Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

Hongyue Dang; Jinying Yang; Jing Li; Xiwu Luan; Yunbo Zhang; Guizhou Gu; Rongrong Xue; Mingyue Zong; Martin G Klotz

doi:10.1128/AEM.01889-12

. 2013 Jan;79(1):121–132. doi: 10.1128/AEM.01889-12

Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

Hongyue Dang ^a,^b,^✉, Jinying Yang ^a, Jing Li ^a, Xiwu Luan ^c,^d, Yunbo Zhang ^e, Guizhou Gu ^e, Rongrong Xue ^e, Mingyue Zong ^e, Martin G Klotz ^a,^f

PMCID: PMC3536100 PMID: 23064334

Abstract

The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N₂ fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH₄⁺ concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N₂ fixation and NH₄⁺ production. Several other environmental factors, including sediment pore water PO₄³⁻ concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery.

INTRODUCTION

Microbial fixation of nitrogen (N) is an important process in the global N biogeochemical cycle (1, 2), providing bioavailable ammonia as the bioenergetically least costly source of nitrogen, which is especially important in oligotrophic ecosystems. The current N budget of the marine N cycle is considered unbalanced caused by N-loss exceeding N-gain. Such an imbalance, if real, would eventually lead to an end of primary production in the oceans, and it has thus been hypothesized that the N-gain due to N₂ fixation in the ocean is underestimated (3, 4). Although the prevailing theory assumes that N₂ fixation ceases in such environments, recent findings suggest that microbial N₂ fixation operates also in marine environments served with bioavailable N from other sources (5–8). Thus, diazotrophic microorganisms may be more widespread and play more important roles in marine environments than previously suggested. Although N₂ fixation (nitrogenase activity) is performed by numerous groups of microorganisms (9) and the nitrogenase reductase-encoding gene, nifH, has been frequently targeted as a functional biomarker for investigating the diazotrophic community in natural environments (10, 11), the diversity and community structure of the nifH-harboring microorganisms are still poorly understood globally, and many important diazotrophs remain to be discovered (12–18).

Deep-sea sediments constitute the largest fraction of the Earth's surface and a vast portion of these sediments are anoxic and thus suitable habitat for microbial N₂ fixation (19); however, marine diazotrophic microbes have been studied mostly in oligotrophic surface waters (see reference 20 and references therein) and certain shallow-water benthic systems, such as salt marshes, mangroves, microbial mats and other coastal environments (21–26). Rarely conducted investigations of the N₂ fixation microbial communities of deep-sea waters and hydrothermal vent fluids have discovered diverse and novel nifH gene sequences (12, 13). Although this correlates with the finding that dissolved N₂ is abundant (0.59 mM) in the deep ocean (12), these environments have been sparsely investigated regarding the diversity and community structure of the diazotrophic microbiota.

Marginal seas are major areas of active biogeochemical cycling processes (27). These areas may absorb a significant fraction of atmospheric CO₂ by high-level primary production and organic matter export to deep oceans and sediments (28, 29), playing an important role in alleviating the ongoing global warming and climate change. However, this capability usually does not operate at full capacity due to the lack of adequate nutrient input for aquatic bioproductivity. Marine N₂ fixation plays an important role in general in marine ecosystem and an even more important role in areas of the tropical and subtropical ocean (30). N₂ fixation, along with atmospheric N deposition, is the only source of new N that can lead to a net sequestration of atmospheric CO₂ in the deep ocean (31). In the marine benthic ecosystem, recent studies indicate that archaea, especially the anaerobic methanotrophic (ANME) archaeal functional guild (32), constitute a great portion of the in situ diazotrophic community and play an important role in supplying newly fixed nitrogen in deep-sea sediments rich in methane hydrates (14–17). Due to the global distribution of methane hydrates in marginal sea sediments (4, 33), the ANME diazotrophs' contribution to the marine and global N₂ fixation cannot be neglected. However, it is currently still unknown whether archaeal diazotrophs are specific to methane-charged environments, such as deep-sea sediments rich in methane hydrates, or widely distributed in sediments even without methane hydrates. The distribution and contribution of archaeal diazotrophs in marine sediments need to be investigated.

The South China Sea (SCS), the largest marginal sea (∼3.5 × 10⁶ km²) in the western Pacific Ocean, is a huge subtropical and tropical oligotrophic water body with usually undetectable nitrate and phosphate in the euphotic layer (34). Since the seawater average N/P is below the Redfield ratio, productivity in the SCS is likely limited by the supply of nitrogenous nutrients (35). Several large rivers, such as the Pearl River and the Mekong River, discharge into the SCS. Riverine input may provide nutrients for phytoplankton growth and bioproduction. However, this input is localized to the estuarine and coastal areas and does not provide the nutrients for the whole oligotrophic SCS (36). Monsoonal wind driving eddies and other upwelling and vertical mixing and diffusing processes may also provide nutrient supply from deep water to surface water in the SCS (37, 38). Thus, sediment microbial N₂ fixation may play an important role in maintaining deep-water nutrient availability and surface water nutrient supply.

Seawater diazotrophic bacteria and N₂ fixation potential have been previously investigated in several areas of SCS (6, 34, 38–42). Sediment diazotrophic microbiota have not yet been investigated even though they may play an important role in providing newly fixed nitrogen to the oligotrophic SCS ecosystem. In addition, evidenced and prospective methane hydrate reservoirs have been identified in the northern South China Sea (nSCS) (43–46), and these areas may sustain productive methane-seep-specific ecosystems, requiring a high supply of nitrogenous nutrients and thus favoring the development of an active microbial diazotroph community. The nSCS provides an ideal ecosystem to investigate the distribution and contribution of sediment microbial diazotrophs in general, and the archaeal diazotrophs in particular, to the marine N cycling. In the present study, numerous sampling stations were selected, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas to cover the major environments characteristic to the nSCS to investigate the diversity, abundance, distribution, community composition, and structure of the sediment diazotrophic microbiota and major environmental factors that influence their ecological features.

MATERIALS AND METHODS

Sample collection and environmental factor measurements.

Sediment samples were collected from 12 stations of the nSCS using a 0.1-m² stainless steel Gray O'Hara box corer or a deep-sea sediment grab sampler during an Open Cruise of R/V Shiyan 3 in August of 2007 (Fig. 1). Only undisturbed samples with clear overlying seawater were collected to ensure the integrity of the surface sediment structures. Replicate surface sediment subcore samples down to a 5-cm depth for microbiological and environmental analyses were taken aseptically with sterile 60-ml syringes (luer end removed), homogenized, and stored in airtight sterile plastic bags at −20°C during the cruise and at −80°C after delivery to the laboratory.

Fig 1 — Map showing the 12 sediment sampling sites in the nSCS. (Modified from Zhou et al. [47] with permission from Springer Science+Business Media.)

Surface sediment temperature was measured on deck inside the sampler once the sediments were collected. Other environmental factors were measured in the laboratory. Sediment organic carbon (OrgC) and organic nitrogen (OrgN) contents were measured with a PE 2400 Series II CHNS/O elemental analyzer (Perkin-Elmer, Norwalk, CT). The sediment contents of water (WC), total organic matter (OM), total phosphorus (TP), inorganic phosphorus (IP), and organic phosphorus (OrgP) were measured according to the method of Danovaro (48). Sediment pore water dissolved nutrient concentrations, such as nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), and phosphate (PO₄³⁻), were measured with a nutrient QUAATRO AutoAnalyzer (Bran+Luebbe, Germany). Sediment pore water dissolved urea concentration was measured according to the method of Grasshoff et al. (49). A Cilas 940L laser granulometer (Company Industrielle des Lasers, France) was used for sediment granularity analysis (Table 1).

