Cold Seep Sediments Research Articles

Unexpected genetic and microbial diversity for arsenic cycling in deep sea cold seep sediments

Cold seeps, where cold hydrocarbon-rich fluid escapes from the seafloor, show strong enrichment of toxic metalloid arsenic (As). The toxicity and mobility of As can be greatly altered by microbial processes that play an important role in global As biogeochemical cycling. However, a global overview of genes and microbes involved in As transformation at seeps remains to be fully unveiled. Using 87 sediment metagenomes and 33 metatranscriptomes derived from 13 globally distributed cold seeps, we show that As detoxification genes (arsM, arsP, arsC1/arsC2, acr3) were prevalent at seeps and more phylogenetically diverse than previously expected. Asgardarchaeota and a variety of unidentified bacterial phyla (e.g. 4484-113, AABM5-125-24 and RBG-13-66-14) may also function as the key players in As transformation. The abundances of As cycling genes and the compositions of As-associated microbiome shifted across different sediment depths or types of cold seep. The energy-conserving arsenate reduction or arsenite oxidation could impact biogeochemical cycling of carbon and nitrogen, via supporting carbon fixation, hydrocarbon degradation and nitrogen fixation. Overall, this study provides a comprehensive overview of As cycling genes and microbes at As-enriched cold seeps, laying a solid foundation for further studies of As cycling in deep sea microbiome at the enzymatic and processual levels.

Metal-Driven Anaerobic Oxidation of Methane as an Important Methane Sink in Methanic Cold Seep Sediments.

Anaerobic oxidation of methane (AOM) coupled with reduction of metal oxides is supposed to be a globally important bioprocess in marine sediments. However, the responsible microorganisms and their contributions to methane budget are not clear in deep sea cold seep sediments. Here, we combined geochemistry, muti-omics, and numerical modeling to study metal-dependent AOM in methanic cold seep sediments in the northern continental slope of the South China Sea. Geochemical data based on methane concentrations, carbon stable isotope, solid-phase sediment analysis, and pore water measurements indicate the occurrence of anaerobic methane oxidation coupled to metal oxides reduction in the methanic zone. The 16S rRNA gene and transcript amplicons, along with metagenomic and metatranscriptomic data suggest that diverse anaerobic methanotrophic archaea (ANME) groups actively mediated methane oxidation in the methanic zone either independently or in syntrophy with, e.g., ETH-SRB1, as potential metal reducers. Modeling results suggest that the estimated rates of methane consumption via Fe-AOM and Mn-AOM were both 0.3 μmol cm-2 year-1, which account for ~3% of total CH4 removal in sediments. Overall, our results highlight metal-driven anaerobic oxidation of methane as an important methane sink in methanic cold seep sediments. IMPORTANCE Anaerobic oxidation of methane (AOM) coupled with reduction of metal oxides is supposed to be a globally important bioprocess in marine sediments. However, the responsible microorganisms and their contributions to methane budget are not clear in deep sea cold seep sediments. Our findings provide a comprehensive view of metal-dependent AOM in the methanic cold seep sediments and uncovered the potential mechanisms for involved microorganisms. High amounts of buried reactive Fe(III)/Mn(IV) minerals could be an important available electron acceptors for AOM. It is estimated that metal-AOM at least contributes 3% of total methane consumption from methanic sediments to the seep. Therefore, this research paper advances our understanding of the role of metal reduction to the global carbon cycle, especially the methane sink.

High Microeukaryotic Diversity in the Cold-Seep Sediment.

Microeukaryotic diversity, community structure, and their regulating mechanisms remain largely unclear in chemosynthetic ecosystems. Here, using high-throughput sequencing data of 18S rRNA genes, we explored microeukaryotic communities from the Haima cold seep in the northern South China Sea. We compared three distinct habitats: active, less active, and non-seep regions, with vertical layers (0-25cm) from sediment cores. The results showed that seep regions harbored more abundant and diverse parasitic microeukaryotes (e.g., Apicomplexa and Syndiniales) as indicator species, compared to nearby non-seep region. Microeukaryotic community heterogeneity was larger between habitats than within habitat, and greatly increased when considering molecular phylogeny, suggesting the local diversification in cold-seep sediments. Microeukaryotic α-diversity at cold seeps was positively increased by metazoan richness and dispersal rate of microeukaryotes, while its β-diversity was promoted by heterogeneous selection mainly from metazoan communities (as potential hosts). Their combined effects led to the significant higher γ-diversity (i.e., total diversity in a region) at cold seeps than non-seep regions, suggesting cold-seep sediment as a hotspot for microeukaryotic diversity. Our study highlights the importance of microeukaryotic parasitism in cold-seep sediment and has implications for the roles of cold seep in maintaining and promoting marine biodiversity.

