Scholarly article on topic 'Microbial diversity in cold seep sediments from the northern South China Sea'

Microbial diversity in cold seep sediments from the northern South China Sea Academic research paper on "Biological sciences"

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{"16S rRNA" / "Microbial diversity" / "Cold seep" / "Marine sediments" / "Northern South China Sea"}

Abstract of research paper on Biological sciences, author of scientific article — Yong Zhang, Xin Su, Fang Chen, Yuanyuan Wang, Lu Jiao, et al.

Abstract South China Sea (SCS) is the largest Western Pacific marginal sea. However, microbial studies have never been performed in the cold seep sediments in the SCS. In 2004, “SONNE” 177 cruise found two cold seep areas with different water depth in the northern SCS. Haiyang 4 area, where the water depth is around 3000 m, has already been confirmed for active seeping on the seafloor, such as microbial mats, authigenic carbonate crusts and bivalves. We investigated microbial abundance and diversity in a 5.55-m sediment core collected from this cold seep area. An integrated approach was employed including geochemistry and 16S rRNA gene phylogenetic analyses. Here, we show that microbial abundance and diversity along with geochemistry profiles of the sediment core revealed a coupled reaction between sulphate reduction and methane oxidation. Acridine orange direct count results showed that microbial abundance ranges from 105 to 106 cells/g sediment (wet weight). The depth-related variation of the abundance showed the same trend as the methane concentration profile. Phylogenetic analysis indicated the presence of sulphate-reducing bacteria and anaerobic methane-oxidizing archaea. The diversity was much higher at the surface, but decreased sharply with depth in response to changes in the geochemical conditions of the sediments, such as methane, sulphate concentration and total organic carbon. Marine Benthic Group B, Chloroflexi and JS1 were predominant phylotypes of the archaeal and bacterial libraries, respectively.

Academic research paper on topic "Microbial diversity in cold seep sediments from the northern South China Sea"

GEOSCIENCE FRONTIERS 3(3) (2012) 301-316

available at China University of Geosciences (Beijing)


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Microbial diversity in cold seep sediments from the northern South China Sea

Yong Zhang a b, Xin Su a b *, Fang Chen c, Yuanyuan Wang a b, Lu Jiao a b, Hailiang Dong a'd, Yongyang Huang c, Hongchen Jiang a'e'*

a State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China

School of Ocean Sciences, China University of Geosciences, Beijing 100083, China c Guangzhou Marine Geology Survey, Guangzhou 510075, China

Department of Geology, Miami University, Oxford, OH 45056, USA e Geomicrobiology Laboratory, Science Institute, China University of Geosciences, Beijing 100083, China

Received 8 August 2011; accepted 23 November 2011 Available online 16 December 2011


16S rRNA; Microbial diversity; Cold seep; Marine sediments; Northern South China Sea

Abstract South China Sea (SCS) is the largest Western Pacific marginal sea. However, microbial studies have never been performed in the cold seep sediments in the SCS. In 2004, "SONNE" 177 cruise found two cold seep areas with different water depth in the northern SCS. Haiyang 4 area, where the water depth is around 3000 m, has already been confirmed for active seeping on the seafloor, such as microbial mats, authigenic carbonate crusts and bivalves. We investigated microbial abundance and diversity in a 5.55-m sediment core collected from this cold seep area. An integrated approach was employed including geochemistry and 16S rRNA gene phylogenetic analyses. Here, we show that microbial abundance and diversity along with geochemistry profiles of the sediment core revealed a coupled reaction between sulphate reduction and methane oxidation. Acridine orange direct count results showed that microbial abundance ranges from 105 to 106 cells/g sediment (wet weight). The depth-related variation of the abundance showed the same trend as the methane concentration profile. Phylogenetic analysis

* Corresponding authors. State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China. Tel.: +86 10 82335403; fax: +86 10 82320065.

E-mail addresses: (X. Su), hongchen.jiang@gmail. com (H. Jiang).

1674-9871 © 2011, China University of Geosciences (Beijing) and Peking University. Production and hosting by Elsevier B.V. All rights reserved.

Peer-review under responsibility of China University of Geosciences (Beijing).

doi: 10.1016/j.gsf.2011.11.014

indicated the presence of sulphate-reducing bacteria and anaerobic methane-oxidizing archaea. The diversity was much higher at the surface, but decreased sharply with depth in response to changes in the geochemical conditions of the sediments, such as methane, sulphate concentration and total organic carbon. Marine Benthic Group B, Chloroflexi and JS1 were predominant phylotypes of the archaeal and bacterial libraries, respectively.

© 2011, China University of Geosciences (Beijing) and Peking University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Cold seep sediments represent one of the most extreme marine conditions and offer unbounded opportunities to discover the interactions between macroorganisms/microbes and geochemical processes. Defining the diversity and distribution of microbial communities in marine cold seep sediments has been a longstanding challenge in the field of microbial ecology and evolution (Li et al., 1999). Cold seeps are characterized by fluids seepage into surface sediments, and the fluids have elevated methane and/ or sulfide concentrations over those of ambient seawater. The first cold seep ecosystem was discovered on the Florida Escarpment in the Gulf of Mexico (Paull et al., 1984). Subsequently, seeping sites were found in different geological settings, including active [e.g. Northern Pacific (Kojima, 2002)] and passive margins [e.g. Florida Escarpment in the Gulf of Mexico (Paull et al., 1984)] at depths ranging from 10 m (Jensen et al., 1992) to at least 7300 m (Jumars and Hessler, 1976).

Microbial chemosynthetic carbon fixation is the basis for the food web at cold seeps. Microbial-driven metabolic processes, such as aerobic oxidation of methane, anaerobic oxidation of methane (AOM), methanogenesis, sulphate reduction, sulfide oxidation, and petroleum hydrocarbon oxidation, have been recognized (Aharon and Fu, 2000; Hinrichs et al., 2000; Formolo et al., 2004; Inagaki et al., 2004; Joye et al., 2004; Niemann et al., 2006). In these processes, AOM presumably coupled to sulphate reduction facilitates formation of carbonates and, in many places, generates extremely high concentrations of hydrogen sulphide in pore waters (Levin, 2005). Methane can also be oxidized aerobically to CO2 by aerobic methanotrophic bacteria that live at the interface between anoxic, methane-rich sediments and oxic seawater Reed et al., 2009). In cold seep environments, microorganisms contribute to the growth of invertebrates as symbionts and play an important role in the sulphur cycle (Kennicutt et al., 1985).

