Seep Sediments Research Articles

Hydrocarbon-degrading microbial populations in permanently cold deep-sea sediments in the NW Atlantic

Permanently cold deep-sea sediments (2500–3500 m water depth) with and without indications of thermogenic hydrocarbon seepage were exposed to naphtha to examine the presence and potential of cold-adapted aerobic hydrocarbon-degrading microbial populations. Monitoring these microcosms for volatile hydrocarbons by GC–MS revealed sediments without in situ hydrocarbons responded more rapidly to naphtha amendment than hydrocarbon seep sediments overall, but seep sediments removed aromatic hydrocarbons benzene, toluene, ethylbenzene and xylene (BTEX) more readily. Naphtha-driven aerobic respiration was more evident in surface sediment (0–20 cmbsf) than deeper anoxic layers (>130 cmbsf) that responded less rapidly. In all cases, enrichment of Gammaproteobacteria included lineages of Oleispira, Pseudomonas, and Alteromonas known to be associated with marine oil spills. On the other hand, taxa known to be prevalent in situ and diagnostic for thermogenic hydrocarbon seepage in deep sea sediment, did not respond to naphtha amendment. This suggests a limited role for these prevalent seep-associated populations in the context of aerobic hydrocarbon biodegradation.

Synergetic effects of chlorinated paraffins and microplastics on microbial communities and nitrogen cycling in deep-sea cold seep sediments

Chlorinated paraffins (CPs) and microplastics (MPs) are commonly found in deep-sea cold seep sediments, where nitrogen cycling processes frequently occur. However, little is known about their combined effects on sedimentary microbial communities and nitrogen cycling in these environments. This study aimed to investigate the synergistic impacts of CPs and MPs on microbial communities and nitrogen cycling in deep-sea cold seep sediments through microcosm experiments. Our results demonstrated that the presence of CPs and MPs induced significant alterations in microbial community composition, promoting the growth of Halomonas. Furthermore, CPs and MPs were found to enhance nitrification, denitrification and anammox processes, which was evidenced by the higher abundance of genes associated with nitrification and denitrification, as well as increased activity of denitrification and anammox in the CPs and MPs-treatment groups compared to the control group. Additionally, the enhanced influence of CPs and MPs on denitrification was expected to promote nitrate-dependent and sulfate-dependent anaerobic oxidation of methane, thereby resulting in less methane released into the environment. These findings shed light on the potential consequences of simultaneous exposure to CPs and MPs on biogeochemical nitrogen cycling in deep-sea cold seep sediments.

Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

Promethearchaeum syntrophicum gen. nov., sp. nov., an anaerobic, obligately syntrophic archaeon, the first isolate of the lineage 'Asgard' archaea, and proposal of the new archaeal phylum Promethearchaeota phyl. nov. and kingdom Promethearchaeati regn. nov.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of 'Asgard' archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300-750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4-30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml-1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with 'Asgard' archaea and more specifically DHVC1/DSAG/MBG-B and 'Lokiarchaeota'/'Lokiarchaeia'. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus 'Lokiarchaeum ossiferum' (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Salinity and nutrient condition as key factors drive the assembly of sediment prokaryotic communities

Despite extensive research on the geographical patterns of microbial communities, our comprehension of the mechanisms underlying their spatial distribution is still limited. Natural ecosystems provide opportunities to investigate the structure, connectivity, and assembly processes of prokaryotic communities. Saline lakes, mangroves, ocean margins, cold seeps, and open oceans as five distinct natural ecosystems exhibit varied levels of salinity and nutrient condition (carbon sources, electron donors, and electron acceptors). Based on the analysis of 197 sets of published 16S rRNA gene amplicon sequencing data on sediment samples of the five habitats, differences in salinity and nutrient conditions were identified to play a critical role in governing the composition, connectivity, and assembly process of prokaryotic communities. Specifically, unique prokaryotic community patterns were observed in these habitats, e.g., mangrove sediment communities were shown to have the highest alpha diversity and the lowest community-level ribosomal RNA gene operon (rrn) copy numbers, compared to the open ocean sediment communities. Positive correlation predominated connections (>80% of total connections) of the prokaryotic microbial networks in the five habitats. Communities within nutrient-rich saline lake and cold seep sediments exhibit the strongest and closest connections. Using the dissimilarity-overlap curve and null model, differences in composition, connectivity, and assembly process were found to be predominantly governed by deterministic forces. These findings enhance our understanding of microbial ecology in typical saline environments and enable us to investigate intricate ecosystems.