Table 1.

Measurements of in situ environmental parameters of the 12 sampling stations in the northern South China Sea

Environmental factor^a	Station
Environmental factor^a	A3	CF6	CF8	CF11	CF14	CF15	E407	E422	E501	E504	E505	E801
Longitude (°E)	114°23.163′	119°30.060′	111°3.710′	114°34.477′	115°12.971′	115°29.373′	120°0.017′	112°0.793′	110°41.835′	111°18.096′	111°29.029′	110°20.410′
Latitude (°N)	21°50.525′	22°0.316′	18°1.908′	19°43.341′	19°54.256′	19°59.581′	18°29.810′	18°0.341′	18°59.995′	19°0.102′	18°59.897′	17°11.450′
Water depth (m)	52	2,441	1,548	1,050	1,220	1,300	1,800	2,456	65	131	154	1,400
Sediment
Temp (°C)	21.9	5.5	5.0	5.2	3.6	3.4	4.7	3.8	20.5	17.7	17.4	3.8
WC (%)	36.92	29.66	27.92	32.25	33.19	36.40	32.51	32.37	18.23	35.22	15.22	27.94
OM (%)	1.89	3.06	2.26	2.10	1.83	2.68	2.33	2.09	1.08	1.79	1.58	3.22
OrgC (%)	1.00	0.63	0.96	1.32	1.32	1.27	0.67	0.94	0.28	0.76	0.47	1.09
OrgN (%)	0.10	0.08	0.12	0.16	0.15	0.17	0.06	0.11	0.03	0.08	0.06	0.14
OrgP (μmol/g)	5.92	3.52	6.17	8.69	7.62	12.62	8.36	4.28	3.11	6.31	2.55	8.55
IP (μmol/g)	19.89	23.52	16.56	17.53	16.56	13.99	16.31	18.64	7.81	17.61	15.33	15.23
TP (μmol/g)	25.81	27.04	22.73	26.22	24.18	26.61	24.67	22.92	10.92	23.92	17.88	23.78
OrgC/OrgN	10.00	7.88	8.00	8.25	8.80	7.47	11.17	8.55	9.33	9.50	7.83	7.79
Sand content (%)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	38.18	0.14	0.62	0.00
Silt content (%)	64.91	63.00	62.93	62.41	62.74	60.75	53.61	59.29	44.46	69.72	75.7	57.94
Clay content (%)	35.09	36.97	37.07	37.59	37.26	39.25	46.39	40.71	17.37	30.14	23.68	42.06
Median grain size (ø)	7.00	7.27	7.12	7.14	7.15	7.28	7.78	7.34	4.75	6.68	6.12	7.58
Mean grain size (ø)	7.27	7.49	7.42	7.42	7.37	7.48	7.74	7.61	5.43	7.01	6.52	7.71
Sorting coefficient	1.65	1.50	1.59	1.60	1.63	1.60	1.56	1.54	2.25	1.69	1.79	1.40
Kurtosis	1.95	1.80	1.86	1.88	1.92	1.88	1.85	1.78	2.74	2.02	2.18	1.65
Skewness	0.95	0.84	0.97	0.93	0.87	0.78	–0.76	0.88	1.94	1.17	1.49	0.78
Sediment pore water
Salinity (‰)	31.5	31.2	31.0	30.0	30.5	31.5	31.5	30.0	30.1	31.0	32.5	31.0
pH	7.17	7.11	7.40	7.16	7.15	7.15	7.06	7.17	7.28	7.13	7.35	7.06
Eh (mv)	–15.0	–11.0	–28.0	–14.0	–13.0	–13.0	–8.0	–14.0	–21.0	–12.0	–27.0	–8.0
DO (μM)	118.75	203.13	128.13	178.13	237.50	140.63	237.50	234.38	115.63	96.88	165.63	215.63
NO₃⁻ (μM)	14.31	3.61	1.46	13.58	14.92	6.43	22.67	17.42	3.95	2.85	6.30	9.05
NO₂⁻ (μM)	0.42	0.83	0.60	1.09	1.19	1.00	1.86	1.53	0.63	2.80	0.56	3.99
NO_x⁻ (μM)^b	14.73	4.44	2.06	14.67	16.11	7.43	24.53	18.95	4.58	5.65	6.86	13.04
NH₄⁺ (μM)	47.11	5.97	54.25	8.22	105.94	13.76	126.68	8.72	525.74	59.11	32.70	475.56
DIN (μM)^c	61.84	10.41	56.31	22.89	122.05	21.19	151.21	27.67	530.32	64.76	39.56	488.60
PO₄³⁻ (μM)	5.49	6.26	6.42	7.54	6.19	6.29	8.46	7.62	5.07	9.08	4.42	14.42
N/P (DIN/PO₄³⁻)	11.26	1.66	8.77	3.04	19.72	3.37	17.87	3.63	104.60	7.13	8.95	33.88
Urea (μM)	1.91	1.00	1.24	1.16	2.07	2.82	0.58	1.58	2.41	1.33	2.57	5.06

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WC, water content; OM, organic matter; OrgC, organic C; OrgN, organic N; OrgP, organic P; IP, inorganic P; TP, total P; ø, the Krumbein phi scale of grain size; Eh, redox potential.

NO_x⁻ was calculated as the sum of NO₂⁻ and NO₃⁻.

The total inorganic N concentration (DIN) was calculated as the sum of NH₄⁺, NO₂⁻, and NO₃⁻.

DNA extraction and nifH gene clone library analyses.

Sediment microbial community DNA was extracted and pooled from replicate subcore samples of each station using a FastPrep DNA extraction kit for soil and a FastPrep-24 cell disrupter (MP Biomedicals, Solon, OH) as described previously (15, 50). DNA concentrations were measured using PicoGreen (Molecular Probes, Eugene, OR) and a Modulus single-tube multimode reader fluorometer (Turner Biosystems, Sunnyvale, CA). Bacterial and archaeal nitrogenase reductase genes (nifH, including anfH and vnfH) were amplified with the primers nifHfw and nifHrv (12, 15, 17). To test the reproducibility of our experimental procedure and to identify any potential small-scale (∼20 cm) spatial variability of the sediment diazotrophic community, two separate nifH gene clone libraries (E407-I and E407-II) were constructed for sampling station E407, each from a distinct subcore DNA sample. PCR product cloning followed previous procedures (15, 50). Cloned gene fragments were reamplified to check the correct size of the DNA inserts using vector primers M13-D and RV-M (51), which were also used for sequencing with an ABI 3770 sequencer (Applied Biosystems, Foster City, CA). The resultant DNA sequences were translated into conceptual NifH protein sequences, and the BLASTp program was used for retrieval of the top-hit sequences from GenBank (52). NifH sequences were grouped into operational taxonomic units (OTU) within 0.05 sequence distance calculated by using the DOTUR program (53). Alignments of the NifH protein sequences, obtained with the program CLUSTAL X version 2.0 (54), were used for inference of phylogeny with PHYLIP version 3.69 (55), as reported previously (15).

Quantification of major microbial groups.