Microbial community structures have been investigated in different typological cold seep areas around the world, such as the Gulf of Mexico (Yan et al., 2006), Hydrate Ridge (Knittel et al., 2005), Mediterranean Sea (Heijs et al., 2007; Omoregie et al., 2009), Nankai Trough (Arakawa et al., 2006), Sagami Bay (Fang et al., 2006; Takishita et al., 2007), and Marmara Sea (Ritt et al., 2010). Those previous studies showed that different cold seep areas harboured characteristic microbial community structures. For example, in Sagami Bay, g-proteobacteria and d-pro-teobacteria were the dominant bacterial phylotypes, and Euryarchaeotes including the anaerobic methane oxidation group (ANME)-2a and ANME-2c were detected (Fang et al., 2006). In Nankai Trough cold seep, Gammaproteobacteria and Deltapro-teobacteria were identified amongst the bacteria from three depths of the sediments, but ANMEs and methanogens were only found in the 600-m deep sediments (Arakawa et al., 2006). In the

tropical Timor Sea methane seep, Alpha-, Delta- and Gammap-roteobacteria and marine group-I (MG-I) were the dominant bacterial and archaeal phylotypes, respectively (Wasmund et al.,

2009). At Hydrate Ridge, there were only four phylogenetic archaelal clusters detected, most of which belonged to ANME (Knittel et al., 2005). In pockmarks and brine seeps of Eastern Mediterranean mud volcanoes, the microbial activity showed strong differences with respect to the rates of AOM and sulphate reduction as well as microbial structures (Omoregie et al., 2009). In the Gulf of Mexico, anaerobic methane-oxidizing communities were influenced by hydrocarbons at the gas hydrate and seep sites, and can be differentiated from those in other normal marine sediments (Yan et al., 2006).

The South China Sea (SCS) is one of the marginal seas around the Pacific Ocean. Tectonically, it is a passive margin setting grading into the SCS Basin and it abuts on the accretionary wedge formed off-shore with the Southwestern Taiwan Island. During the past decades, a number of geophysical and geological cruises were performed in this area, and old and/or active methane seepages were found in the Dongsha area of the SCS (Suess, 2005). These cruises also showed the presence of gas hydrates, and gas hydrate samples were obtained by drilling in the Shenhu area of the northern SCS in 2007 (Zhang et al., 2007).

During the past decades, a number of geophysical, geological, biological and geochemical evidences for the presence of gas hydrates, and cold seeps or fluid and methane vents have been found in the Haiyang 4 Site. Bottom simulating reflectors (BSRs) were found in this region (Song et al., 2001). Methane-derived cold seep carbonates and presence of microbes in these carbonates were reported by Chen (Chen et al., 2005) and Su et al. (2008). Cold seep bivalve communities and bacterial mats were observed by seafloor camera surveys during the Chinese "Haiyang 4" cruises and SO177 cruise (Fig. 1); and an alive bivalve sample was obtained by "Haiyang 4" in 2004 and abundant dead bivalve shells were colleted by "SO177" in 2005 (Suess, 2005; Huang et al., 2008). The SO177 cruise also detected high concentrations of methane, sulphate-methane-interface (SMI), and negative chloride ion anomalies in sediment cores (TV-guided multi-core and gravity cores) from the "Haiyang 4" Site area (Suess, 2005; Huang et al., 2008).

A few studies have examined microbial communities in the SCS. The results showed that archaeal and bacterial diversities of the SCS sediments were similar to those in other deep-sea sediments (Xu et al., 2004; Jiang et al., 2007; Li et al., 2008a, b; Zhang et al.,

2010). However, little is known about the microbial community composition and its correlation with geochemical conditions in cold seep sediments in the SCS. This knowledge is of great importance for understanding the biogeochemical processes in globe cold seep ecosystems. The objective of this study was therefore to study microbial communities in sediments of a gravity piston core (Core DSH-1) collected from the cold seep in the northern SCS.

119°00' 119°10' 119o20' 119°30' 119°40' 119°50'E

■......till I

-3750 -3250 -2750 -2250 -1750 -1250

Depth (m)

Figure 1 Haiyang 4 area in the northern South China Sea (adapted from Suess, 2005). Abyssal dead seep bivalve communities (white shells) and light yellow coloured bacterial mats (adapted from Huang et al., 2008). The marked star showed the location of gravity cores (DSH-1 and SO177-GC9).

2. Materials and methods

2.1. Study site and sample collection

The study site is located at the end of the Formosa Canyon (FC) in the Southwestern Taiwan Basin in the northern SCS. The FC has developed along the northwest part of the LRTF [Luzon—Ryukyu Transform Fault (Hsu et al., 1998, 2004)]. The abyssal plain in and adjacent to the FC, with a water depth ranging from 2900 m on flanks to 3400 m at the centre of the FC channel (Fig. 1), is covered with sediments characterized by silty clay interbedded with turbidities. Since the first evidence of cold seeps was discovered by the Chinese research vessel "Haiyang 4", this area was named as "Haiyang 4 Area" by the "SONNE" 177 cruise (SO177), a cruise of the cooperative research project between Chinese and German leading marine research institutions, i.e. the Guangzhou Marine Geological Survey and Leibniz-Institut für Meereswissenschaften Kiel (Suess, 2005; Huang et al., 2008).

Among a number of SO177 gravity cores in the "Haiyang 4 Area", the SO177 Core GC 9 (21°18'26.52"N, 119°11'49.62"E, 3009 m below sea level) is a ~ 850-cm-long and consists mainly of silty clay interbedded with turbidite layers. Evidences of fluid and

methane vent in this core were found by the SO177 cruise. For example, the SMI took place at the depth about 600 cm below seafloor (cmbsf), high concentrations of methane (8000 mmol/L) and Cl anomaly (540 mmol/L) were present at a depth of about 500 cmbsf and at the base of the core (Suess, 2005; Huang et al., 2008).

In summer 2006, a 5.55-m sediment gravity piston core DSH-1 was collected from the water depth of 3016 m during the "Haiyang 4" cruise. Core DSH-1 is located close to the SO177 Core GC 9 (Fig. 1). Lithologically, Core DSH-1 is composed of silty clay interbedded with turbidite layers, which was well corresponded to the SO177 Core GC 9, however, it is 300 cm shorter than SO177 Core GC 9. Onboard geochemical analyses showed a sharp increase of head-space methane concentration from 2.1 to 20.4 mmol/L at a depth of about 400 cmbsf at Core DSH-1 (Su et al., 2007), indicating the presence of SMI. Such relative near seafloor surface occurrence of SMI provided evidence for methane seepage in this core.

Core DSH-1 was immediately dissected into 50 cm sections onboard as soon as it was retrieved. The first 5 cm of each section was cut for microbial analyses. Approximately 1 cm3 sediments were taken from the top of the 5-cm sections with a syringe and added into a sterilized bottle with 9 mL filtered (0.22-mm) seawater. A 4% (v/v) formaldehyde solution was added to the

bottle to fix the cells and the bottle was stored at 4 °C until cell counting. Then the 5-cm sections of sediments were stored in liquid nitrogen for microbial molecular work. After return to the lab, samples for microbial molecular work were stored in liquid nitrogen at the Guangzhou Marine Geological Survey for three months, and then transported with dry ice to the Geomicrobiology Laboratory of China University of Geosciences (Beijing) and then stored in a —80 °C freezer until further analysis.

2.2. Geochemical analyses

Headspace methane concentration in the Core DSH-1 sediments was measured using HP5890 Series II Gas Chromatography onboard of the "Haiyang 4" during the cruise (Su et al., 2007).

