The diversification and potential function of microbiome in sediment-water interface of methane seeps in South China Sea

Abstract

The sediment-water interfaces of cold seeps play important roles in nutrient transportation between seafloor and deep-water column. Microorganisms are the key actors of biogeochemical processes in this interface. However, the knowledge of the microbiome in this interface are limited. Here we studied the microbial diversity and potential metabolic functions by 16S rRNA gene amplicon sequencing at sediment-water interface of two active cold seeps in the northern slope of South China Sea, Lingshui and Site F cold seeps. The microbial diversity and potential functions in the two cold seeps are obviously different. The microbial diversity of Lingshui interface areas, is found to be relatively low. Microbes associated with methane consumption are enriched, possibly due to the large and continuous eruptions of methane fluids. Methane consumption is mainly mediated by aerobic oxidation and denitrifying anaerobic methane oxidation (DAMO). The microbial diversity in Site F is higher than Lingshui. Fluids from seepage of Site F are mitigated by methanotrophic bacteria at the cyclical oxic-hypoxic fluctuating interface where intense redox cycling of carbon, sulfur, and nitrogen compounds occurs. The primary modes of microbial methane consumption are aerobic methane oxidation, along with DAMO, sulfate-dependent anaerobic methane oxidation (SAMO). To sum up, anaerobic oxidation of methane (AOM) may be underestimated in cold seep interface microenvironments. Our findings highlight the significance of AOM and interdependence between microorganisms and their environments in the interface microenvironments, providing insights into the biogeochemical processes that govern these unique ecological systems.

Keywords: DAMO; cold seeps; methane metabolism; oxic-hypoxic shifting; sediment-water interface.

FIGURE 1

Sampling locations. (A) Site F and Lingshui cold seeps location in the South China Sea; (B) Conceptual graph of Site F and Lingshui representative microenvironment interface; (C,D) Pictures of sampling sites and organisms sampled during the present study. Representative photos of sampling habitats in Site F (C) and Lingshui (D), respectively. All photographs were taken by ROV Kexue.

FIGURE 2

The NMDS analysis of archaeal (A) and bacterial (B) community and the compositions of archaeal (C) and bacterial (D) community in the order level in Site F and Lingshui cold seeps.

FIGURE 3

Community compositions at family level across different microenvironments at Lingshui and Site F. (A) Relative abundances of subgroups of methanogens and ANME. The orange bars on the right show the total percentages of methane-metabolizing archaea accounted for the total archaea. (B) Relative abundances of the subgroups of methanotrophic bacteria. The pink bars next to the barchart on the right show the methanotrophic percentages accounted for total bacteria. (C) Relative abundances of the dominant subgroups of putative denitrifying bacteria. The light orange/green bars on the right show the total percentages of putative aerobic/anaerobic denitrifiers accounted for total bacteria. (D) Relative abundances of the dominant subgroups of putative sulfate-reducing bacteria. The right viridis bars show the total percentages of SRB accounted for total bacteria. (E) Relative abundances of the dominant subgroups of putative sulfur-oxidizing bacteria (SOB). The right purple bars show the SOB percentages accounted for total bacteria. In all figures, F and L indicate the Site F and Lingshui.

FIGURE 4

The heatmap of functional predictions of archaea and bacteria in sediment-water interface of Lingshui and Site F cold seeps.

FIGURE 5

Distance-based redundancy analysis (dbRDA) plot of the forward selection based on environmental variables (methane, DIC, nitrite, nitrate, ammonia and dissolved oxygen) fitted to the taxonomic composition of bacterial and archaeal communities (at the class level). The significant explanatory variables are showed (red vectors). Microbial taxa are indicated by yellow squares and labeled with black words.

FIGURE 6

Co-occurrence networks and their topological features based on the total prokaryotic communities in the high-methane-supply, low-methane-supply, and control groups. The size of nodes (OTUs) is related to the relative sequence abundance of OTUs. Large modules with ≥10 nodes are shown in different colors, while the small modules are shown in gray color.

FIGURE 7

Web model coupled to carbon/sulfur/nitrogen metabolic process at the interface of Site F by the microbial 16S rRNA gene analyses [During exoenzymatic hydrolysis, detrital organic matter is buried at the interface as macromolecules (MM), then broken down to low molecular weight matters (LMW). Fermentation further degrades LMW to short chain fatty acids such as acetate, and hydrogen, which would act as substrates to produce methane (Zhuang et al., 2019a; Joye, 2020). Meanwhile, fluid seepage from the deep sediment layers delivers additional methane to the interface. At the cyclical oxic-hypoxic fluctuating interfaces, when shifting to aerobic state, methane oxidizing bacteria (dominant by Methylococcales) mediate the aerobic/anaerobic methane oxidation, while AOA mediate ammonia (NH₃) oxidation to produce nitrate/nitrite and SOB mediate hydrogen sulfide (H₂S) oxidation to produce sulfate, which provide the reaction substrates for anaerobic methane oxidation. When anaerobic, the anaerobic methane oxidation coupled with SAMO/DAMO produce carbon dioxide and reductive substances such as hydrogen sulfide/ammonia. The serial numbers represent different metabolic microbial groups. ➀ Fermentation microbiomes, mainly Flavobacteriales, Bacteroidales and so on (Zhuang et al., 2019a,b); ➁ Methanogens, mainly including g_Candidatus Methanomethylicus, g_Methanobacterium, g_Methanimicrococcus (Zhuang et al., 2019a; Joye, 2020); ➂ Aerobic methane oxidation microbiomes, such as Methylomonadaceae, Methyloligellaceae; while AOM microbiomes, not only ANMEs, maybe also including Methylomonadaceae; ➃ AOM coupled SAMO, the prevalent SRB microbiomes are Desulfocapsaceae, Geopsychrobacteraceae, Desulfobulbaceae; AOM coupled DAMO, the predominant aerobic denitrifiers are Pseudomonadaceae, and anaerobic denitrifiers are Alteromonadaceae, Pseudoalteromonadaceae; ➄ Ammonia-oxidizing microorganisms, including Nitrosopumilaceae, Nitrososphaeraceae and so on; SOB principally including Sulfurovaceae, Rhodobacteraceae].