Discovery of prenylated indole alkaloid and natural xanthone from cold-seep sediment derived fungus Talaromyces funiculosus SD-523

Discovery of prenylated indole alkaloid and natural xanthone from cold-seep sediment derived fungus Talaromyces funiculosus SD-523

Disentangling the Functional Role of Fungi in Cold Seep Sediment.

Cold seeps are biological oases of the deep sea fueled by methane, sulfates, nitrates, and other inorganic sources of energy. Chemolithoautotrophic bacteria and archaea dominate seep sediment, and their diversity and biogeochemical functions are well established. Fungi are likewise diverse, metabolically versatile, and known for their ability to capture and oxidize methane. Still, no study has ever explored the functional role of the mycobiota in the cold seep biome. To assess the complex role of fungi and fill in the gaps, we performed network analysis on 147 samples to disentangle fungal-prokaryotic interactions (fungal 18S and prokaryotic 16S) in the Haima cold seep region. We demonstrated that fungi are central species with high connectivity at the epicenter of prokaryotic networks, reduce their random-attack vulnerability by 60%, and enhance information transfer efficiency by 15%. We then scavenged a global metagenomic and metatranscriptomic data set from 10 cold seep regions for fungal genes of interest (hydrophobins, cytochrome P450s, and ligninolytic family of enzymes); this is the first study to report active transcription of 2,500+ fungal genes in the cold seep sediment. The genera Fusarium and Moniliella were of notable importance and directly correlated with high methane abundance in the sulfate-methane transition zone (SMTZ), likely due to their ability to degrade and solubilize methane and oils. Overall, our results highlight the essential yet overlooked contribution of fungi to cold seep biological networks and the role of fungi in regulating cold seep biogeochemistry. IMPORTANCE The challenges we face when analyzing eukaryotic metagenomic and metatranscriptomic data sets have hindered our understanding of cold seep fungi and microbial eukaryotes. This fact does not make the mycobiota any less critical in mediating cold seep biogeochemistry. On the contrary, many fungal genera can oxidize and solubilize methane, produce methane, and play a unique role in nutrient recycling via saprotrophic enzymatic activity. In this study, we used network analysis to uncover key fungal-prokaryotic interactions that can mediate methane biogeochemistry and metagenomics and metatranscriptomics to report that fungi are transcriptionally active in the cold seep sediment. With concerns over rising methane levels and cold seeps being a pivotal source of global methane input, our holistic understanding of methane biogeochemistry with all domains of life is essential. We ultimately encourage scientists to utilize state-of-the-art tools and multifaceted approaches to uncover the role of microeukaryotic organisms in understudied systems.

The effects of organic matter and anaerobic oxidation of methane on the microbial sulfate reduction in cold seeps

Cold seep sediments are dominated by intensive microbial sulfate reduction coupled to anaerobic oxidation of methane. However, the contribution proportion between this process and the role of organic matter has remained enigmatic. Here, pore water data combined with PROFILE model, fluxes of sulfate and methane concentration calculated from Fick's first law, and δ34SSO4 and δ18OSO4 of pore water sulfate were studied to reconstruct co-occurring microbial organoclastic sulfate reduction and anaerobic oxidation of methane coupled with sulfate reduction in methane seep sediments collected from South China Sea. The sulfate concentration profiles of C9 and C14 in Qiongdongnan Basin generally show quasilinear depletion with depth. Reaction-transport modeling provided close fits to concentration data. δ18OSO4 and δ34SSO4 increase fastest with sediment depth above 400 cmbsf and slowest below that depth. The values of methane flux are always lower than those of total sulfate reduction of sulfate diffusive flux at GC-10, GC-9, GC-11 and HD319 sites in Taixinan Basin. Besides, positions of sulfate methane transition zone in all study sites are approximately ~400 to 800 centimeters below seafloor. These results showed that microbial sulfate reduction in sediments is mainly controlled by intense anaerobic oxidation of methane, but there is a certain relationship with organic matter metabolism process. This emphasizes that traditional redox order of bacterial respiration is highly simplified, where, in sediments such as these seeps, all of these microbial sulfate reduction processes can occur together with complex couplings between them.

