Environmental heterogeneity shapes the C and S cycling-associated microbial community in Haima's cold seeps

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.

Methane seeps are chemosynthetic ecosystems in the deep-sea environment. Microbial community structures have been extensively studied in the seepage-affected sediments and investigation in the water column above the seeping sites is still lacking. In this study, prokaryotic communities in the bottom water about 50 cm from the seabed at methane seeps with various seepage intensities in Haima, South China Sea were comparatively studied by using 16S ribosomal RNA gene sequencing. These sites were assigned based on their distinct methane content levels and seafloor landscapes as the non-seepage (NS) site, low-intensity seepage (LIS) site, and high-intensity seepage (HIS) site. The abundances of the dominant phyla Proteobacteria, Bacteroidetes, and Actinobacteria differed significantly between NS and the two seepage sites (p < 0.05). Alpha diversity differed among the three sites with the HIS site showing the lowest community diversity. Principal component analysis revealed highly divergent bacterial community structures at three sites. Many environmental variables including temperature, alkalinity, pH, methane, dissolved organic carbon (DOC), and inorganic nutrients were measured. Redundancy analysis indicated that methane content is the key environmental factor driving bacterial community variation (p = 0.001). Linear discriminant analysis effect size analysis identified various differentially enriched genera at the LIS and HIS sites. Phylogenetic analysis revealed close phylogenetic relationship among the operational taxonomic units of these genera with known oil-degrading species, indicating oil seepage may occur at the Haima cold seeps. Co-occurrence networks indicated that the strength of microbial interactions was weakest at the HIS site. This study represents a comprehensive comparison of microbial profiles in the water column of cold seeps in the SCS, revealing that the seepage intensity has a strong impact on bacterial community dynamics.

Deep-sea cold seeps harbor unique microbial communities that play crucial roles in biogeochemical cycles and possess potential biotechnological applications. Herein, we report the isolation, characterization, and genomic analysis of a novel Bacillus species, Bacillus haimaensis sp. nov. (type strain CSS-39T, CCTCC M20241382), obtained from sediments collected at a depth of 1,350 m in the Haima cold seep, South China Sea. Phylogenomic analysis, revealing an average nucleotide identity of 87.78% and a digital DNA-DNA hybridization value of 34.0% with its closest relative B. tianshenii DSM 25879T, confirms the taxonomic novelty of the genus Bacillus. The complete 4.54 Mb genome of B. haimaensis reveals adaptations to the cold seep environment, including enhanced nutrient acquisition capabilities and stress response mechanisms. Comparative genomic analysis identifies 27 unique gene clusters related to spore germination and sulfate assimilation, suggesting specialized metabolic strategies for this extreme habitat. Furthermore, six biosynthetic gene clusters, including a novel lassopeptide cluster, indicate a potential for secondary metabolite production. Phenotypic characterization demonstrates the strain's ability to utilize diverse carbon sources and tolerate a wide range of environmental conditions. Our findings provide insights into microbial adaptations to deep-sea cold seeps and highlight the potential of B. haimaensis for biotechnological applications in bioremediation and natural product discovery. This study expands our understanding of microbial diversity in extreme marine environments and offers a new model bacterium for investigating bacterial adaptations to deep-sea ecosystems.IMPORTANCEThe discovery of Bacillus haimaensis sp. nov. in the Haima cold seep of the South China Sea represents a significant advancement in our understanding of microbial adaptations to extreme marine environments. This novel species exhibits remarkable metabolic versatility and unique genomic features, providing insights into bacterial survival strategies in nutrient-variable, high-pressure deep-sea ecosystems. Comprehensive genomic analysis reveals distinctive biosynthetic gene clusters, suggesting untapped potential for discovering novel natural product. Furthermore, B. haimaensis exhibits promising capabilities for aromatic compound degradation, indicating potential applications in marine bioremediation. This work not only expands our knowledge of microbial diversity in understudied deep-sea habitats but also highlights the biotechnological promise of extremophiles. The adaptive mechanisms elucidated in B. haimaensis, particularly those related to sporulation and sulfate assimilation, contribute to our broader understanding of microbial ecology in cold seeps and may inform future research on climate change impacts on deep-sea ecosystems.

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.

