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Background: The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy-the utilization of organic carbon as energy source but inorganic carbon as sole carbon source-has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8)., Results: To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13 C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13 C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13 C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility., Conclusion: We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. Video Abstract., (© 2024. The Author(s).)

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 (δ 13 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 δ 13 C values indicate active methane metabolism. Sedimentary CL- and IPL-crenarchaeol were exclusively derived from the water column, as indicated by non-variable δ 13 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 TEX 86 , 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., (© 2024 The Authors. Geobiology published by John Wiley & Sons Ltd.)

Metagenomic analysis has facilitated prediction of a variety of carbon utilization potentials by uncultivated archaea including degradation of protein, which is a wide-spread carbon polymer in marine sediments. However, the activity of detrital catabolic protein degradation is mostly unknown for the vast majority of archaea. Here, we show actively executed protein catabolism in three archaeal phyla (uncultivated Thermoplasmata, SG8-5; Bathyarchaeota subgroup 15; Lokiarchaeota subgroup 2c) by RNA- and lipid-stable isotope probing in incubations with different marine sediments. However, highly abundant potential protein degraders Thermoprofundales (MBG-D) and Lokiarchaeota subgroup 3 were not incorporating 13 C-label from protein during incubations. Nonetheless, we found that the pathway for protein utilization was present in metagenome associated genomes (MAGs) of active and inactive archaea. This finding was supported by screening extracellular peptidases in 180 archaeal MAGs, which appeared to be widespread but not correlated to organisms actively executing this process in our incubations. Thus, our results have important implications: (i) multiple low-abundant archaeal groups are actually catabolic protein degraders; (ii) the functional role of widespread extracellular peptidases is not an optimal tool to identify protein catabolism, and (iii) catabolic degradation of sedimentary protein is not a common feature of the abundant archaeal community in temperate and permanently cold marine sediments., (© 2022. The Author(s).)

Asgard is a recently discovered archaeal superphylum, closely linked to the emergence of eukaryotes. Among Asgard archaea, Lokiarchaeota are abundant in marine sediments, but their in situ activities are largely unknown except for Candidatus 'Prometheoarchaeum syntrophicum'. Here, we tracked the activity of Lokiarchaeota in incubations with Helgoland mud area sediments (North Sea) by stable isotope probing (SIP) with organic polymers, 13 C-labelled inorganic carbon, fermentation intermediates and proteins. Within the active archaea, we detected members of the Lokiarchaeota class Loki-3, which appeared to mixotrophically participate in the degradation of lignin and humic acids while assimilating CO 2 , or heterotrophically used lactate. In contrast, members of the Lokiarchaeota class Loki-2 utilized protein and inorganic carbon, and degraded bacterial biomass formed in incubations. Metagenomic analysis revealed pathways for lactate degradation, and involvement in aromatic compound degradation in Loki-3, while the less globally distributed Loki-2 instead rely on protein degradation. We conclude that Lokiarchaeotal subgroups vary in their metabolic capabilities despite overlaps in their genomic equipment, and suggest that these subgroups occupy different ecologic niches in marine sediments.

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., (Copyright © 2018 American Society for Microbiology.)

Anaerobic oxidation of methane (AOM) was shown to reduce methane emissions by over 50% in freshwater systems, its main natural contributor to the atmosphere. In these environments iron oxides can become main agents for AOM, but the underlying mechanism for this process has remained enigmatic. By conducting anoxic slurry incubations with lake sediments amended with 13 C-labeled methane and naturally abundant iron oxides the process was evidenced by significant 13 C-enrichment of the dissolved inorganic carbon pool and most pronounced when poorly reactive iron minerals such as magnetite and hematite were applied. Methane incorporation into biomass was apparent by strong uptake of 13 C into fatty acids indicative of methanotrophic bacteria, associated with increasing copy numbers of the functional methane monooxygenase pmoA gene. Archaea were not directly involved in full methane oxidation, but their crucial participation, likely being mediators in electron transfer, was indicated by specific inhibition of their activity that fully stopped iron-coupled AOM. By contrast, inhibition of sulfur cycling increased 13 C-methane turnover, pointing to sulfur species involvement in a competing process. Our findings suggest that the mechanism of iron-coupled AOM is accomplished by a complex microbe-mineral reaction network, being likely representative of many similar but hidden interactions sustaining life under highly reducing low energy conditions.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed., (Copyright © 2015, American Association for the Advancement of Science.)

Marine microorganisms adapt to their habitat by structural modification of their membrane lipids. This concept is the basis of numerous molecular proxies used for paleoenvironmental reconstruction. Archaeal tetraether lipids from ubiquitous marine planktonic archaea are particularly abundant, well preserved in the sedimentary record and used in several molecular proxies. We here introduce the direct, extraction-free analysis of these compounds in intact sediment core sections using laser desorption ionization (LDI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LDI FTICR-MS can detect the target lipids in single submillimeter-sized spots on sediment sections, equivalent to a sample mass in the nanogram range, and could thus pave the way for biomarker-based reconstruction of past environments and ecosystems at subannual to decadal resolution. We demonstrate that ratios of selected archaeal tetraethers acquired by LDI FTICR-MS are highly correlated with values obtained by conventional liquid chromatography/MS protocols. The ratio of the major archaeal lipids, caldarchaeol and crenarchaeol, analyzed in a 6.2-cm intact section of Mediterranean sapropel S1 at 250-µm resolution (∼ 4-y temporal resolution), provides an unprecedented view of the fine-scale patchiness of sedimentary biomarker distributions and the processes involved in proxy signal formation. Temporal variations of this lipid ratio indicate a strong influence of the ∼ 200-y de Vries solar cycle on reconstructed sea surface temperatures with possible amplitudes of several degrees, and suggest signal amplification by a complex interplay of ecological and environmental factors. Laser-based biomarker analysis of geological samples has the potential to revolutionize molecular stratigraphic studies of paleoenvironments.

