjiang Estuary were separated into <20, 20-32, 32-63, 63-125 and >125 µm fractions, and analyzed for GDGTs as well as total organic carbon (TOC), stable isotopic composition (δ13C) of TOC and lignin phenols, to investigate the size and spatial distributions of GDGTs and the particle size effects on GDGTs proxies in this large river delta-front estuary. The concentrations of isoprenoidal GDGTs (isoGDGTs) were higher in the finest fractions and in off-estuary sites. On the contrary, branched GDGTs (brGDGTs) were high not only in the finest fractions but in coarser fractions (>32 µm fractions), and thus at both near- and off-estuary sites. The branched and isoprenoid tetraether (BIT) index increased with increasing grain size, and decreased sharply from the estuary (~0.52) to the shelf (~0.16). BrGDGTs were positively correlated with crenarcheaol in both high and low BIT regions. The brGDGTIIIa/IIa ratios in all size fractions were <0.59, further indicating that the brGDGTs were mainly derived from terrestrial input with minimum in-situ production. Fractional TOC source assignments derived from the BIT index was significantly positively correlated with the fractions of terrestrial OC from a mixing model based on δ13C-TOC and lignin contents, indicating that BIT may track a broader pool of terrestrial OC than just soil OC. This work provides novel, yet preliminary insights into the size fractionated distribution characteristics of GDGTs and the applicability of BIT as a proxy for OC sources in estuarine sediments. More work is needed to further clarify the particle size effects on other GDGTs proxies in estuarine systems.

The Exploration of the Thermococcus barophilus Lipidome Reveals the Widest Variety of Phosphoglycolipids in Thermococcales.

One of the most distinctive characteristics of archaea is their unique lipids. While the general nature of archaeal lipids has been linked to their tolerance to extreme conditions, little is known about the diversity of lipidic structures archaea are able to synthesize, which hinders the elucidation of the physicochemical properties of their cell membrane. In an effort to widen the known lipid repertoire of the piezophilic and hyperthermophilic model archaeon Thermococcus barophilus, we comprehensively characterized its intact polar lipid (IPL), core lipid (CL), and polar head group compositions using a combination of cutting-edge liquid chromatography and mass spectrometric ionization systems. We tentatively identified 82 different IPLs based on five distinct CLs and 10 polar head group derivatives of phosphatidylhexoses, including compounds reported here for the first time, e.g., di-N-acetylhexosamine phosphatidylhexose-bearing lipids. Despite having extended the knowledge on the lipidome, our results also indicate that the majority of T. barophilus lipids remain inaccessible to current analytical procedures and that improvements in lipid extraction and analysis are still required. This expanded yet incomplete lipidome nonetheless opens new avenues for understanding the physiology, physicochemical properties, and organization of the membrane in this archaeon as well as other archaea.

Evidence for Enzymatic Backbone Methylation of the Main Membrane Lipids in the Archaeon Methanomassiliicoccus luminyensis.

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing, as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis, the only available isolate known to produce these compounds, via stable isotope labeling with [methyl-13C]methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. IMPORTANCE Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis, these lipids are the result of methyl transfer(s) from an S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.

Marine Group II Euryarchaeota Contribute to the Archaeal Lipid Pool in Northwestern Pacific Ocean Surface Waters.

Planktonic archaea include predominantly Marine Group I Thaumarchaeota (MG I) and Marine Group II Euryarchaeota (MG II), which play important roles in the oceanic carbon cycle. MG I produce specific lipids called isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs), which are being used in the sea surface temperature proxy named TEX86. Although MG II may be the most abundant planktonic archaeal group in surface water, their lipid composition remains poorly characterized because of the lack of cultured representatives. Circumstantial evidence from previous studies of marine suspended particulate matter suggests that MG II may produce both GDGTs and archaeol-based lipids. In this study, integration of the 16S rRNA gene quantification and sequencing and lipid analysis demonstrated that MG II contributed significantly to the pool of archaeal tetraether lipids in samples collected from MG II-dominated surface waters of the Northwestern Pacific Ocean (NWPO). The archaeal lipid composition in MG II-dominated NWPO waters differed significantly from that of known MG I cultures, containing relatively more 2G-OH-, 2G- and 1G- GDGTs, especially in their acyclic form. Lipid composition in NWPO waters was also markedly different from MG I-dominated surface water samples collected in the East China Sea. GDGTs from MG II-dominated samples seemed to respond to temperature similarly to GDGTs from the MG I-dominated samples, which calls for further study using pure cultures to determine the exact impact of MG II on GDGT-based proxies.