Potential coupling of microbial methane, nitrogen, and sulphur cycling in the Okinawa Trough cold seep sediments.

The Okinawa Trough (OT) is a back-arc basin with a wide distribution of active cold seep systems. However, our understanding of the metabolic function of microbial communities in the cold seep sediments of the OT remains limited. In this study, we investigated the vertical profiles of functional genes involved in methane, nitrogen, and sulphur cycling in the cold seep sediments of the OT. Furthermore, we explored the possible coupling mechanisms between these biogeochemical cycles. The study revealed that the majority of genes associated with the nitrogen and sulphur cycles were most abundant in the surface sediment layers. However, only the key genes responsible for sulphur disproportionation (sor), nitrogen fixation (nifDKH), and methane metabolism (mcrABG) were more prevalent within sulfate-methane transition zone (SMTZ). Significant positive correlations (P < 0.05) were observed between functional genes involved in sulphur oxidation, thiosulphate disproportionation with denitrification, and dissimilatory nitrate reduction to ammonium (DNRA), as well as between AOM/methanogenesis and nitrogen fixation, and between sulphur disproportionation and AOM. A genome of Filomicrobium (class Alphaproteobacteria) has demonstrated potential in chemoautotrophic activities, particularly in coupling DNRA and denitrification with sulphur oxidation. Additionally, the characterized sulfate reducers such as Syntrophobacterales have been found to be capable of utilizing nitrate as an electron acceptor. The predominant methanogenic/methanotrophic groups in the OT sediments were identified as H2-dependent methylotrophic methanogens (Methanomassiliicoccales and Methanofastidiosales) and ANME-1a. This study offered a thorough understanding of microbial ecosystems in the OT cold seep sediments, emphasizing their contribution to nutrient cycling.IMPORTANCEThe Okinawa Trough (OT) is a back-arc basin formed by extension within the continental lithosphere behind the Ryukyu Trench arc system. Cold seeps are widespread in the OT. While some studies have explored microbial communities in OT cold seep sediments, their metabolic potential remains largely unknown. In this study, we used metagenomic analysis to enhance comprehension of the microbial community's role in nutrient cycling and proposed hypotheses on the coupling process and mechanisms involved in biogeochemical cycles. It was revealed that multiple metabolic pathways can be performed by a single organism or microbes that interact with each other to carry out various biogeochemical cycling. This data set provided a genomic road map on microbial nutrient cycling in OT sediment microbial communities.

A vast repertoire of secondary metabolites potentially influences community dynamics and biogeochemical processes in cold seeps.

In deep-sea cold seeps, microbial communities thrive on the geological seepage of hydrocarbons and inorganic compounds, differing from photosynthetically driven ecosystems. However, their biosynthetic capabilities remain largely unexplored. Here, we analyzed 81 metagenomes, 33 metatranscriptomes, and 7 metabolomes derived from nine different cold seep areas to investigate their secondary metabolites. Cold seep microbiomes encode diverse and abundant biosynthetic gene clusters (BGCs). Most BGCs are affiliated with understudied bacteria and archaea, including key mediators of methane and sulfur cycling. The BGCs encode diverse antimicrobial compounds that potentially shape community dynamics and various metabolites predicted to influence biogeochemical cycling. BGCs from key players are widely distributed and highly expressed, with their abundance and expression levels varying with sediment depth. Sediment metabolomics reveals unique natural products, highlighting uncharted chemical potential and confirming BGC activity in these sediments. Overall, these results demonstrate that cold seep sediments serve as a reservoir of hidden natural products and sheds light on microbial adaptation in chemosynthetically driven ecosystems.