The abundances of sediment bacteria, archaea, nifH-harboring microorganisms, ANME diazotrophs, and methanogenic and methanotrophic archaea were quantified by using real-time fluorescent quantitative PCR (qPCR) methods with group-specific primers targeting, respectively, the 16S rRNA, nifH, nifD (encoding the α-subunit of nitrogenase molybdenum-iron protein), and mcrA (encoding the α-subunit of methyl-coenzyme M reductase) genes (see Table S1 in the supplemental material). All qPCR assays were carried out in triplicate with an ABI Prism 7500 sequence detection system (Applied Biosystems) using the SYBR green qPCR protocols (17, 56). Reaction conditions of qPCR were optimized with reference plasmids carrying the respective target genes constructed in this and previous studies (15, 51, 56). In all qPCR experiments, fluorescence reads were carried out at 72 or 80°C, and negative controls lacking template DNA were subjected to the same qPCR procedures to detect any possible contamination or carryover. Agarose gel electrophoresis and melting-curve analysis were routinely used to confirm the specificity of the qPCRs. Melting curves were obtained at 60 to 95°C, with a read every 1°C and holding for 1 s between reads. The resultant qPCR data were analyzed with the second-derivative maximum method using the ABI Prism 7500 SDS software (version 1.4; Applied Biosystems) (56, 57).

For the qPCR assays, standard curves were generated by serial dilution of reference plasmids containing target 16S rRNA, nifH, nifD, or mcrA gene fragments as the insert, with plasmids extracted from E. coli hosts using a Mini plasmid kit (Qiagen, Valencia, CA) and linearized with an endonuclease specific for the vector region. Concentrations of linearized plasmid DNAs were measured using PicoGreen and a Modulus single-tube multimode reader fluorometer. The ranges of the reference plasmid copy numbers used for standard curve constructions were shown in Table S2 in the supplemental material, along with the efficiency and sensitivity of the individual qPCR standard curves.

Statistical analyses.

The coverage of each nifH clone library was calculated as C = [1 − (n₁/N)] × 100, where n₁ is the number of unique OTU and N is the total number of clones in a library (58). Indices of gene diversity (Shannon-Wiener [H] and Simpson [D]) and evenness (J) were calculated using the OTU data (50). Rarefaction analysis and two nonparametric richness estimators, the abundance-based coverage estimator (S_ACE) and the bias-corrected Chao1 (S_Chao1), were calculated using DOTUR (53).

Community classification of the nifH-carrying microbial assemblages was performed with Fast UniFrac environmental clustering and principal coordinate analyses (PCoA) (59). Correlations between the nifH-harboring microbial assemblages and environmental factors were determined by canonical correspondence analysis (CCA) using the software Canoco (version 4.5; Microcomputer Power, Ithaca, NY) according to previously described procedures (50). Weighted NifH OTU and class data were used to identify the most significant environmental factors that had the strongest influence on the community structure and spatial distribution of the nifH-harboring microbial assemblages in the nSCS.

Pearson correlation analyses (significance level α = 0.05) of the abundance of sediment 16S rRNA, nifH, nifD, and mcrA genes with environmental factors were performed with the statistics software MINITAB (release 13.32; Minitab, State College, PA) as detailed previously (56, 57).

Nucleic acid sequence accession numbers.

The nifH sequences determined have been deposited in GenBank under accession numbers HQ223480 to HQ224498 and JQ412939 to JQ413029.

RESULTS

Site description.

Our sampling sites represent most of the typical sedimentary environments of the nSCS (Fig. 1), including the Pearl River estuary (station A3), coastal and offshore sites close to Hainan Island (E501, E504, and E505, <200-m water depth) and deep-water sites (>1,000-m water depth) close to Taiwan Island (CF6), Luzon Island (E407), Dongsha Islands (CF11, CF14, and CF15), Xisha Trough (CF8 and E422), and Xisha Islands (E801). Methane hydrates have previously been discovered in the Shenhu area southwest of Dongsha Islands (46). The Xisha Trough, Jiulong methane reef area southwest of Taiwan Island, and Bijia'nan basin northwest of Luzon Island were previously identified as gas hydrate prospective areas (43–45). Several of our sampling sites were located in or near these previously identified or perspective methane hydrate reservoirs (see Fig. 1 for details).

Diversity of the sediment nifH genes.

The two nifH gene clone libraries (E407-I and E407-II) constructed from separate sediment subcore samples of station E407 had similar OTU diversity based on rarefaction analysis (see Fig. S1 in the supplemental material). Community classification using Fast UniFrac environmental clustering (see Fig. S2 in the supplemental material) and PCoA (see Fig. S3 in the supplemental material) revealed that these two clone libraries were similar, indicating both the reproducibility of our experimental procedures and the negligible within-site variability of the sediment nifH-harboring microbial community at this station. Thus, these two nifH clone libraries were combined into a single library. The high similarity of the nifH clone libraries from E407 distinct subcore samples also suggests that the other sites have similar negligible intrasite variability, making intersite comparisons statistically reliable and meaningful.

Of the 12 nifH clone libraries constructed for the nSCS sediment samples, 1195 clones were identified to contain a valid nifH gene fragment, resulting in 1110 unique DNA sequences, 826 unique protein sequences and 441 OTU. The values of library coverage (C) ranged from 43.6 to 66.3% (Table 2), which together with rarefaction analysis (see Fig. S1 in the supplemental material) indicated a very high diversity of the nifH gene sequences in the sediments of the nSCS. The coastal site E501 had the lowest diversity and the offshore site E505 had the highest OTU diversity, based on all of the diversity indices (H, 1/D, and J). The S_ACE and S_Chao1 richness estimators are consistent to the above results for site E505. However, these two estimators showed that the expected OTU richness of site E501 were higher than some of the other sites (Table 2).

Table 2.

Biodiversity and predicted richness of the sediment nifH gene sequences obtained from the sampling stations of the northern South China Sea^a

Station	No. of clones	No. of unique gene sequences	No. of OTU	C (%)	H	1/D	J	S_ACE	S_Chao1
A3	91	86	52	59.3	5.17	27.67	0.91	186.83	112.55
CF6	96	89	53	63.5	5.34	42.62	0.93	136.14	112.50
CF8	80	79	47	60.0	5.17	35.51	0.93	101.30	109.00
CF11	85	74	50	61.2	5.29	43.54	0.94	129.05	94.00
CF14	89	86	56	51.7	5.42	43.51	0.93	232.32	206.50
CF15	93	92	55	61.3	5.45	49.74	0.94	136.96	107.50
E407	172	170	85	66.3	5.72	34.36	0.89	257.28	182.24
E422	87	85	54	51.7	5.31	36.32	0.92	212.91	177.00
E501	102	92	53	65.7	5.14	25.63	0.90	144.36	102.58
E504	110	102	65	61.8	5.74	68.91	0.95	140.88	136.75
E505	101	95	73	43.6	5.96	93.52	0.96	271.17	206.00
E801	89	85	54	56.2	5.44	55.94	0.95	145.98	177.50

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The OTU of the diazotrophic microbial NifH sequences were determined at a 0.05 distance cutoff using the DOTUR program. The coverage (C), Shannon-Weiner (H), Simpson (D), and evenness (J) indices and the S_ACE and S_Chao1 richness estimators were calculated using the OTU data.

Phylogeny of the NifH protein sequences.

