Archaeal Lipid Biomarkers Research Articles

Archaeal lipid biomarkers in near-surface sediments at a giant colony of the bivalve Calyptogena: Molecular records of a massive methane release event associated with methane hydrate dissociation

To investigate the possibility that dissociation of subsurface methane hydrate (MH) in the eastern Nankai Trough, offshore of Japan, led to the formation of a giant colony of the bivalve Calyptogena (currently mostly dead), the carbon isotope ratios (δ13C) of archaeal lipids and methane were measured in near-surface core sediments at Daini-Tenryu Knoll. The irregular variation of porewater methane δ13C with depth (from −75 ‰ to −26 ‰) suggested that originally low δ13C microbial methane was degraded in different proportions by anaerobic methane oxidation. Consistent with this inference, biomarkers of anaerobic methanotrophic archaea (ANME), namely, crocetane (2,6,11,15-tetramethylhexadecane), PMI (2,6,10,15,19-pentamethylicosane), and diethers (archaeol and hydroxyarchaeols), were detected in lipid extracts. The low diether δ13C values (−121 ‰ to −104 ‰) were characteristic of ANME, but less variable than the methane δ13C values, and the relationships between diethers and methane δ13C values deviated from regression lines derived using worldwide data from modern methane seep sites. In contrast, δ13C values of the ANME source methane predicted from those regression lines and the diether δ13C values agreed well with methane δ13C values in MH samples obtained by nearby deep drilling. This result strongly suggests that most of the diethers were produced by ANME that proliferated during a past massive methane release event associated with MH dissociation. The crocetane δ13C value, measured in a mixture with phytane and estimated from the correlation of the δ13C of the mixture with the mole fraction of crocetane, was about −127 ‰. More than half of the PMI δ13C values were greater than −100 ‰, suggesting the background presence of fossil PMI from methanogens.

Methane Index: Towards a quantitative archaeal lipid biomarker proxy for reconstructing marine sedimentary methane fluxes

In marine sediments, anaerobic methane oxidation (AOM) carried out by consortia of methanotrophic archaea (ANME groups) and sulfate reducing bacteria (SRB) represents a major sink for methane (40–290 Tg of C yr−1). The variability of seafloor methane seepage and AOM is crucial to sedimentary biogeochemistry and global carbon/sulfur cycling, yet poorly constrained for Earth’s history. Based on the rationale that a major group of ANME preferentially synthesizes certain tetraether lipids, Methane Index (MI) measured from sedimentary archaeal lipid biomarkers called GDGTs (glycerol dialkyl glycerol tetraethers) has been widely used as a qualitative indicator for methane-impact on sediments. However, it was unclear whether small amounts of diagenetic methane in porewater could cause high MI values or that high methane flux associated with, for example cold seeps, is required. Since not all ANME groups synthesize GDGTs, another contested issue is whether this index is broadly representative of AOM. Here, we use modern porewater and sedimentary lipid profiles from the world’s ocean to further explore MI’s potential to address these questions. The new data show that MI is quantitatively linked to sedimentary methane diffusive flux and the depth of sulfate-methane transition zone (SMTZ) which is the diagenetic front where AOM activity is mostly concentrated. In particular, high MI values (>0.3–0.5) are strongly associated with high methane fluxes (>∼0.1 mmol m−2 d−1) and shallow SMTZ depths (<1–3 m) commonly linked to gas hydrate dissociations. Sensitivity analyses suggest that when the variability of several key parameters (e.g., seawater sulfate concentration, geothermal gradient, and sediment porosity) is small, modern MI – methane flux and MI – SMTZ depth relationships can be applied to reconstruct methane history and diagenetic zonation of the geological past. Although only Group I of ANME archaea synthesizes GDGTs, the co-occurrence of AOM biomarkers (e.g., crocetane, PMI, and hydroxyarchaeol) that are attributed to ANME-2 or ANME-3 and high MI values suggest that MI is broadly representative of AOM. Lastly, we applied MI to probe the methane venting history by using new data from a Cascadia Margin site (Hydrate Ridge, IODP U1328) since the penultimate interglacial, and reinterpreted recently reported MI data from three Southern Ocean sites (ODP 1168, 1170, and IODP U1356) during the late Oligocene – early Miocene. Overall, these applications demonstrate that MI can serve as a sensitive and quantitative proxy to help broadly establish Cenozoic and Mesozoic history of marine methane biogeochemical cycling.