Due to the lack of pore water data for Core DSH-1, the pore water data from Core GC 9 (Suess, 2005; Huang et al., 2008) were taken as reference. However, it should be noticed that the SMI in Core DSH-1 is about 200 cmbsf shallower than that in Core GC 9. According to the SO177 report (Suess, 2005), pore water was extracted by pressure filtration using a PE-squeezer on board of the SO177 cruise. The squeezer was operated with argon at a pressure gradually increasing up to 5 bar. Depending on the porosity and compressibility of the sediments, up to 30 mL of pore water were retrieved from each core section (30 cm). The pore water samples were filtered through 0.2mm cellulose acetate membrane filters and analyzed during the cruise for alkalinity, chloride, ammonia, phosphate, hydrogen sulfide, silicate, methane, sulphate, and bromide using previously published methods (Table 1) (Suess, 2005).

Total organic carbon (TOC) content was determined on shore in the geochemical laboratory of the School of Ocean Sciences, China University of Geosciences (Beijing) according to the modified procedures described elsewhere (Liu, 1996). Briefly, about 0.5 g sediment was dried in air and mixed into 10 mL potassium dichromate buffer solution (1/6 x 0.400 mol). The resulting slurry was then heated at 175 °C for 10 min, followed by addition of 5 mL phosphoric acid buffer (acid:H2O = 1:1). Titration was performed using 0.2 mol ferrisulfas solution with sodium diphenylamine sulfonate as an indicator.

2.3. Total microbial cell counts

Total microbial cells in the sediments were counted by using the method of acridine orange direct count (AODC) as previously described (Bottomley, 1994).

2.4. DNA extraction, PCR and phylogenetic analyses

Based on the profiles of pore water chemistry and AODC results, three sediment subsamples (DSH1, 5—10 cm depth at the top;

DSH9, 400—405 cm depth in the middle, and DSH12, 550—555 cm depth at the bottom) were selected to correlate microbial community structure with these geochemical results. Community DNA was extracted from 0.5 to 1.0 g sediments by using UltraClean soil DNA extraction kit (MoBio, Solana Beach, Calif., USA) according to the manufacturer's instructions.

16S rRNA genes were amplified with primers Arch21F and Arch958R (DeLong, 1992) for archaea and with primers Bac27F and Univ1492R (Lane, 1991) for bacteria, respectively. PCR conditions are as following: initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 and 54 °C for 30 s, and extension at 72 °C for 2 and 1.5 min (for bacteria and archaea, respectively); and a final extension at 72 °C for 10 min. All PCRs were run in triplicate. PCR products were examined by electrophoresis on a 1% agarose gel.

PCR products were purified using a Gel Spin DNA purification kit (AXYGEN, USA), then ligated into the pGEM-T Easy Vector (Promega, USA) and transformed into Escherichia coli JM109 competent cells. Six clone libraries (three each for bacteria and archaea) were constructed. Colonies were randomly selected and analyzed for the 16S rRNA gene inserts. Sequencing reactions were carried out with primers Arch21F and Bac27F for archaea and bacteria with the BigDye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, USA). The 16S rRNA gene sequences were determined with an ABI 3730 automated sequencer. Nucleotide sequences were assembled and edited by using Sequencer v.4.8 (GeneCodes, Ann Arbor, MI).

The potential presence of chimeric sequences was examined with Bellerophon (Huber et al., 2004). The secondary-structures of all obtained sequences were analyzed using the Vienna RNA Package (Hofacker, 2003). Potential chimeric sequences were removed. Operational taxonomic units (OTUs) were determined using DOTUR (Schloss and Handelsman, 2005) with a 97% cutoff value. Based on dissimilar distance and pairwise comparisons, neighbour-joining phylogenetic trees were constructed from with the Jukes—Cantor distance model using the molecular evolutionary genetics analysis (MEGA) program, version 4.1. Bootstrap replications of 1000 were assessed.

2.5. Statistical analysis

The coverage (C) was derived from the equation C = 1—(n/N), where n is the number of clones that occurred only once, and N is the total number of clones examined (Mullins et al., 1995). Rarefaction curves were calculated by the DOTUR program (Schloss and Handelsman, 2005).

Table 1 Methods used for geochemical analyses.

Constituent Method Reference

Chloride Titration Gieskes et al., 1991

Total organic carbon Titration Liu, 1996

Hydrogen sulphide Spectrophotometry Grasshoff et al., 1999

Methane Gas chromatography Niewohner et al., 1998

Sulphate, chloride Ion chromatography


Note: Methods for pore water analyses were cited from the SO177 report (Suess, 2005).

2.6. Nucleotide sequence accession numbers

The sequences determined in this study were deposited in the GenBank database under accession numbers GU475193-GU475255 and GU475256-GU475365 for archaea and bacteria, respectively.

3. Results

3.1. Geochemical results

Headspace methane concentration was about 2.1 mmol/L at the top of the core, and increased with depth, reached to >20.4 mmol/L at the bottom of the core (Table 2). According to the "SONNE" 177 cruise report (Suess, 2005; Huang et al., 2008), sulphate concentration showed a decreasing trend with depth, from 29.1 mmol/L at the top to 12.6 mmol/L at the bottom of the core (Table 2). Correspondingly, hydrogen sulfide concentration increased from zero above 250 cm to 1.6 mmol/L at the 550 cm depth (Table 2). The concentration of Cl" was approximately 550 mmol/L above 450 cm and slightly decreased below this depth (Table 2). TOC content ranged from 0.5% to 0.7% (dry weight) (Table 2).

3.2. Microbial abundance

AODC results showed that the abundance decreased from 2.3 x 106 cells/g (wet weight) near the top to 0.5 x 106 cells/g at 200 cm depth, followed by an increase to the maximal value of 9.6 106 cells/g near the bottom. The trend corresponded to the methane concentration variation throughout the core (Fig. 2E).

3.3. Clone library analyses

A total of 213 archaeal 16S rRNA gene clone sequences (104, 77, and 32 for DSH1A, DSH9A, and DSH12A, respectively) and 182 bacterial 16S rRNA gene clone sequences (112, 33, and 37 for DSH1B, DSH9B, and DSH12B, respectively) were subjected to phylogenetic analyses. The numbers of the sampled clones represented 43.8%—100% coverage for the clone libraries (Table 3). Rarefaction curves indicated that the number of OTUs for the archaeal library for the middle and bottom samples nearly reached saturation (Fig. 3A), but it was not enough for the surface sample, especially for the bacterial library (Fig. 3B).

~200 -

D 400 -

600 LIZ_,__. _

0 40 0 2 540 560

Sulfate (mmol/L) H2S (mmol/L) ci" (mmol/L)

CH4 (mmol/L) 106 (cells/g) w(TOC) (%)

Figure 2 Depth profiles of sulphate (A), hydrogen sulfide (B), chloride ion (C), methane (D) concentrations in pore water, microbial abundance in the sediments as determined by AODC (E), and total organic carbon content (F) (pore water data based on Suess, 2005).

Archaeal diversity decreased rapidly with depth along the core, from fifty-four OTUs at the top to five and four OTUs at the middle and bottom, respectively (Table 3). Bacterial diversity showed a similar trend with depth. In the top sediments, eighty-five OTUs were observed, but the number of OTUs decreased to fifteen and ten (Table 3) for the middle and bottom sediment samples, respectively. Among the identified groups, marine benthic group (MBG)-B and halobacteria (21.2% each) were the predominant groups for archaea (Table 3) and Proteobacteria (25.9%) for bacteria (Table 3) for the top sediment sample. With

Table 2 Geochemical parameters of the methane seepage core DSH-1 in the northern SCS.