The obtained 1,110 distinct nifH gene sequences shared 40.2 to 99.8% identity with one another. Nearly half of these DNA sequences (49.5%) did not have a match with known sequences from GenBank, except for the primer regions. The sequences that did have matches were 64.8 to 98.0% identical to the closest-match GenBank nifH sequences. However, all of the corresponding protein sequences deduced from the 1,110 nifH gene sequences showed various degrees of identity (42.1 to 100.0%) with known GenBank NifH sequences. The nSCS sediment NifH sequences were highly variable, and they shared 25.2 to 100.0% sequence identity with one another. Numerous sequences had quite high identities (≥90%) with NifH sequences from bacterial or archaeal isolates, such as those affiliated with Alphaproteobacteria (Bradyrhizobium), Betaproteobacteria (Azoarcus), Gammaproteobacteria (Allochromatium, Azotobacter, Pseudomonas, Teredinibacter, and Vibrio), Deltaproteobacteria (Desulfatibacillum, Desulfobacterium, Desulfovibrio, Desulfurivibrio, Desulfuromonas, Geobacter, and Pelobacter), Chlorobi (Chlorobaculum, Chloroherpeton, and Prosthecochloris), Firmicutes (Clostridium, Desulfitobacterium, Desulfotomaculum, Paenibacillus, and Turicibacter), and methanogenic Euryarchaeota (Methanococcoides, Methanosarcina, Methanosphaerula, and “Candidatus Methanoregula”). Some sequences shared moderate identities (≥80%) with NifH sequences from other bacterial or archaeal isolates, such as those affiliated with Bacteroidetes (“Candidatus Azobacteroides”), Verrucomicrobia (Coraliomargarita and Verrucomicrobiae), Chlorobi (Chlorobium), Deltaproteobacteria (Desulfonatronospira), Firmicutes (Alkaliphilus, Butyrivibrio, Ethanoligenens, Eubacterium, and Syntrophothermus), and methanogenic Euryarchaeota (Methanohalophilus and Methanothermobacter). Half of the nSCS sediment NifH sequences (50.5%) shared lower identities (≤70%) with their best-match GenBank NifH sequences.

The constructed phylogenetic tree putatively revealed a total of nine NifH classes (Fig. 2; also see Fig. S4 in the supplemental material), including all of the previously known NifH clusters (15) and several previously unidentified NifH clusters. A new putative classification schema for the NifH clusters emerged from our current phylogenetic analysis.

A total of 144 nSCS NifH OTU were affiliated within class I (Fig. 2; also see Fig. S4 in the supplemental material), containing NifH sequences from both bacteria and archaea and alternative nitrogenase reductase sequences encoded by anfH and vnfH (12). Several NifH clusters were identified in this class, including previously defined clusters I, II, III, and IIIx, part (“subcluster A”) of cluster IV (15), and two previously unknown clusters with NifH sequences exclusively from nSCS sediments (Fig. 2; also see Fig. S4 in the supplemental material). Our class I sequences were related to NifH sequences from diverse N₂-fixing bacterial and archaeal isolates, indicating that these nifH-harboring microorganisms might be functional diazotrophs. The related GenBank environmental sequences were obtained from soils, rhizospheres of rice, soybean, mangrove, salt marsh, and seagrass, deep-sea methane seep sediments, and hydrothermal vent environments. We also obtained one sequence (E422-68) that was affiliated with the ANME NifH cluster (“cluster IIIx”) previously identified exclusively from deep-sea methane seep sediments (14–16).

A total of 115 nSCS NifH OTU were affiliated within class II (Fig. 2; also see Fig. S4 in the supplemental material), which was originally classified as part (“subcluster B”) of NifH “cluster IV” in previous literature (9). Our NifH sequences, along with a few sequences from methane-hydrate-bearing deep-sea sediments of the Okhotsk Sea and the deep-sea hydrothermal vent environment of Juan de Fuca Ridge (12, 15), constituted the majority of the class II NifH sequences. The separation of these environmental sequences from the sequences obtained from bacterial and archaeal isolates indicated the novelty of these marine NifH sequences (see Fig. S4 in the supplemental material).

A total of 99 nSCS NifH OTU were affiliated within class III (Fig. 2; also see Fig. S4 in the supplemental material), which was defined as “cluster V” in a previous study (15). Sequences in this class were exclusively detected from sediments of the Okhotsk Sea and nSCS (15), except for NifH-2 from the non-N₂-fixing Methanocella paludicola SANAE (GenBank accession number BAI60977) and one Lake Vallentunasjon sequence that might be mistakenly classified as a chlorophyllide reductase-like sequence in the original publication (60).

Seventeen nSCS NifH OTU were affiliated within class IV (Fig. 2; also see Fig. S4 in the supplemental material), which comprised a newly defined cluster in the present study. Sequence E407-II-40 was related to some NifH sequences from Firmicutes (Dialister and Selenomonas), while all of the other nSCS sequences in this class were distantly related to the NifH paralogues of methanogenic Euryarchaeota (Methanoculleus, Methanoplanus, Methanoregula, Methanosphaerula, and Methanospirillum).

Eleven nSCS NifH OTU were affiliated within class V (Fig. 2; also see Fig. S4 in the supplemental material), which was classified as part (“subcluster A”) of NifH “cluster VI” in a previous study (15). Our environmental sequences in this class were related to NifH sequences of anaerobic methanogenic Euryarchaeota (Methanosarcina and Methanospirillum), Deltaproteobacteria (Desulfatibacillum and Desulfobacterium), and Firmicutes (Desulfotomaculum). Within this class, strain Methanosarcina acetivorans C2A was a diazotrophic archaeon (61), and strain Desulfatibacillum alkenivorans AK-01 might be capable of carrying out N₂ fixation (62). Thus, the microorganisms that carried the class V NifH sequences may be functional diazotrophs.

Forty-three nSCS NifH OTU were affiliated within class VI (Fig. 2; also see Fig. S4 in the supplemental material), which was classified as part (“subcluster B”) of NifH “cluster VI” in a previous study (15). Our NifH sequences, along with a few sequences from the methane-hydrate-bearing deep-sea sediments of the Okhotsk Sea (15), constituted the majority of the class VI NifH sequences. This class also includes some NifH sequences from Fusobacteria isolates (Fusobacterium) (63).

Four nSCS NifH OTU were affiliated within classes VII, VIII, and IX, respectively (Fig. 2; also see Fig. S4 in the supplemental material), forming three newly defined NifH clusters. The NifH sequence from Clostridium cellulolyticum H10 is the only known sequence that is affiliated with one of these classes.

Spatial distribution of the nifH-harboring microbial assemblages.

Multivariate statistical analyses indicated that different nSCS sediment environments contained distinct nifH-harboring microbial assemblages. Both Fast UniFrac all-environment P test significance (P = 0.000) and UniFrac significance (P ≤ 0.001) statistics indicated a significant difference among the nSCS sediment nifH-harboring microbial assemblages. The heterogeneous distribution of the nifH-harboring microbial communities was confirmed via Fast UniFrac PCoA (Fig. 3), environmental clustering (see Fig. S5 in the supplemental material), and CCA (Fig. 4). Environmental variables in the first two CCA dimensions (CCA1 and CCA2) explained 26.0% of the total variance in the nifH-harboring microbial community composition and 28.0% of the cumulative variance of the nifH-harboring microbial community-environment relationship (Fig. 4a). Sediment temperature was identified as the only significant environmental factor (P = 0.008; 1,000 Monte Carlo permutations) that contributed the most to the heterogeneous community structure and spatial distribution of the nSCS sediment nifH-harboring microbial assemblages. This environmental factor alone provided 12.3% of the total CCA explanatory power.

Fig 3 — Ordination diagram of the Fast UniFrac weighted and normalized PCoA analysis of the nSCS sediment *nifH*-harboring microbial assemblages as revealed by using the NifH protein sequence data. Shown is the plot of the first two principal coordinate axes (P1 and P2) for PCoA and the distributions of *nifH*-harboring microbial assemblages (designated with the sampling station names) in response to these axes.

Fig 4 — CCA ordination plots for the first two principal dimensions of the relationship between the *in situ* environmental parameters of the nSCS and the distribution of the sediment *nifH*-harboring microbial assemblages as analyzed by using data of the NifH OTU (a) and the NifH classes (b). Correlations between environmental variables and CCA axes are represented by the length and angle of arrows (environmental factor vectors). Covarying variables, such as NH₄⁺ and DIN (r = 0.9993), and OrgC and OrgN (r = 0.9680), were checked to minimize colinearity in the CCA analyses.

