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Detailed knowledge of the extent of post-genetic modifications affecting shallow submarine hydrocarbons fueled from the deep subsurface is fundamental for evaluating source and reservoir properties. We investigated gases from a submarine high-flux seepage site in the anoxic Eastern Black Sea in order to elucidate molecular and isotopic alterations of low-molecular-weight hydrocarbons (LMWHC) associated with upward migration through the sediment and precipitation of shallow gas hydrates. For this, near-surface sediment pressure cores and free gas venting from the seafloor were collected using autoclave technology at the Batumi seep area at 845 m water depth within the gas hydrate stability zone. Vent gas, gas from pressure core degassing, and from hydrate dissociation were strongly dominated by methane (> 99.85 mol.% of ∑[C1-C4, CO2]). Molecular ratios of LMWHC (C1/[C2 + C3] > 1000) and stable isotopic compositions of methane (δ13C = - 53.5‰ V-PDB; D/H around - 175‰ SMOW) indicated predominant microbial methane formation. C1/C2+ ratios and stable isotopic compositions of LMWHC distinguished three gas types prevailing in the seepage area. Vent gas discharged into bottom waters was depleted in methane by > 0.03 mol.% (∑[C1-C4, CO2]) relative to the other gas types and the virtual lack of 14C-CH4 indicated a negligible input of methane from degradation of fresh organic matter. Of all gas types analyzed, vent gas was least affected by molecular fractionation, thus, its origin from the deep subsurface rather than from decomposing hydrates in near-surface sediments is likely. As a result of the anaerobic oxidation of methane, LMWHC in pressure cores in top sediments included smaller methane fractions [0.03 mol.% ∑(C1-C4, CO2)] than gas released from pressure cores of more deeply buried sediments, where the fraction of methane was maximal due to its preferential incorporation in hydrate lattices. No indications for stable carbon isotopic fractionations of methane during hydrate crystallization from vent gas were found. Enrichments of 14C-CH4 (1.4 pMC) in short cores relative to lower abundances (max. 0.6 pMC) in gas from long cores and gas hydrates substantiates recent methanogenesis utilizing modern organic matter deposited in top sediments of this high-flux hydrocarbon seep area. © 2009 Elsevier B.V. All rights reserved.

The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. Organic acids like acetate are common in these ecosystems, but their role in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we develop an isotope-exchange 'clock' based on the abiotic equilibration of H-isotopes between acetate and water, which can be used to define the maximum in situ acetate residence time. We apply this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we find that acetate residence times are <1 million years and calculated a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates the utility of isotope-exchange clocks in determining residence times of biomolecules with possible applications to other environments., (© 2024. The Author(s).)

Reactive iron (Fe R ) serves as an important sink of organic carbon (OC) in marine surface sediments, which preserves approximately 20% of total OC (TOC) as reactive iron-bound OC (Fe R -OC). However, the fate of Fe R -OC in subseafloor sediments and its availability to microorganisms, remain undetermined. Here, we reconstructed continuous Fe R -OC records in two sediment cores of the northern South China Sea encompassing the suboxic to methanic biogeochemical zones and reaching a maximum age of ~100 kyr. The downcore Fe R -OC contributes a relatively stable proportion of 13.3 ± 3.2% to TOC. However, distinctly lower values of less than 5% of TOC, accompanied by notable 13 C depletion of Fe R -OC, are observed in the sulfate-methane transition zone (SMTZ). Fe R -OC is suggested to be remobilized by microbially mediated reductive dissolution of Fe R and subsequently remineralized, the flux of which is 18-30% of the methane consumption in the SMTZ. The global reservoir of Fe R -OC in microbially active Quaternary marine sediments could be 19-46 times the size of the atmospheric carbon pool. Thus, the Fe R -OC pool may support subseafloor microorganisms and contribute to regulating Earth's carbon cycle., (© 2024. The Author(s).)

Organic matter (OM) transformations in marine sediments play a crucial role in the global carbon cycle. However, secondary production and priming have been ignored in marine biogeochemistry. By incubating shelf sediments with various 13 C-labeled algal substrates for 400 days, we show that ~65% of the lipids and ~20% of the proteins were mineralized by numerically minor heterotrophic bacteria as revealed by RNA stable isotope probing. Up to 11% of carbon from the algal lipids was transformed into the biomass of secondary producers as indicated by 13 C incorporation in amino acids. This biomass turned over throughout the experiment, corresponding to dynamic microbial shifts. Algal lipid addition accelerated indigenous OM degradation by 2.5 to 6 times. This priming was driven by diverse heterotrophic bacteria and sulfur- and iron-cycling bacteria and, in turn, resulted in extra secondary production, which exceeded that stimulated by added substrates. These interactions between degradation, secondary production, and priming govern the eventual fate of OM in marine 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 (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.)