Targeting methanotrophs and isolation of a novel psychrophilic Methylobacter species from a terrestrial Arctic alkaline methane seep in Lagoon Pingo, Central Spitsbergen (78° N)

The microbial diversity associated with terrestrial groundwater seepage through permafrost soils is tightly coupled to the geochemistry of these fluids. Terrestrial alkaline methane seeps from Lagoon Pingo, Central Spitsbergen (78°N) in Norway, with methane-saturated and oxygen-limited groundwater discharge providing a potential habitat for methanotrophy. Here, we report on the microbial community’s comparative analyses and distribution patterns at two sites close to Lagoon Pingo’s methane emission source. To target methane-oxidizing bacteria from this system, we analysed the microbial community pattern of replicate samples from two sections near the main methane seepage source. DNA extraction, metabarcoding and subsequent sequencing of 16S rRNA genes revealed microbial communities where the major prokaryotic phyla were Pseudomonadota (42–47%), Gemmatimonadota (4–14%) and Actinobacteriota (7–11%). Among the Pseudomonadota, members of the genus Methylobacter were present at relative abundances between 1.6 and 4.7%. Enrichment targeting the methane oxidising bacteria was set up using methane seep sediments as inoculum and methane as the sole carbon and energy source, and this resulted in the isolation of a novel psychrophilic methane oxidizer, LS7-T4AT. The optimum growth temperature for the isolate was 13 °C and the pH optimum was 8.0. The morphology of cells was short rods, and TEM analysis revealed intracytoplasmic membranes arranged in stacks, a distinctive feature for Type I methanotrophs in the family Methylomonadaceae of the class Gammaproteobacteria. The strain belongs to the genus Methylobacter based on high 16S rRNA gene similarity to the psychrophilic species of Methylobacter psychrophilus Z-0021T (98.95%), the psychrophilic strain Methylobacter sp. strain S3L5C (99.00%), and the Arctic mesophilic species of Methylobacter tundripaludum SV96T (99.06%). The genome size of LS7-T4AT was 4,338,157 bp with a G + C content of 47.93%. The average nucleotide identities (ANIb) of strain LS7-T4AT to 10 isolated strains of genus Methylobacter were between 75.54 and 85.51%, lower than the species threshold of 95%. The strain LS7-T4AT represents a novel Arctic species, distinct from other members of the genus Methylobacter, for which the name Methylobacter svalbardensis sp. nov. is proposed. The type of strain is LS7-T4AT (DSMZ:114308, JCM:39463).

Speciation and distribution of arsenic in cold seep sediments of the South China Sea

Arsenic (As) is an abundant metalloid in marine environments, while the biogeochemical cycling of As in cold seeps remains poorly understood. We characterized the speciation of As and investigated controls of As distribution in cold seeps of South China Sea. High methane concentrations (0.2–5.5 mmol/L) and rapid sulfate depletion were observed in the seepage. Dissolved inorganic arsenic (DIAs) was enriched in the porewater ranging from 7.5 to 23.5 μg/L. As in the solid phase ranged from 2.9 to 22.6 μg/g, and sulfide mineral-bound As dominated the total arsenic (TAs) pool, followed by iron (manganese, aluminum) oxide-bound As. The significant correlations between porewater Fe2+ and DIAs reflect the controls of iron on DIAs release. Incubation experiments showed that adsorption to the solid phase and sulfate reduction activity affected the bioavailability and removal of DIAs, suggesting that multiple processes regulate the speciation and transformation of As in seep sediments.

Pseudallenes A and B, new sulfur-containing ovalicin sesquiterpenoid derivatives with antimicrobial activity from the deep-sea cold seep sediment-derived fungus Pseudallescheria boydii CS-793.