Correlation of the nSCS sediment nifH-harboring microbial assemblages with environmental variables was further analyzed with CCA using the NifH class data. Environmental variables in the first two CCA dimensions (CCA1 and CCA2) explained 68.1% of the total variance in the sediment nifH-harboring microbial community composition and 69.7% of the cumulative variance of the nifH-harboring microbial community-environment relationship (Fig. 4b). Water depth (P = 0.035), sediment pore water PO₄³⁻ concentration (P = 0.051), and sediment temperature (P = 0.071) were identified as the only significant environmental factors, providing 11.5, 15.4, and 19.2% of the total CCA explanatory power, respectively. All the other environmental factors analyzed were not found by the CCA to be significant (P > 0.100) in their respective contributions to the heterogeneous spatial distribution of the sediment nifH-harboring microbial assemblages in the nSCS. The CCA showed that the sediment microorganisms that harbored NifH sequences from distinct classes (Fig. 2; also see Fig. S4 in the supplemental material) responded differently to the prevalent in situ environmental factors (Fig. 4b). However, the two putatively functional diazotrophic groups that harbored the class I and class V NifH sequences might have a similar environmental requirement that was different from the other nifH-harboring microbial groups (Fig. 4b).

Abundance of nifH-harboring microorganisms.

Melting curve analyses of the amplified genes (16S rRNA, nifH, nifD, and mcrA) confirmed that the fluorescence signals were obtained from specific PCR products of our qPCR quantifications. Standard curves generated using plasmids containing cloned target gene fragments to relate the threshold cycle (C_T) to the gene copy number revealed linearity (R² ≥ 0.981) over several orders of magnitude of the standard plasmid DNA concentrations (see Table S2 in the supplemental material). The obtained high correlation coefficients and similar slopes indicated high primer hybridization and extension efficiencies (see Table S2 in the supplemental material), making comparison of the different genes' abundances reliable.

The qPCR results showed heterogeneous distributions of the sediment bacterial and archaeal 16S rRNA gene abundances in the nSCS, with the shallow water sites usually harboring higher gene abundances for both bacteria and archaea (Table 3). In addition, the determined copy numbers of the bacterial 16S rRNA genes were much higher than those of the archaeal 16S rRNA genes (averaging 23.5:1).

Table 3.

Abundance of 16S rRNA, nifH, nifD, and mcrA genes in sediments of the 12 sampling stations of the northern South China Sea

Sampling station	Mean no. of target genes g of sediment⁻¹ (SE)
Sampling station	Bacterial 16S rRNA	Archaeal 16S rRNA	Total nifH	ANME nifH	ANME nifD	Total mcrA	Subgroup a-b mcrA	Subgroup c-d mcrA	Subgroup e mcrA	Subgroup f mcrA
A3	1.52 × 10¹¹ (3.84 × 10¹⁰)	5.47 × 10⁹ (4.29 × 10⁸)	2.63 × 10⁷ (1.25 × 10⁶)	ND^a	ND	6.80 × 10⁵ 4.18 × 10⁴	ND	ND	4.45 × 10⁴ (2.91 × 10²)	2.36 × 10³ (1.61 × 10²)
CF6	2.68 × 10¹⁰ (3.57 × 10⁹)	2.42 × 10⁹ (8.43 × 10⁷)	2.04 × 10⁶ (1.76 × 10³)	1.07 × 10³ (2.69 × 10)	1.42 × 10³ (6.45 × 10)	4.58 × 10⁵ 1.02 × 10⁴	1.69 × 10³ (1.19 × 10²)	1.62 × 10³ (3.02 × 10)	ND	1.86 × 10³ (1.79 × 10²)
CF8	2.82 × 10¹⁰ (1.37 × 10⁹)	1.65 × 10⁹ (1.59 × 10⁸)	8.50 × 10⁵ (7.19 × 10⁴)	ND	ND	2.75 × 10⁵ 6.92 × 10³	2.56 × 10 (2.22)	ND	ND	4.81 × 10² (1.64 × 10)
CF11	6.17 × 10¹⁰ (1.51 × 10¹⁰)	1.20 × 10⁹ (8.42 × 10⁷)	1.60 × 10⁶ (5.46 × 10⁴)	3.31 × 10³ (1.15 × 10²)	3.59 × 10³ (5.79 × 10)	3.05 × 10⁵ 1.71 × 10⁴	1.26 × 10² (5.91)	4.27 × 10³ (3.98 × 10²)	2.55 × 10³ (2.09 × 10²)	3.53 × 10² (1.91 × 10)
CF14	4.93 × 10¹⁰ (2.30 × 10⁹)	1.16 × 10⁹ (3.07 × 10⁷)	3.17 × 10⁶ (2.64 × 10⁵)	4.59 × 10³ (2.24 × 10²)	3.89 × 10³ (6.06 × 10)	5.78 × 10⁵ 4.44 × 10⁴	2.40 × 10² (2.26 × 10)	3.87 × 10³ (2.75 × 10)	1.37 × 10³ (7.81)	6.62 × 10² (5.66 × 10)
CF15	1.30 × 10¹¹ (5.59 × 10⁹)	4.95 × 10⁹ (2.67 × 10⁷)	1.35 × 10⁶ (1.22 × 10⁵)	8.16 × 10³ (4.65 × 10²)	5.84 × 10³ (1.69 × 10²)	3.30 × 10⁵ 1.90 × 10⁴	4.39 × 10² (1.59 × 10)	5.58 × 10³ (1.39 × 10²)	3.23 × 10³ (1.89 × 10)	1.79 × 10³ (1.63 × 10²)
E407	2.80 × 10¹⁰ (2.49 × 10⁹)	1.51 × 10⁹ (4.53 × 10⁷)	9.83 × 10⁵ (3.58 × 10⁴)	5.94 × 10² (3.97 × 10)	3.24 × 10² (3.03 × 10)	5.12 × 10⁵ 5.81 × 10³	3.87 × 10² (1.10 × 10)	3.67 × 10² (9.45)	2.32 × 10³ (2.99 × 10)	3.36 × 10³ (3.71 × 10)
E422	3.05 × 10¹⁰ (2.31 × 10⁹)	1.05 × 10⁹ (7.62 × 10⁷)	1.64 × 10⁶ (2.95 × 10⁴)	3.88 × 10² (3.29 × 10)	1.55 × 10³ (6.88 × 10)	3.50 × 10⁵ 2.52 × 10⁴	7.84 × 10² (6.82 × 10)	4.97 × 10² (2.18 × 10)	2.09 × 10³ (1.58 × 10²)	4.47 × 10² (3.95 × 10)
E501	9.39 × 10¹⁰ (1.01 × 10¹⁰)	6.03 × 10⁹ (4.42 × 10⁸)	5.65 × 10⁷ (3.79 × 10⁶)	ND	ND	9.57 × 10⁵ 4.62 × 10⁴	ND	ND	4.78 × 10³ (1.36 × 10²)	ND
E504	1.44 × 10¹¹ (5.15 × 10¹⁰)	1.73 × 10¹⁰ (9.52 × 10⁸)	1.20 × 10⁷ (1.09 × 10⁶)	ND	ND	1.83 × 10⁶ 9.04 × 10⁴	1.12 × 10² (6.24)	ND	3.97 × 10³ (3.89 × 10²)	ND
E505	6.07 × 10¹⁰ (5.33 × 10⁹)	5.48 × 10⁹ (2.34 × 10⁸)	4.34 × 10⁶ (3.40 × 10⁴)	ND	ND	1.61 × 10⁶ 3.67 × 10⁴	1.43 × 10² (1.06 × 10)	ND	3.48 × 10³ (9.64 × 10)	ND
E801	3.13 × 10¹⁰ (2.19 × 10⁹)	1.32 × 10⁹ (2.34 × 10⁷)	3.18 × 10⁶ (3.97 × 10⁴)	ND	ND	7.41 × 10⁵ 5.33 × 10⁴	ND	ND	ND	ND

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ND, not detectable.