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 (δ 13 C) 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 δ 13 C-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The production and anaerobic oxidation of methane (AOM) by microorganisms is widespread in organic-rich deep subseafloor sediments. Yet, the organisms that carry out these processes remain largely unknown. Here we identify members of the methane-cycling microbial community in deep subsurface, hydrate-containing sediments of the Peru Trench by targeting functional genes of the alpha subunit of methyl coenzyme M reductase ( mcrA ). The mcrA profile reveals a distinct community zonation that partially matches the zonation of methane oxidizing and -producing activity inferred from sulfate and methane concentrations and carbon-isotopic compositions of methane and dissolved inorganic carbon (DIC). Mcr A appears absent from sulfate-rich sediments that are devoid of methane, but mcr A sequences belonging to putatively methane-oxidizing ANME-1a-b occur from the zone of methane oxidation to several meters into the methanogenesis zone. A sister group of ANME-1a-b, referred to as ANME-1d, and members of putatively aceticlastic Methanothrix (formerly Methanosaeta ) occur throughout the remaining methanogenesis zone. Analyses of 16S rRNA and mcr A-mRNA indicate that the methane-cycling community is alive throughout (rRNA to 230 mbsf) and active in at least parts of the sediment column (mRNA at 44 mbsf). Carbon-isotopic depletions of methane relative to DIC (-80 to -86‰) suggest mostly methane production by CO 2 reduction and thus seem at odds with the widespread detection of ANME-1 and Methanothrix . We explain this apparent contradiction based on recent insights into the metabolisms of both ANME-1 and Methanothricaceae , which indicate the potential for methanogenetic growth by CO 2 reduction in both groups., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lever, Alperin, Hinrichs and Teske.)

Background: A large proportion of prokaryotic microbes in marine sediments remains uncultured, hindering our understanding of their ecological functions and metabolic features. Recent environmental metagenomic studies suggested that many of these uncultured microbes contribute to the degradation of organic matter, accompanied by acetogenesis, but the supporting experimental evidence is limited., Results: Estuarine sediments were incubated with different types of organic matters under anaerobic conditions, and the increase of uncultured bacterial populations was monitored. We found that (1) lignin stimulated the increase of uncultured bacteria within the class Dehalococcoidia. Their ability to metabolize lignin was further supported by the presence of genes associated with a nearly complete degradation pathway of phenolic monomers in the Dehalococcoidia metagenome-assembled genomes (MAGs). (2) The addition of cellulose stimulated the increase of bacteria in the phylum Ca. Fermentibacterota and family Fibrobacterales, a high copy number of genes encoding extracellular endoglucanase or/and 1,4-beta-cellobiosidase for cellulose decomposition and multiple sugar transporters were present in their MAGs. (3) Uncultured lineages in the order Bacteroidales and the family Leptospiraceae were enriched by the addition of casein and oleic acid, respectively, a high copy number of genes encoding extracellular peptidases, and the complete β-oxidation pathway were found in those MAGs of Bacteroidales and Leptospiraceae, respectively. (4) The growth of unclassified bacteria of the order Clostridiales was found after the addition of both casein and cellulose. Their MAGs contained multiple copies of genes for extracellular peptidases and endoglucanase. Additionally, 13 C-labeled acetate was produced in the incubations when 13 C-labeled dissolved inorganic carbon was provided., Conclusions: Our results provide new insights into the roles of microorganisms during organic carbon degradation in anaerobic estuarine sediments and suggest that these macro and single molecular organic carbons support the persistence and increase of uncultivated bacteria. Acetogenesis is an additional important microbial process alongside organic carbon degradation. Video Abstract., (© 2023. The Author(s).)

The exact drivers for the end-Permian mass extinction (EPME) remain controversial. Here we focus on a ~10,000 yr record from the marine type section at Meishan, China, preceding and covering the onset of the EPME. Analyses of polyaromatic hydrocarbons at sampling intervals representing 1.5-6.3 yr reveal recurrent pulses of wildfires in the terrestrial realm. Massive input pulses of soil-derived organic matter and clastic materials into the oceans are indicated by patterns of C 2 -dibenzofuran, C 30 hopane and aluminum. Importantly, in the ~2,000 years preceding the main phase of the EPME, we observe a clearly defined sequence of wildfires, soil weathering, and euxinia provoked by the fertilization of the marine environment with soil-derived nutrients. Euxinia is indicated by sulfur and iron concentrations. Our study suggests that, in South China, centennial scale processes led to a collapse of the terrestrial ecosystem ~300 yr (120-480 yr; ± 2 s.d.) before the onset of the EPME and that this collapse induced euxinic conditions in the ocean, ultimately resulting in the demise of marine ecosystems., (© 2023. The Author(s).)

