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Marine Environmental Research

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Microbial diversity of two cold seep systems in gas hydrate-bearingsediments in the South China Sea

Hongpeng Cuia,b, Xin Sua,b,∗, Fang Chenc, Melanie Hollandd, Shengxiong Yangc,Jinqiang Liangc,∗∗, Pibo Suc, Hailiang Dongb,e, Weiguo Houb

a School of Ocean Sciences, China University of Geosciences, Beijing, 100083, Chinab State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing, 100083, ChinacGuangzhou Marine Geological Survey, Guangzhou, 510075, ChinadGeotek Ltd. United Kingdom, Daventry, NN118PB, UKe Department of Geology and Environmental Earth Science, Miami University, OH, 45056, USA

A R T I C L E I N F O

Keywords:ArchaeaBacteriaCommunityGas hydrateSedimentsAnaerobic oxidation of methaneSulfate methane transition zone

A B S T R A C T

Cold seep is a unique habitat for microorganisms in deep marine sediments, and microbial communities andbiogeochemical processes are still poorly understood, especially in relation to hydrate-bearing geo-systems. Inthis study, two cold seep systems were sampled and microbial diversity was studied at Site GMGS2-08 in thenorthern part of the South China Sea (SCS) during the GMGS2 gas hydrate expedition. The current cold seepsystem was composed of a sulfate methane transition zone (SMTZ) and an upper gas hydrate zone (UGHZ). Theburied cold seep system was composed of an authigenic carbonate zone (ACZ) and a lower gas hydrate zone(LGHZ). These drill core samples provided an excellent opportunity for analyzing the microbial abundance anddiversity based on quantitative polymerase chain reaction (qPCR) and high-throughput 16S rRNA gene se-quencing. Compared to previous studies, the high relative abundance of ANME-1b, a clade of anaerobic me-thanotrophic archaea (ANME), may perform anaerobic oxidation of methane (AOM) in collaboration withANME-2c and Desulfobacteraceae in the SMTZ, and the high relative abundances of Hadesarchaea, ANME-1barchaea and Aerophobetes bacteria were found in the gas hydrate zone (GHZ) at Site GMGS2-08. ANME-1b,detected in the GHZ, might mainly mediate the AOM process, and the process might occur in a wide depth rangewithin the LGHZ. Moreover, bacterial communities were significantly different between the GHZ and non-GHZsediments. In the ACZ, archaeal communities were different between the two samples from the upper and thelower layers, while bacterial communities shared similarities. Overall, this new record of cold seep microbialdiversity at Site GMGS2-08 showed the complexity of the interaction between biogeochemical reactions andenvironmental conditions.

1. Introduction

Cold seeps usually develop in continental margin sediments, wheresedimentary organic matter is progressively degraded, and transformedinto deeply buried hydrocarbons, such as methane (Vigneron et al.,2013). Cold seeps often contain low-temperature fluids rich in hydro-carbons (primarily methane) and hydrogen sulfide, and then rise to theseafloor, forming oases of elevated microbial biomass and variousfaunal assemblages (Jørgensen and Boetius, 2007; Niu et al., 2017).Due to this distinct biogeochemistry dominated by fluid flow and hy-drocarbon transport, microbes such as methanotrophic and methano-genic archaea, hydrocarbon degraders and sulfate-reducing bacteria are

usually the key functional groups at such cold seep ecosystems(Pachiadaki and Kormas, 2013). The sulfate methane transition zone(SMTZ) is an important region in the sediments where the methanerising from below and the sulfate diffusing from above form a regionsuitable for anaerobic methanotrophy, where anaerobic oxidation ofmethane (AOM) and sulfate reduction (SR) have often been detectedwithin the SMTZ (Boetius et al., 2000; Orphan et al., 2002; Harrisonet al., 2009; Knittel and Boetius, 2009; Lin et al., 2014). The AOMprocess consumes a large fraction of methane in marine sediments, andis responsible for a significant increase in alkalinity, dissolved inorganiccarbon, and sulfide, as well as promotion of the precipitation of au-thigenic carbonates and iron sulfides, which influence the landscape of

https://doi.org/10.1016/j.marenvres.2019.01.009Received 26 July 2018; Received in revised form 29 December 2018; Accepted 14 January 2019

∗ Corresponding author. School of Ocean Sciences, China University of Geosciences, Beijing, 100083, China.∗∗ Corresponding author. Guangzhou Marine Geological Survey, Guangzhou, 510075, China.E-mail addresses: xsu@cugb.edu.cn (X. Su), ljinqiang@hydz.cn (J. Liang).

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the seafloor. Discovery of authigenic carbonates often indicates me-thane release from seeps (Suess, 2014; Feng and Chen, 2015; Masonet al., 2015; Chen et al., 2016; Lin et al., 2016).

To date, studies of microbial communities related to gas hydrate-bearing sediments have been investigated in many places, which in-clude the Cascadia Margin (Bidle et al., 1999; Marchesi et al., 2001;Lanoil et al., 2005), the Hydrate Ridge (Knittel et al., 2005), the Gulf ofMexico (Lanoil et al., 2001; Mills et al., 2003; Yan et al., 2006), thePacific Ocean Margin (Inagaki et al., 2006), the Nankai Trough (Reedet al., 2002; Colwell et al., 2004; Arakawa et al., 2006; Nunoura et al.,2012; Katayama et al., 2016), the Andaman Sea (Briggs et al., 2012),the Ulleung Basin (Briggs et al., 2013; Lee et al., 2013; Cho et al., 2017),and the South China Sea (SCS) (Jiao et al., 2015). These studies mainlyfocused on archaeal and bacterial communities in the gas hydrate zone(GHZ) or the AOM-SR process in the SMTZ. Previous studies haveshown that the AOM-SR process is usually mediated by a consortium ofanaerobic methanotrophic archaea (ANME) and sulfate reducing bac-teria (SRB). Three types of ANME (ANME-1, -2, and -3) have been de-scribed to date, and they appear to occupy distinct ecological niches:ANME-1 and ANME-2 are widespread in many different environments,but ANME-3 is mainly present in mud volcanoes (Boetius et al., 2000;Orphan et al., 2002; Knittel et al., 2005; Knittel and Boetius, 2009; Linet al., 2014; Niu et al., 2017; Timmers et al., 2017). Moreover, ANME-1is frequently found as single cells, indicating that ANME-1 may performAOM process without a bacterial partner (Orphan et al., 2002; Knittelet al., 2005; Maignien et al., 2012; Stokke et al., 2012). Furthermore,ANME-1 might be involved in methane production via reversal of themethanogenic pathway (Harrison et al., 2009; Lloyd et al., 2011; Choet al., 2017). However, ANME-1 lacks the genes encoding N5, N10-me-thylene- tetrahydromethanopterin (H4MPT) reductase (mer) and theenzymes of the dissimilatory sulfate reduction pathway such as ade-nylyltransferase (sat), APS reductase (apr) or dissimilatory sulfite re-ductase (dsr) which are needed during methanogenesis and sulfate re-duction (Meyerdierks et al., 2010; Stokke et al., 2012; Timmers et al.,2017).

Microbial communities associated with cold seeps usually displayhigh density and unique diversity. Archaea and bacteria are crucial inthe biogeochemistry of cold seep systems (Boetius and Suess, 2004;Hein et al., 2006; Suess et al., 2014; Case et al., 2015; Cui et al., 2016;Niu et al., 2017). The gas hydrate dissociation enhanced the fluid fluxesin the cold seeps, which may influence the microbial abundance anddiversity (Hein et al., 2006; Yanagawa et al., 2014; Chen et al., 2016).At present, the microbial communities in the cold seeps related to the

gas hydrate are not as yet fully understood, especially in the SCS. TheGMGS2 gas hydrate drilling expedition was carried out in the SCS,performed by Guangzhou Marine Geological Survey (GMGS) in co-operation with Fugro N.V. (Netherlands) and Geotek Ltd. (UK) usingthe geotechnical drillship M/V REM Etive from June to September in2013 (Zhang et al., 2015). During Expedition GMGS2, Site GMGS2-08was shown to have a deep authigenic carbonate layer (including fos-silized macrofauna) with underlying gas hydrate, which was inter-preted to be a buried cold seep. A gas hydrate layer and SMTZ above theburied cold seep showed that the area was a current cold seep (Zhanget al., 2015; Chen et al., 2016; Lin et al., 2016; Kuang et al., 2018). Onthis basis, the main objective of this study was to investigate microbialdiversity in the two hydrate-related cold seep systems (current andburied) by using high-throughput sequencing of the 16S rRNA gene. Wealso extend the analysis to predict functional genes might be related tomethanogenesis and sulfate metabolism by using Phylogenetic In-vestigation of Communities by Reconstruction of Unobserved States(PICRUSt) at Site GMGS2-08.