Archaeal lipid biomarker as a tool to constrain the origin of methane at ancient methane seeps: Insight into subsurface fluid flow in the geological past

The origin of methane discharged at ancient methane seeps is a key to understand subsurface fluid-flow processes in sedimentary basins. The carbon isotopic composition of methane-seep carbonates can be used to estimate the origin of methane, but mixing of multiple carbon sources results in ambiguous interpretation. We here constrained the origin of methane at ancient seeps by using the carbon isotopic composition and the isotope fractionation of lipid biomarkers of anaerobic methane-oxidizing archaea. We estimated the carbon-isotope fractionation between methane and biomarkers as the offset between their δ13C values at modern seep sites in the Japan Sea and other regions of the world. The biomarker pentamethylicosane (PMI) extracted from a modern seep carbonate of the Japan Sea shows a 44‰ offset from the seep methane. On the basis of literature data, we calculated the carbon-isotope offset as mostly ~20–60‰ for PMI at modern seeps across the world. We also assessed the relationship of the δ13C values between methane and biomarkers by regression analysis on the data taken at the modern seeps, obtaining regression formulas to estimate the δ13C values of methane from those of biomarkers. For ancient seeps, we examined Miocene to Pleistocene methane-seep carbonate rocks collected from Japan Sea sediments. PMI extracted from these ancient carbonates have δ13C values ranging from −137‰ to −93‰. Using the δ13C values of PMI and the carbon-isotope offset and regression formulas, we estimated that the methane at the ancient seeps was mainly of biogenic origin and migrated from the shallow subsurface.

Assessing the carbon assimilation and production of benthic archaeal lipid biomarkers using lipid-RIP

Abstract Distributional patterns of intact polar lipids (IPLs) suggest that archaea are abundant in marine subsurface sediments. However, the production of archaeal lipids remains largely unresolved. We performed a lipid radioisotope probing (lipid-RIP) experiment using 14C-bicarbonate and 2-14C-acetate with surface and methanogenic marine sediments from the Rhone delta (Mediterranean Sea) to investigate mechanisms of carbon assimilation and to assess biomass production of benthic archaea. The direct determination of carbon assimilation rates into diagnostic IPLs in the experiments with 14C-bicarbonate were up to 9 times higher than in the incubations with 2-14C-acetate, implying that autotrophy is an important carbon pathway for the benthic archaea in the Rhone delta sediments. Production rates of polyglycosidic archaeols (AR) were one to two orders of magnitude higher than those of mono- and polyglycosidic glycerol dialkyl glycerol tetraethers (GDGTs), suggesting that the former IPLs are good biomarkers for active benthic archaea in marine sediments. In contrast, the low production rates of the monoglycosidic AR and GDGTs, indicate that a large fraction of these IPLs may represent fossil sedimentary remains from the water column. Unexpectedly, the lipid production rates of AR and GDGT core lipids (CLs) were similar to those of polyglycosidic IPLs. Considering the relatively short period of incubation (21 days), this suggests that CLs may be actively synthesized by benthic archaea in marine sediments and are not exclusively formed from the degradation of IPL precursors.

A combined lipidomic and 16S rRNA gene amplicon sequencing approach reveals archaeal sources of intact polar lipids in the stratified Black Sea water column.