Genesis and preservation of authigenic magnetite and greigite in the cold seep sediments, Bay of Bengal

We report a new mechanism of authigenic greigite and magnetite formation and preservation in an anomalous “sulfide-rich” interval in the cold (methane) seep sediments from the Bay of Bengal. Rock magnetic proxies (SIRM/χlf, ARM/SIRM) coupled with microscopic (TEM, SEM) observations provided compelling evidence for the presence of nanoscale authigenic magnetite and greigite particles in the methanic sediments. Multiple peaks of SIRM/χlf coupled with high Hcr (>53 mT) and DJH values suggest significant contribution from authigenic greigite particles to the bulk sediment magnetism. High ARM/SIRM in the sulfide-rich intervals within methanic zone suggest that nanoscale authigenic magnetite formation is closely linked with the microbial iron reduction process fuelled by anaerobic oxidation of methane (AOM). We proposed that enrichment of reactive secondary iron (oxyhydr) oxides through selective leaching of Fe2+ during diagenetic dissolution and maghemitization of detrital magnetic particles and iron-sulfide mineral oxidation at the paleo-SMTZ fronts, hindered the pyritization processes and favoured microbial iron reduction which led to the formation and preservation of authigenic greigite and magnetite in methanic sediments.

Evolutionary ecology of microbial populations inhabiting deep sea sediments associated with cold seeps

Deep sea cold seep sediments host abundant and diverse microbial populations that significantly influence biogeochemical cycles. While numerous studies have revealed their community structure and functional capabilities, little is known about genetic heterogeneity within species. Here, we examine intraspecies diversity patterns of 39 abundant species identified in sediment layers down to 430 cm below the sea floor across six cold seep sites. These populations are grouped as aerobic methane-oxidizing bacteria, anaerobic methanotrophic archaea and sulfate-reducing bacteria. Different evolutionary trajectories are observed at the genomic level among these physiologically and phylogenetically diverse populations, with generally low rates of homologous recombination and strong purifying selection. Functional genes related to methane (pmoA and mcrA) and sulfate (dsrA) metabolisms are under strong purifying selection in most species investigated. These genes differ in evolutionary trajectories across phylogenetic clades but are functionally conserved across sites. Intrapopulation diversification of genomes and their mcrA and dsrA genes is depth-dependent and subject to different selection pressure throughout the sediment column redox zones at different sites. These results highlight the interplay between ecological processes and the evolution of key bacteria and archaea in deep sea cold seep extreme environments, shedding light on microbial adaptation in the subseafloor biosphere.

Emestrin-type thiodiketopiperazines from Aspergillus nidulans SD-531, a fungus obtained from the deep-sea sediment of cold seep in the South China Sea

Three new emestrin-type thiodiketopiperazines including didethio-11a-methylthioemestrin (1), 7′-epi-didethio-11a-methylthioemestrin (2), and 2″-desmethyl-MPC1001F (3), along with three known analogues emestrin (4), dethiosecoemestrin (5), and emestrin H (6), were isolated and identified from the culture extract of Aspergillus nidulans SD-531, a fungus obtained from the deep-sea cold seep sediment sample collected from the South China Sea. Their structures and absolute configurations were determined by spectroscopic analysis and TDDFT-ECD calculations. Compounds 3–6 showed antimicrobial activity against some of the tested strains, while compounds 3–5 had cytotoxic activity against the tumor cell line Huh 7.5. Compound 4 exhibited the strongest cytotoxic activity with an IC50 value of 0.25 μM, which was better than that of the positive control sorafenib.

Molecular evidence for the production of labile, sulfur-bearing dissolved organic matter in the seep sediments of the South China Sea

Cold seeps with methane-rich fluids leaking out of the seafloor usually support massive biomass of chemosynthetic organisms and associated fauna. A substantial amount of methane is converted to dissolved inorganic carbon by microbial metabolism, and this process also releases dissolved organic matter (DOM) into pore water. Here, pore water samples from “Haima cold seeps” sediments and the non-seep reference sediments in the northern South China Sea were analyzed for optical properties and molecular compositions of pore water DOM. Our results showed that the relative abundance of protein-like DOM, H/Cwa and molecular lability boundary percentage (MLBL%) in the seep sediments were significantly higher than those in the reference sediments, indicating that more labile DOM related to unsaturated aliphatic compounds is produced in the seep sediments. Spearman's correlation of the fluoresce and molecular data suggested that the humic-like components (C1 and C2) mainly constituted the refractory compounds (CRAM, highly unsaturated and aromatics compounds). In contrast, the protein-like component (C3) had high H/C ratios featuring high degree of DOM lability. The amount of S-containing formulas (CHOS and CHONS) was greatly elevated in the seep sediments, likely caused by abiotic and biotic sulfurization of DOM in the sulfidic environment. Although the abiotic sulfurization was proposed to have a stabilizing effect on organic matter, our results implied that the biotic sulfurization in the cold seep sediments would increase DOM lability. Overall, the labile DOM accumulated in the seep sediments is closely linked to methane oxidation, which not only support heterotrophic communities and but also likely have an impact on carbon and sulfur cycling in the sediments and the ocean.