2. Materials and methods

2.1. Site description and sampling

The GMGS2 drilling area was located in a vicinity of two seafloorbathymetric highs in the middle of the Taixinan Basin and 13 sites weredrilled during this expedition (Fig. 1), which included 10 logging-while-drilling and 3 downhole wire line logging pilot holes. Site GMGS2-08was drilled and cored to a depth of 100m below seafloor (mbsf) in awater depth of 798m. The resistivity and gamma-ray indicated ex-istence of two layers of gas hydrate in the sediments (Fig. S1) (Zhanget al., 2014, 2015). The current cold seep system contained some light-yellow authigenic carbonates in the upper sediments and an upper gashydrate zone (UGHZ) at depth from 8 to 23 mbsf. The buried cold seepsystem was composed of a massive authigenic carbonate zone (ACZ)from 58 to 63 mbsf and a lower gas hydrate zone (LGHZ) from 65 to 98mbsf (Sha et al., 2015; Zhang et al., 2015; Zhuang et al., 2016). Thedepth of the SMTZ at Site GMGS2-08, determined by sulfate and me-thane profiles, occurs between 1.0 and 7.0 mbsf and the sulfate-me-thane interface (SMI) occurs at about 5.0 mbsf (Fig. S1) (Zhuang et al.,2016; Kuang et al., 2018).

Drill cores were sampled for porewater analysis; samples for mi-crobial analysis were taken adjacent to these geochemical samples. 22sediment samples were selected for microbial analysis (Table S1) and

Fig. 1. Location of the Site GMGS2-08 in the northern SCS (Zhang et al., 2015; Lin et al., 2016).

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each sample was cut into 15-cm-long sections, stored in sealed nitrogen-flushed aluminum foil bags, and stored at −20 °C. At the end of theexpedition, all the samples were transported on dry ice to the Geomi-crobiology Laboratory of China University of Geosciences (Beijing). Thesamples were trimmed off contaminated materials on a clean bench,and stored in a −80 °C freezer until further analysis.

2.2. DNA extraction and PCR amplification

DNA was extracted from 22 sediment samples using the Soil DNAIsolation Kit (MP) following the manufacturer's instructions. The DNAfrom each replicate was mixed in equal amounts to create a compositeDNA sample. Microbial sequencing was performed using the MiSeqIllumina platform at Meiji Biotechnology Company (Shanghai, China).Fragments of 16S rRNA genes from the 15 different depth layers (TableS1) were amplified using the primer combinations of 524F and 958R forarchaea and 338F and 806R for bacteria by the polymerase chain re-action (PCR) (Table S2). Three replicates were conducted with thecomposite DNA template for the PCR reaction and the triplicate PCRproducts were mixed and checked by gel electrophoresis. Each PCRmixture contained 4 μL of 5× FastPfu buffer, 2 μL of 2.5mM dNTPs,0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase, and 10 ng oftemplate DNA in a total volume of 20 μL. The archaeal 16S rRNA genewas amplified by PCR using a thermal profile of 94 °C for 5min, fol-lowed by 32 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2minand a final extension at 72 °C for 10min. For the bacteria, the PCRprogram was 95 °C for 3min; 27 cycles of 30 s at 95 °C; 30 s at 55 °C, and45 s at 72 °C; and final extension for 10min at 72 °C.

2.3. qPCR

Genes subject to quantitative polymerase chain reaction (qPCR)analysis in the 22 sediment samples (Table S1) included archaeal,bacterial, ANME-1 and ANME-2 16S rRNA genes and a functional genedsrA. qPCR amplification was performed using an ABI Prism 7500 se-quence detection system (Applied Biosystems, USA), and all the reac-tions were in a volume of 20 μl containing 10 μL of SYBR Premix Ex Taq(TaKaRa, Japan), 2 μL of DNA, 0.4 μL of each primer, 0.4 μL ROX Re-ference Dye II and an appropriate volume of ddH2O. Primers and an-nealing temperatures were according to the manufacturer's re-commendations (Table S2). Each reaction was conducted in triplicate.The standards for archaeal and bacterial 16S rRNA genes were preparedfrom nearly full length sequences amplified from the crude extractsusing domain specific primers (21F/958R for archaea and 27F/1492Rfor bacteria) and the standards for ANME-1, ANME-2c 16S rRNA anddsrA genes were prepared from the cloned amplicons of these targetgroups. Plasmids were extracted and serial diluted at 1:10 to generate astandard templates and amplified to measure the threshold cycle (CT)for a known concentration with R2 values greater than 0.99.

2.4. Sequencing and bioinformatics

Raw fastq files were demultiplexed and quality filtered using QIIME(Caporaso et al., 2010). Operational Taxonomic Units (OTUs) wereclustered with a 97% similarity cutoff using UPARSE (version 7.1) andchimeric sequences were identified and removed using UCHIME. Onerepresentative sequence was picked for each OTU by selecting the mostabundant sequence in that OTU and these representative sequenceswere analyzed by RDP Classifier against the SILVA 16S rRNA databaseusing a confidence threshold of 70% (Amato et al., 2013). In alphadiversity analysis, alpha diversity parameters such as Chao 1 andShannon were estimated using MOTHUR (Schloss et al., 2009). In betadiversity analysis, non-metric multidimensionnal scaling (NMDS) ana-lysis was performed to visually interpret the microbial communitystructure among the different layers, which was based on the weightedUniFrac distance for data relating to 16S rRNA genes data. One-way

analysis of similarity (ANOSIM) was performed in MOTHUR to testwhether there was a significant difference in the microbial communitycomposition among the different zones. Furthermore, the Mann-Whitney U (Wilcoxon rank-sum) test was used to reveal which organ-isms were responsible for the dissimilarity observed in communitycomposition among the zones. To identify the KEGG Orthologs relatedto the methanogenesis and sulfate metabolism pathways, we used theKEGG database based on the archaeal and bacterial 16S rRNA genes byPICRUSt (Langille et al., 2013). The raw reads were deposited into theNCBI Sequence Read Archive (SRA) database under accession number:PRJNA414008, PRJNA414009.

3. Result

3.1. Quantification of gene abundance

The abundances of the archaeal and bacterial 16S rRNA genesranged from 4.1× 104 to 8.6×106 copies/g wet weight sediment andfrom 7.4×105 to 2.0× 108 copies/g wet weight sediment, respec-tively. The abundances of the ANME-1 and ANME-2c 16S rRNA genesranged from 4.1× 103 to 5.9×106 copies/g wet weight sediment andfrom 1.5×102 to 1.1× 105 copies/g wet weight sediment, respec-tively. The functional gene dsrA ranged from 3.1× 103 to 2.6× 105

copies/g wet weight sediment. Generally, these genes abundances(except for the ANME-2c 16S rRNA gene) peaked at the SMI (5.0 mbsf),and decreased with depth. Moreover, the ANME-1, ANME-2c 16S rRNAand dsrA genes abundances peaked in the UGHZ (12.3 mbsf), and thearchaeal, bacterial, ANME-1 16S rRNA and dsrA genes abundances in-creased with depth in the LGHZ (Fig. 2).

3.2. Archaeal and bacterial diversity

A total of 474369 and 499648 high-quality sequences from archaeaand bacteria were obtained by Miseq sequencing for 15 sedimentsamples, which were assigned to 316 total archaeal OTUs and 3650total bacterial OTUs, respectively. Diversity indices of all samples areshown in Table S3. The archaeal richness indices Chao 1 and Shannonranged from 37.0 to 235.4 and 0.5 to 3.5, respectively. The bacterialrichness indices Chao 1 and Shannon ranged from 241.3 to 1369.0 and3.3 to 6.2, respectively. In general, the archaeal richness indices Chao 1and Shannon were higher in the LGHZ with an average of 199.4 and3.3, and lower in the ACZ with an average of 57.5 and 2.2. The bacterialrichness indices Chao 1 and Shannon were higher in the SMTZ with anaverage of 1198.4 and 5.6, and lower in the UGHZ with an average of537.4 and 4.0.