Pseudallenes A and B (1 and 2), the new and rare examples of sulfur-containing ovalicin derivatives, along with three known analogues 3-5, were isolated and identified from the culture extract of Pseudallescheria boydii CS-793, a fungus obtained from the deep-sea cold seep sediments. Their structures were established by detailed interpretation of NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis confirmed and established the structures and absolute configurations of compounds 1-3, thus providing the first characterized crystal structure of an ovalicin-type sesquiterpenoid. In the antimicrobial assays, compounds 1-3 showed broad-spectrum inhibitory activities against several plant pathogens with MIC values ranging from 2 to 16 μg/mL.

Physiological and metabolic insights into the first cultured anaerobic representative of deep-sea Planctomycetes bacteria.

Planctomycetes bacteria are ubiquitously distributed across various biospheres and play key roles in global element cycles. However, few deep-sea Planctomycetes members have been cultivated, limiting our understanding of Planctomycetes in the deep biosphere. Here, we have successfully cultured a novel strain of Planctomycetes (strain ZRK32) from a deep-sea cold seep sediment. Our genomic, physiological, and phylogenetic analyses indicate that strain ZRK32 is a novel species, which we propose be named: Poriferisphaera heterotrophicis. We show that strain ZRK32 replicates using a budding mode of division. Based on the combined results from growth assays and transcriptomic analyses, we found that rich nutrients, or supplementation with NO3- or NH4+ promoted the growth of strain ZRK32 by facilitating energy production through the tricarboxylic acid cycle and the Embden-Meyerhof-Parnas glycolysis pathway. Moreover, supplementation with NO3- or NH4+ induced strain ZRK32 to release a bacteriophage in a chronic manner, without host cell lysis. This bacteriophage then enabled strain ZRK32, and another marine bacterium that we studied, to metabolize nitrogen through the function of auxiliary metabolic genes. Overall, these findings expand our understanding of deep-sea Planctomycetes bacteria, while highlighting their ability to metabolize nitrogen when reprogrammed by chronic viruses.

Physiological and metabolic insights into the first cultured anaerobic representative of deep-sea Planctomycetes bacteria

Planctomycetes bacteria are ubiquitously distributed across various biospheres and play key roles in global element cycles. However, few deep-sea Planctomycetes members have been cultivated, limiting our understanding of Planctomycetes in the deep biosphere. Here, we have successfully cultured a novel strain of Planctomycetes (strain ZRK32) from a deep-sea cold seep sediment. Our genomic, physiological, and phylogenetic analyses indicate that strain ZRK32 is a novel species, which we propose be named: Poriferisphaera heterotrophicis. We show that strain ZRK32 replicates using a budding mode of division. Based on the combined results from growth assays and transcriptomic analyses, we found that rich nutrients, or supplementation with NO3- or NH4+ promoted the growth of strain ZRK32 by facilitating energy production through the tricarboxylic acid cycle and the Embden-Meyerhof-Parnas glycolysis pathway. Moreover, supplementation with NO3- or NH4+ induced strain ZRK32 to release a bacteriophage in a chronic manner, without host cell lysis. This bacteriophage then enabled strain ZRK32, and another marine bacterium that we studied, to metabolize nitrogen through the function of auxiliary metabolic genes. Overall, these findings expand our understanding of deep-sea Planctomycetes bacteria, while highlighting their ability to metabolize nitrogen when reprogrammed by chronic viruses.

Geochemistry of iron and trace metals in seep carbonates of the middle Okinawa Trough impacted by hydrothermal plumes