Isoprenoid Quinones Resolve the Stratification of Redox Processes in a Biogeochemical Continuum from the Photic Zone to Deep Anoxic Sediments of the Black Sea.

The stratified water column of the Black Sea serves as a model ecosystem for studying the interactions of microorganisms with major biogeochemical cycles. Here, we provide detailed analysis of isoprenoid quinones to study microbial redox processes in the ocean. In a continuum from the photic zone through the chemocline into deep anoxic sediments of the southern Black Sea, diagnostic quinones and inorganic geochemical parameters indicate niche segregation between redox processes and corresponding shifts in microbial community composition. Quinones specific for oxygenic photosynthesis and aerobic respiration dominate oxic waters, while quinones associated with thaumarchaeal ammonia oxidation and bacterial methanotrophy, respectively, dominate a narrow interval in suboxic waters. Quinone distributions indicate highest metabolic diversity within the anoxic zone, with anoxygenic photosynthesis being a major process in its photic layer. In the dark anoxic layer, quinone profiles indicate the occurrence of bacterial sulfur and nitrogen cycling, archaeal methanogenesis, and archaeal methanotrophy. Multiple novel ubiquinone isomers, possibly originating from unidentified intra-aerobic anaerobes, occur in this zone. The respiration modes found in the anoxic zone continue into shallow subsurface sediments, but quinone abundances rapidly decrease within the upper 50 cm below the sea floor, reflecting the transition to lower energy availability. In the deep subseafloor sediments, quinone distributions and geochemical profiles indicate archaeal methanogenesis/methanotrophy and potentially bacterial fermentative metabolisms. We observed that sedimentary quinone distributions track lithology, which supports prior hypotheses that deep biosphere community composition and metabolisms are determined by environmental conditions during sediment deposition.IMPORTANCE Microorganisms play crucial roles in global biogeochemical cycles, yet we have only a fragmentary understanding of the diversity of microorganisms and their metabolisms, as the majority remains uncultured. Thus, culture-independent approaches are critical for determining microbial diversity and active metabolic processes. In order to resolve the stratification of microbial communities in the Black Sea, we comprehensively analyzed redox process-specific isoprenoid quinone biomarkers in a unique continuous record from the photic zone through the chemocline into anoxic subsurface sediments. We describe an unprecedented quinone diversity that allowed us to detect distinct biogeochemical processes, including oxygenic photosynthesis, archaeal ammonia oxidation, aerobic methanotrophy, and anoxygenic photosynthesis in defined geochemical zones.

Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin.

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed "Mat Mound") were characterized by porewater geochemistry of methane, C2-C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates.

Pelolinea submarina gen. nov., sp. nov., an anaerobic, filamentous bacterium of the phylum Chloroflexi isolated from subseafloor sediment.

A novel, anaerobic filamentous bacterium, strain MO-CFX1(T), was isolated from a methanogenic community, which was originally established from subseafloor sediments collected from off the Shimokita Peninsula, Japan. Cells were non-spore-forming, non-motile, Gram-stain-negative and filamentous. The filaments were longer than 10 µm and 130-150 nm in width. Growth of the strain was observed at 10-37 °C (optimum 25-30 °C), at pH 5.5-8.5 (optimum pH 7.0) and in 0-50 g NaCl l(-1) (optimum 15 g NaCl l(-1)). The strain was able to grow with a number of carbohydrates in the presence of yeast extract. The major cellular fatty acids were monounsaturated C18 : 1ω9, C16 : 1ω7 and saturated C18 : 0 and C16 : 0. The intact polar lipids of the strain were dominated by diacylglyceride and sphingolipid core lipid structures with monoglycosidic, mixed phosphomonoglycosidic and fatty-acid-modified monoglycosidic polar head groups. The G+C content of the genomic DNA was 52.4 mol%. Based on the comparative 16S rRNA gene sequence analysis, strain MO-CFX1(T) was affiliated with the class Anaerolineae within the phylum Chloroflexi and was most closely related to Leptolinea tardivitalis YMTK-2(T) (sequence identity of 91.0 %). Based on phenotypic and genetic properties of the novel isolate, we propose a novel species representing a new genus Pelolinea submarina gen. nov., sp. nov., for strain MO-CFX1(T) ( = JCM 17238(T), = KCTC 5975(T)). This is the first formal description, to our knowledge, of an isolate of the phylum Chloroflexi from the deep-sea sedimentary environment.