Taxonomic analysis showed that the relative abundances of majorarchaeal and bacterial groups at Site GMGS2-08 (Fig. 3A). MBGB(Marine Benthic Group B) (23.2%), ANME (20.0%), Bathyarchaeota(previously referred to as MCG) (18.1%), Hadesarchaea (previouslyreferred to as SAGMEG) (18.0%), MBGD (Marine Benthic Group D)(4.4%), Group C3 (4.2%), and MHVG (Marine Hydrothermal VentGroup) (1.7%) were the major archaeal groups. Firmicutes (15.5%),Chloroflexi (15.1%), Actinobacteria (12.8%), Gammaproteobacteria(12.3%), Aerophobetes (6.5%), Bacteroidetes (5.7%), Betaproteo-bacteria (5.4%), Deltaproteobacteria (5.3%), Alphaproteobacteria(5.0%), JS1 (3.3%), Acidobacteria (1.9%), and Cyanobacteria (1.3%)were the major bacterial groups. The relative abundances of major ar-chaeal and bacterial community compositions of the 15 samples alongthe vertical depth were summarized in Fig. 3B. In the SMTZ, ANME(3.4–91.5%), MBGB (5.0–52.2%), Hadesarchaea (0.1–26.1%), Bath-yarchaeota (1.9–18.9%), Group C3 (0.4–8.0%), and MBGD (0.1–5.4%)were the dominant archaeal groups. Firmicutes (5.6–37.5%), Chloro-flexi (7.5–23.1%), Deltaproteobacteria (0.8–19.2%), Actinobacteria(9.0–17.4%), Bacteroidetes (4.5–12.4%), and JS1 (1.2–8.4%) were thedominant bacterial groups. In the UGHZ, Hadesarchaea (0.1–46.7%),Bathyarchaeota (0.8–37.4%), MBGB (7.4–32.3%), and MBGD

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Fig. 2. Copy numbers of the archaeal, bacterial, ANME-1, ANME-2c 16S rRNA and dsrA genes populations of Site GMGS2-08. Two blue-shaded regions are the upperand lower gas hydrate zones. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Relative abundances of major archaeal and bacterial groups (A) and the community compositions (B) in the sediments of Site GMGS2-08. Both figures showmicrobial taxa with relative abundance>0.5% of the total community.

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(0.1–16.3%) were the dominant archaeal members in most layers; ad-ditionally, a high relative abundance of ANME (96.4%) was detected at12.3 mbsf. For bacteria, Betaproteobacteria (0.2–32.3%), Actino-bacteria (5.5–31.0%), Aerophobetes (2.3–26.6%), Chloroflexi(9.7–25.0%), Deltaproteobacteria (0.4–23.6%), and Gammaproteo-bacteria (1.7–13.1%) were the dominant bacterial members. In theACZ, MBGB (47.0%), MHVG (9.4%), and Thaumarchaeota (7.3%) weredominant archaeal groups in the upper ACZ, and Hadesarchaea(48.5%), and Bathyarchaeota (35.9%) were the predominant archaealgroups in the lower ACZ. Firmicutes (31.3–32.5%), Actinobacteria(13.9–15.3%), Bacteroidetes (9.8–13.7%), and Chloroflexi (6.2–8.0%)were the predominant bacterial groups in the ACZ. In the LGHZ,Bathyarchaeota (19.8–28.2%), MBGB (13.8–26.6%), ANME(21.1–25.7%), and MBGD (4.4–6.7%) were the dominant archaealgroups, while Hadesarchaea (2.6–10.4%) accounted for low relativeabundance compared to the UGHZ. Gammaproteobacteria(19.8–67.5%), Aerophobetes (0.2–25.4%), Chloroflexi (3.7–25.0%),Betaproteobacteria (4.0–14.8%), Deltaproteobacteria (1.0–11.0%), andActinobacteria (0.6–10.2%) were the dominant bacterial members inthe LGHZ.

NMDS and ANOSIM tests were performed to visualize the re-lationship of the microbial communities in the 15 samples along thevertical depth. Based on the weighted UniFrac distance, NMDS gave asatisfactory representation of the archaeal (stress= 0.0597) and bac-terial (stress= 0.0797) data (Fig. 4). Results showed that the archaealcommunities from most of the samples of SMTZ and LGHZ were clus-tered together, and two samples of SMI (5.0 mbsf) and UGHZ (12.3mbsf) had similar compositions, while two samples of ACZ displayedrelatively large variations. In general, bacterial communities from theUGHZ and LGHZ were distinct from those of the SMTZ and ACZ. InANOSIM analysis, the archaeal communities were not significantlydifferent among the four zones (ANOSIM, P > 0.05), while significantdifferences for bacterial communities were discovered between UGHZand SMTZ (ANOSIM, P=0.027), between UGHZ and ACZ (ANOSIM,P=0.005), and between LGHZ and SMTZ (ANOSIM, P= 0.017),especially between the GHZ (UGHZ and LGHZ) and non-GHZ (SMTZ,ACZ and Other) (ANOSIM, P= 0.001) (Table 1). Furthermore, Mann-Whitney U test analysis elucidated the taxa responsible for these bac-terial community differences between the GHZ and non-GHZ samples(Fig. 5). The relative abundances of Aerophobetes and Gammaproteo-bacteria were significantly higher in the GHZ samples than in the non-GHZ samples, while the relative abundances of Clostridia and Bacilli(Firmicutes phylum), Bacteroidia (Bacteroidetes phylum), Acid-obacteria, and Cyanobacteria were significantly higher in the non-GHZ

samples (Mann-Whitney U test, P < 0.05).

3.3. Predictive functional analysis

The PICRUSt predictions revealed that the archaeal 16S rRNA genewas linked to 36 and 12 KEGG Orthologs for the enzymes involved inmethanogenesis and sulfate pathways, respectively, which included thecentral enzymes for a complete methane-oxidizing pathway from CH4

to CO2 and the enzyme of dissimilatory sulfite reductase (sat). Thebacterial 16S rRNA gene was linked to 23 orthologs for the enzymesinvolved sulfate pathway, such as the enzymes of adenylyltransferase(sat), APS reductase (apr) and dissimilatory sulfite reductase (dsr)(Tables S4, S5, S6).

4. Discussion

4.1. Microbial community in the SMTZ

Previous studies revealed that Hadesarchaea, Group C3, ANME,MBGB, Bathyarchaeota and MBGD were often the dominant archaealmembers, and JS1, Chloroflexi, Deltaproteobacteria andPlanctomycetes were usually the dominant bacterial members in theSMTZ (Table S7). Most of these archaea and bacteria were detected inthe SMTZ of Site GMGS2-08, while, the unique characteristics were thehigh relative abundance of ANME archaea (3.4–91.5%), and the lowrelative abundance of Planctomycetes bacteria (< 0.5%).

The members of ANME and Deltaproteobacteria are usually de-tected within the SMTZ, and are involved in the AOM-SR process(Orphan et al., 2002; Biddle et al., 2006; Inagaki et al., 2006; Harrisonet al., 2009; Hamdan et al., 2011). In this study, the ANME sequencesincluded ANME-1b, ANME-2c, ANME-1a and ANME-2a. ANME-1bdominated the ANME cluster, accounting for 85.3% of the ANME se-quences, and Desulfobacteraceae was the dominant cluster of Delta-proteobacteria, accounting for 73.3% of the Deltaproteobacteria se-quences. The relative abundances of ANME-1b and Desulfobacteraceaeincreased with depth and peaked at the SMI (5.0 mbsf), accounting for91.4% and 17.3% of the archaeal and bacterial sequences, respectively.Moreover, qPCR also revealed the ANME-1, ANME-2c 16S rRNA anddsrA genes abundances peaked at the SMI (5.0 mbsf) (Fig. 2).

ANME-1b has been detected in the SMTZ of the gas hydrate-bearingsites UBGH2-10 and AT1-GT1/C from Ulleung Basin and NankaiTrough, accounting for 42.6% and 0.25% of the archaeal sequences,respectively (Lee et al., 2013; Katayama et al., 2016). At Site GMGS2-08, ANME-1b represented 91.4% of the total archaeal sequences at the

Fig. 4. The NMDS plots for archaeal (A) and bacterial (B) community structure among the 15 sediment samples using the weighted UniFrac distance based on OTUlevel.