For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that electrospray-ionization Orbitraps and other mass spectrometers widely used in the life sciences can be fine-tuned for high-precision isotope ratio analysis. Since isotope patterns form everywhere in nature based on well-understood principles, intramolecular isotope measurements allow unique insights into a fascinating range of research topics. This Perspective introduces a wider readership to current topics in stable isotope research with the aim of discussing how soft-ionization mass spectrometry coupled with ultrahigh mass resolution can enable long-envisioned progress. We highlight novel prospects of observing isotopes in intact polar compounds and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology.

Methods that unambiguously prove microbial plastic degradation and allow for quantification of degradation rates are necessary to constrain the influence of microbial degradation on the marine plastic budget. We developed an assay based on stable isotope tracer techniques to determine microbial plastic mineralization rates in liquid medium on a lab scale. For the experiments, 13 C-labeled polyethylene ( 13 C-PE) particles (irradiated with UV-light to mimic exposure of floating plastic to sunlight) were incubated in liquid medium with Rhodococcus ruber as a model organism for proof of principle. The transfer of 13 C from 13 C-PE into the gaseous and dissolved CO 2 pools translated to microbially mediated mineralization rates of up to 1.2 % yr -1 of the added PE. After incubation, we also found highly 13 C-enriched membrane fatty acids of R. ruber including compounds involved in cellular stress responses. We demonstrated that isotope tracer techniques are a valuable tool to detect and quantify microbial plastic degradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mud volcanoes transport deep fluidized sediment and their microbial communities and thus provide a window into the deep biosphere. However, mud volcanoes are commonly sampled at the surface and not probed at greater depths, with the consequence that their internal geochemistry and microbiology remain hidden from view. Urania Basin, a hypersaline seafloor basin in the Mediterranean, harbors a mud volcano that erupts fluidized mud into the brine. The vertical mud pipe was amenable to shipboard Niskin bottle and multicorer sampling and provided an opportunity to investigate the downward sequence of bacterial and archaeal communities of the Urania Basin brine, fluid mud layers and consolidated subsurface sediments using 16S rRNA gene sequencing. These microbial communities show characteristic, habitat-related trends as they change throughout the sample series, from extremely halophilic bacteria (KB1) and archaea ( Halodesulfoarchaeum spp.) in the brine, toward moderately halophilic and thermophilic endospore-forming bacteria and uncultured archaeal lineages in the mud fluid, and finally ending in aromatics-oxidizing bacteria, uncultured spore formers, and heterotrophic subsurface archaea (Thermoplasmatales, Bathyarchaeota, and Lokiarcheota) in the deep subsurface sediment at the bottom of the mud volcano. Since these bacterial and archaeal lineages are mostly anaerobic heterotrophic fermenters, the microbial ecosystem in the brine and fluidized mud functions as a layered fermenter for the degradation of sedimentary biomass and hydrocarbons. By spreading spore-forming, thermophilic Firmicutes during eruptions, the Urania Basin mud volcano likely functions as a source of endospores that occur widely in cold seafloor sediments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lazar, Schmidt, Elvert, Heuer, Hinrichs and Teske.)

The relatively stable Holocene climate was preceded by a pronounced event of abrupt warming in the Northern Hemisphere, the termination of the Younger Dryas (YD) cold period 1,2 . Although this transition has been intensively studied, its imprint on low-latitude ocean temperature is still controversial and its effects on sub-annual to decadal climate variability remain poorly understood 1,3,4 . Sea surface temperature (SST) variability at these timescales in the tropical Atlantic is expected to intensify under current and future global warming and has considerable consequences for environmental conditions in Africa and South America, and for tropical Pacific climate 5-8 . Here we present a 100-µm-resolution record obtained by mass spectrometry imaging (MSI) of long-chain alkenones in sediments from the Cariaco Basin 9-11 and find that annually averaged SST remained stable during the transition into the Holocene. However, seasonality increased more than twofold and approached modern values of 1.6 °C, probably driven by the position and/or annual range of the Intertropical Convergence Zone (ITCZ). We further observe that interannual variability intensified during the early Holocene. Our results demonstrate that sub-decadal-scale SST variability in the tropical Atlantic is sensitive to abrupt changes in climate background, such as those witnessed during the most recent glacial to interglacial transition., (© 2022. The Author(s).)

The greenhouse gas methane (CH 4 ) is of pivotal importance for Earth's climate system and as a human energy source. A significant fraction of this CH 4 is produced by anaerobic Archaea . Here, we describe the first CH 4 production by facultative anaerobic wood-rot fungi during growth on hydroxylated/carboxylated aromatic compounds, including lignin and lignite. The amount of CH 4 produced by fungi is positively correlated with the amount of CH 3 Cl produced during the rapid growth period of the fungus. Biochemical, genetic, and stable isotopic tracer analyses reveal the existence of a novel halomethane-dependent fungal CH 4 production pathway during the degradation of phenol and benzoic acid monomers and polymers and utilization of cyclic sugars. Even though this halomethane-dependent pathway may only play a side role in anaerobic fungal activity, it could represent a globally significant, previously overlooked source of biogenic CH 4 in natural ecosystems. IMPORTANCE Here, we demonstrate that wood-rot fungi produce methane anaerobically without the involvement of methanogenic archaea via a new, halomethane-dependent pathway. These findings of an anaerobic fungal methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tourte, Coffinet, Wörmer, Lipp, Hinrichs and Oger.)

Consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria mediate the anaerobic oxidation of methane (AOM) in marine sediments. However, even sediment-free cultures contain a substantial number of additional microorganisms not directly related to AOM. To track the heterotrophic activity of these community members and their possible relationship with AOM, we amended meso- (37°C) and thermophilic (50°C) AOM cultures (dominated by ANME-1 archaea and their partner bacteria of the Seep-SRB2 clade or Candidatus Desulfofervidus auxilii) with L-leucine-3- 13 C ( 13 C-leu). Various microbial lipids incorporated the labeled carbon from this amino acid, independent of the presence of methane as an energy source, specifically bacterial fatty acids, such as iso and anteiso -branched C 15:0 and C 17:0 , as well as unsaturated C 18:1ω9 and C 18:1ω7 . In natural methane-rich environments, these bacterial fatty acids are strongly 13 C-depleted. We, therefore, suggest that those fatty acids are produced by ancillary bacteria that grow on 13 C-depleted necromass or cell exudates/lysates of the AOM core communities. Candidates that likely benefit from AOM biomass are heterotrophic bacterial members of the Spirochetes and Anaerolineae-known to produce abundant branched fatty acids and present in all the AOM enrichment cultures. For archaeal lipids, we observed minor 13 C-incorporation, but still suggesting some 13 C-leu anabolism. Based on their relatively high abundance in the culture, the most probable archaeal candidates are Bathyarchaeota, Thermoplasmatales, and Lokiarchaeota. The identified heterotrophic bacterial and archaeal ancillary members are likely key players in organic carbon recycling in anoxic marine sediments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor SE declared a past co-authorship with one of the authors, GW., (Copyright © 2022 Zhu, Wegener, Hinrichs and Elvert.)

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea-type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn-glycerol 1-phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O-acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first-time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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 - 13 C]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.

A fourth of the global seabed sediment volume is buried at depths where temperatures exceed 80 °C, a previously proposed thermal barrier for life in the subsurface. Here, we demonstrate, utilizing an extensive suite of radiotracer experiments, the prevalence of active methanogenic and sulfate-reducing populations in deeply buried marine sediment from the Nankai Trough subduction zone, heated to extreme temperature (up to ~120 °C). The small microbial community subsisted with high potential cell-specific rates of energy metabolism, which approach the rates of active surface sediments and laboratory cultures. Our discovery is in stark contrast to the extremely low metabolic rates otherwise observed in the deep subseafloor. As cells appear to invest most of their energy to repair thermal cell damage in the hot sediment, they are forced to balance delicately between subsistence near the upper temperature limit for life and a rich supply of substrates and energy from thermally driven reactions of the sedimentary organic matter., (© 2022. The Author(s).)

A mechanistic understanding of formation pathways of low-molecular-weight hydrocarbons is relevant for disciplines such as atmospheric chemistry, geology, and astrobiology. The patterns of stable carbon isotopic compositions (δ 13 C) of hydrocarbons are commonly used to distinguish biological, thermogenic, and abiotic sources. Here, we report unusual isotope patterns of nonmethane hydrocarbons in hydrothermally heated sediments of the Guaymas Basin; these nonmethane hydrocarbons are notably 13 C-enriched relative to sedimentary organic matter and display an isotope pattern that is reversed relative to thermogenic hydrocarbons (i.e., δ 13 C ethane > δ 13 C propane > δ 13 C n -butane > δ 13 C n -pentane). We hypothesized that this pattern results from abiotic reductive conversion of volatile fatty acids, which were isotopically enriched due to prior equilibration of their carboxyl carbon with dissolved inorganic carbon. This hypothesis was tested by hydrous pyrolysis experiments with isotopically labeled substrates at 350 °C and 400 bar that demonstrated 1) the exchange of carboxyl carbon of C 2 to C 5 volatile fatty acids with 13 C-bicarbonate and 2) the incorporation of 13 C from 13 C-2-acetic acid into ethane and propane. Collectively, our results reveal an abiotic formation pathway for nonmethane hydrocarbons, which may be sufficiently active in organic-rich, geothermally heated sediments and petroleum systems to affect isotopic compositions of nonmethane hydrocarbons., Competing Interests: The authors declare no competing interest.