Geochemistry of iron and trace metals in seep carbonates and its dependence on anaerobic oxidation of methane (AOM)-driven diagenesis in cold seeps are not well documented. Here we characterize geochemistry of Fe and trace metals in carbonate nodules collected at an active cold seep in the middle Okinawa Trough (OT) impacted by hydrothermal plumes. The carbonate nodules in the studied core are dominated by aragonite even at sulfate-low core bottom. Most aragonite at the core bottom may have formed much earlier close to the seafloor under high sulfate concentration. Additionally, porewater at the depth may not be developed to a condition for the formation of calcite dominating over aragonite due to constant downward replenishment of sulfate across through shallow SMTZ (40 cm below sediment surface). As indicated by δ13C and δ18O of the carbonates, anaerobic oxidation of thermogenic methane from gas hydrate dissociation is the dominant fluid source for carbonate precipitation. It can be inferred from the existence of vivianite that Fe reduction coupled to AOM (Fe-AOM) may have ever been prevalent at the hydrothermal Fe-impacted seep site. Enhanced inputs of Mo, Co, Cu, Ni, and Zn associated with hydrothermal Fe-oxyhydroxides-organic colloids is the main source for their high enrichment in the carbonate nodules, and clay minerals and AOM-related microbial organics may have played an important role in their partitioning in the nodules. Uranium enrichment in the carbonate nodules is mainly through enhanced U delivery associated with Fe-oxyhydroxides-scavenged organic colloids and additional U enrichment induced by U(VI) reduction. U(IV) incorporation into carbonate lattices dominates U partitioning in the carbonate nodules over the upper 60 cm of the seep sediments, but the incorporation is substantially dampened at depth due to an increase in concentration of dissolved inorganic carbon, which consequently may have rendered U(IV) adsorption on clay minerals as the important U sink in the nodules. Our results suggest that the exceptional enrichments of Co, Cu, Ni, Zn, Mo, and U in seep carbonates may be used as a paleo-proxy to differentiate cold-seep carbonates whose formation has been co-impacted by hydrothermal plumes.

Phylogenetic diversity of functional genes in deep-sea cold seeps: a novel perspective on metagenomics

BackgroundLeakages of cold, methane-rich fluids from subsurface reservoirs to the sea floor are termed cold seeps. Recent exploration of the deep sea has shed new light on the microbial communities in cold seeps. However, conventional metagenomic methods largely rely on reference databases and neglect the phylogeny of functional genes.ResultsIn this study, we developed the REMIRGE program to retrieve the full-length functional genes from shotgun metagenomic reads and fully explored the phylogenetic diversity in cold seep sediments. The abundance and diversity of functional genes involved in the methane, sulfur, and nitrogen cycles differed in the non-seep site and five cold seep sites. In one Haima cold seep site, the divergence of functional groups was observed at the centimeter scale of sediment depths, with the surface layer potentially acting as a reservoir of microbial species and functions. Additionally, positive correlations were found between specific gene sequence clusters of relevant genes, indicating coupling occurred within specific functional groups.ConclusionREMIRGE revealed divergent phylogenetic diversity of functional groups and functional pathway preferences in a deep-sea cold seep at finer scales, which could not be detected by conventional methods. Our work highlights that phylogenetic information is conducive to more comprehensive functional profiles, and REMIRGE has the potential to uncover more new insights from shotgun metagenomic data.8PNdJCR5n5eFCnFqsf6X4UVideo

Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation.

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

Effects of microplastics on cold seep sediment prokaryotic communities

Cold seep sediments are an important reservoir of microplastics (MPs) whose impact on the structure and function of prokaryotic community is not well understood. In this study, the impact of 0.2% and 1% (w/w) polyethylene (PE), polystyrene (PS), and polypropylene (PP) MPs on the cold seep sediment prokaryotic community was investigated in a 120-day laboratory incubation experiment. The results revealed that exposure to MPs altered sedimentary chemical properties in a type- and concentration-dependent manner. Furthermore, MPs significantly altered the structure of bacterial community, with some MPs degradation-associated bacterial phyla significantly increasing (p < 0.05). However, in the case of archaea, the changes in the structure of microbial community were less pronounced (p > 0.05). Co-occurrence network analysis revealed that the addition of MPs reduced the network complexity, while PICRUSt2 and FAPROTAX analyses suggested that 0.2% PP and 1% PS MPs had the most significant effects on the nitrogen and carbon cycles (p < 0.05). Overall, this study provides new insights into the effects of MPs on the structure and function of microbial communities in cold seep sediments.