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SMI (5.0mbsf) and this high relative abundance has not been reportedin the other gas hydrate-bearing sites. ANME at around the SMTZ haspreviously been detected in methane-seep sediments, and many studiesdemonstrated that the ANME/SRB ratio was 1:3 in the ANME-2 domi-nated sediments (Orcutt and Meile, 2008), however, the ratio was16–86:1 in the ANME-1 dominated sediments of the Nyegga (Roalkvamet al., 2011) and the ratio was 6:1 in the ANME-1 dominated sedimentsof the Ulleung Basin (Lee et al., 2013). In this study, the abundance ofthe ANME-1 16S rRNA gene was 10–100 times higher than ANME-2c16S rRNA and dsrA genes abundances within the SMTZ. Therefore, atSite GMGS2-08, ANME-1b may perform AOM in partinership withANME-2c and Desulfobacteraceae in the SMTZ.

Beside the SMTZ, Bathyarchaeota and MBGD were also dominant inthe UGHZ and LGHZ. Lloyd et al. (2013) suggested that Bathyarchaeotaand MBGD can produce peptidases (protein-degrading enzymes) andmay be capable of metabolizing protein, which might indicate thatheterotrophy is an important metabolic mode for these groups. Chlor-oflexi and Actinobacteria were widely distributed in the 15 samples.

Chloroflexi has usually been detected in anoxic and organic rich en-vironments such as sediments, subsurfaces and deep-sea hydrothermalsediments (Fry et al., 2008; Flores et al., 2012; Lv et al., 2017), and thepredominant subgroup at Site GMGS2-08 was the class Dehalococcoidia(Phylum Chloroflexi), which is known to be involved in the reductivedegradation of substituted aromatic hydrocarbons (Fennell et al., 2004;Wasmund et al., 2014; Pöritz et al., 2015). Actinobacteria is potentialdegraders of cellulose, hemicellulose and chitin (Yilmaz et al., 2015),and this group may be involved in organic material breakdown at SiteGMGS2-08.

4.2. Microbial community in the GHZ

Compared to previous studies investigating archaeal and bacterialcommunities in the GHZ (Table S8), at Site GMGS2-08, archaea of theBathyarchaeota, MBGB, MBGD, ANME and bacteria of the Chloroflexi,Actinobacteria, Proteobacteria (Beta, Delta and Gamma), JS1 were alsopredominant in the GHZ. However, the unique characteristics of the

Table 1Significance tests of the archaeal and bacterial community structures at the OTU level between the different zones based on weighted UniFrac matrix by usingANOSIM. (* 0.01 < P value ≤ 0.05; ** 0.001 < P value ≤ 0.01; ***P value ≤ 0.001).

Zones Archaea Bacteria

r P r P

UGHZ-SMTZ 0.131 0.305 0.594 0.027*UGHZ-ACZ 0.071 0.445 0.999 0.005**LGHZ-SMTZ 0.036 0.426 0.754 0.017*LGHZ-ACZ 0.500 0.083 0.999 0.098SMTZ-ACZ 0.473 0.092 0.018 0.441UGHZ-LGHZ 0.296 0.114 −0.056 0.559(UGHZ + LGHZ)-(SMTZ + ACZ + Other) −0.020 0.547 0.698 0.001***

Fig. 5. Significant bacterial differences obtained by Mann-Whitney U test at the class level between the GHZ (UGHZ and LGHZ; blue) and non-GHZ (SMTZ, ACZ andOther; orange) are represented by asterisks. (* 0.01 < P value ≤ 0.05; ** 0.001 < P value ≤ 0.01; ***P value ≤ 0.001). (For interpretation of the references tocolour in this figure legend, the reader is referred to the Web version of this article.)

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archaeal and bacterial communities in the GHZ at Site GMGS2-08 werethe high relative abundances of Hadesarchaea, ANME-1b archaea andAerophobetes bacteria, and the low relative abundances of Plancto-mycetes and Firmicutes bacteria.

At present, the high relative abundance of Hadesarchaea was onlyretrieved from the GHZ in SCS (not only sediments from this study, butalso from other GHZ in SCS sites, as shown in our laboratory's un-published data). It has recently been reported that Hadesarchaea isprobably not involved in methanogenesis and is thought to be involvedin the oxidation of carbon monoxide coupled with H2O reduction(Baker et al., 2016). Hadesarchaea was the dominant archaeal group inthe UGHZ (representing 30.4% of total archaeal sequences). However,the relative abundance of Hadesarchaea was low in the LGHZ (re-presenting 5.1% of the total archaeal sequences), and more environ-mental data is still needed to give a reasonable explanation for thisdistribution.

It is noteable that ANME-1b had a high relative abundance of 75.8%at the top of the UGHZ (12.3 mbsf) and 10.8–20.3% in all three samplesin the LGHZ. To the best of our knowledge, such a high relativeabundance of ANME-1b has not yet been found in other GHZ from gashydrate-bearing sites: ANME-1b was detected in the GHZ (300 mbsf) atsite AT1-GT1/C of the Nankai Trough, but with a relative abundance ofonly 5.6% (Katayama et al., 2016).

Previous studies of in situ methane production by normal metha-nogens (e.g., Methanosarcina, Methanomicrobia, Methanobacteriaceae)showed that the TOC required for methanogenesis was usually greaterthan 0.5% and the hydrate saturation generated was generally 1–2%(Waseda, 1998; Klauda and Sandler, 2005), while for the formation ofhigh-saturation gas hydrate, favorable geologic conditions were neededto allow methane-rich fluids to migrate along fractures into the GHZ(Wallmann et al., 2012; Sha et al., 2015). At Site GMGS2-08, the TOCcontents ranges from 0.52% to 1.49% (Fig. S1) (Chen et al., 2016), andthe relative abundances of the typical methanogens were very low,accounting for less than 0.5% of the total archaeal sequences, while thesaturations of gas hydrate were in the range of 30–50% (Zhang et al.,2015). Faults, gas chimneys and diapiric structures were visible inseismic data (Li et al., 2013; Zhang et al., 2014; Sha et al., 2015).Therefore, at Site GMGS2-08, the evidence is in favor of the migrationof methane-bearing fluids, to form the high-saturation gas hydrate.

In view of the above discussions, there are two possibilities to ex-plain the high relative abundance of ANME-1b was detected in theUGHZ and LGHZ: 1. ANME-1b might take the place of normal metha-nogens and be involved with methane production as several studiesproposed that ANME-1 might switch to methanogenic metabolism inmethanogenic and gas hydrate-bearing environments (Lloyd et al.,2011; Yanagawa et al., 2011; Bertram et al., 2013; Cho et al., 2017); 2.ANME-1b mainly participated in the AOM process in the GHZ. Webelieve that the second possibility is more relevant based on two ar-guments. First, ANME-1 is often identified in sulfate-depleted sedimentswithout a (closely associated) bacterial partner, and could performAOM process alone (Maignien et al., 2012; Stokke et al., 2012; Niuet al., 2017; Timmers et al., 2017). Second, the archaeal communitiesshowed similar composition among most samples from the SMTZ andLGHZ. The two samples from the SMI (5.0 mbsf) and the upper of theUGHZ (12.3 mbsf) also shown similarities (Fig. 4). Furthermore, a highrelative abundance of MBGB was detected in the SMTZ, UGHZ andLGHZ. Previous studies proposed that MBGB might assimilate sedi-mentary organic matter as a carbon source while performing AOMprocess for energy generation and possibly partner with ANME duringthe AOM process (Biddle et al., 2006; Inagaki et al., 2006; Sørensen andTeske, 2006; Lee et al., 2013; Aoki et al., 2014). Thus, ANME-1b may bedriving the AOM process, and the wide distribution of ANME-1b in theLGHZ indicates the AOM process may take place over a wide depthrange, which might imply that more methane-rich fluids were suppliedfrom underneath the LGHZ or massive gas hydrate dissociation wasoccurring in the LGHZ. Furthermore, the archaeal richness indices Chao

1 and Shannon showed relatively high values in the LGHZ, whichsuggested the archaeal groups may be active.