Turnover of microbial lipids in the deep biosphere and growth of benthic archaeal populations.

Deep subseafloor sediments host a microbial biosphere with unknown impact on global biogeochemical cycles. This study tests previous evidence based on microbial intact polar lipids (IPLs) as proxies of live biomass, suggesting that Archaea dominate the marine sedimentary biosphere. We devised a sensitive radiotracer assay to measure the decay rate of ([(14)C]glucosyl)-diphytanylglyceroldiether (GlcDGD) as an analog of archaeal IPLs in continental margin sediments. The degradation kinetics were incorporated in model simulations that constrained the fossil fraction of subseafloor IPLs and rates of archaeal turnover. Simulating the top 1 km in a generic continental margin sediment column, we estimated degradation rate constants of GlcDGD being one to two orders of magnitude lower than those of bacterial IPLs, with half-lives of GlcDGD increasing with depth to 310 ky. Given estimated microbial community turnover times of 1.6-73 ky in sediments deeper than 1 m, 50-96% of archaeal IPLs represent fossil signals. Consequently, previous lipid-based estimates of global subseafloor biomass probably are too high, and the widely observed dominance of archaeal IPLs does not rule out a deep biosphere dominated by Bacteria. Reverse modeling of existing concentration profiles suggest that archaeal IPL synthesis rates decline from around 1,000 pg⋅mL(-1) sediment⋅y(-1) at the surface to 0.2 pg⋅mL(-1)⋅y(-1) at 1 km depth, equivalent to production of 7 × 10(5) to 140 archaeal cells⋅mL(-1) sediment⋅y(-1), respectively. These constraints on microbial growth are an important step toward understanding the relationship between the deep biosphere and the carbon cycle.

Assessing production of the ubiquitous archaeal diglycosyl tetraether lipids in marine subsurface sediment using intramolecular stable isotope probing.

The membrane lipids diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2G-GDGTs) in marine subsurface sediments are believed to originate from uncultivated benthic archaea, yet the production of 2G-GDGTs from subseafloor samples has not been demonstrated in vitro. In order to validate sedimentary biosynthesis of 2G-GDGTs, we performed a stable carbon isotope probing experiment on a subseafloor sample with six different (13) C-labelled substrates (bicarbonate, methane, acetate, leucine, glucose and Spirulina platensis biomass). After 468 days of anoxic incubation, only glucose and S. platensis resulted in label uptake in lipid moieties of 2G-GDGTs, indicating incorporation of carbon from these organic substrates. The hydrophobic moieties of 2G-GDGTs showed minimal label incorporation, with up to 4‰ (13) C enrichment detected in crenarchaeol-derived tricyclic biphytane from the S. platensis-supplemented slurries. The 2G-GDGT-derived glucose or glycerol moieties also showed (13) C incorporation (Δδ(13) C = 18-38‰) in the incubations with glucose or S. platensis, consistent with a lipid salvage mechanism utilized by marine benthic archaea to produce new 2G-GDGTs. The production rates were nevertheless rather slow, even when labile organic matter was supplied. The 2G-GDGT turnover times of 1700-20,500 years were much longer than those estimated for subseafloor microbial communities, implying that sedimentary 2G-GDGTs as biomarkers of benthic archaea are cumulative records of past and present generations.

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.

Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids.

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ(13)C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ(13)C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the δ(13)C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in (13)C relative to the corresponding major biphytane. This (13)C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (∼15‰) than in those from benthic archaea (<7‰). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms.

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.

Significant contribution of Archaea to extant biomass in marine subsurface sediments.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells. Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (ref. 1) and 303 Pg (ref. 3) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem. Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells, are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

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.

Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru.

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100-2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.