Sample number Depth (cm) CH4 (mmol/L) SO|" (mmol/L) H2S (mmol/L) Cl" (mmol/L) w(TOC) (%)

DSH1 5— -10 2.05 29.1 0 555 0.51

DSH2 50— 55 2.32 28.22 0 551 0.51

DSH3 100— 105 2.53 28.31 0 558 0.56

DSH4 150— 155 2.61 28.7 0 556 0.65

DSH5 200— 205 1.59 28.63 0 557 0.58

DSH6 250— -255 1.77 28.64 0 556 0.79

DSH7 300— 205 2.49 28.46 14.80 554 0.61

DSH8 350— 355 3.74 28.36 17.20 554 0.61

DSH9 400— 405 5.45 25.75 5.60 556 0.50

DSH10 450— -455 8.08 21.35 1276.80 554 0.55

DSH11 500— 505 12.73 15.27 1824.00 546 0.59

DSH12 550— 555 20.34 12.62 1612.00 549 0.56

Note: SO2", H2S and Cl" results cited from the SO177 report (Huang et al., 2008).

Table 3 Phylogenetic affiliations and compositions of archaeal and bacterial 16S rRNA gene clones retrieved from the methane seepage

sediments in the northern SCS.

Clone library DSH1 DSH9 DSH12


Depth (cm) 5—10 400—405 550—555

No. of clone sequences 104 112 77 33 32 37

No. of OTUsa 54 85 5 15 4 10

Coverage (%) 69.2 43.8 100 81.9 100 83.8


ANME-1 2 (6.3%)b

Halobacteriales 22 (21.2%)

MBG-D 22 (21.2%) 12 (15.6%)

SAGMEG 2 (1.9%) 14 (18.2%)

ADL 8 (25%)

UEC-1 10 (9.6%)

UEC-2 13 (12.5%)


MBG-B 1 (0.96%) 30 (38.9%) 20 (62.5%)

MBG-C 16 (15.4%) 5 (6.5%)

MCG 9 (8.7%)

MGI 4 (3.8%) 2 (6.3%)

C3 5 (4.8%) 16 (20.8%)


Alphaproteobacteria 3 (2.7%)

Gammaproteobacteria 4 (3.6%) 1 (2.7%)

Epsilonproteobacteria 7 (6.3%)

Deltaproteobacteria 15 (13.4%) 1 (2.7%)

Acidobacteria 1 (0.9%)

Actinobacteria 1 (0.9%) 1 (3%) 1 (2.7%)

Chlorobi 5 (4.5%)

Chloroflexi 6 (5.4%) 26 (78.8%) 8 (21.6%)

Firmicutes 4 (3.6%)

JS1 2 (1.8%) 5 (15.2%) 26 (70.3%)

OP3 4 (3.6%)

OP8 4 (3.6%)

OP11 16 (14.3%)

Planctomycetes 17 (15.2%) 1 (3%)

Spirochaetes 3 (2.7%)

TM6 3 (2.7%)

Verrucomicrobia 3 (2.7%)

WS3 4 (3.6%)

Unclassified 10 (8.9%)

a Number of different OTU identified at the phylotype level.

b The number outside the parenthesis represents the number of clone sequences in the library, and the number inside the parenthesis is the relative abundance of clones affiliated with each major group in the clone libraries.

Number of sequenced clones Number of sequenced clones

Figure 3 Relative richness of archaea (A) and bacteria (B) shown through rarefaction analyses in the methane seepage sediments from the northern SCS.

— DSH1A73 (GU475229)

-Eastern Mediterranean sdiments clone 113A67 (EF687634)

r DSH1A96 (GU475235)

Gulf of Mexico Sediments clone SURF-GC205-Arc10 (DQ521761) DSH1A59 (GU475220) 4 Mangrove Soil clone MKCSM-G5 (DQ363833) DSH1A1 (GU475193) 3 Skan Bay sediments clone SBAK-shallow-42 (DQ640173) DSH1A139 (GU475245) DSH1A16 (GU475202) 2 Pacific Ocean Margin sediments clone ODP1244A5.5 (AB177232) "I_| DSH1A38 (GU475213)

10^ South China Sea sediments clone MD2896-A30 (EU048661) 75i South China Sea sediments clone (EU385832)

"" "iNankai Trough ODP Leg 190 sediments clone NANK-A83 (AY436525) DSH9A14 (GU475249) 12 99 r DSH1A17 (GU475203) 4 60— DSH1A39 (GU475214)

Skan Bay sediments clone SBAK-mid-07 (DQ640150) Skan Bay sediments clone SBAK-mid-69 (DQ640229) Okhotsk coastal sediments clone 0HKA1.30 (AB094526) Skan Bay sediments clone SBAK-mid-68 (DQ640228) DSH1A13 (GU475200) 3 DSH1A46 (GU475217) • DSH1-1A66 (GU475225)

r DSH1A33 (GU475210) 5

Peru continental shelf sediments 1H5_E05 (DQ301986)

-DSH1A130 (GU475243) \r DSH1A8 (GU475195)

P-South China Sea sediments clone SCS-QBS-A31 (EF104088) ]_| DSH1A116 (GU475238) 2

99- Santa Barbara Basin sediments clone A050A02 (FJ455912)

Cascadia Margin ODP Leg 892b sediments clone V.8.ArB20 (AY367348) DSH12A17 (GU475255) 2 Marine sediments clone BA1b1 (AF134382)

¡Ir D I2T- H

100 92

j—DSH1A78 (GU475231)

T- Iheya Basin hydrothermal vent clone pMC1A4 (AB019754) -DSH1A15 (GU475201) 4

51 58 r DS

67 "nT- :

„„ L Me

Mid-Atlantic Ridge sediments clone pIR3AG06 (AY354120) -DSH1A67 (GU475226) DSH1A21 (GU475206) 2 DSH1A9 (GU475196) 6

DSH1A18 (GU475204) 5 Mediterranean Cold Seep sediments clone Kazan-2A-04/BC19-2A-04 (AY591982)

-Eastern Mediterranean sediments clone Urania-2A-34 (AY627512)

IP-DSH1A91 (GU475233) 2

|j- Baby Bare Seamount sediments clone FS266-25A-03 (AY704379) T- DSH1A10 (GU475197)

100-DSH1A127 (GU475242)

'-South China Sea sediments clone MD2896-A21 (EU048656)

-DSH1A37 (GU475212)

-DSH1A138 (GU475244)

-Juan de Fuca Ridge active hydrothermal field clone FZ1aA153 (AY165971)

-Eastern Mediterranean volcano clone 104A11 (EF687522)

-DSH1A65 (GU475224)

-DSH1A12 (GU475199)

-DSH1A69 (GU475228)

-DSH1A63 (GU475222)

-DSH1A104 (GU475236) 2

-ODP Leg 201 sediments clone 4H3_ar22 (DQ302023)

-DSH1A64 (GU475223) - DSH1A30 (GU475209) 3

L Santa Barbara Basin sediments clone A163B09 (FJ455952) Cheetham Salt Works sediments clone CSW6.14.5 (AY498648) DSH12A7 (GU475254) 8

100 95

15 L <5

100 It

100 'Tibetan Lakes sediments clone CEHLW-A11 (FJ155649) -Pacific Ocean Margin sediments clone ODP1227A5.28 (AB177032)

|-DSH1A112 (GU475237)

di-DSH1A118 (GU475239)

53H1-South China Sea sediments clone MD2896-A37 (EU385611)

q- DSH9A15 (GU475250) 14 69T- Peru continental shelf sediments clone 1H5_D09 (DQ301983)


- Aquifex pyrophilus (M83548)

Figure 4 Neighbour-joining tree (partial sequences, ~700 bp) showing the phylogenetic relationships of archaeal 16S rRNA gene sequences cloned in the methane seepage sediments from the northern SCS. Bootstrap values of >50% (for 1000 iterations) are shown. Aquifex pyrophilus is used as the out-group. One representative clone type within each phylotype is shown and the number of clones within each phylotype is shown at the end (after the GenBank accession number). If there is only one clone sequence in a given phylotype, the number "1" is not shown.