Microbial research related to Haima cold seep from the surfacemarine sediments (to 8.3 mbsf) in SCS showed that the ANME-1b wasdivided into three subgroups (ANME-1bI, ANME-1bII and ANME-1bIII)with different distribution patterns, and the authors suggested thatANME-1bII contained sequences only retrieved from the SCS (Niu et al.,2017). We compared our eight OTUs belonging to ANME-1b with theirsequences, and phylogenetic analysis also showed three subgroups (Fig.S2). OTU42 (ANME-1bIII) was dominant above the SMI (5.0 mbsf) andin the three samples of the LGHZ. OTU447 (ANME-1bII) was the majorcomponent of the community at the SMI (5.0 mbsf) and in the upperUGHZ (12.3 mbsf). OTU93 (ANME-1bII) was common in the upper ofUGHZ (12.3 mbsf) and the relative abundance of OTU93 increased withdepth in the LGHZ. However, ANME-1bI (OTUs 108 & 137) was onlyobserved, and in low relative abundance, in the lower part of the SMTZ(6.5 mbsf) and in the upper portion of the LGHZ (69.3 mbsf) (Fig. S3).Overall, the different distribution patterns of the ANME-1b subgroupsstrongly indicate that they may possess diverse metabolic capabilities atSite GMGS2-08.

Moreover, it was surprising that the dsrA gene was detected belowthe SMTZ, especially in the UGHZ and LGHZ (Fig. 2), because sulfate asthe electron acceptor for active SRB was not present in the deep layers.At Site GMGS2-08, the detectable sulfate in the UGHZ and LGHZ waslikely due to core contamination by drilling fluids. This is consistentwith a previous study on microbial diversity of the marine sediments atODP Site 1227 from the Peru margin (Blazejak and Schippers, 2011),which also observed the dsrA gene in layers where sulfate was not de-tectable in the pore water, and the authors suggested that sulfide oxi-dation occurred with reactive iron or manganese oxides as oxidant andthe low amounts of sulfate might be constantly consumed by SRB indeeply buried sediments, thus it remained undetectable in the porewater. Taken together, the wide vertical distribution of the dsrA geneindicated that functionally diverse potential SRB would populate thesubseafloor sediments in deeper zones as well as around the SMTZ andmight be involved in the biogeochemical cycles of organic matter andinorganic energy and elemental sources (Nunoura et al., 2016).

Mann-Whitney U test analysis demonstrated that Clostridia andBacilli (Firmicutes phylum), Bacteroidia (Bacteroidetes phylum),Acidobacteria and Cyanobacteria were observed significantly higher inthe non-GHZ at Site GMGS2-08 (Mann-Whitney U test, P < 0.05)(Fig. 5). Studies have reported that Clostridia can depolymerize starch,chitin, xylan, and cellulose, and Bacillus sp. may play a role in long-chain-paraffin biodegradation (Wiegel et al., 2006; Choi and Lee,2013). Bacteroidetes is normally linked to fermentative metabolism oflabile high molecular weight organic matter, especially under sulfate-reducing conditions, and has the capacity to degrade polysaccharides(Llobet-Brossa et al., 1998; Acosta-González et al., 2013; Hanreichet al., 2013). Comparative genomics revealed broad carbon utilizationcapabilities for Acidobacteria, including the competence to metabolizeC1 compounds and polysaccharide breakdown (cellulose, xylan)(Wegner and Liesack, 2017). Cyanobacteria is involved in nitrogenmetabolism (Luter et al., 2014).

Mann-Whitney U test analysis demonstrated that Aerophobetes andGammaproteobacteria were found significantly higher in the GHZ thanin the non-GHZ at Site GMGS2-08 (Mann-Whitney U test, P < 0.05)(Fig. 5). A recent genomic study suggested that the Aerophobetesbacterium TCS1 has a versatile metabolism and might be involved inglycolysis and pyruvate fermentation (Wang et al., 2016). Gammapro-teobacteria has been considered as potential key player in biode-gradation of oil contaminants in the marine environment (Hazen et al.,2010; Gao et al., 2015), and Pseudomonas, Acinetobacter and Alter-omonadales were the three dominant clusters at Site GMGS2-08.Pseudomonas and Acinetobacter are often associated with oil-degradingprocesses (Kostka et al., 2011; Dawar and Aggarwal, 2015). Alter-omonadales has potential roles in polysaccharide biodegradation and

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carbohydrate metabolism (Edwards et al., 2010). Furthermore, Bur-kholderiales (Phylum Proteobacteria) was also dominant at the bottomof the UGHZ (20.5 mbsf) and LGHZ (93.7 mbsf), accounting for 32.3%and 14.8% of the total bacterial sequences, respectively. Burkholder-iales can use both autotrophic and heterotrophic carbon fixation me-chanisms in isolated deep biosphere environments where concentra-tions of different carbon substrates fluctuate over time, and harbors thepotential for catabolism of aromatic compounds (Pérez-Pantoja et al.,2012; Magnabosco et al., 2016).

Su et al. (2010) suggested that the microbial communities might bedifferent when methane takes different physical forms, such as sedi-mentary layers with solid hydrate or in zones with only dissolved me-thane. At Site GMGS2-08, significant bacterial community differenceswere found between the GHZ and non-GHZ samples and would supportthis suggestion. As was mentioned in the preceding section, the me-thane fluids likely migrated along fractures into the GHZ at SiteGMGS2-08, and many studies have found that some microbes mightalso utilize the substances in migrating fluids as their energy resources(Seewald, 2003; Yanagawa et al., 2014). Thus, the methane fluidsmight affect the sediments characteristics and then the dominant bac-terial groups occurring in the GHZ may be nourished. Moreover, thedominant bacterial groups presented a variety of distribution trends inthe GHZ. For example, in the LGHZ, the relative abundances of Gam-maproteobacteria increased with depth and Aerophobetes decreasedwith depth, while this pattern was not found in the UGHZ. This dif-ferent distribution pattern might be related to the composition of fluids,the amount of fluids supplied and duration of fluid flow. Further in-vestigations are needed to expand our understanding of the microbialdiversity and processes related to cold seep systems in gas hydrate-bearing sediments.

4.3. Microbial communities in the ACZ

Previous studies of microbial community analysis related to authi-genic carbonates have taken place on seafloor samples of isolated car-bonate blocks with sizes from centimeters to tens of meters as well ascontinuous pavements. The microbial communities in such environ-ments have been generally found to be dominated by microbial taxaperforming AOM-SR process, such as ANME, Deltaproteobacteria,Epsilonproteobacterial, Helicobacteraceae and Thiotrichaceae (Marlowet al., 2014a; b; Case et al., 2015). In the two samples of the ACZ at SiteGMGS2-08, archaeal communities were quite different, while the bac-terial communities were similar, with a very low relative abundances ofmicroorganisms related to the AOM-SR process. These results might bedue to relic DNA from AOM-SR-associated organisms degraded withincarbonates during the long periods of carbonate burial. Alternatively,some factors may play a role in controlling the composition of themicrobial community in the ACZ at Site GMGS2-08, as Case et al.(2015) suggested that carbonates host distinct, diverse, and dynamicmicrobial assemblages rather than passively record a time-integratedhistory of seep microorganisms.

5. Conclusions

Site GMGS2-08 obtained cores contained two cold seep systems. Thecurrent cold seep system comprised the SMTZ and UGHZ, and theburied cold seep system was composed of the ACZ and LGHZ. This workmainly presented the genes abundances as well as the archaeal andbacterial 16S rRNA genes diversity among the four zones at SiteGMGS2-08. Distinct from the microbial communities in other studies atgas hydrate-bearing sites, the high relative abundance of ANME-1b wasdetected in the SMTZ, and the high relative abundances ofHadesarchaea, ANME-1b archaea and Aerophobetes bacteria werefound in the GHZ at Site GMGS2-08. In the SMTZ, ANME-1b mayperform AOM process with ANME-2c and Desulfobacteraceae, and theANME-1, ANME-2c 16S rRNA and dsrA genes abundances peaked at the

SMI (5.0 mbsf). In the UGHZ and LGHZ, the high relative abundance ofANME-1b was mainly involved in the AOM process, and the processmight occur in a wide depth range within the LGHZ, as all three samplescontained ANME-1b. Phylogenetic analysis further revealed that thethree subgroups of ANME-1b were distributed in different patterns inthe SMTZ, UGHZ and LGHZ, which indicated ANME-1b possesses di-verse metabolic capabilities at Site GMGS2-08. Furthermore, significantdifferences in bacterial communities were discovered between the GHZand non-GHZ. In the buried cold seep system, our study provides thefirst insight into the microbial communities in deep seep-associatedauthigenic carbonates, and archaeal communities were different be-tween the upper and lower samples of the ACZ, while bacterial com-munities had similar composition.

Acknowledgments

We thank the crew and scientists of the GMGS2 expedition for theirsupports in sediment samples and analysis. This research was supportedby the China Geological Survey project for South China Sea Gas HydrateResource Exploration (Grant No. DD20160211) and the FundamentalResearch Funds for the Central Universities-the China University ofGeosciences (Beijing) (Grant No. 2-9-2016-143).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.marenvres.2019.01.009.