Using chemical compositions of sediments to constrain methane seepage dynamics: A case study from Haima cold seeps of the South China Sea

Cold seeps and hydrothermal vents are seafloor habitats fueled by subsurface energy sources. Both habitat types coexist in Guaymas Basin in the Gulf of California, providing an opportunity to compare microbial communities with distinct physiologies adapted to different thermal regimes. Hydrothermally active sites in the southern Guaymas Basin axial valley, and cold seep sites at Octopus Mound, a carbonate mound with abundant methanotrophic cold seep fauna at the Central Seep location on the northern off-axis flanking regions, show consistent geochemical and microbial differences between hot, temperate, cold seep, and background sites. The changing microbial actors include autotrophic and heterotrophic bacterial and archaeal lineages that catalyze sulfur, nitrogen, and methane cycling, organic matter degradation, and hydrocarbon oxidation. Thermal, biogeochemical, and microbiological characteristics of the sampling locations indicate that sediment thermal regime and seep-derived or hydrothermal energy sources structure the microbial communities at the sediment surface.

Methane (CH4) as a powerful greenhouse gas is the second largest contributor to global climate warming. Mangrove sediments are an important natural source of biogenic CH4 with rich organic carbon (C) and diverse sulfur (S) compounds, ideally for studying CH4 and S cycling processes and coupling mechanisms. Here we sampled mangrove sediment cores and analyzed their key microbial groups, key environmental factors and possible coupling mechanisms for CH4 and S cycling by metagenome sequencing approaches. Our results showed that Methanomicrobiales and Methanophagales were predominant methanogens, Methanospirareceae was a representative of anaerobic methanotrophic archaea (ANME), and Desulfobacteraceae and Desulfobulbaceae were abundant sulfate-reducing bacteria (SRB), while Ectothiorhodospiraceae, Chromatiaceae and Comamonadaceae were dominant S-oxidizers. Correlation network analysis revealed positive interactions among methanogens, ANME and SRB. Also, metagenome-assembled genome (MAG) analysis indicated interspecies hydrogen transfer and extracellular electron exchange via conductive pili, flagella, and cytochromes were potential coupling mechanisms between methanogens and SRB. ANME could form consortia with SRB by intermediate metabolites (e.g., acetate) and/or direct interspecies electron transfer (e.g., flagella, pili, cytochromes). Furthermore, methanogen MAGs encoded thiosulfate oxidation and partial sulfate reduction pathways, while the ANME MAGs possessed potentials for S disproportionation and incomplete sulfate reduction. Additionally, SO42-, total sulfur, moisture content and salinity were important environmental factors affecting the microbial community structure and gene families involved in CH4 and S cycling. This study provides novel insights into coupling mechanisms of CH4 and S cycling processes in mangrove sediments, having important implications for mitigating global warming.

Microbes have obligate requirements for trace metals in metalloenzymes that catalyse important biogeochemical reactions. In anoxic methane- and sulphide-rich environments, microbes may have unique adaptations for metal acquisition and utilization because of decreased bioavailability as a result of metal sulphide precipitation. However, micronutrient cycling is largely unexplored in cold (≤ 10°C) and sulphidic (> 1 mM ΣH(2)S) deep-sea methane seep ecosystems. We investigated trace metal geochemistry and microbial metal utilization in methane seeps offshore Oregon and California, USA, and report dissolved concentrations of nickel (0.5-270 nM), cobalt (0.5-6 nM), molybdenum (10-5600 nM) and tungsten (0.3-8 nM) in Hydrate Ridge sediment porewaters. Despite low levels of cobalt and tungsten, metagenomic and metaproteomic data suggest that microbial consortia catalysing anaerobic oxidation of methane (AOM) utilize both scarce micronutrients in addition to nickel and molybdenum. Genetic machinery for cobalt-containing vitamin B12 biosynthesis was present in both anaerobic methanotrophic archaea (ANME) and sulphate-reducing bacteria. Proteins affiliated with the tungsten-containing form of formylmethanofuran dehydrogenase were expressed in ANME from two seep ecosystems, the first evidence for expression of a tungstoenzyme in psychrophilic microorganisms. Overall, our data suggest that AOM consortia use specialized biochemical strategies to overcome the challenges of metal availability in sulphidic environments.

Novel use of unique minerals to reveal an intensified methane seep during the last glacial period in the South China Sea

Characterization of marine microbial communities around an Arctic seabed hydrocarbon seep at Scott Inlet, Baffin Bay

In situ detection of the fine scale heterogeneity of active cold seep environment of the Formosa Ridge, the South China Sea