Lipidomic diversity and proxy implications of archaea from cold seep sediments of the South China Sea

Cold seeps on the continental margins are characterized by intense microbial activities that consume a large portion of methane by anaerobic methanotrophic archaea (ANME) through anaerobic oxidation of methane (AOM). Although ANMEs are known to contain unique ether lipids that may have an important function in marine carbon cycling, their full lipidomic profiles and functional distribution in particular cold-seep settings are still poorly characterized. Here, we combined the 16S rRNA gene sequencing and lipidomic approaches to analyze archaeal communities and their lipids in cold seep sediments with distinct methane supplies from the South China Sea. The archaeal community was dominated by ANME-1 in the moderate seepage area with strong methane emission. Low seepage area presented higher archaeal diversity covering Lokiarchaeia, Bathyarchaeia, and Thermoplasmata. A total of 55 core lipids (CLs) and intact polar lipids (IPLs) of archaea were identified, which included glycerol dialkyl glycerol tetraethers (GDGTs), hydroxy-GDGTs (OH-GDGTs), archaeol (AR), hydroxyarchaeol (OH-AR), and dihydroxyarchaeol (2OH-AR). Diverse polar headgroups constituted the archaeal IPLs. High concentrations of dissolved inorganic carbon (DIC) with depleted δ13CDIC and high methane index (MI) values based on both CLs (MICL) and IPLs (MIIPL) indicate that ANMEs were active in the moderate seepage area. The ANME-2 and ANME-3 clades were characterized by enhanced glycosidic and phosphoric diether lipids production, indicating their potential role in coupling carbon and phosphurus cycling in cold seep ecosystems. ANME-1, though representing a smaller proportion of total archaea than ANME-2 and ANME-3 in the low seepage area, showed a positive correlation with MIIPL, indicating a different mechanism contributing to the IPL-GDGT pool. This also suggests that MIIPL could be a sensitive index to trace AOM activities performed by ANME-1. Overall, our study expands the understanding of the archaeal lipid composition in the cold seep and improves the application of MI using intact polar lipids that potentially link to extent ANME activities.

Marine Cold Seeps As A Gateway Of Deep Subsurface Extremophiles To The Seafloor

In the Earth’s deep subsurface life is comprised exclusively of microorganisms, and estimates indicate 12-45% of the global prokaryotic biomass, on the order of 1029 microbes, is found in subseafloor sediments. Investigating how this enormous microbial biomass is maintained in the extreme habitats below seafloor is critical for understanding the rules of life in the deep biosphere. Furthermore, Earth’s subseafloor habitats often present analog environments detected in other planets such as the recently discovered “ocean worlds”, i.e., planetary bodies in our solar system which consist of large subsurface oceans including Saturn’s moons Titan and Enceladus and Jupiter’s moon Europa. Therefore, investigating life in and beneath Earth’s oceans remains at the forefront of the current astrobiological research endeavors. Despite the inhospitable nature of the subseafloor sedimentary realm, active microbial populations including bacteria capable of transforming into dormant endospores have been demonstrated to inhabit deeply buried anoxic sediments and oil reservoirs, permeable ocean crust, and around hydrothermal vents. These extreme habitats often remain physically connected to the seafloor by unique geological features such as marine cold seeps that transmit hydrocarbon-rich fluids originating in deep sediment layers. It remains unclear how fluid migration in cold seeps influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. In this study, we addressed this knowledge gap by analyzing over 180 marine surficial sediments from the Gulf of Mexico and the Monterey Bay to assess whether hydrocarbon fluid migration serves as a mechanism for the dispersal of subsurface extremophiles and their introduction into the seabed via cold seeps. Seafloor samples were collected either by piston coring or ROV-operated push coring and were stored at -20°C upon collection. Presence of hydrocarbons in the piston core sediments wa characterized by gas chromatography mass spectrometry and fluorescence spectroscopy whereas gas seepage was determined in the ROV push cores by visual confirmation of gas bubbles emanating from the seafloor. Sediment microbiome composition was determined by high throughput 16S rRNA gene amplicon sequencing. Metabolic diversity was assessed via a genome-centric metagenomics approach aided by shotgun metagenomic sequencing of selected samples. Additionally, viable bacterial endospore communities were investigated from a subset of over 120 of the above samples by allowing endospore germination using a high-temperature incubation assay followed by amplicon sequencing. While 132 of the piston core sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the bacterial lineages Atribacterota and Aminicenantia and the archaeal lineage Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum, in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of non-respiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. In addition, eight different lineages of anaerobic bacterial endospores activated by sediment incubation assays were strongly associated with hydrocarbon-bearing sediments. These lineages were most closely related to Clostridiales previously detected in oil reservoirs from around the world. These results cumulatively point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.