References

Acosta-González, A., Rosselló-Móra, R., Marqués, S., 2013. Characterization of theanaerobic microbial community in oil-polluted subtidal sediments: aromatic biode-gradation potential after the Prestige oil spill. Environ. Microbiol. 15, 77–92.

Amato, K.R., Yeoman, C.J., Kent, A., Righini, N., Carbonero, F., Estrada, A., Gaskins, H.R.,Stumpf, R.M., Yildirim, S., Torralba, M., 2013. Habitat degradation impacts blackhowler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J. 7, 1344.

Aoki, M., Ehara, M., Saito, Y., Yoshioka, H., Miyazaki, M., Saito, Y., Miyashita, A.,Kawakami, S., Yamaguchi, T., Ohashi, A., 2014. A long-term cultivation of ananaerobic methane-oxidizing microbial community from deep-sea methane-seep se-diment using a continuous-flow bioreactor. PLoS One 9, e105356.

Arakawa, S., Sato, T., Yoshida, Y., Usami, R., Kato, C., 2006. Comparison of the microbialdiversity in cold-seep sediments from different depths in the Nankai Trough. J. Gen.Appl. Microbiol. 52, 47–54.

Baker, B.J., Saw, J.H., Lind, A.E., Lazar, C.S., Hinrichs, K.-U., Teske, A.P., Ettema, T.J.,2016. Genomic inference of the metabolism of cosmopolitan subsurface Archaea,Hadesarchaea. Nat. Microbiol. 1, 16002.

Bertram, S., Blumenberg, M., Michaelis, W., Siegert, M., Krüger, M., Seifert, R., 2013.Methanogenic capabilities of ANME-archaea deduced from 13C-labelling approaches.Environ. Microbiol. 15, 2384–2393.

Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R., Fredricks,H.F., Elvert, M., Kelly, T.J., Schrag, D.P., 2006. Heterotrophic Archaea dominatesedimentary subsurface ecosystems off Peru. P. Natl. Acad. Sci. USA. 103,3846–3851.

Bidle, K.A., Kastner, M., Bartlett, D.H., 1999. A phylogenetic analysis of microbial com-munities associated with methane hydrate containing marine fluids and sediments inthe Cascadia margin (ODP site 892B). FEMS Microbiol. Lett. 177, 101–108.

Blazejak, A., Schippers, A., 2011. Real-time PCR quantification and diversity analysis ofthe functional genes aprA and dsrA of sulfate-reducing prokaryotes in marine sedi-ments of the Peru continental margin and the Black Sea. Front. Microbiol. 2.

Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., 2000. A marine microbial con-sortium apparently mediating anaerobic oxidation of methane. Nature 407, 623.

Boetius, A., Suess, E., 2004. Hydrate Ridge: a natural laboratory for the study of microbiallife fueled by methane from near-surface gas hydrates. Chem. Geol. 205 (3–4),291–310.

Briggs, B.R., Graw, M., Brodie, E.L., Bahk, J.-J., Kim, S.-H., Hyun, J.-H., Kim, J.-H., Torres,M., Colwell, F.S., 2013. Microbial distributions detected by an oligonucleotide mi-croarray across geochemical zones associated with methane in marine sedimentsfrom the Ulleung Basin. Mar. Petrol. Geol. 47, 147–154.

Briggs, B.R., Inagaki, F., Morono, Y., Futagami, T., Huguet, C., Rosell-Mele, A., Lorenson,T.D., Colwell, F.S., 2012. Bacterial dominance in subseafloor sediments characterizedby methane hydrates. FEMS Microbiol. Ecol. 81, 88–98.

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K.,Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., 2010. QIIME allows analysis ofhigh-throughput community sequencing data. Nat. Methods 7, 335.

Case, D.H., Pasulka, A.L., Marlow, J.J., Grupe, B.M., Levin, L.A., Orphan, V.J., 2015.Methane seep carbonates host distinct, diverse, and dynamic microbial assemblages.

H. Cui et al. Marine Environmental Research 144 (2019) 230–239

237

mBio 6 e01348-01315.Chen, F., Hu, Y., Feng, D., Zhang, X., Cheng, S., Cao, J., Lu, H., Chen, D., 2016. Evidence

of intense methane seepages from molybdenum enrichments in gas hydrate-bearingsediments of the northern South China Sea. Chem. Geol. 443, 173–181.

Cho, H., Hyun, J.-H., You, O.-R., Kim, M., Kim, S.-H., Choi, D.-L., Green, S.J., Kostka, J.E.,2017. Microbial community structure associated with biogeochemical processes inthe sulfate-methane transition zone (SMTZ) of gas-hydrate-bearing sediment of theUlleung Basin, east sea. Geomicrobiol. J. 34, 207–219.

Choi, Y.J., Lee, S.Y., 2013. Microbial production of short-chain alkanes. Nature 502, 571.Colwell, F., Matsumoto, R., Reed, D., 2004. A review of the gas hydrates, geology, and

biology of the Nankai Trough. Chem. Geol. 205, 391–404.Cui, H., Su, X., Chen, F., Wei, S., Chen, S., Wang, J., 2016. Vertical distribution of archaeal

communities in cold seep sediments from the jiulong methane reef area in the southChina sea. Biosci. J. 32.

Dawar, C., Aggarwal, R.K., 2015. Draft genome sequence of hydrocarbon-degradingPseudomonas putida strain KG-4, isolated from soil samples collected from Krishna-Godavari basin in India. Genome Announc. 3 e00590-00515.

Edwards, J.L., Smith, D.L., Connolly, J., McDonald, J.E., Cox, M.J., Joint, I., Edwards, C.,McCarthy, A.J., 2010. Identification of carbohydrate metabolism genes in the me-tagenome of a marine biofilm community shown to be dominated byGammaproteobacteria and Bacteroidetes. Genes 1, 371–384.

Feng, D., Chen, D., 2015. Authigenic carbonates from an active cold seep of the northernSouth China Sea: new insights into fluid sources and past seepage activity. Deep-sea.Res. Pt. II: Top. Stud. Oceanogr. 122, 74–83.

Fennell, D.E., Nijenhuis, I., Wilson, S.F., Zinder, S.H., Häggblom, M.M., 2004.Dehalococcoides ethenogenes strain 195 reductively dechlorinates diverse chlori-nated aromatic pollutants. Environ. Sci. Technol. 38, 2075–2081.

Flores, G., Shakya, M., Meneghin, J., Yang, Z., Seewald, J., Geoff Wheat, C., Podar, M.,REYSENBACH, A.L., 2012. Inter-field variability in the microbial communities ofhydrothermal vent deposits from a back-arc basin. Geobiology 10, 333–346.

Fry, J.C., Parkes, R.J., Cragg, B.A., Weightman, A.J., Webster, G., 2008. Prokaryoticbiodiversity and activity in the deep subseafloor biosphere. FEMS Microbiol. Ecol. 66,181–196.

Gao, X., Gao, W., Cui, Z., Han, B., Yang, P., Sun, C., Zheng, L., 2015. Biodiversity anddegradation potential of oil-degrading bacteria isolated from deep-sea sediments ofSouth Mid-Atlantic Ridge. Mar. Pollut. Bull. 97, 373–380.

Hamdan, L.J., Gillevet, P.M., Pohlman, J.W., Sikaroodi, M., Greinert, J., Coffin, R.B.,2011. Diversity and biogeochemical structuring of bacterial communities across thePorangahau ridge accretionary prism, New Zealand. FEMS Microbiol. Ecol. 77,518–532.

Hanreich, A., Schimpf, U., Zakrzewski, M., Schlüter, A., Benndorf, D., Heyer, R., Rapp, E.,Pühler, A., Reichl, U., Klocke, M., 2013. Metagenome and metaproteome analyses ofmicrobial communities in mesophilic biogas-producing anaerobic batch fermenta-tions indicate concerted plant carbohydrate degradation. Syst. Appl. Microbiol. 36,330–338.

Harrison, B.K., Zhang, H., Berelson, W., Orphan, V.J., 2009. Variations in archaeal andbacterial diversity associated with the sulfate-methane transition zone in continentalmargin sediments (Santa Barbara Basin, California). Appl. Environ. Microbiol. 75,1487–1499.