The characterization of metabolically active fungal isolates within the deep marine subsurface will alter current ecosystem models and living biomass estimates that are limited to bacterial and archaeal populations. Although marine fungi have been studied for over fifty years, a detailed description of fungal populations within the deep subsurface is lacking. Fungi possess metabolic pathways capable of utilizing previously considered non-bioavailable energy reserves. Therefore, metabolically active fungi would occupy a unique niche within subsurface ecosystems, with the potential to provide an organic carbon source for heterotrophic prokaryotic populations from the transformation of non-bioavailable energy into substrates, as well as from the fungal necromass itself. These organic carbon sources are not currently being considered in subsurface energy budgets. Sediments from South Pacific Gyre subsurface, one of the most energy-limited environments on Earth, were collected during the Integrated Ocean Drilling Program Expedition 329. Anoxic and oxic sediment slurry enrichments using fresh sediment were used to isolate multiple fungal strains in media types that varied in organic carbon substrates and concentration. Metabolically active and dormant fungal populations were also determined from nucleic acids extracted from in situ cryopreserved South Pacific Gyre sediments. For further characterization of physical growth parameters, two isolates were chosen based on their representation of the whole South Pacific Gyre fungal community. Results from this study show that fungi have adapted to be metabolically active and key community members in South Pacific Gyre sediments and potentially within global biogeochemical cycles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kiel Reese, Sobol, Bowles and Hinrichs.)

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8&#95;13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe 2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Microbial life in marine sediment contributes substantially to global biomass and is a crucial component of the Earth system. Subseafloor sediment includes both aerobic and anaerobic microbial ecosystems, which persist on very low fluxes of bioavailable energy over geologic time. However, the taxonomic diversity of the marine sedimentary microbial biome and the spatial distribution of that diversity have been poorly constrained on a global scale. We investigated 299 globally distributed sediment core samples from 40 different sites at depths of 0.1 to 678 m below the seafloor. We obtained ∼47 million 16S ribosomal RNA (rRNA) gene sequences using consistent clean subsampling and experimental procedures, which enabled accurate and unbiased comparison of all samples. Statistical analysis reveals significant correlations between taxonomic composition, sedimentary organic carbon concentration, and presence or absence of dissolved oxygen. Extrapolation with two fitted species-area relationship models indicates taxonomic richness in marine sediment to be 7.85 × 10 3 to 6.10 × 10 5 and 3.28 × 10 4 to 2.46 × 10 6 amplicon sequence variants for Archaea and Bacteria, respectively. This richness is comparable to the richness in topsoil and the richness in seawater, indicating that Bacteria are more diverse than Archaea in Earth's global biosphere., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Microbial mats from alkaline hot springs in the Yellowstone National Park are ideal natural laboratories to study photosynthetic life under extreme conditions, as well as the nuanced interactions of oxygenic and anoxygenic phototrophs. They represent distinctive examples of chlorophototroph (i.e., chlorophyll or bacteriochlorophyll-based phototroph) diversity, and several novel phototrophs have been first described in these systems, all confined in space, coexisting and competing for niches defined by parameters such as light, oxygen, or temperature. In a novel approach, we employed mass spectrometry imaging of chloropigments, quinones, and intact polar lipids (IPLs) to describe the spatial distribution of different groups of chlorophototrophs along the ~ 1 cm thick microbial mat at 75 µm resolution and in the top ~ 1.5 mm green part of the mat at 25 µm resolution. We observed a fine-tuned sequence of oxygenic and anoxygenic chlorophototrophs with distinctive biomarker signatures populating the microbial mat. The transition of oxic to anoxic conditions is characterized by an accumulation of biomarkers indicative of anoxygenic phototrophy. It is also identified as a clear boundary for different species and ecotypes, which adjust their biomarker inventory, particularly the interplay of quinones and chloropigments, to prevailing conditions. Colocalization of the different biomarker groups led to the identification of characteristic IPL signatures and indicates that glycosidic diether glycerolipids are diagnostic for anoxygenic phototrophs in this mat system. The zoom-in into the upper green part further reveals how oxygenic and anoxygenic phototrophs share this microenvironment and informs on subtle, microscale adjustments in lipid composition of Synechococcus spp., (© 2020 The Authors. Geobiology published by John Wiley & Sons Ltd.)

Dual stable isotope probing has been used to infer rates of microbial biomass production and modes of carbon fixation. In order to validate this approach for assessing archaeal production, the methanogenic archaeon Methanosarcina barkeri was grown either with H 2 , acetate or methanol with D 2 O and 13 C-dissolved inorganic carbon (DIC). Our results revealed unexpectedly low D incorporation into lipids, with the net fraction of water-derived hydrogen amounting to 0.357 ± 0.042, 0.226 ± 0.003 and 0.393 ± 0.029 for growth on H 2 /CO 2 , acetate and methanol respectively. The variability in net water H assimilation into lipids during the growth of M. barkeri on different substrates is possibly attributed to different Gibbs free energy yields, such that higher energy yield promoted the exchange of hydrogen between medium water and lipids. Because NADPH likely serves as the portal for H transfer, increased NADPH production and/or turnover associated with high energy yield may explain the apparent differences in net water H assimilation into lipids. The variable DIC and water H incorporation into M. barkeri lipids imply systematic, metabolic patterns of isotope incorporation and suggest that the ratio of 13 C-DIC versus D 2 O assimilation in environmental samples may serve as a proxy for microbial energetics in addition to microbial production and carbon assimilation pathways., (© 2020 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 TEX 86 . 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., (Copyright © 2020 Ma, Coffinet, Lipp, Hinrichs and Zhang.)