80 DSH9A1 (GU475247) 30 74 DSH12A5 (GU475253) 20

54 100

Nankai Trough sediments clone KM-r-0.28 (AB214523) DSH1A19 (GU475205)

Northwestern Atlantic Ocean sediments clone CRA8-27cm (AF119128) DSH1A61 (GU475221) 2

100 71

Antarctic bathypelagic sediments clone PS2ARC16 (EF069362) DSH1A93 (GU475234) 2

99 Iheya North field sediments clone IARC-10 (AB175585) Mangrove Soil clone MKCST-C7 (DQ363808) Tibetan Lakes sediments clone GHLW-A14 (FJ155594) South Dakota Wind Cave clone WCA46 (AY217531) DSH12A2 (GU475252) 2

91 DSH1A2 (GU475194) 10

68 Nankai Trough, ODP Leg 190 sediments clone NANK-A84 (AY436515) L Nankai Trough, ODP Leg 190 sediments clone NANK-A106 (AY436518) DSH9A21 (GU475251) 5

Forearc Basin sediments clone MA-A1-1 (AY093446) DSH1A57 (GU475219) 2 L DSH1A83 (GU475232) 2

-DSH1A147 (GU475246)

DSH1A28 (GU475208)

100 DSH1A41 (GU475215) 2

Pacific Ocean Margin sediments clone ODP1251A1.8 (AB177267) DSH1A11 (GU475198) 3 DSH9A9 (GU475248) 15

100 Pacific Ocean Margin sediments clone ODP1230A33.09 (AB177118) DSH1A122 (GU475240)

-Northwestern Atlantic Ocean sediments clone APA2-17cm (AF119135)

99 DSH1A34 (GU475211) 2 91 Tidal flat sediments clone BS1-1-8 (AY396655) DSH1A68 (GU475227) ■ Mangrove Soil clone MKCSM-A7 (DQ363775) DSH1A56 (GU475218) ! Tidal flat sediments clone BS1-1-38 (AY396673) 100 DSH1A123 (GU475241)

Okhotsk coastal sediments clone OHKA1.5 (AB094517) 100 DSH1A77 (GU475230)

l-Skan Bay sediments clone SBAK-deep-53 (DQ640190)

— DSH1A26 (GU475207)

— Pacific Ocean Margin sediments clone ODP1227A1.22 (AB177001) DSH1A44 (GU475216)

100 Eastern Mediterranean sediments clone Urania-2A-09 (AY627488) -Aquifex pyrophilus (M83548)

Figure 4 (Continued).

depth, the MBG-B group became predominant, and reached 38.9% and 62.5% for the middle and bottom samples, respectively. For bacteria, Chloroflexi and JS1 groups became dominant, and reached 78.8% and 70.3% for the middle and bottom samples, respectively.

All the retrieved archaeal clone sequences were classified into seven euryarchaeotal and six crenarchaeotal groups, respectively (Table 3 and Fig. 4).

(1) Crenarchaeota. One hundred and eight clones were clustered into the Crenarchaeota, which consisted of five major groups:

MBG-B and -C (Vetriani et al., 1999), miscellaneous cren-archaeotic group (MCG) (Takai and Horikoshi, 1999), marine group I (MGI) (DeLong, 1992), and C3 (Inagaki et al., 2006). Fifty-one clones (23.6% of the archaeal library) were affiliated with MBG-B (synonymous with the deep-sea archaeal group, DSAG), and they were related to clone sequences retrieved from the deep-sea sediments of northwestern Atlantic Ocean (Vetriani et al., 1999) and the methane seep sediments of Nankai Trough (Nunoura et al., 2006). Twenty-one clones belonged to MBG-C and the closest relatives were those retrieved from the Forearc Basin sediments (Reed et al.,

2002) and the Nankai Trough sediments (Newberry et al., 2004). Nine clones were grouped into the MCG group. They were related to the sediment clones from the Okhotsk coast (Inagaki et al., 2003), Nankai Trough (Newberry et al., 2004), Eastern Mediterranean (Heijs et al., 2008), and Skan Bay (Kendall et al., 2007). Six clones were grouped into MGI and the reference sequences were retrieved from the Iheya North field hydrothermal vent (Nakagawa et al., 2005) and the Antarctic bathypelagic sediments (Gillan and Danis, 2007),

the Tibetan lake (FJ155594) sediments, and mangrove soil (DQ363808). Twenty-one clones belonged to the C3 group. They were related to the sequences recovered from the Site 1251 and 1230 of the ODP Pacific Ocean Margin sediments (Inagaki et al., 2006).

(2) Euryarchaeota. A total of 105 clones clustered into the Eur-yarchaeota, which consisted of seven phylotypes: MBG-D (Vetriani et al., 1999), ANME-1 (Hinrichs et al., 1999), Halobacteriales (Heijs et al., 2007), South African Gold Mine

DSH1B5 (GU475259) -DSH1B107 (GU475313)

■DSH1B110 (GU475315) DSH1B60 (GU475294)

DSH1B115 (GU475318)

99 1 Southern Okinawa Trough sediments p763_b_4.46 (AB305515) DSH1B54 (GU475288)

Pacific Ocean Margin sediments clone ODP1230B3.14 (AB177191)

98 | DSH12B69 (GU475364) Pacific Ocean Margin se SrDSH1B6 (GU475260) 2

"L South China Sea surface sediments clone MD2896-B8 (EU048668)

86 _| DSH1B119 (GU475321)

92lpacific Ocean Margin sediments clone ODP1230B1.02 (AB177127) - DSH1B121 (GU475323) -DSH1B16 (GU475268)

■ DSH1B55 (GU475289)

-DSH1B26 (GU475274)

■ DSH1B17 (GU475269) 100 L_ Desulfocapsa sp. CBII115 (DQ831556) - Desulfuromusa sp. A601 (EU283459)

J00|DSH1B108 (GU475314)

Uncultured clone 0DP1230B12.11 (AB177148)

-DSH1B70 (GU475298)

- DSH1B143 (GU475338) -DSH1B7 (GU475261) 3

-DSH1B9 (GU475263)

100 I—Helicobacter sp. (AB188787) DSH1B8 (GU475262) 2

— Lau Basin hydrothermal vents clone pLM5B-21 (AB247855)

DSH1B127 (GU475327)

100 I-Sulfitobacter sp. DHVB8 (FJ848891)

DSH1B28 (GU475276) - Coxiella sp. Ax29_G2 (EF092201) — DSH1B133 (GU475331) Thioalkalivibrio sp. JT58-36 (AB189351) DSH1B18 (GU475270)

-DSH1B94 (GU475309)

I DSH12B17 (GU475361)

100 L Psychrobacter sp. 228(130zx) (AM403661)

-Aquifex pyrophilus (M83548) H

S S ro CD

Figure 5 Phylogenetic analysis of the bacterial 16S rRNA gene sequences in the methane seepage sediments from northern SCS. The same algorithms as those for the archaeal tree were used. Aquifex pyrophilus is used as the out-group. Panel A is the first bacterial subtree showing the Proteobacteria. (B) This figure is the second subtree showing the non-Proteobacteria.