Five new verrucosidin derivatives from Penicillium polonicum, a deep-sea cold-seep sediment isolated fungus

Five new verrucosidin derivatives, poloncosidins G–K (1–5), were isolated from the deep sea cold-seep sediment-derived fungus Penicillium polonicum CS-252. Their planar structures were elucidated by discreet analysis of the NMR spectroscopic and HRESIMS spectrometric data. The absolute configurations of compounds 1–5 were deduced from the combination of the modified Mosher's method and quantum chemical calculations of their ECD and NMR (with DP4+ probability analysis) data. The antimicrobial activities against several human- and aquatic-pathogenic bacteria of all the isolated compounds were evaluated and the structure-bioactivity relationship was briefly discussed.

Methane supply drives prokaryotic community assembly and networks at cold seeps of the South China Sea.

Marine cold seeps are unique chemosynthetic habitats fuelled by deeply sourced hydrocarbon-rich fluids discharged at the seafloor. Through oxidizing methane and other hydrocarbons, microorganisms inhabiting cold seeps supply subsurface-derived energy to higher trophic levels, sustaining highly productive oases of life in the deep sea. Despite the central role of microbiota in mediating biogeochemical cycles, the factors that govern the assembly and network of prokaryotic communities in cold seeps remain poorly understood. Here we analysed the geochemical and microbiological profiles of 11 different sediment cores from two spatially distant cold seeps of the South China Sea. We show that prokaryotic communities belonging to the same methane-supply regimes (high-methane-supply, low-methane-supply and non-seep control sediments) had a highly similar community structure, regardless of geographical location, seep-associated biota (mussel, clam, microbial mat) and sediment depth. Methane supply appeared to drive the niche partitioning of anaerobic methanotrophic archaea (ANME) at the regional scale, with ANME-1 accounting for >60% sequence abundance of ANME in the high-methane-supply sediments, while ANME-2 dominated (>90%) the low-methane-supply sediments. Increasing methane supply enhanced the contribution of environmental selection but lessened the contributions of dispersal limitation and drift to overall community assembly. High methane supply, moreover, promoted a more tightly connected, less stable prokaryotic network dominated by positive correlations. Together, these results provide a potentially new framework for understanding the niches and network interplay of prokaryotic communities across different methane seepage regimes in cold-seep sediments.

Interactions of Microplastics and Methane Seepage in the Deep-Sea Environment

Microplastics (MPs) are important exempla of the Anthropocene and are exerting an increasing impact on Earth’s carbon cycle. The huge imbalance between the MPs floating on the marine surface and those that are estimated to have been introduced into the ocean necessitates a detailed assessment of marine MP sinks. Here, we demonstrate that cold seep sediments, which are characterized by methane fluid seepage and a chemosynthetic ecosystem, effectively capture and accommodate small-scale (< 100 μm) MPs, with 16 types of MPs being detected. The abundance of MPs in the surface of the sediment is higher in methane-seepage locations than in non-seepage areas. Methane seepage is beneficial to the accumulation, fragmentation, increased diversity, and aging of MPs. In turn, the rough surfaces of MPs contribute to the sequestration of the electron acceptor ferric oxide, which is associated with the anaerobic oxidation of methane (AOM). The efficiency of the AOM determines whether the seeping methane (which has a greenhouse effect 83 times greater than that of CO2 over a 20-year period) can enter the atmosphere, which is important to the global methane cycle, since the deep-sea environment is regarded as the largest methane reservoir associated with natural gas hydrates.

A deep-sea sulfate-reducing bacterium generates zero-valent sulfur via metabolizing thiosulfate.

Zero-valent sulfur (ZVS) is a crucial intermediate in the sulfur geobiochemical circulation and is widespread in deep-sea cold seeps. Sulfur-oxidizing bacteria are thought to be the major contributors to the formation of ZVS. However, ZVS production mediated by sulfate-reducing bacteria (SRB) has rarely been reported. In this study, we isolated and cultured a typical SRB designated Oceanidesulfovibrio marinus CS1 from deep-sea cold seep sediment in the South China Sea. We show that O. marinus CS1 forms ZVS in the medium supplemented with thiosulfate. Proteomic and protein activity assays revealed that thiosulfate reductase (PhsA) and the sulfide:quinone oxidoreductase (SQR) played key roles in driving ZVS formation in O. marinus CS1. During this process, thiosulfate firstly was reduced by PhsA to form sulfide, then sulfide was oxidized by SQR to produce ZVS. The expressions of PhsA and SQR were significantly upregulated when O. marinus CS1 was cultured in a deep-sea cold seep, strongly indicating that strain CS1 might form ZVS in the deep-sea environment. Notably, homologs of phsA and sqr were widely identified from microbes living in sediments of deep-sea cold seep in the South China Sea by the metagenomic analysis. We thus propose that SRB containing phsA and sqr genes potentially contribute to the formation of ZVS in deep-sea cold seep environments.