Sediment-hosted CO 2 -rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO 2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments., Competing Interests: The authors have declared that no competing interests exist.

Marine microalgae sequester as much CO 2 into carbohydrates as terrestrial plants. Polymeric carbohydrates (i.e., glycans) provide carbon for heterotrophic organisms and constitute a carbon sink in the global oceans. The quantitative contributions of different algal glycans to cycling and sequestration of carbon remain unknown, partly because of the analytical challenge to quantify glycans in complex biological matrices. Here, we quantified a glycan structural type using a recently developed biocatalytic strategy, which involves laminarinase enzymes that specifically cleave the algal glycan laminarin into readily analyzable fragments. We measured laminarin along transects in the Arctic, Atlantic, and Pacific oceans and during three time series in the North Sea. These data revealed a median of 26 ± 17% laminarin within the particulate organic carbon pool. The observed correlation between chlorophyll and laminarin suggests an annual production of algal laminarin of 12 ± 8 gigatons: that is, approximately three times the annual atmospheric carbon dioxide increase by fossil fuel burning. Moreover, our data revealed that laminarin accounted for up to 50% of organic carbon in sinking diatom-containing particles, thus substantially contributing to carbon export from surface waters. Spatially and temporally variable laminarin concentrations in the sunlit ocean are driven by light availability. Collectively, these observations highlight the prominent ecological role and biogeochemical function of laminarin in oceanic carbon export and energy flow to higher trophic levels., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The flux of methane, a potent greenhouse gas, from the seabed is largely controlled by anaerobic oxidation of methane (AOM) coupled to sulfate reduction (S-AOM) in the sulfate methane transition (SMT). S-AOM is estimated to oxidize 90% of the methane produced in marine sediments and is mediated by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria. An additional methane sink, i.e., iron oxide coupled AOM (Fe-AOM), has been suggested to be active in the methanic zone of marine sediments. Geochemical signatures below the SMT such as high dissolved iron, low to undetectable sulfate and high methane concentrations, together with the presence of iron oxides are taken as prerequisites for this process. So far, Fe-AOM has neither been proven in marine sediments nor have the governing key microorganisms been identified. Here, using a multidisciplinary approach, we show that Fe-AOM occurs in iron oxide-rich methanic sediments of the Helgoland Mud Area (North Sea). When sulfate reduction was inhibited, different iron oxides facilitated AOM in long-term sediment slurry incubations but manganese oxide did not. Especially magnetite triggered substantial Fe-AOM activity and caused an enrichment of ANME-2a archaea. Methane oxidation rates of 0.095 ± 0.03 nmol cm -3 d -1 attributable to Fe-AOM were obtained in short-term radiotracer experiments. The decoupling of AOM from sulfate reduction in the methanic zone further corroborated that AOM was iron oxide-driven below the SMT. Thus, our findings prove that Fe-AOM occurs in methanic marine sediments containing mineral-bound ferric iron and is a previously overlooked but likely important component in the global methane budget. This process has the potential to sustain microbial life in the deep biosphere., (Copyright © 2020 Aromokeye, Kulkarni, Elvert, Wegener, Henkel, Coffinet, Eickhorst, Oni, Richter-Heitmann, Schnakenberg, Taubner, Wunder, Yin, Zhu, Hinrichs, Kasten and Friedrich.)

Marine sediments host an unexpectedly large microbial biosphere, suggesting unique microbial mechanisms for surviving burial and slow metabolic turnover. Although dormancy is generally considered an important survival strategy, its specific role in subsurface sediments remains unclear. We quantified dormant bacterial endospores in 331 marine sediment samples from diverse depositional types and geographical origins. The abundance of endospores relative to vegetative cells increased with burial depth and endospores became dominant below 25 m, with an estimated population of 2.5 × 10 28 to 1.9 × 10 29 endospores in the uppermost kilometer of sediment and a corresponding biomass carbon of 4.6 to 35 Pg surpassing that of vegetative cells. Our data further identify distinct endospore subgroups with divergent resistance to burial and aging. Endospores may shape the deep biosphere by providing a core population for colonization of new habitats and/or through low-frequency germination to sustain slow growth in this environment.