Archaea are important players in marine biogeochemical cycles, and their membrane lipids are useful biomarkers in environmental and geobiological studies. However, many archaeal groups remain uncultured and their lipid composition unknown. Here, we aim to expand the knowledge on archaeal lipid biomarkers and determine the potential sources of those lipids in the water column of the euxinic Black Sea. The archaeal community was evaluated by 16S rRNA gene amplicon sequencing and by quantitative PCR. The archaeal intact polar lipids (IPLs) were investigated by ultra‐high‐pressure liquid chromatography coupled to high‐resolution mass spectrometry. Our study revealed both a complex archaeal community and large changes with water depth in the IPL assemblages. In the oxic/upper suboxic waters (<105 m), the archaeal community was dominated by marine group (MG) I Thaumarchaeota, coinciding with a higher relative abundance of hexose phosphohexose crenarchaeol, a known marker for Thaumarchaeota. In the suboxic waters (80–110 m), MGI Nitrosopumilus sp. dominated and produced predominantly monohexose glycerol dibiphytanyl glycerol tetraethers (GDGTs) and hydroxy‐GDGTs. Two clades of MGII Euryarchaeota were present in the oxic and upper suboxic zones in much lower abundances, preventing the detection of their specific IPLs. In the deep sulfidic waters (>110 m), archaea belonging to the DPANN Woesearchaeota, Bathyarchaeota, and ANME‐1b clades dominated. Correlation analyses suggest that the IPLs GDGT‐0, GDGT‐1, and GDGT‐2 with two phosphatidylglycerol (PG) head groups and archaeol with a PG, phosphatidylethanolamine, and phosphatidylserine head groups were produced by ANME‐1b archaea. Bathyarchaeota represented 55% of the archaea in the deeper part of the euxinic zone and likely produces archaeol with phospho‐dihexose and hexose‐glucuronic acid head groups.

Inference on Paleoclimate Change Using Microbial Habitat Preference in Arctic Holocene Sediments

The present study combines data of microbial assemblages with high-resolution paleoceanographic records from Core GC1 recovered in the Chukchi Sea. For the first time, we have demonstrated that microbial habitat preferences are closely linked to Holocene paleoclimate records, and found geological, geochemical, and microbiological evidence for the inference of the sulphate-methane transition zone (SMTZ) in the Chukchi Sea. In Core GC1, the layer of maximum crenarchaeol concentration was localized surrounding the SMTZ. The vertically distributed predominant populations of Gammaproteobacteria and Marine Group II Euryarchaeota (MG-II) were consistent with patterns of the known global SMTZs. MG-II was the most prominent archaeal group, even within the layer of elevated concentrations of crenarchaeol, an archaeal lipid biomarker most commonly used for Marine Group I Thaumarchaeota (MG-I). The distribution of MG-I and MG-II in Core GC1, as opposed to the potential contribution of MG-I to the marine tetraether lipid pool, suggests that the application of glycerol dibiphytanyl glycerol tetraethers (GDGT)-based proxies needs to be carefully considered in the subsurface sediments owing to the many unknowns of crenarchaeol. In conclusion, microbiological profiles integrated with geological records seem to be useful for tracking microbial habitat preference, which reflect climate-triggered changes from the paleodepositional environment.

Methanogen Biomarkers in the Discontinuous Permafrost Zone of Stordalen, Sweden

ABSTRACTPermafrost peatlands are both an important source of atmospheric CH4 and a substantial sink for atmospheric CO2. Climate change can affect this balance, with higher temperatures resulting in the conversion of permafrost soils to wetlands and associated accelerated mineralisation and increased CH4 emission. To better understand the impact of such processes on methanogen populations, we investigated the anaerobic decay of soil carbon in a low Arctic, discontinuous permafrost peatland. Cores were collected monthly from sedge and Sphagnum mires in north Sweden during the summer of 2006. We determined CH4 concentrations and production potentials, together with variations in the size of the methanogenic community as indicated by concentrations of archaeal lipid biomarkers (phosphorylated archaeol, archaeol and hydroxyarchaeol). Concentrations of methanogen biomarkers generally were higher at the sedge site, increased with depth for all sites and months, and were usually below the detection limits in shallow (&lt;10 cm) Sphagnum peat. The distribution of biomarkers reflects the strong influence of water table depth on anaerobic conditions and methanogen populations, while differences in biomarker concentrations can be explained by differences in vegetation cover and pH. However, methanogen populations inferred from biomarker data show a decoupling from in‐situ CH4 production over the season and from CH4 production potential, suggesting that other factors such as the availability of labile organic substrates can influence methanogen abundance. Archaeal lipid biomarkers appear to offer a potential new means to investigate permafrost biogeochemical processes but the interpretation of signals remains complex. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Lipid biomarkers for anaerobic oxidation of methane and sulphate reduction in cold seep sediments of Nyegga pockmarks (Norwegian margin): discrepancies in contents and carbon isotope signatures