The total nifH gene abundance also showed a distributional heterogeneity in the nSCS sediments, where the shallow water sites usually harbored higher abundances (Table 3). The “cluster IIIx” nifH genes of the ANME diazotrophic group (15) were only detected in the sampling stations of methane hydrate or its prospective areas (CF6, CF11, CF14, CF15, E407, and E422), with quite low abundance (3.88 × 10² to 8.16 × 10³ copies g of sediment⁻¹). Similarly, the nifD genes specific to the ANME diazotrophs (17) were also detected only in these sampling stations with low abundance (3.24 × 10² to 5.84 × 10³ copies g of sediment⁻¹). Previous studies indicate that the ANME diazotrophs are exclusively associated with the ANME-2c subgroup (15–17). In the current study, the abundance of the ANME-2c subgroup-specific mcrA genes (i.e., mcrA subgroup c-d in Table 3) showed very similar spatial distribution to the ANME diazotrophic nifH and nifD distributions in the nSCS sediments. The consistency of the qPCR quantification results from all of these three biomarker genes indicated that the nSCS deep-sea sediments did harbor ANME diazotrophs, and they occurred only in methane hydrate or its prospective areas (Table 3).

The total archaeal mcrA gene abundance also showed a distributional heterogeneity: station E504 had the highest gene copy number (1.83 × 10⁶ copies g of sediment⁻¹), and station CF8 had the lowest (2.75 × 10⁵ copies g of sediment⁻¹) (Table 3). The subgroup-specific mcrA genes that targeted the ANME subgroups a-b, e, and f, respectively, also showed spatially heterogeneous distributions in the sediments of the nSCS (Table 3).

The abundances of both sediment bacteria and archaea significantly correlated positively with sediment temperature and negatively with water depth and sediment pore water dissolved oxygen content (DO) (see Table S3 in the supplemental material). The abundance of the nifH-harboring microbes significantly correlated positively with sediment temperature, sediment pore water concentrations of NH₄⁺ and DIN, and the sediment pore water N/P ratio and negatively with water depth, sediment inorganic phosphorus (IP), total phosphorus (TP), and organic matter (OM) contents. Moreover, most of the sedimentological parameters measured in the current study were also found to correlate with the abundance of the nifH-harboring microbes (see Table S3 in the supplemental material). The abundance of the sediment ANME diazotrophs significantly correlated positively with the sediment organic carbon, organic nitrogen, and organic phosphorus contents (OrgC, OrgN, and OrgP, respectively) in the nSCS, as decoded by all three subgroup-specific target genes: ANME nifH, nifD, and subgroup c-d mcrA (see Table S3 in the supplemental material).

The abundance of the sediment total archaeal methanogens and ANME methanotrophs detected by the total mcrA genes significantly correlated positively with sediment temperature and negatively with water depth and sediment clay content in the nSCS (see Table S3 in the supplemental material). The abundance of each of the ANME subgroups detected by subgroup-specific mcrA genes was correlated with distinct environmental factors, such as water depth (positively) for mcrA subgroup a-b, sediment OrgC, OrgN, and OrgP (positively) for mcrA subgroup c-d, sediment temperature (positively) for mcrA subgroup e, and sediment skewness (negatively) for mcrA subgroup f (see Table S3 in the supplemental material).

DISCUSSION

Diversity and novelty of the nSCS nifH gene sequences.

Diverse and novel nifH sequences were obtained in the current study (Table 2, Fig. 2; also see Fig. S1 and S4 in the supplemental material), expanding the NifH phylogeny from the previously defined seven clusters (15) to the presently defined nine putative classes (Fig. 2; also see Fig. S4 in the supplemental material). Most of the newly defined classes were established due to the discovery of novel nifH sequences from the sediments of the nSCS. Our new NifH classification schema is more accurate than the previously defined ones (15). For example, the division of the NifH sequences of the previously defined “cluster IV” and their inclusion in two distinct classes (class I and class II) separately in our new NifH classification schema are consistent with the fact that the NifH sequences from the previous “cluster IV” are polyphyletic (9, 15).

The great diversity and novelty of the nifH sequences obtained from the nSCS marine sediments are highly unexpected as environmental nifH genes have been investigated for several decades (10). Technically, the success of our detection of a variety of novel nifH sequences and classes can be attributed to the use of the new nifH PCR primers designed by Mehta et al. (12). The unique environment of nSCS sediments and the previous neglect of sediment diazotrophic microbial communities may also have contributed to the finding of novel nifH sequences and classes (Fig. 2 and see Fig. S4 in the supplemental material). However, it should be borne in mind that probably not all of the detected nifH sequences came from active diazotrophic microorganisms. For example, most of the NifH sequences from the originally defined “cluster IV” are not well characterized, and some are found not to be involved in N₂ fixation (9, 17, 64). Although it is currently unknown whether the microorganisms that carry the novel nifH sequences from most of our newly defined classes are functional for active N₂ fixation in natural environments, our study extends the diversity and evolutionary complexity of the nifH gene sequences. Recent genome sequencing of various bacterial and archaeal isolates provides further evidence of the diversity and novelty of the nifH sequences in the microbial world (see Fig. S4 in the supplemental material for some of the nifH sequences from microbial genomes). Furthermore, some nifH genes are found to be located on plasmids (65). Rapid evolution and high frequency of horizontal gene transfer of the nifH genes, even at the interdomain level between bacteria and archaea (9), may favor diversity and novelty of the nifH sequences, especially in the sediment environment, where intimate syntrophic interactions between bacteria and archaea, such as the consortia formed between sulfate-reducing bacteria and ANME (14, 16), may facilitate microbial gene transfer. The deep ocean is a source of abundant nitrate (66). Sediment N₂ fixation by diverse nifH-harboring bacteria and archaea may contribute significantly to this large nutrient reservoir.

Environmental influence on the sediment nifH-harboring microbial assemblages.

Abundant bacteria and archaea were detected in the marine sediments of the nSCS, with bacterial 16S rRNA gene copies being predominant over those of archaea (Table 3). Using one of the most efficient and successful methods (FastPrep) for sediment DNA extraction (67), determined 16S rRNA gene copy numbers of bacteria were much higher than those of archaea (averaging 23.5:1). Even with consideration of various 16S rRNA gene copy numbers in bacterial and archaeal genomes and the potential for unequal DNA extraction and primer specificity for the molecular detection (68, 69), the large ratio suggests that bacteria dominate quantitatively over archaea in the surface sediments of the nSCS.

The correlation of the total bacterial and archaeal abundances with sediment temperature (positively), sediment pore water DO (negatively), and water depth (negatively) (see Table S3 in the supplemental material) suggests that the sediment bacterial and archaeal abundances decrease from coastal to deep-sea environments, likely controlled by sediment in situ temperature and pore water DO. These results also indicate that anaerobic bacteria and archaea may be the major constituents of the microbiota in the surface sediments (down to a 5-cm depth) of the nSCS.