The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments into the hydrosphere. AOM is largely mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), and has mainly been investigated in deep-sea sediments. Here we studied methane seepage at four spots located at 12 m water depth in coastal, organic carbon depleted permeable sands off the Island of Elba (Italy). We combined biogeochemical measurements, sequencing-based community analyses and in situ hybridization to investigate the microbial communities of this environment. Increased alkalinity, formation of free sulfide and nearly stoichiometric methane oxidation and sulfate reduction rates up to 200 nmol g-1 day-1 indicated the predominance of sulfate-coupled AOM. With up to 40 cm thickness the zones of AOM activity were unusually large and occurred in deeper sediment horizons (20–50 cm below seafloor) as compared to diffusion-dominated deep-sea seeps, which is likely caused by advective flow of pore water due to the shallow water depth and permeability of the sands. Hydrodynamic forces also may be responsible for the substantial phylogenetic and unprecedented morphological diversity of AOM consortia inhabiting these sands, including the clades ANME-1a/b, ANME-2a/b/c, ANME-3, and their partner bacteria SEEP-SRB1a and SEEP-SRB2. High microbial dispersal, the availability of diverse energy sources and high habitat heterogeneity might explain that the emission spots shared few microbial taxa, despite their physical proximity. Although the biogeochemistry of this shallow methane seep was very different to that of deep-sea seeps, their key functional taxa were very closely related, which supports the global dispersal of key taxa and underlines strong selection by methane as the predominant energy source. Mesophilic, methane-fueled ecosystems in shallow-water permeable sediments may comprise distinct microbial habitats due to their unique biogeochemical and physical characteristics. To link AOM phylotypes with seep habitats and to enable future meta-analyses we thus propose that seep environment ontology needs to be further specified.

The anaerobic oxidation of methane (AOM) is an important sink of methane that plays a significant role in global warming. AOM was first found to be coupled with sulfate reduction and mediated by anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). ANME, often forming consortia with SRB, are phylogenetically related to methanogenic archaea. ANME-1 is even able to produce methane. Subsequently, it has been found that AOM can also be coupled with denitrification. The known microbes responsible for this process are Candidatus Methylomirabilis oxyfera (M. oxyfera) and Candidatus Methanoperedens nitroreducens (M. nitroreducens). Candidatus Methylomirabilis oxyfera belongs to the NC10 bacteria, can catalyze nitrite reduction through an “intra-aerobic” pathway, and may catalyze AOM through an aerobic methane oxidation pathway. However, M. nitroreducens, which is affiliated with ANME-2d archaea, may be able to catalyze AOM through the reverse methanogenesis pathway. Moreover, manganese (Mn4+) and iron (Fe3+) can also be used as electron acceptors of AOM. This review summarizes the mechanisms and associated microbes of AOM. It also discusses recent progress in some unclear key issues about AOM, including ANME-1 in hypersaline environments, the effect of oxygen on M. oxyfera, and the relationship of M. nitroreducens with ANME.

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.

Gulf of Mexico sediments harbor numerous hydrocarbon seeps associated with high sedimentation rates and thermal maturation of organic matter. These ecosystems host abundant and diverse microbial communities that directly or indirectly metabolize components of the emitted fluid. To investigate microbial function and activities in these ecosystems, metabolic potential (metagenomic) and gene expression (metatranscriptomic) analyses of two cold seep areas of the Gulf of Mexico were carried out. Seeps emitting biogenic methane harbored microbial communities dominated by archaeal anaerobic methane oxidizers of phylogenetic group 1 (ANME-1), whereas seeps producing fluids containing a complex mixture of thermogenic hydrocarbons were dominated by ANME-2 lineages. Metatranscriptome measurements in both communities indicated high levels of expression of genes for methane metabolism despite their distinct microbial communities and hydrocarbon composition. In contrast, the transcription level of sulfur cycle genes was quite different. In the thermogenic seep community, high levels of transcripts indicative of syntrophic anaerobic oxidation of methane (AOM) coupled to sulfate reduction were detected. This syntrophic partnership between the dominant ANME-2 and sulfate reducers potentially involves direct electron transfer through multiheme cytochromes. In the biogenic methane seep, genes from an ANME-1 lineage that are potentially involved in polysulfide reduction were highly expressed, suggesting a novel bacterium-independent anaerobic methane oxidation pathway coupled to polysulfide reduction. The observed divergence in AOM activities provides a new model for bacterium-independent AOM and emphasizes the variation that exists in AOM pathways between different ANME lineages. IMPORTANCE Cold seep sediments are complex and widespread marine ecosystems emitting large amounts of methane, a potent greenhouse gas, and other hydrocarbons. Within these sediments, microbial communities play crucial roles in production and degradation of hydrocarbons, modulating oil and gas emissions to seawater. Despite this ecological importance, our understanding of microbial functions and methane oxidation pathways in cold seep ecosystems is poor. Based on gene expression profiling of environmental seep sediment samples, the present work showed that (i) the composition of the emitted fluids shapes the microbial community in general and the anaerobic methanotroph community specifically and (ii) AOM by ANME-2 in this seep may be coupled to sulfate reduction by Deltaproteobacteria by electron transfer through multiheme cytochromes, whereas AOM by ANME-1 lineages in this seep may involve a different, bacterium-independent pathway, coupling methane oxidation to elemental sulfur/polysulfide reduction.