Distributions and carbon isotopic compositions of microbial lipid biomarkers were investigated in sediment cores from the G11 and G12 pockmarks in the Nyegga sector of the Storegga Slide on the mid-Norwegian margin to explore differences in depth zonation, type and carbon assimilation mode of anaerobic methane-oxidizing archaea (ANMEs) and associated sulphate-reducing bacteria responsible for anaerobic oxidation of methane (AOM) in these cold seep environments. While the G11 site is characterised by black reduced sediments colonized by gastropods and Siboglinidae tubeworms, the G12 site has black reduced sediments devoid of fauna but surrounded by a peripheral occurrence of gastropods and white filamentous microbial mats. At both sites, bulk sediments contained abundant archaeal and bacterial lipid biomarkers substantially depleted in 13C, consisting mainly of isoprenoidal hydrocarbons and dialkyl glycerol diethers, fatty acids and non-isoprenoidal monoalkylglycerol ethers. At the G11 site, down-core profiles revealed that lipid biomarkers were in maximum abundance from 10 cm depth to the core bottom at 16 cm depth, associated with δ13C values of −57 to −136‰. At the G12 site, by contrast, lipid biomarkers were in high abundance in the upper 5 cm sediment layer, associated with δ13C values of −43 to −133‰. This suggests that, as expected from the benthic fauna characteristics of the sites, AOM takes place mainly at depth in the G11 pockmark but just below the seafloor in the G12 pockmark. These patterns can be explained largely by variable fluid flow rates. Furthermore, at both sites, a dominance of ANME-2 archaea accompanied by their bacterial partners is inferred based on lipid biomarker distributions and carbon isotope signatures, which is in agreement with recently published DNA analyses for the G11 pockmark. However, the present data reveal high discrepancies in the contents and δ13C values for both archaeal and bacterial lipid profiles, implying the possible involvement of at least two distinct AOM-related microbial consortia at the inferred AOM depth zonation of G11 and G12 pockmark sediments. In both sediment cores, the δ13C profiles for most archaeal lipids suggest a direct assimilation of dissolved inorganic carbon (DIC) in addition to methane by ANMEs (chemoautotrophy); constant and highly depleted δ13C profiles for PMI:3, an archaeal lipid biomarker presumably related to ANME-2, suggest a direct assimilation of 13C-depleted methane-derived carbon via AOM (methanotrophy). Evidently, the common approach of investigating lipid biomarker contents and δ13C signatures in cold seep sediments does not suffice to precisely discriminate between the carbon assimilation mode for each ANME archaeal group and associated bacteria. Rather, this needs to be combined with further specific labelling studies including different carbon sources (methane carbon, methane-derived organic intermediates and DIC) in order to unravel the metabolic pathways of each microbial consortium involved in AOM (ANME-1 vs. ANME-2 vs. ANME-3 archaeal group and associated bacteria).

Identification of isoprenoid glycosidic glycerol dibiphytanol diethers and indications for their biosynthetic origin

A series of archaeal lipid biomarkers, the isoprenoid glycerol dibiphytanol diethers (GDDs), was recently described and proposed to represent either biosynthetic intermediates or diagenetic products of the relatively more abundant glycerol dibiphytanyl glycerol tetraether (GDGT) lipids (Liu, X.-L., Lipp, J.S., Schröder, J.M., Summons, R.E., Hinrichs, K.-U., 2012. Isoprenoid glycerol dialkanol diethers: a series of novel lipids in marine sediments. Organic Geochemistry 43, 50–55). Here we report a novel series of polar lipids comprising a glycosidic head group and GDD core lipids with varying cycloalkyl distributions (1G-GDDs), which were found in estuarine and hot spring sediments, as well as in a pure culture of the mesophilic thaumarchaeon Nitrosopumilus maritimus. 1G-GDDs represented up to 4% of the corresponding monoglycosidic GDGTs (1G-GDGTs). The distinct cycloalkyl distribution patterns of these intact polar lipids (IPLs) and detection of 1G-GDDs in an archaeal culture suggest a biosynthetic source of 1G-GDDs rather than formation exclusively via diagenetic removal of a glycerol moiety from 1G-GDGTs.