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

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (10 2 to 10 3 cells cm -3 ) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Members of the archaeal phylum Bathyarchaeota are among the most abundant microorganisms on Earth. Although versatile metabolic capabilities such as acetogenesis, methanogenesis, and fermentation have been suggested for bathyarchaeotal members, no direct confirmation of these metabolic functions has been achieved through growth of Bathyarchaeota in the laboratory. Here we demonstrate, on the basis of gene-copy numbers and probing of archaeal lipids, the growth of Bathyarchaeota subgroup Bathy-8 in enrichments of estuarine sediments with the biopolymer lignin. Other organic substrates (casein, oleic acid, cellulose, and phenol) did not significantly stimulate growth of Bathyarchaeota Meanwhile, putative bathyarchaeotal tetraether lipids incorporated 13 C from 13 C-bicarbonate only when added in concert with lignin. Our results are consistent with organoautotrophic growth of a bathyarchaeotal group with lignin as an energy source and bicarbonate as a carbon source and shed light into the cycling of one of Earth's most abundant biopolymers in anoxic marine sediment., Competing Interests: The authors declare no conflict of interest.

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.)

Temperature exerts a first-order control on microbial populations, which constantly adjust the fluidity and permeability of their cell membrane lipids to minimize loss of energy by ion diffusion across the membrane. Analytical advances in liquid chromatography coupled to mass spectrometry have allowed the detection of a stunning diversity of bacterial and archaeal lipids in extreme environments such as hot springs, hydrothermal vents and deep subsurface marine sediments. Here, we investigated a thermal gradient from 18 to 101°C across a marine sediment field and tested the hypothesis that cell membrane lipids provide a major biochemical basis for the bioenergetics of archaea and bacteria under heat stress. This paper features a detailed lipidomics approach with the focus on membrane lipid structure-function. Membrane lipids analyzed here include polar lipids of bacteria and polar and core lipids of archaea. Reflecting the low permeability of their ether-linked isoprenoids, we found that archaeal polar lipids generally dominate over bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids. A close examination of archaeal and bacterial lipids revealed a membrane quandary: not only low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. It is possible that these configurations toward a more fluidized membrane at elevated temperatures are counterbalanced by the high abundance of archaeal glycolipids and bacterial sphingolipids, which could reduce membrane permeability through strong intermolecular hydrogen bonding. Our results provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems.

Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia-oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane-spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The unique geochemistry of marine shallow-water hydrothermal systems promotes the establishment of diverse microbial communities with a range of metabolic pathways. In contrast to deep-sea vents, shallow-water vents not only support chemosynthesis, but also phototrophic primary production due to the availability of light. However, comprehensive studies targeting the predominant biogeochemical processes are rare, and consequently a holistic understanding of the functioning of these ecosystems is currently lacking. To this end, we combined stable isotope probing of lipid biomarkers with an analysis of the bacterial communities to investigate if chemoautotrophy, in parallel to photoautotrophy, plays an important role in autotrophic carbon fixation and to identify the key players. The study was carried out at a marine shallow-water hydrothermal system located at 5 m water depth off Dominica Island (Lesser Antilles), characterized by up to 55°C warm hydrothermal fluids that contain high amounts of dissolved Fe 2+ . Analysis of the bacterial diversity revealed Anaerolineae of the Chloroflexi as the most abundant bacterial class. Furthermore, the presence of key players involved in iron cycling generally known from deep-sea hydrothermal vents (e.g., Zetaproteobacteria and Geothermobacter ), supported the importance of iron-driven redox processes in this hydrothermal system. Uptake of 13 C-bicarbonate into bacterial fatty acids under light and dark conditions revealed active photo- and chemoautotrophic communities, with chemoautotrophy accounting for up to 65% of the observed autotrophic carbon fixation. Relatively increased 13 C-incorporation in the dark allowed the classification of ai C 15:0 , C 15:0 , and i C 16:0 as potential lipid biomarkers for bacterial chemoautotrophy in this ecosystem. Highest total 13 C-incorporation into fatty acids took place at the sediment surface, but chemosynthesis was found to be active down to 8 cm sediment depth. In conclusion, this study highlights the relative importance of chemoautotrophy compared to photoautotrophy in a shallow-water hydrothermal system, emphasizing chemosynthesis as a prominent process for biomass production in marine coastal environments influenced by hydrothermalism.

Ocean margin sediments have been considered as important sources of dissolved organic carbon (DOC) to the deep ocean, yet the contribution from advective settings has just started to be acknowledged. Here we present evidence showing that near-surface heating of sediment in the Guaymas Basin, a young extensional depression, causes mass production and discharge of reactive dissolved organic matter (DOM). In the sediment heated up to ~100 °C, we found unexpectedly low DOC concentrations in the pore waters, reflecting the combined effect of thermal desorption and advective fluid flow. Heating experiments suggested DOC production to be a rapid, abiotic process with the DOC concentration increasing exponentially with temperature. The high proportions of total hydrolyzable amino acids and presence of chemical species affiliated with activated hydrocarbons, carbohydrates and peptides indicate high reactivity of the DOM. Model simulation suggests that at the local scale, near-surface heating of sediment creates short and massive DOC discharge events that elevate the bottom-water DOC concentration. Because of the heterogeneous distribution of high heat flow areas, the expulsion of reactive DOM is spotty at any given time. We conclude that hydrothermal heating of young rift sediments alter deep-ocean budgets of bioavailable DOM, creating organic-rich habitats for benthic life.