99 .DSH9B1 (GU475341) 5 — DSH12B13 (GU475360) 3

(GU475343) 5 DSH9B23 (GU475351) 2

r DSH9B16 (GU475348) 2 ji Pacific Ocean Margin sediments clone ODP1244B5.17 (AB177303)

n-DSH12B8 (GU475359)

(GU475349) 4

67 i-DSH1


100 f DSH9B12 (GU475347) 2 -L DSH9B52 (GU475354)

Salt marsh clone SIMO-1820 (AY711186) DSH1B128 (GU475328) 2

-I |—— Gulf of Mexico continental slope clone IODP1319B11.35 (AB433056)

4 L-|_I—DSH12B3 (GU'™"' "

I Santa Barbara Ba — DSH9B46 (GU

Gulf of MeXico clone IODP1320B92.4 (AB433088)

_I—DSH12B3 (GU475358) 3

""9p Santa Barbara Basin sediments clone 5BAV_A12arb (EU181483) DSH9B46 (GU475353) _r DSH9B38 (GU475352) 2

-DSH1B118 (GU475320) -Ocean crust clone P9X2b3A11 (EU491096) - DSH1B31 (GU475277)

- DSH1B42 (GU475282J 2 _

_t DSH1B14 (GU475267) 2

Q|~98i-Hydrothermal vent.done Sc-NB09 (AB193925)

_DSH1B104 (GU475312) -DSH1B44 (GU475283) _Sediments clone 661229 (DQ404778)

-DSH1B68 (GU475297J

, Mangrove soil clone MSB-3D10 (DQ811922)

_DSH1B34 (GU475278) 2

_DSH1B135 (GU475333)

98 |-DSH1B10 (GU475264) 2

I-Hypersaline clone 02D2Z37

I-DSH1B51 (GU475286)

-J-DSH1B59 (GU475293) 3

T-DSH1B87 (GU475306) 3

)8 | DSH9B2 (GU475342)

—I_Geodermatophilus sp. (X92363)

| DSH12B74 (GU475365)

-77^71 Propionibacterium acnes (AB538431)

100 100 |-DSH1B72 (GU475299)

78 I-1_Freshwater pond clone MVS-86 (DQ676424)

0 I- DSH1B84 (GU4/5305)

-L Mud volcano clone 113B496 (EF687501)

-DSH1B57 (GU475291) 2

_DSH1B138 (GU475334)

-DSH1B20 (GU475271) 2

_South China Sea sediments clone MD2896-B88 (EU385702)

,-DSH1B38 (GU475280)

Marine clone JdFBGBact_44 (DQ070831) DSH1B120 (GU475322)

_ DSH1B122 (GU475324)

-DSH1B100 (GU475310)

_ DSH1B132 (GU475330)

100 I— DSH1B141 (GU475337) 2 —-LPer ------ " ' '

Peru Margin sediments clone 0DP1230B10.05


0 .— DSH1B73 (GU475300) -L Holophaga sp. JT58-6 (AB189335) .— Cascadia Margin sediment clone 0DP1251B3.20 (AB177335)

{j-DSH1B114 (GU475317) i

31 Sc' iU ™ 171 1 1

South China Sea sediments clone MD2902-B70 (EU385888) 93 I DSH1B58 (GU475292) 100 IXisha Trough sediments clone MD2902-B12 (EU048619)

-10-DSH1B123 (GU475325) 2

. DSH1B124 (GU475326)

100 . DSH,1Bi2 (GU475265) 4

Pacific Ocean Margin sediments clone ODP1251B1.4 (AB177300)

_100 |— DSH1B63 (GU475296) 2

1_ Cascadia Margin clone

- DSH1B77 (GU475303)



, DSH9B5 (GU475344) 2 'SHIB" (GU475303) J Pacific Ocean Margin sediments clone ODP1244B5.17 (AB177250) 1DSH12B2 (GU475357) 23

_ DSH9B7 (GU475345) 3

-A forearc basin sediments clone MB-B2-103 (AY093469)

_ DSH12B26 (GU475362) | DSH1B24 (GU475272) 2 1DSH12B53 (GU475363) 2 -DSH1B13 (GU475266)

_Northern Bering Sea sediments clone 038E63 (EU925847)

I-DSH1B78 (GU475304)

nn L Southern Okinawa Trough hydrothermal sediments clone p763_b_7.01 (AB305517)

99 I-DSH1B139 (GU475335)

- South China Sea sediments clone MD2898-B25 (EU386066)

49 (GU475285) 2 (GU475319)

_DSH1B62 (GU475295)

DSH1B53 (GU475287) |— DSH1B146 (GU475340)

1 Deep-sea sediments done MD2896-1m.71 (DQ996969) 100 |—DSH1B4 (GU475258)

-1 Shelf sediments clone SC3-8 (DQ289943)

100 rDSH1B130 (GU475329)

H_South China Sea clone MD2894-B42 (EU386035)

DSH1B46 (GU475284)

_DSH1B91 (GU475308)

-DSH1B140 (GU475336)

South China Sea clone MD2902-B96 (EU385904)

_DSH9B8 (GU475346)

_DSH1B111 (GU475316) 2

_DSH1B75 (GU475302)

DSH1B25 (GU475273) 2

_DSH1 B102 (GU475311)

DSH1B134 (GU475332) DSH1B2 (GU475256) 2 Cascadia Margin ODP1251B8.4 (AB177345)

-DSH1B36 (GU475279) 2

_DSH1B39 (GU475281)


_Arabian Sea sediments clone PL 98 (FJ2O8498)

DSH1B74 (GU475301)

Arabian Sea sediments clone PL 73 (EU445348)

-DSH1B27 (GU475275)

Boulder Creek river clone Bol26 (AY193177) DSH1B3 (GU475257)

Aquifers clone HDBW-WB69 (AB237732) DSH1B89 (GU475307)

Aquifex pyrophilus (M83548)

South China Sea sediments clone MD2896-B236 (EU385812)

Figure 5 (Continued).