Phylogenetically and catabolically diverse diazotrophs reside in deep-sea cold seep sediments

Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance.

Deep-Sea In Situ Insights into the Formation of Zero-Valent Sulfur Driven by a Bacterial Thiosulfate Oxidation Pathway.

ABSTRACTZero-valent sulfur (ZVS) distributes widely in the deep-sea cold seep, which is an important immediate in the sulfur cycle of cold seep. In our previous work, we described a novel thiosulfate oxidation pathway determined by thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) mediating the conversion of thiosulfate to ZVS in the deep-sea cold seep bacterium Erythrobacter flavus 21-3. However, the occurrence and ecological role of this pathway in the deep-sea cold seep were obscure. Here, we cultured E. flavus 21-3 in the deep-sea cold seep for 10 days and demonstrated its capability of forming ZVS in the in situ field. Based on proteomic, stoichiometric analyses and microscopic observation, we found that this thiosulfate oxidation pathway benefited E. flavus 21-3 to adapt the cold seep conditions. Notably, ~25% metagenomes assembled genomes derived from the shallow sediments of cold seeps contained both tsdA and soxB, where presented abundant sulfur metabolism-related genes and active sulfur cycle. Our results suggested that the thiosulfate oxidation pathway determined by TsdA and SoxB existed across many bacteria inhabiting in the cold seep and frequently used by microbes to take part in the active cold seep biogeochemical sulfur cycle.

Verrucosidin Derivatives from the Deep Sea Cold-Seep-Derived Fungus Penicillium polonicum CS-252.

Six novel verrucosidin derivatives, namely, poloncosidins A–F (1–6), together with one known analogue (7), were isolated and identified from the deep-sea-derived fungus Penicillium polonicum CS-252, which was obtained from cold-seep sediments collected in the South China Sea at a depth of 1183 m. Their structures were mainly established on the basis of a detailed interpretation of NMR spectroscopic and mass spectrometric data. The relative and absolute configurations of compounds 1–6 were determined by ECD calculations and a DP4+ probability analysis. Compounds 1–5 represent the first examples of verrucosidins with a 2,5-dihydrofuran ring which is uncommon among the known analogues. These compounds exhibited inhibitory activities against several human and aquatic pathogens with MIC values ranging from 4 to 32 μg/mL.

Diversity of Anaerobic Methane Oxidizers in the Cold Seep Sediments of the Okinawa Trough.

Active cold seeps in the Okinawa Trough (OT) have been widely identified, but the sediment microbial communities associated with these sites are still poorly understood. Here, we investigated the distribution and biomass of the microbial communities, particularly those associated with the anaerobic oxidation of methane (AOM), in sediments from an active cold seep in the mid-Okinawa Trough. Methane-oxidizing archaea, including ANME-1a, ANME-1b, ANME-2a/b, ANME-2c, and ANME-3, were detected in the OT cold seep sediments. Vertical stratification of anaerobic methanotrophic archaea (ANME) communities was observed in the following order: ANME-3, ANME-1a, and ANME-1b. In addition, the abundance of methyl coenzyme M reductase A (mcrA) genes corresponded to high levels of dissolved iron, suggesting that methane-metabolizing archaea might participate in iron reduction coupled to methane oxidation (Fe-AOM) in the OT cold seep. Furthermore, the relative abundance of ANME-1a was strongly related to the concentration of dissolved iron, indicating that ANME-1a is a key microbial player for Fe-AOM in the OT cold seep sediments. Co-occurrence analysis revealed that methane-metabolizing microbial communities were mainly associated with heterotrophic microorganisms, such as JS1, Bathy-1, and Bathy-15.

Revealing the Response of Microbial Communities to Micro(Nano)Plastics Exposure in Cold Seep Sediment

Revealing the Response of Microbial Communities to Micro(Nano)Plastics Exposure in Cold Seep Sediment

Revealing the Response of Microbial Communities to Polyethylenemicro(Nano)Plastics Exposure in Cold Seep Sediment

Revealing the Response of Microbial Communities to Polyethylenemicro(Nano)Plastics Exposure in Cold Seep Sediment