Hazen, T.C., Dubinsky, E.A., DeSantis, T.Z., Andersen, G.L., Piceno, Y.M., Singh, N.,Jansson, J.K., Probst, A., Borglin, S.E., Fortney, J.L., 2010. Deep-sea oil plume en-riches indigenous oil-degrading bacteria. Science 330, 204–208.

Hein, J.R., Normark, W.R., McIntyre, B.R., Lorenson, T.D., Powell, C.L., 2006.Methanogenic calcite, 13C-depleted bivalve shells, and gas hydrate from a mudvolcano offshore southern California. Geology 34 (2), 109–112.

Inagaki, F., Nunoura, T., Nakagawa, S., Teske, A., Lever, M., Lauer, A., Suzuki, M., Takai,K., Delwiche, M., Colwell, F.S., 2006. Biogeographical distribution and diversity ofmicrobes in methane hydrate-bearing deep marine sediments on the Pacific OceanMargin. P. Natl. Acad. Sci. USA. 103, 2815–2820.

Jørgensen, B.B., Boetius, A., 2007. Feast and famine–microbial life in the deep-sea bed.Nat. Rev. Microbiol. 5, 770.

Jiao, L., Su, X., Wang, Y., Jiang, H., Zhang, Y., Chen, F., 2015. Microbial diversity in thehydrate-containing and-free surface sediments in the Shenhu area, South China Sea.Geosci. Front. 6, 627–633.

Katayama, T., Yoshioka, H., Takahashi, H.A., Amo, M., Fujii, T., Sakata, S., 2016. Changesin microbial communities associated with gas hydrates in subseafloor sediments fromthe Nankai Trough. FEMS Microbiol. Ecol. 92.

Klauda, J.B., Sandler, S.I., 2005. Global distribution of methane hydrate in ocean sedi-ment. Energy Fuels 19, 459–470.

Knittel, K., Boetius, A., 2009. Anaerobic oxidation of methane: progress with an unknownprocess. Annu. Rev. Microbiol. 63, 311–334.

Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R., 2005. Diversity and distributionof methanotrophic archaea at cold seeps. Appl. Environ. Microbiol. 71, 467–479.

Kostka, J.E., Prakash, O., Overholt, W.A., Green, S.J., Freyer, G., Canion, A., Delgardio, J.,Norton, N., Hazen, T.C., Huettel, M., 2011. Hydrocarbon-degrading bacteria and thebacterial community response in Gulf of Mexico beach sands impacted by theDeepwater Horizon oil spill. Appl. Environ. Microbiol. 77, 7962–7974.

Kuang, Z., Fang, Y., Liang, J., Lu, J., Wang, L., 2018. Geomorphological-geological-geo-physical signatures of high-flux fluid flows in the eastern Pearl River Mouth Basin andeffects on gas hydrate accumulation. Sci. China Earth Sci. 61 (7), 914–924.

Langille, M.G., Zaneveld, J., Caporaso, J.G., McDonald, D., Knights, D., Reyes, J.A.,Clemente, J.C., Burkepile, D.E., Thurber, R.L.V., Knight, R., 2013. Predictive func-tional profiling of microbial communities using 16S rRNA marker gene sequences.Nat. Biotechnol. 31 (9), 814.

Lanoil, B.D., La Duc, M.T., Wright, M., Kastner, M., Nealson, K.H., Bartlett, D., 2005.Archaeal diversity in ODP legacy borehole 892b and associated seawater and

sediments of the Cascadia Margin. FEMS Microbiol. Ecol. 54, 167–177.Lanoil, B.D., Sassen, R., La Duc, M.T., Sweet, S.T., Nealson, K.H., 2001. Bacteria and

ArchaeaPhysically associated with Gulf of Mexico gas hydrates. Appl. Environ.Microbiol. 67, 5143–5153.

Lee, J.-W., Kwon, K.K., Azizi, A., Oh, H.-M., Kim, W., Bahk, J.-J., Lee, D.-H., Lee, J.-H.,2013. Microbial community structures of methane hydrate-bearing sediments in theUlleung Basin, east sea of Korea. Mar. Petrol. Geol. 47, 136–146.

Li, L., Lei, X., Zhang, X., Sha, Z., 2013. Gas hydrate and associated free gas in the DongshaArea of northern South China Sea. Mar. Petrol. Geol. 39, 92–101.

Lin, L.H., Wu, L.W., Cheng, T.W., Tu, W.X., Lin, J.R., Yang, T.F., Chen, P.C., Wang, Y.,Wang, P.L., 2014. Distributions and assemblages of microbial communities along asediment core retrieved from a potential hydrate-bearing region offshore south-western Taiwan. J. Asian Earth Sci. 92, 276–292.

Lin, Q., Wang, J., Algeo, T.J., Su, P., Hu, G., 2016. Formation mechanism of authigenicgypsum in marine methane hydrate settings: evidence from the northern South ChinaSea. Deep-sea. Res. Pt. I: Oceanogr. Res. Pap. 115, 210–220.

Llobet-Brossa, E., Rosselló-Mora, R., Amann, R., 1998. Microbial community compositionof Wadden Sea sediments as revealed by fluorescence in situ hybridization. Appl.Environ. Microbiol. 64, 2691–2696.

Lloyd, K.G., Alperin, M.J., Teske, A., 2011. Environmental evidence for net methaneproduction and oxidation in putative ANaerobic MEthanotrophic (ANME) archaea.Environ. Microbiol. 13, 2548–2564.

Lloyd, K.G., Schreiber, L., Petersen, D.G., Kjeldsen, K.U., Lever, M.A., Steen, A.D.,Stepanauskas, R., Richter, M., Kleindienst, S., Lenk, S., 2013. Predominant archaea inmarine sediments degrade detrital proteins. Nature 496, 215.

Luter, H.M., Gibb, K., Webster, N.S., 2014. Eutrophication has no short-term effect on theCymbastela stipitata holobiont. Front. Microbiol. 5, 216.

Lv, B., Cui, Y., Tian, W., Feng, D., 2017. Composition and influencing factors of bacterialcommunities in ballast tank sediments: implications for ballast water and sedimentmanagement. Mar. Environ. Res. 132, 14–22.

Magnabosco, C., Ryan, K., Lau, M., Kuloyo, O., Kieft, T., van Heerden, E., Onstott, T.,2016. A metagenomic window into carbon metabolism at 3 km depth in Precambriancontinental crust. ISME J. 10, 730–741.

Maignien, L., Parkes, R.J., Cragg, B., Niemann, H., Knittel, K., Coulon, S., Akhmetzhanov,A., Boon, N., 2012. Anaerobic oxidation of methane in hypersaline cold seep sedi-ments. FEMS Microbiol. Ecol. 83, 214–231.

Marchesi, J.R., Weightman, A.J., Cragg, B.A., Parkes, R.J., Fry, J.C., 2001. Methanogenand bacterial diversity and distribution in deep gas hydrate sediments from theCascadia Margin as revealed by 16S rRNA molecular analysis. FEMS Microbiol. Ecol.34, 221–228.

Marlow, J.J., Steele, J.A., Case, D.H., Connon, S.A., Levin, L.A., Orphan, V.J., 2014a.Microbial abundance and diversity patterns associated with sediments and carbo-nates from the methane seep environments of Hydrate Ridge, OR. Frontiers Marine.Sci. 1, 44.

Marlow, J.J., Steele, J.A., Ziebis, W., Thurber, A.R., Levin, L.A., Orphan, V.J., 2014b.Carbonate-hosted methanotrophy represents an unrecognized methane sink in thedeep sea. Nat. Commun. 5, 5094.

Mason, O.U., Case, D.H., Naehr, T.H., Lee, R.W., Thomas, R.B., Bailey, J.V., Orphan, V.J.,2015. Comparison of archaeal and bacterial diversity in methane seep carbonatenodules and host sediments, Eel River Basin and Hydrate Ridge, USA. Microb. Ecol.70, 766–784.

Meyerdierks, A., Kube, M., Kostadinov, I., Teeling, H., Glöckner, F.O., Reinhardt, R.,Amann, R., 2010. Metagenome and mRNA expression analyses of anaerobic metha-notrophic archaea of the ANME-1 group. Environ. Microbiol. 12, 422–439.

Mills, H.J., Hodges, C., Wilson, K., MacDonald, I.R., Sobecky, P.A., 2003. Microbial di-versity in sediments associated with surface-breaching gas hydrate mounds in theGulf of Mexico. FEMS Microbiol. Ecol. 46, 39–52.