Euryarchaeotic Group (SAGMEG) (Takai et al., 2001), Antarctic deep lake (ADL) (Burns et al., 2004), and two Uncultured Euryarchaeotic Clusters (UECs). Thirty-three clones (15.5% of the archaeal library) were grouped into MBG-D. They were related to the clones retrieved from the sediments in Eastern Mediterranean (Omoregie et al., 2008), Okhotsk (Inagaki et al., 2003), SCS, Skan Bay (Kendall et al., 2007), Gulf of Mexico (Lloyd et al., 2006) and the Cascadia and Peru Margins (Inagaki et al., 2006). Sixteen clones belonged to the SAGMEG and grouped with those retrieved from the Peru Margin (Sorensen and Teske, 2006) and the SCS sediments. Twenty-two clones were grouped into Hal-obacteriales and they were related to the clones retrieved from Mediterranean (Heijs et al., 2007, 2008), the Iheya Basin hydrothermal vent (Nakagawa et al., 2005), Mid-Atlantic Ridge (Nercessian et al., 2005), and the Baby Bare Seamount sediments (Huber et al., 2006). Two sequences belonging to the ANME-1 group were related to clones obtained from the Cascadia Margin (Lanoil et al., 2005) and methane-consuming sediments from the Eel River basin (Hinrichs et al., 1999). Twenty-three clones formed the two UECs. They all belonged to the DSH1A clone library. Ten clones formed the UEC-1 group and they were related to the clones recovered from SCS (Jiang et al., 2007), Peru continental shelf (Sorensen and Teske, 2006), and Santa Barbara Basin sediments (Harrison et al., 2009). Fourteen clones were grouped into the EUC-2 group and were related to clone sequences from the SCS (Li et al., 2008a), Juan de Fuca Ridge active hydrothermal field (Schrenk et al., 2003), Eastern Mediterranean Volcano (Omoregie et al., 2008), and Santa Barbara Basin (Harrison et al., 2009).

One hundred and eighty-two bacterial clones could be classified into fifteen phylogenetic groups (Table 3), among which Proteobacteria, Chloroflexi, and JS1 (Rochelle et al., 1994) were the dominant groups in the top, middle and bottom samples, respectively (Fig. 5).

Proteobacteria accounted for 17% of the whole bacterial library and consisted of four distinct subdivisions: Alpha-, Delta-, Gamma-, and Epsilonproteobacteria (Table 3 and Fig. 5A). Most of these sequences were obtained from the top sediment sample. Three clone sequences were affiliated with the Alphaproteobac-teria group, and they were related to Sulfitobacter sp. and one clone sequence (AB247855) retrieved from Lau Basin hydrothermal vents. Sixteen sequences were grouped into the Deltap-roteobacteria, and they were affiliated with a sulphate-reducing bacterium (Desulfuromusa sp.) at the >90% similarity level. Most of the Deltaproteobacteria clones were related to clone sequences retrieved from the sediments from Southern Okinawa Trough, Pacific Ocean Margin, and SCS. One clone sequence (GU475269) was (94% similarity) related to a sulphate-reducing bacterial (SRB) clone (Desulfocapsa sp., DQ831556) detected from marine sediments. However, other Deltaproteobacteria sequences were also related to this clone sequence (DQ831556) and another sulphate-reducing bacterium (Desulfuromusa sp., EU283459) at >90% similarity level. In addition, other reference clone sequences from the GenBank (such as AB177191) were retrieved from the Peru Margin methane hydrate sediments which was also related to the SRB Desulfobacterium aniline (Inagaki et al., 2006). Therefore, SRB-related sequences were abundant within this core.

Seven sequences were grouped into the Epsilonproteobacteria, which were related to sequences of Helicobacter sp. retrieved

from the Sagami Bay (Fang et al., 2006) and the Peru Margin sediments (Inagaki et al., 2006). Five sequences were related to the Coxiella sp., Thioalkalivibrio sp. and Psychrobacter sp., and they were classified into the Gammaproteobacteia group. Chloroflexi and JS1 were the dominant groups (accounting for 78.8% and 70.3%, respectively) in the middle and bottom samples. Thirty-seven clone sequences were affiliated with Chloroflexi, and they were related to the sediment clones isolated from the Gulf of Mexico (Nunoura et al., 2009), Santa Barbara Basin (Harrison et al., 2009), Pacific Ocean Margin (Inagaki et al., 2006), and SCS (Li et al., 2008b). Thirty-three sequences were affiliated with JS1, and they were closely related to sequences retrieved from the methane hydrate-bearing sediments in Nankai Trough (Reed et al., 2002) and Cascadia Margin (Inagaki et al., 2006).

Besides the clones affiliated with the three predominant phylogenetic groups above, other clones were affiliated with several minor groups, such as Acidobacteria, Actinobacteria, Chlorobi, Firmicutes, Planctomycetes, Spirochaetes, Verrucomi-crobia, OP3, OP8, OP11, TM6 and WS3, and unclassified groups, each accounting for a few percent of the total (Fig. 5B and Table 3). Most of these clones were retrieved from the top sample except that two clone sequences from the middle sediment sample were grouped into Actinobacteria and Planctomycetes, and that one clone sequence from the bottom sample was grouped into Actinobacteria.

4. Discussion and conclusions

4.1. Low microbial abundance

Microbial abundance in the DSH-1 core was lower than other SCS areas, which were typically around 107 cells/g counted by AODC (Jiang et al., 2007) and 109 cells/g counted by fluorescence in situ hybridization (FISH) (Li et al., 2008a), and a few other cold seep sediments, such as Nile Deep Sea Fan, which was typically around 109 cells/g counted by FISH (Omoregie et al., 2009). But the microbial abundance in the DSH-1 core was a little higher than another site (<1.5 x 106 cells/g sediments), which retrieved from the shallow water cold seep area far from the seep vent in the Dongsha area (Su et al., 2007).

w(TOC) content can reach 11% in organic-rich sediments (Patience et al., 1990), in contrast with <1% in organic-poor sediments (Coolen et al., 2002; Newberry et al., 2004). It is proposed that microbial abundance in marine sediments is related to the concentration of TOC (Parkes et al., 2000). The relatively low abundance of microbes at the study site may be as a result of the low total w(TOC) concentration (from 0.49% to 0.65%, Table 2).

In addition, the depth-related variation of the microbial abundance showed the same trend as the headspace methane concentration profile (Fig. 2D and E). This positive correlation suggested that microbes seem to be sensitive to variations of methane concentration and they may be using methane as an energy source.

4.2. Microbial diversity and biogeochemical processes 4.2.1. High microbial diversity

High archaeal and bacterial diversity reflected in ten phylotypes of archaea and fifteen phylotypes of bacteria (Table 3) detected at the 5—10 cm depth in this core. In contrast, at the Qiongdongnan Basin sediments of the SCS, just two bacterial phylotypes and six archaeal phylotypes were detected (Jiang et al., 2007). In Xisha Trough at

about 10 cmbsf, eight bacterial and seven archaeal phylotypes were detected (Li et al., 2008b). At Shenhu (Zhang et al., 2010) and Nansha (Li et al., 2008a) surface sediments, there were six archaeal and seven bacterial phylotypes were detected, respectively.