The correlation of the total nifH-harboring microbial abundance with sediment temperature (positively), water depth (negatively), and most of the sedimentological parameters (see Table S3 in the supplemental material) indicates that the surface sediment nifH-harboring microbial abundance decreases from coastal to deep-sea environments, likely controlled by in situ sediment temperature and sedimentological conditions. CCA also indicated that sediment temperature was an important environmental factor influencing the community structure and spatial distribution of the sediment nifH-harboring microbial assemblages in the nSCS (Fig. 4). It has been found previously that temperature is the most important environmental factor influencing the spatial distribution of seawater N₂-fixing cyanobacteria in the oceans (70). Thus, temperature may be a universally important environmental factor controlling the distribution of the nifH-harboring microbial community in both water columns and sediments in marine environments.

The abundance of the sediment total nifH genes also correlated positively with the sediment pore water N/P ratio and concentrations of NH₄⁺ and DIN (see Table S3 in the supplemental material), indicating that the sediment nifH-harboring microorganisms might be active in N₂ fixation and NH₄⁺ production in the nSCS. We also found that the abundance of the sediment total nifH genes correlated negatively with sediment inorganic P (IP), total P (TP), and organic matter (OM) contents (see Table S3 in the supplemental material). This might indicate that the sediment nifH-harboring microbial population actively consumed sediment phosphorus and organic matter. However, this explanation needs to be viewed with caution. Most of the sediment P might be tightly absorbed or bound to sediment particles or minerals and thus might not be the soluble reactive phosphorus for microbial utilization. Similarly, most deep-sea sediment organic matter might be old and recalcitrant to microbial utilization. Therefore, whether the negative correlations of the sediment nifH-harboring microorganism abundance with the sediment inorganic P, total P, and organic matter contents might indicate a cause-effect relationship needs to be further investigated.

CCA identified sediment pore water PO₄³⁻ concentration as a key environmental factor influencing the community structure and spatial distribution of the nifH-harboring microorganisms in the sediments of the nSCS (Fig. 4b). This is meaningful since pore water PO₄³⁻ is the major form of soluble reactive phosphorus utilizable by microorganisms. The nSCS has experienced some dramatic change during the last several decades, mainly caused by intensified anthropogenic activities. Recent studies indicated that the bioproductivity of nSCS might be limited by P in estuarine and coastal areas (71, 72). Our data showed that the two shallowest sampling sites, A3 and E501, indeed had quite low sediment pore water PO₄³⁻ concentrations (Table 1). It is interesting to discover that the availability of sediment pore water PO₄³⁻ may have an important influence on the sediment nifH-harboring microbial community structure and distribution and thus potentially on the sediment nitrogen-fixing activity.

Deep-sea methane seep-specific ANME-2c diazotrophic archaea.

Methane seeps, mainly formed in gas hydrate-bearing sediments of marginal seas, sustain significant chemosynthetic ecosystems (73). The characteristic high biomass and productivity of these ecosystems, driven by rapid C and S microbial transformations, may concomitantly require a large supply of N nutrients (15, 56). Recent investigations indicate that N₂-fixing microorganisms, especially archaea in the ANME-2c subgroup, may play a critical role in supplying newly fixed nitrogen to these ecosystems (14–17). These studies also indicate that the distribution of the ANME-2c subgroup diazotrophs may be methane seep specific, seemingly absent in other nonseep marine environments (15–17). The results of the present study support this hypothesis.

Our quantification results of universal and subgroup-specific qPCR determinations of the mcrA genes indicated that archaeal methanogens were present in all of the sediment environments of the nSCS, while distribution of the specific ANME subgroups varied and might be controlled by distinct environmental factors (Table 3; also see Table S3 in the supplemental material). These results also indicated that only the abundance of subgroup c-d mcrA genes correlated with the abundances of the “cluster IIIx” nifH and nifD genes (Table 3), suggesting that only the ANME-2c archaea and not the archaea from other ANME subgroups were diazotrophs. This finding agrees with previous studies (14–17). Correlation analyses using the mcrA subgroup-specific qPCR data indicated that the ANME-2c diazotrophs had a distribution pattern and environment relationship distinct from those of the other nondiazotrophic ANME subgroups (see Table S3 in the supplemental material). ANME-2c diazotroph abundance significantly correlated with sediment OrgC, OrgN, and OrgP for all three “cluster IIIx”-specific target genes: ANME nifH, nifD, and subgroup c-d mcrA (see Table S3 in the supplemental material). Although ANME activity is usually thought of as a chemolithoautotrophic process, certain organic substrates may provide optimal growth and performances of anaerobic methane oxidation and N₂ fixation for the syntrophic consortia of ANME archaea and sulfate-reducing bacteria (14, 16, 74).

Quantifications via qPCR targeting the “cluster IIIx”-specific nifH, nifD, and mcrA genes confirmed the existence of the ANME-2c diazotrophs in the marine sediments of the methane hydrate-bearing or prospective areas in the nSCS (Table 3). Although only one ANME-2c diazotroph-related nifH sequence (E422-68, GenBank accession no. HQ224110) was obtained in the present study (see Fig. S4 in the supplemental material), the rare occurrence of the ANME nifH sequences in our clone libraries is not a surprise. The ANME-2c diazotrophs might exist with very low abundance in the marine sediments and their corresponding nifH sequences might be diluted out in the clone libraries due to the overwhelmingly high diversity and abundance of the other nifH sequences in the nSCS sediments (Table 2, Fig. 2; also see Fig. S1 and S4 in the supplemental material). The nSCS methane hydrates are buried very deeply in the sediments (>150 m below the seafloor surface), and the nSCS methane seeps are characterized by micro gas venting with conduit/channel diameters of only 200 to 600 μm (46, 75). The deep burial of methane hydrates and the low level of methane gas venting make the intensity of the methane supply very low and also the methane seeps as localized features in the surface sediments of the nSCS. Although some of our sediment sampling sites were in or near the methane hydrate-bearing or prospective areas, they were likely not located exactly on the main venting channels due to the difficulty of on-board deep-sea sediment sampling. A low supply of methane might sustain low ANME diazotrophic populations, as detected by our qPCR measurements (Table 3). Our current data indicated that the distribution of the ANME-2c diazotrophs was associated with the presence of methane seeps, possibly caused by the dependence of ANME archaea on the methane supply for their ecophysiological activities. Our current data, together with the results from other recent studies (14–17), support the hypothesis that ANME-2c diazotrophs reside exclusively in the deep-sea methane seep sediments and that their presence has diagnostic value for the discovery of deep-sea methane hydrate reservoirs.

Supplementary Material

Supplemental material

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ACKNOWLEDGMENTS

This study was supported by China NSFC grants 91028011 (H.D.) and 41076091 (H.D. and M.G.K.), National Key Basic Research Program of China grants 2013CB955700 (H.D.) and 2007CB411702 (X.L.), Fundamental Research Funds for the Central Universities of China grant 09CX05005A (H.D.), and U.S. NSF grants 0541797 and 0948202 (M.G.K.).

The sediment samples used in this study were collected during the 2007 South China Sea Open Cruise by R/V Shiyan 3, South China Sea Institute of Oceanology, CAS. We thank two anonymous reviewers for their constructive comments and Jian Ren, Shuai Wang, Longsheng Cheng, Songbing Yan, Zihan Chen, Wei Wei, and Guanhang Liu for their assistance in the project.

Footnotes

Published ahead of print 12 October 2012

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01889-12.