Distribution and isotopic signatures of archaeal lipid biomarkers associated with gas hydrate occurrences on the northern Cascadia Margin

We have investigated the distributions and carbon isotopic compositions of archaeal membrane lipids in gas-hydrate-bearing sediments collected from the northern Cascadia Margin offshore from Vancouver Island (Sites U1327 and U1328) by the R/V JOIDES Resolution during IODP Expedition 311. Archaeal lipid biomarkers, including glycerol dialkyl glycerol tetraethers (GDGTs), tend to become abundant below 100mbsf (meters below sea floor). Tricyclic biphytane (BP[3]; which is a robust biomarker derived from GDGT), crenarchaeol, and other BPs exhibit δ13C values of ca. −20‰, and become abundant between 130 and 230mbsf at Site U1328. In this depth range, concentrations of ammonium and phosphate in interstitial waters also increase, suggesting that a larger population and higher activity of heterotrophic community consisting of crenarchaeota and other archaea decompose the sedimentary organic matter, thereby liberating ammonium and phosphate. Such crenarchaeotic activity can produce other metabolic products such as molecular hydrogen by fermentation of organic matter during diagenesis. Furthermore, near the organic matter decomposition zone (130 to 230mbsf), a probable methanogen biomarker (13C-depleted BP[1] with δ13C values as low as −48.8‰) becomes abundant, indicating that methanogens utilize these diagenetic products. The molecular and isotopic distributions of archaeal lipid biomarkers indicate that the archaeal community plays an important role in the biogeochemical cycles of deep-sea sediments, including both methanogenesis and nutrient recycling.

Supercritical fluid extraction of bacterial and archaeal lipid biomarkers from anaerobically digested sludge.

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile.

Biomarkers, chemistry and microbiology show chemoautotrophy in a multilayer chemocline in the Cariaco Basin

The Cariaco Basin is the world's largest truly marine anoxic basin. We have conducted a comprehensive multidisciplinary investigation of the water column (42–750m) bracketing the redox boundary (a 250-m thick “chemocline”) of the Cariaco Basin to evaluate linkages between lipid biomarkers, distributions of major dissolved chemical species, and the microbial community and associated redox processes. Our multidimensional data set includes: hydrography, water column chemistry, microbial distributions and rates, and lipid biomarkers. Multivariant statistical analysis of this data set partitions the investigated water column into 5 distinct zones, each characterized by different chemistries, microbiologies and biomarker compositions. The core of this chemocline is a 25-m thick suboxic zone where both dissolved oxygen and sulfide were below detection limits, bacterial and archaeal cell numbers and the rate of chemoautotrophic (dark) carbon fixation are elevated, and dissolved chemical species and bacterial and archaeal lipid biomarkers are indicative of tightly coupled cycles of carbon, nitrogen, and sulfur through chemoautotrophy.

Methane Index: A tetraether archaeal lipid biomarker indicator for detecting the instability of marine gas hydrates

Gas hydrates represent one of the largest pools of readily exchangeable carbon on Earth's surface. Releases of the greenhouse gas methane from hydrates are proposed to be responsible for climate change at numerous events in geological history. Many of these inferred events, however, were based on carbonate carbon isotopes which are susceptible to diagenetic alterations. Here we propose a molecular fossil proxy, i.e., the “Methane Index (MI)”, to detect and document the destabilization and dissociation of marine gas hydrates. MI consists of the relative distribution of glycerol dibiphytanyl glycerol tetraethers (GDGTs), the core membrane lipids of archaea. The rational behind MI is that in hydrate-impacted environments, the pool of archaeal tetraether lipids is dominated by GDGT-1, -2 and -3 due to the large contribution of signals from the methanotrophic archaeal community. Our study in the Gulf of Mexico cold-seep sediments demonstrates a correlation between MI and the compound-specific carbon isotope of GDGTs, which is strong evidence supporting the MI-methane consumption relationship. Preliminary applications of MI in a number of hydrate-impacted and/or methane-rich environments show diagnostic MI values, corroborating the idea that MI may serve as a robust indicator for hydrate dissociation that is useful for studies of global carbon cycling and paleoclimate change.