In Western Pacific, like Nankai Trough cold seep surface sediments, only Proteobacteria, Spirochaetaceae—Cytophaga, gram-positive bacteria, an unknown group bacteria, MGI, and ANME archaea were detected (Arakawa et al., 2006). In Sagami Bay, three bacterial and a few archaeal groups were found (Fang et al., 2006). In Japan Trench, three phylotypes of bacteria, Pro-teobacteria, Cytophaga, and gram-positive bacteria, and MGI and some methanogenic archaea were detected (Li et al., 1999). At Florida Escarpment surface sediments, five phylotypes of bacteria and one group of Methanosarcinales and 3% Crenarchaeota were discovered (Reed et al., 2006). In the Gulf of Mexico hydrocarbon seep, several bacterial phylotypes Proteobacteria, Firmicutes and Actinobacteria and archaeal phylotypes Methanosarcinales and Thermoplasmales were found (Mills et al., 2003).

We were unable to explain the differences at the present time, and could only roughly assume that the high microbial diversity in studied core might be controlled by much complex geochemical or micro-ecological conditions in the cold seep area of the northern SCS.

4.2.2. Microbial biogeochemical processes

According to pore water data from the referenced Core GC 9 and headspace methane concentration of Core DSH, the profiles of sulphate, hydrogen sulfide and methane (Fig. 2A, B and C) in this core suggested that there may be a coupled biogeochemical process: sulphate reduction and AOM below the water-sediment

interface, which is consistent with the previous findings in the same area (Suess, 2005; Huang et al., 2008). Although AOM is still an unclear process, field and laboratory studies have provided ample evidence that AOM can be mediated by a structured consortium consisting of the ANME group and SRB (Knittel et al., 2005) or mediated by ANME-1 alone (Knittel and Boetius, 2009). The syntrophic consortium of SRB and anaerobic methane oxidizers is typically present in gas hydrate communities (Boetius et al., 2000; Orphan et al., 2001). However, these two groups appear to be present at the study site. For example, certain clone sequences of the ANME group (DSH12A17 in Fig. 4) and SRB-related sequences (Fig. 5A) were detected at the bottom sediments, where the methane concentration was high. This coexistence of the ANME- and SRB-related clones indicated that sulphate reduction might be coupled with AOM at this depth in the core.

Archaeal group MBG-B was originally found at deep-sea sediments (Vetriani et al., 1999) and hydrothermal vent sites (Takai and Horikoshi, 1999) and in present in a number of deep subsurface and hydrothermal vent sites (Takai and Horikoshi, 1999). Subsequently, this group was reported from deep sediments in hydrate zones at Nankai Trough, Peru continental shelf, and the sea of Okhotsk (Reed et al., 2002; Inagaki et al., 2003; Sorensen and Teske, 2006; Dang et al., 2009, 2010). Inagaki et al. (2006) showed that clone libraries from hydrate-bearing sites of the Cascadia and Peru Margins were dominated by MBG-B especially in the sulphate reducing zone in shallow sediments above gas hydrates. Thus, this group was proposed to play an important role in biogeochemical processes such as sulphate reduction and methane oxidation (Biddle et al., 2006). In this study, the percentage of the MBG-B increased with depth, and

Figure 6 Relative abundance of main taxonomic groups of 16S rRNA gene identified in archaeal (A) and bacterial libraries (B).

reached 38.9% and 62.5% in the middle and bottom (Fig. 6A) of the core, respectively. Simultaneously, at these depths the chloridion showed a negative abnormity (less than seawater average value) and the methane concentration showed a sharp increase (Fig. 2C and D). These microbial and the geochemical parameters illustrated that the geochemical environments was comparable to the gas hydrate sediments in Cascadia and Peru Margins (Inagaki et al., 2006). There might be an upward methane diffusion from the sediments below.

Bacterial major groups were Chloroflexi and candidate division JS1. Chloroflexi was dominant in the middle sample and was a significant fraction in the bottom sample (Fig. 6B). This phylum has been recognized as a typical bacterial cluster containing a number of diverse environmental clones (Hugenholtz et al., 1998). The 16S rRNA gene sequences obtained from marine sediments are frequently present within the classes Anaerolineae, Caldilineae, 'Dehalococcoidetes', and the unclassified subphylum IV (Reed et al., 2002; Inagaki et al., 2003, 2006; Kormas et al., 2003; Parkes et al., 2005). Within these classes, only a few representatives are obtained in culture, and thus characteristics about their metabolic functions remain limited. However, based on relatedness of clone sequences to cultures, Inagaki et al. (2006) inferred that closely related members of heterotrophic Chloro-flexi living in marine sediments may grow syntrophically with hydrogenotrophic Chloroflexi species or other hydrogenotrophic microbes such as methanogens. Albeit speculative, the dominance of Chloroflexi clones in our study may be due to the presence of abundant methane at the investigated locations. Webster et al. (2007) showed that Chloroflexi and candidate division JS1 seem to be well suited to life in the subsurface and are often associated with organic-rich sediments. JS1 was another predominant group of the bacterial libraries, especially at the bottom sample where there was abundant methane (Fig. 2D and B). This group has previously been found in methane-rich subsurface sediments where gas hydrates are present (Inagaki et al., 2006), suggesting that these bacteria may prefer sedimentary habitats with high concentrations of methane associated with hydrates (Inagaki et al., 2006). This discovery suggests that some members of this division may be associated with methanogenic consortia and that others are adapted to or can tolerate high pressures (Webster et al., 2004). However, representatives of this group have not been cultivated as yet, and thus their metabolic pathways remain unknown (Blazejak and Schippers, 2010). Only indirect evidence from stable-isotope probing exists suggesting that members of this clade are able to incorporate acetate and glucose (or glucose metabolite) under anaerobic, sulphate-reducing conditions (Webster et al., 2006b).

Previous studies (Inagaki et al., 2006; Webster et al., 2006a) indicated that Chloroflexi and JS1 bacteria occur widely in organic-rich deep marine sediments. TOC in this core was much lower (Table 2) than other sediments (Webster et al., 2006a, and references in this article). But Chloroflexi and JS1 bacteria were abundant below 400 cm in this core. That might indicate that the presence of these two groups bacteria have no direct relation with TOC content. Microbial communities can be stratified in deep marine sediments, and surrounding geochemical and geological settings strongly affect the community structure.

As discussed above, although the metabolic functions of the MBG-B group of archaea, and Chloroflexi and JS1 of bacteria are largely unknown, the recognition of microbial populations that consistently occur in the presence of methane hydrates serves as a starting point for defining their ecological and biogeochemical significances (Inagaki et al., 2006). So the retrievability of these

uncharacterized phylogenetic clades in our study might further corroborate that metabolic functions of these microorganisms are related to methane seepage.

In conclusion, our data indicated a high microbial diversity in the cold seep at the northern SCS, especially in the surface sediments, in contrast with other areas of the SCS, and with other cold seeps worldwide. The major groups were consistent with those previously detected in various cold seep environments, including methane/organic-rich and/or putative gas hydrate-bearing marine sediments (Inagaki et al., 2006). Some unique phylotypes were also present in sediments studied. Combined geochemical and microbial data suggested a coupled reaction of sulphate reduction and methane oxidation at this methane seepage site.


This research was supported by the National Program on Key Basic

Research Project (973 Program) (Grant No. 2009CB219502),

National Special Foundation (Grant No. GZH200200203-02-01),

and Non-profit Industry Financial Program of Ministry of Land

and Resources of the PRC (Grant No. 200811014-02).


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