The Phylogeny, Metabolic Potentials, and Environmental Adaptation of an Anaerobe, Abyssisolibacter sp. M8S5, Isolated from Cold Seep Sediments of the South China Sea.

Bacillota are widely distributed in various environments, owing to their versatile metabolic capabilities and remarkable adaptation strategies. Recent studies reported that Bacillota species were highly enriched in cold seep sediments, but their metabolic capabilities, ecological functions, and adaption mechanisms in the cold seep habitats remained obscure. In this study, we conducted a systematic analysis of the complete genome of a novel Bacillota bacterium strain M8S5, which we isolated from cold seep sediments of the South China Sea at a depth of 1151 m. Phylogenetically, strain M8S5 was affiliated with the genus Abyssisolibacter within the phylum Bacillota. Metabolically, M8S5 is predicted to utilize various carbon and nitrogen sources, including chitin, cellulose, peptide/oligopeptide, amino acids, ethanolamine, and spermidine/putrescine. The pathways of histidine and proline biosynthesis were largely incomplete in strain M8S5, implying that its survival strictly depends on histidine- and proline-related organic matter enriched in the cold seep ecosystems. On the other hand, strain M8S5 contained the genes encoding a variety of extracellular peptidases, e.g., the S8, S11, and C25 families, suggesting its capabilities for extracellular protein degradation. Moreover, we identified a series of anaerobic respiratory genes, such as glycine reductase genes, in strain M8S5, which may allow it to survive in the anaerobic sediments of cold seep environments. Many genes associated with osmoprotectants (e.g., glycine betaine, proline, and trehalose), transporters, molecular chaperones, and reactive oxygen species-scavenging proteins as well as spore formation may contribute to its high-pressure and low-temperature adaptations. These findings regarding the versatile metabolic potentials and multiple adaptation strategies of strain M8S5 will expand our understanding of the Bacillota species in cold seep sediments and their potential roles in the biogeochemical cycling of deep marine ecosystems.

Lipids and Terpenoids from the Deep-Sea Fungus Trichoderma lixii R22 and Their Antagonism against Two Wheat Pathogens.

Five new lipids, tricholixins A-E (1-5), and two known terpenoids, brasilane A (6) and harzianone A (7), were discovered from a deep-sea strain (R22) of the fungus Trichoderma lixii isolated from the cold seep sediments of the South China Sea. Their structures and relative configurations were identified by meticulous analysis of MS and IR as well as NMR data. The absolute configuration of 5 was ascertained by dimolybdenum-induced ECD data in particular. Compounds 1 and 2 represent the only two new butenolides from marine-derived Trichoderma, and they further add to the structural diversity of these molecules. Although 6 has been reported from a basidiomycete previously, it is the first brasilane aminoglycoside of Trichoderma origin. During the assay against wheat-pathogenic fungi, both 1 and 2 inhibited Fusarium graminearum with an MIC value of 25.0 μg/mL, and 6 suppressed Gaeumannomyces graminis with an MIC value of 12.5 μg/mL. Moreover, the three isolates also showed low toxicity to the brine shrimp Artemia salina.