Ammonia-oxidizing Archaea in Kamchatka Hot Springs

Mounting evidence suggests that ammonia-oxidizing archaea (AOA) may play important roles in nitrogen cycling in geothermal environments. In this study, the diversity, distribution and ecological significance of AOA in terrestrial hot springs in Kamchatka (Far East Russia) were explored using amoA genes complemented by analysis of glycerol dialkyl glycerol tetraethers (GDGTs) of archaea. PCR amplification of functional genes (amoA) from AOA and ammonia-oxidizing bacteria (AOB) was performed on microbial mats/streamers and sediments collected from three hot springs (42°C to 87°C and pH 5.5-7.0). No amoA genes of AOB were detected. The amoA genes of AOA formed three distinct phylogenetic clusters with Cluster 3 representing the majority (∼59%) of OTUs. Some of the sequences from Cluster 3 were closely related to those from acidic soil environments, which is consistent with the predominance of low pH (<7.0) in these hot springs. Species richness (estimated by Chao1) was more frequently higher at temperatures below 75°C than above it, indicating that AOA may be favored in the moderately high temperature environments. Quantitative PCR of 16S rRNA genes showed that crenarchaeota counted for up to 80% of total archaea. S-LIBSHUFF separated all samples into two phylogenetic groups. The profiles of GDGTs were well separated among the studied springs, suggesting a spatial patterning of archaeal lipid biomarkers. However, this patterning did not correlate significantly with variation in archaeal amoA, suggesting that AOA are not the predominant archaeal group in these springs producing the observed GDGTs.

Palaeo methane-seepage history traced by biomarker patterns in a carbonate crust, Nile deep-sea fan (Eastern Mediterranean Sea)

We investigated lipid biomarkers in three depth-intervals of a methane-derived carbonate crust collected from a pockmark in the Nile Deep-Sea fan sampled during the Nautinil cruise of the R/V l'Atalante. A diverse set of 13C-depleted archaeal and bacterial lipids were detected along this crust with δ 13C values as low as − 118‰. This provides evidence that anaerobic oxidation of methane was the main process that induced the formation of this carbonate crust. Archaeal lipid biomarker distributions revealed the presence of archaea belonging both to the ANME-1 and ANME-2 phylogenetic clusters, with the former predominating. The distribution and compound specific δ 13C values of AOM-derived lipid biomarkers showed no significant variations with depth. These results infer that the archaeal community structure and thus the local seepage environment remained relatively stable during the 5000 kyr period of the carbonate crust precipitation.

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California

Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.

Extended hydroxyarchaeol, a novel lipid biomarker for anaerobic methanotrophy in cold seepage habitats

A new archaeal lipid biomarker, C 20,25 sn-2, C-7 hydroxy isoprenoid glycerol diether, has been identified in methane-related carbonates from six methane seep locations. The molecular structure resembles that of sn-2 hydroxyarchaeol but the hydroxy group-bearing moiety at the sn-2 position comprises a regular C 25 instead of a C 20 isoprenoid chain. Compound specific stable carbon isotope measurements revealed δ 13C values ranging from −96‰ to −129‰, which are similar to δ 13C values of sn-2 hydroxyarchaeol. This supports the origin of the novel compound from archaea involved in the anaerobic oxidation of methane. It also suggests a common source, i.e. archaea within the ANME-2 group, which is consistent with the previously reported 16S rDNA data for the same samples. The frequent appearance of the novel sn-2 isoprenoidal hydroxy glycerol diether in cold seepage sediments with high salinity environments, and its distinct structural similarities with membrane lipids of halophilic and methanogenic microbes, suggests that ANME-2 methanotrophic archaea thriving at elevated salinity are the possible biological source for this specific lipid.