Rationale: Studies of archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in the environment and cultures have exclusively focused on compounds with fully saturated alkyl moieties. Here we report a number of novel unsaturated GDGTs (uns-GDGTs) whose alkyl chains contain up to six double bonds and zero to two cyclopentyl moieties., Methods: The identification of these lipids was achieved via comparison of lipid distribution before and after hydrogenation, characteristic retention time patterns, and diagnostic ions using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and ether cleavage products using gas chromatography/mass spectrometry (GC/MS). Isomerism resulting from different unsaturation patterns in the alkyl moieties was observed and specific positions of double bonds in the biphytene and biphytadiene moieties were tentatively assigned., Results: Uns-GDGTs were detected in sediment and microbial mat samples as both core lipids (CLs) and intact polar lipids (IPLs) associated with mono- or diglycosyl or phosphatidylglycerol headgroups. However, these lipids were overlooked in past investigations because conventional methods for archaeal lipid analysis are unsuitable for uns-GDGTs. Samples from distinct marine environments (Black Sea, Cariaco Basin, Discovery Basin, Eastern Mediterranean Sea, upwelling area off NW Africa, and seep sites off Crimea and Pakistan) were screened for uns-GDGTs using a new LC/MS protocol. The results show that uns-GDGTs contribute significantly to the archaeal lipid pool in anoxic methane-rich environments (Black Sea, Cariaco Basin, and both seep sites) but they were barely detected in the oxic or hypersaline settings., Conclusions: The characteristic distribution of uns-GDGTs implies that they are attractive targets for future studies aiming at the chemotaxonomy of uncultivated archaea and regulation of uns-GDGT biosynthesis., (Copyright © 2014 John Wiley & Sons, Ltd.)

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., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Novel cardiolipins from Archaea were detected by screening the intact polar lipid (IPL) composition of microbial communities associated with methane seepage in deep-sea sediments from the Pakistan margin by high-performance liquid chromatography electrospray ionization mass spectrometry. A series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol, BPG) represented 0.5% to 5% of total archaeal IPLs. These molecules are similar to the recently described cardiolipin analogues with four phytanyl chains from extreme halophilic archaea. It is worth noting that cardiolipin analogues from the seep archaeal communities are composed of four isoprenoidal chains, which may contain differences in chain length (20 and 25 carbon atoms) and degrees of unsaturation and the presence of a hydroxyl group. Two novel diether lipids, structurally related to the BPGs, are described and interpreted as degradation products of archaeal cardiolipin analogues. Since archaeal communities in seep sediments are dominated by anaerobic methanotrophs, our observations have implications for characterizing structural components of archaeal membranes, in which BPGs are presumed to contribute to modulation of cell permeability properties. Whether BPGs facilitate interspecies interaction in syntrophic methanotrophic consortia remains to be tested.

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., (Copyright © 2010 John Wiley & Sons, Ltd.)

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5-6 months amended with either (13)CH(4) or H(13)CO(3)(-). In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of (13)CH(4) or (13)CO(2) were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by (13)CO(2), but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25-1.3 mol% of the methane oxidized.

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.

Paclele Mici is a terrestrial mud volcano field located in the Carpathian Mountains (Romania), where thermal alteration of sedimentary organic compounds leads to methane, higher hydrocarbons and other petroleum compounds that are continuously released into the environment. The hydrocarbons represent potential substrates for microorganisms. We studied lipid biomarkers, stable isotope ratios, the effect of substrate (methane, other organic compounds) addition and 16S rRNA genes to gain insights into the hitherto unknown microbial community at this site. Quantitative real-time polymerase chain reaction analysis demonstrated that bacteria were much more abundant than archaea. Phylogenetic analyses of 16S rDNA clone sequences indicated the presence of bacterial and archaeal lineages generally associated with the methane cycle (methanogens, aerobic and anaerobic methanotrophs), the sulfur cycle (sulfate reducers), and groups linked to the anaerobic degradation of alkanes or aromatic hydrocarbons. The presence of sulfate reducers, methanogens and methanotrophs in this habitat was also confirmed by concurrent surveys of lipid biomarkers and their isotopic signatures. Incubation experiments with several common and complex substrates revealed the potential of the indigenous microbial community for sulfate reduction, methanogenesis and aerobic methanotrophy. Additionally, consistently to the detection of methane-oxidizing archaea (ANME) and 13C-depleted archaeal lipids, a weak but significant activity of anaerobic methane oxidation was measured by radiotracer techniques and in vitro. This survey is the first to report the presence and activity of ANME in a terrestrial environment.

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.