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[B14] 14. Pernthaler A, Dekas AE, Brown CT, Goffredi SK, Embaye T, Orphan VJ. 2008. Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics. Proc. Natl. Acad. Sci. U. S. A. 105:7052–7057 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Dang HY, Luan XW, Zhao JY, Li J. 2009. Diverse and novel nifH and nifH-like gene sequences in the deep-sea methane seep sediments of the Okhotsk Sea. Appl. Environ. Microbiol. 75:2238–2245 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Dekas AE, Poretsky RS, Orphan VJ. 2009. Deep-sea archaea fix and share nitrogen in methane-consuming microbial consortia. Science 326:422–426 [DOI] [PubMed] [Google Scholar]

[B17] 17. Miyazaki J, Higa R, Toki T, Ashi J, Tsunogai U, Nunoura T, Imachi H, Takai K. 2009. Molecular characterization of potential nitrogen fixation by anaerobic methane-oxidizing archaea in the methane seep sediments at the number 8 Kumano Knoll in the Kumano Basin, offshore of Japan. Appl. Environ. Microbiol. 75:7153–7162 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Gaby JC, Buckley DH. 2011. A global census of nitrogenase diversity. Environ. Microbiol. 13:1790–1799 [DOI] [PubMed] [Google Scholar]

[B19] 19. Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. U. S. A. 95:6578–6583 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Zehr JP, Kudela RM. 2011. Nitrogen cycle of the open ocean: from genes to ecosystems. Annu. Rev. Mar. Sci. 3:197–225 [DOI] [PubMed] [Google Scholar]

[B21] 21. Zehr JP, Mellon M, Braun S, Litaker W. 1995. Diversity of heterotrophic nitrogen fixation genes in a marine cyanobacterial mat. Appl. Environ. Microbiol. 61:2527–2532 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Burns JA, Zehr JP, Capone DG. 2002. Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments. Microb. Ecol. 44:336–343 [DOI] [PubMed] [Google Scholar]

[B23] 23. Flores-Mireles AL, Winans SC, Holguin G. 2007. Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Appl. Environ. Microbiol. 73:7308–7321 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Charpy L, Palinska KA, Casareto B, Langlade MJ, Suzuki Y, Abed RM, Golubic S. 2010. Dinitrogen-fixing cyanobacteria in microbial mats of two shallow coral reef ecosystems. Microb. Ecol. 59:174–186 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Gamble MD, Bagwell CE, Larocque J, Bergholz PW, Lovell CR. 2010. Seasonal variability of diazotroph assemblages associated with the rhizosphere of the salt marsh cordgrass, Spartina alterniflora. Microb. Ecol. 59:253–265 [DOI] [PubMed] [Google Scholar]

[B26] 26. Severin I, Acinas SG, Stal LJ. 2010. Diversity of nitrogen-fixing bacteria in cyanobacterial mats. FEMS Microbiol. Ecol. 73:514–525 [DOI] [PubMed] [Google Scholar]

[B27] 27. Walsh JJ. 1991. Importance of continental margins in the marine biogeochemical cycling of carbon and nitrogen. Nature 350:53–55 [Google Scholar]

[B28] 28. Bauer JE, Druffel ERM. 1998. Ocean margins as a significant source of organic matter to the deep open ocean. Nature 392:482–485 [Google Scholar]

[B29] 29. Subramaniam A, Yager PL, Carpenter EJ, Mahaffey C, Björkman K, Cooley S, Kustka AB, Montoya JP, Sañudo-Wilhelmy SA, Shipe R, Capone DG. 2008. Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean. Proc. Natl. Acad. Sci. U. S. A. 105:10460–10465 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B31] 31. Sohm JA, Webb EA, Capone DG. 2011. Emerging patterns of marine nitrogen fixation. Nat. Rev. Microbiol. 9:499–508 [DOI] [PubMed] [Google Scholar]

[B32] 32. Knittel K, Boetius A. 2009. Anaerobic oxidation of methane: progress with an unknown process. Annu. Rev. Microbiol. 63:311–334 [DOI] [PubMed] [Google Scholar]

[B33] 33. Hester KC, Brewer PG. 2009. Clathrate hydrates in nature. Annu. Rev. Mar. Sci. 1:303–327 [DOI] [PubMed] [Google Scholar]

[B34] 34. Moisander PH, Beinart RA, Voss M, Zehr JP. 2008. Diversity and abundance of diazotrophic microorganisms in the South China Sea during intermonsoon. ISME J. 2:954–967 [DOI] [PubMed] [Google Scholar]

[B35] 35. Wu J, Chung SW, Wen LS, Liu KK, Chen YL, Chen H, Karl DM. 2003. Dissolved inorganic phosphorus, dissolved iron, and Trichodesmium in the oligotrophic South China Sea. Global Biogeochem. Cycles 17:1008 [Google Scholar]

[B36] 36. Yin KD, Qian Wu P-YMCS, Chen JC, Huang LM, Song XY, Jian WJ. 2001. Shift from P to N limitation of phytoplankton growth across the Pearl River estuarine plume during summer. Mar. Ecol. Prog. Ser. 221:17–28 [Google Scholar]

[B37] 37. Chen YL, Chen H, Karl DM, Takahashi M. 2004. Nitrogen modulates phytoplankton growth in spring in the South China Sea. Cont. Shelf Res. 24:527–541 [Google Scholar]

[B38] 38. Zhang Y, Zhao ZH, Sun J, Jiao NZ. 2011. Diversity and distribution of diazotrophic communities in the South China Sea deep basin with mesoscale cyclonic eddy perturbations. FEMS Microbiol. Ecol. 78:417–427 [DOI] [PubMed] [Google Scholar]

[B39] 39. Chou W, Chen YL, Sheu DD, Shih Y, Han C, Cho CL, Tseng C, Yang Y. 2006. Estimated net community production during the summertime at the SEATS time-series study site, northern South China Sea: implications for nitrogen fixation. Geophys. Res. Lett. 33:L22610 [Google Scholar]

[B40] 40. Voss M, Bombar D, Natalie L, Dippner JW. 2006. Riverine influence on nitrogen fixation in the upwelling region off Vietnam, South China Sea. Geophys. Res. Lett. 33:L07604 [Google Scholar]

[B41] 41. Chen YL, Chen H, Tuo S, Ohki K. 2008. Seasonal dynamics of new production from Trichodesmium N₂ fixation and nitrate uptake in the upstream Kuroshio and South China Sea basin. Limnol. Oceanogr. 53:1705–1721 [Google Scholar]

[B42] 42. Bombar D, Moisander PH, Dippner JW, Foster RA, Voss M, Karfeld B, Zehr JP. 2011. Distribution of diazotrophic microorganisms and nifH gene expression in the Mekong River plume during intermonsoon. Mar. Ecol. Prog. Ser. 424:39–52 [Google Scholar]

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PERMALINK

Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

Hongyue Dang

Jinying Yang

Jing Li

Xiwu Luan

Yunbo Zhang

Guizhou Gu

Rongrong Xue

Mingyue Zong

Martin G Klotz

Abstract

INTRODUCTION

MATERIALS AND METHODS

Sample collection and environmental factor measurements.

Fig 1.

Table 1.

DNA extraction and nifH gene clone library analyses.

Quantification of major microbial groups.

Statistical analyses.

Nucleic acid sequence accession numbers.

RESULTS

Site description.

Diversity of the sediment nifH genes.

Table 2.

Phylogeny of the NifH protein sequences.

Fig 2.

Spatial distribution of the nifH-harboring microbial assemblages.

Fig 3.

Fig 4.

Abundance of nifH-harboring microorganisms.

Table 3.

DISCUSSION

Diversity and novelty of the nSCS nifH gene sequences.

Environmental influence on the sediment nifH-harboring microbial assemblages.

Deep-sea methane seep-specific ANME-2c diazotrophic archaea.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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