Carbonate formation by anaerobic oxidation of methane: Evidence from lipid biomarker and fossil 16S rDNA

Carbonate chimneys and other carbonate structures occur widespread in the Gulf of Cadiz and probably reflect the presence of cold seeps and associated release of methane in the geological past, possibly in the Early Pleistocene, but it is unclear under what conditions and by which processes these carbonates were formed. We studied a fossil methane-related carbonate crust collected from the Kidd mud volcano in the gulf. Concentrations of microbial lipids, their stable carbon isotope composition, sequences of fossil 16S rRNA genes of anaerobic methanotrophic archaea in combination with mineralogical and carbon and oxygen isotopic composition of carbonate were obtained for seven different horizons of the crust. This combination of organic and inorganic geochemical techniques with molecular ecological methods gave a consistent view on processes resulting in the formation of the crust and indicated that it took place in two phases and in a downward direction. Archaeal lipid biomarkers and fossil 16S rRNA gene sequence data revealed the dominance of archaeal ANME-2 group and elevated methane partial pressures during the formation of the top part of the crust. The lower part of the carbonate was likely formed in an environment with reduced methane fluxes as revealed by the dominance of fossil remains of ANME-1 archaea. The combination of these methods can be used as an effective tool to reconstruct in unprecedented detail the palaeo-biogeochemical processes resulting in the formation of carbonate fabrics. This interdisciplinary strategy may also be applied for other fossil methane-derived carbonates, generating new concepts and knowledge about past methane-related carbonate systems.

Microbial ecology of the stratified water column of the Black Sea as revealed by a comprehensive biomarker study

The stratified water column of the Black Sea is partitioned into oxic, suboxic, and euxinic zones, each characterized by different biogeochemical processes and by distinct microbial communities. In 2003, we collected particulate matter by large volume in situ filtration at the highest resolution to date for lipid biomarker analysis and bacterioplankton for enumeration of major prokaryotic groups. Abundances of several prokaryotic groups were estimated using CARD-FISH probes specific for Bacteria, Archaea ( Crenarchaeota and Euryarchaeota), epsilonproteobacteria (mainly sulfide oxidizers) and sulfate reducing bacteria. We also measured a wide range of bacterial and archaeal lipid biomarkers. Depth distributions of diagnostic biomarkers are matched with zonation of microbial processes, including aerobic bacterial oxidation of methane, oxidation of ammonium by bacteria and archaea, metal reduction, and sulfide oxidation at the chemocline, and bacterial sulfate reduction and anaerobic oxidation of methane by archaea in the anoxic zone. Cell densities for archaea and sulfate reducing bacteria are estimated based on water column biomarker concentrations and compared with CARD-FISH results.

Microbial community in a sediment-hosted CO 2 lake of the southern Okinawa Trough hydrothermal system

Increasing levels of CO2 in the atmosphere are expected to cause climatic change with negative effects on the earth's ecosystems and human society. Consequently, a variety of CO2 disposal options are discussed, including injection into the deep ocean. Because the dissolution of CO2 in seawater will decrease ambient pH considerably, negative consequences for deep-water ecosystems have been predicted. Hence, ecosystems associated with natural CO2 reservoirs in the deep sea, and the dynamics of gaseous, liquid, and solid CO2 in such environments, are of great interest to science and society. We report here a biogeochemical and microbiological characterization of a microbial community inhabiting deep-sea sediments overlying a natural CO2 lake at the Yonaguni Knoll IV hydrothermal field, southern Okinawa Trough. We found high abundances (>10(9) cm(-3)) of microbial cells in sediment pavements above the CO2 lake, decreasing to strikingly low cell numbers (10(7) cm(-3)) at the liquid CO2/CO2-hydrate interface. The key groups in these sediments were as follows: (i) the anaerobic methanotrophic archaea ANME-2c and the Eel-2 group of Deltaproteobacteria and (ii) sulfur-metabolizing chemolithotrophs within the Gamma- and Epsilonproteobacteria. The detection of functional genes related to one-carbon assimilation and the presence of highly 13C-depleted archaeal and bacterial lipid biomarkers suggest that microorganisms assimilating CO2 and/or CH4 dominate the liquid CO2 and CO2-hydrate-bearing sediments. Clearly, the Yonaguni Knoll is an exceptional natural laboratory for the study of consequences of CO2 disposal as well as of natural CO2 reservoirs as potential microbial habitats on early Earth and other celestial bodies.