Niu, M., Fan, X., Zhuang, G., Liang, Q., Wang, F., 2017. Methane-metabolizing microbialcommunities in sediments of the Haima cold seep area, northwest slope of the SouthChina Sea. FEMS Microbiol. Ecol. 93.

Nunoura, T., Takaki, Y., Kazama, H., Hirai, M., Ashi, J., Imachi, H., Takai, K., 2012.Microbial diversity in deep-sea methane seep sediments presented by SSU rRNA genetag sequencing. Microb. Environ. 27, 382–390.

Nunoura, T., Takaki, Y., Shimamura, S., Kakuta, J., Kazama, H., Hirai, M., Masui, N.,Tomaru, H., Morono, Y., Imachi, H., 2016. Variance and potential niche separation ofmicrobial communities in subseafloor sediments off Shimokita Peninsula, Japan.Environ. Microbiol. 18, 1889–1906.

Orcutt, B., Meile, C., 2008. Constraints on mechanisms and rates of anaerobic oxidation ofmethane by microbial consortia: process-based modeling of ANME-2 archaea andsulfate reducing bacteria interactions. Biogeosci. Discuss. 5, 1933–1967.

Orphan, V.J., House, C.H., Hinrichs, K.-U., McKeegan, K.D., DeLong, E.F., 2002. Multiplearchaeal groups mediate methane oxidation in anoxic cold seep sediments. P. Natl.Acad. Sci. USA. 99, 7663–7668.

Pérez-Pantoja, D., Donoso, R., Agulló, L., Córdova, M., Seeger, M., Pieper, D.H., González,B., 2012. Genomic analysis of the potential for aromatic compounds biodegradationin Burkholderiales. Environ. Microbiol. 14, 1091–1117.

Pöritz, M., Schiffmann, C.L., Hause, G., Heinemann, U., Seifert, J., Jehmlich, N., vonBergen, M., Nijenhuis, I., Lechner, U., 2015. Dehalococcoides mccartyi strain DCMB5respires a broad spectrum of chlorinated aromatic compounds. Appl. Environ.Microbiol. 81, 587–596.

Pachiadaki, M.G., Kormas, K.A., 2013. Interconnectivity vs. isolation of prokaryoticcommunities in European deep-sea mud volcanoes. Biogeosciences 10, 2821.

Reed, D.W., Fujita, Y., Delwiche, M.E., Blackwelder, D.B., Sheridan, P.P., Uchida, T.,Colwell, F.S., 2002. Microbial communities from methane hydrate-bearing deepmarine sediments in a forearc basin. Appl. Environ. Microbiol. 68, 3759–3770.

Roalkvam, I., Jørgensen, S.L., Chen, Y., Stokke, R., Dahle, H., Hocking, W.P., Lanzén, A.,

H. Cui et al. Marine Environmental Research 144 (2019) 230–239

238

Haflidason, H., Steen, I.H., 2011. New insight into stratification of anaerobic me-thanotrophs in cold seep sediments. FEMS Microbiol. Ecol. 78, 233–243.

Sørensen, K.B., Teske, A., 2006. Stratified communities of active archaea in deep marinesubsurface sediments. Appl. Environ. Microbiol. 72, 4596–4603.

Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B.,Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J., 2009. Introducing mo-thur: open-source, platform-independent, community-supported software for de-scribing and comparing microbial communities. Appl. Environ. Microbiol. 75,7537–7541.

Seewald, J.S., 2003. Organic-inorganic interactions in petroleum-producing sedimentarybasins. Nature 426, 327.

Sha, Z., Liang, J., Zhang, G., Yang, S., Lu, J., Zhang, Z., McConnell, D.R., Humphrey, G.,2015. A seepage gas hydrate system in northern South China Sea: seismic and welllog interpretations. Mar. Geol. 366, 69–78.

Stokke, R., Roalkvam, I., Lanzen, A., Haflidason, H., Steen, I.H., 2012. Integrated meta-genomic and metaproteomic analyses of an ANME-1-dominated community inmarine cold seep sediments. Environ. Microbiol. 14, 1333–1346.

Suess, E., 2014. Marine cold seeps and their manifestations: geological control, biogeo-chemical criteria and environmental conditions. Int. J. Earth Sci. 103, 1889–1916.

Su, X., Chen, F., Zhang, Y., Wang, Y., Jiao, L., 2010. Geomicrobiology as a new tool forexploration of marine gas hydrates. Geoscience 24, 409–423 (In Chinese).

Timmers, P.H., Welte, C.U., Koehorst, J.J., Plugge, C.M., Jetten, M.S., Stams, A.J., 2017.Reverse methanogenesis and respiration in methanotrophic archaea. Archaea 2017.

Vigneron, A., Cruaud, P., Pignet, P., Caprais, J.-C., Cambon-Bonavita, M.-A., Godfroy, A.,Toffin, L., 2013. Archaeal and anaerobic methane oxidizer communities in the SonoraMargin cold seeps, Guaymas Basin (Gulf of California). ISME J. 7, 1595–1608.

Wallmann, K., Pinero, E., Burwicz, E., Haeckel, M., Hensen, C., Dale, A., Ruepke, L., 2012.The global inventory of methane hydrate in marine sediments: a theoretical ap-proach. Energies 5, 2449–2498.

Wang, Y., Gao, Z.-M., Li, J.-T., Bougouffa, S., Tian, R.M., Bajic, V.B., Qian, P.-Y., 2016.Draft genome of an Aerophobetes bacterium reveals a facultative lifestyle in deep-seaanaerobic sediments. Sci. Bull. 61, 1176–1186.

Waseda, A., 1998. Organic carbon content, bacterial methanogenesis, and accumulationprocesses of gas hydrates in marine sediments. Geochem. J. 32, 143–157.

Wasmund, K., Schreiber, L., Lloyd, K.G., Petersen, D.G., Schramm, A., Stepanauskas, R.,Jørgensen, B.B., Adrian, L., 2014. Genome sequencing of a single cell of the widelydistributed marine subsurface Dehalococcoidia, phylum Chloroflexi. ISME J. 8, 383.

Wegner, C.-E., Liesack, W., 2017. Unexpected dominance of elusive Acidobacteria in earlyindustrial soft coal slags. Front. Microbiol. 8, 1023.

Wiegel, J., Tanner, R., Rainey, F.A., 2006. An Introduction to the Family Clostridiaceae,the Prokaryotes. Springer, pp. 654–678.

Yan, T., Ye, Q., Zhou, J., Zhang, C.L., 2006. Diversity of functional genes for methano-trophs in sediments associated with gas hydrates and hydrocarbon seeps in the Gulf ofMexico. FEMS Microbiol. Ecol. 57, 251–259.

Yanagawa, K., Kouduka, M., Nakamura, Y., Hachikubo, A., Tomaru, H., Suzuki, Y., 2014.Distinct microbial communities thriving in gas hydrate-associated sediments from theeastern Japan Sea. J. Asian Earth Sci. 90, 243–249.

Yanagawa, K., Sunamura, M., Lever, M.A., Morono, Y., Hiruta, A., Ishizaki, O.,Matsumoto, R., Urabe, T., Inagaki, F., 2011. Niche separation of methanotrophicarchaea (ANME-1 and-2) in methane-seep sediments of the eastern Japan Sea off-shore Joetsu. Geomicrobiol. J. 28, 118–129.

Yilmaz, P., Yarza, P., Rapp, J.Z., Glöckner, F.O., 2015. Expanding the world of marinebacterial and archaeal clades. Front. Microbiol. 6.

Zhang, G., Liang, J., Lu, J.a., Yang, S., Zhang, M., Holland, M., Schultheiss, P., Su, X., Sha,Z., Xu, H., 2015. Geological features, controlling factors and potential prospects ofthe gas hydrate occurrence in the east part of the Pearl River Mouth Basin, SouthChina Sea. Mar. Petrol. Geol. 67, 356–367.

Zhang, G., Yang, S., Zhang, M., Liang, J., Lu, J., Holland, M., Schultheiss, P., Team, G.,2014. GMGS2 expedition investigates rich and complex gas hydrate environment inthe South China Sea. Fire in the Ice 14, 1–5.

Zhuang, C., Chen, F., Cheng, S., Lu, H., Wu, C., Cao, J., Duan, X., 2016. Light carbonisotope events of foraminifera attributed to methane release from gas hydrates on thecontinental slope, northeastern South China Sea. Sci. China Earth Sci. 59, 1981–1995.

H. Cui et al. Marine Environmental Research 144 (2019) 230–239

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