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal-associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal-sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ∼20 million years of depositional history., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In the deep ocean, the conversion of methane into derived carbon and energy drives the establishment of diverse faunal communities. Yet specific biological mechanisms underlying the introduction of methane-derived carbon into the food web remain poorly described, due to a lack of cultured representative deep-sea methanotrophic prokaryotes. Here, the response of the deep-sea aerobic methanotroph Methyloprofundus sedimenti to methane starvation and recovery was characterized. By combining lipid analysis, RNA analysis, and electron cryotomography, it was shown that M. sedimenti undergoes discrete cellular shifts in response to methane starvation, including changes in headgroup-specific fatty acid saturation levels, and reductions in cytoplasmic storage granules. Methane starvation is associated with a significant increase in the abundance of gene transcripts pertinent to methane oxidation. Methane reintroduction to starved cells stimulates a rapid, transient extracellular accumulation of methanol, revealing a way in which methane-derived carbon may be routed to community members. This study provides new understanding of methanotrophic responses to methane starvation and recovery, and lays the initial groundwork to develop Methyloprofundus as a model chemosynthesizing bacterium from the deep sea., (© 2016 John Wiley & Sons Ltd.)

Membrane lipids of marine planktonic archaea have provided unique insights into archaeal ecology and paleoceanography. However, past studies of archaeal lipids in suspended particulate matter (SPM) and sediments mainly focused on a small class of fully saturated glycerol dibiphytanyl glycerol tetraether (GDGT) homologues identified decades ago. The apparent low structural diversity of GDGTs is in strong contrast to the high diversity of metabolism and taxonomy among planktonic archaea. Furthermore, adaptation of archaeal lipids in the deep ocean remains poorly constrained. We report the archaeal lipidome in SPM from diverse oceanic regimes. We extend the known inventory of planktonic archaeal lipids to include numerous unsaturated archaeal ether lipids (uns-AELs). We further reveal (i) different thermal regulations and polar headgroup compositions of membrane lipids between the epipelagic (≤ 100 m) and deep (>100 m) populations of archaea, (ii) stratification of unsaturated GDGTs with varying redox conditions, and (iii) enrichment of tetra-unsaturated archaeol and fully saturated GDGTs in epipelagic and deep oxygenated waters, respectively. Such stratified lipid patterns are consistent with the typical distribution of archaeal phylotypes in marine environments. We, thus, provide an ecological context for GDGT-based paleoclimatology and bring about the potential use of uns-AELs as biomarkers for planktonic Euryarchaeota., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sulfate reduction is the predominant anaerobic microbial process of organic matter mineralization in marine sediments, with recent studies revealing that sulfate reduction not only occurs in sulfate-rich sediments, but even extends to deeper, methanogenic sediments at very low background concentrations of sulfate. Using samples retrieved off the Shimokita Peninsula, Japan, during the Integrated Ocean Drilling Program (IODP) Expedition 337, we measured potential sulfate reduction rates by slurry incubations with 35 S-labeled sulfate in deep methanogenic sediments between 1276.75 and 2456.75 meters below the seafloor. Potential sulfate reduction rates were generally extremely low (mostly below 0.1 pmol cm -3 d -1 ) but showed elevated values (up to 1.8 pmol cm -3 d -1 ) in a coal-bearing interval (Unit III). A measured increase in hydrogenase activity in the coal-bearing horizons coincided with this local increase in potential sulfate reduction rates. This paired enzymatic response suggests that hydrogen is a potentially important electron donor for sulfate reduction in the deep coalbed biosphere. By contrast, no stimulation of sulfate reduction rates was observed in treatments where methane was added as an electron donor. In the deep coalbeds, small amounts of sulfate might be provided by a cryptic sulfur cycle. The isotopically very heavy pyrites (δ 34 S = +43‰) found in this horizon is consistent with its formation via microbial sulfate reduction that has been continuously utilizing a small, increasingly 34 S-enriched sulfate reservoir over geologic time scales. Although our results do not represent in-situ activity, and the sulfate reducers might only have persisted in a dormant, spore-like state, our findings show that organisms capable of sulfate reduction have survived in deep methanogenic sediments over more than 20 Ma. This highlights the ability of sulfate-reducers to persist over geological timespans even in sulfate-depleted environments. Our study moreover represents the deepest evidence of a potential for sulfate reduction in marine sediments to date.