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Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands Bao-lan Hu a , Li-dong Shen a , Xu Lian a , Qun Zhu b , Shuai Liu a , Qian Huang a , Zhan-fei He a , Sha Geng a , Dong-qing Cheng b , Li-ping Lou a , Xiang-yang Xu a , Ping Zheng a , and Yun-feng He a,1 a Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China; and b Department of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China Edited by Donald E. Canfield, University of Southern Denmark, Odense, Denmark, and approved February 11, 2014 (received for review October 2, 2013) The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by Candidatus Methylomirabilis oxyfera(M. oxyfera). Here, evi- dence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measure- ments, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable iso- tope experiments confirmed the occurrence of n-damo in the ex- amined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO 2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and partic- ulate methane monooxygenase genes demonstrated that M. oxy- fera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10 7 gene copies per gram of dry soil. It is estimated that 0.51 g of CH 4 m 2 per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents pre- viously unidentified confirmation that the n-damo process is a pre- viously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution. activity | methane cycle | overlooked methane sink N itrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered process that was reported to be per- formed by Candidatus Methylomirabilis oxyfera(M. oxyfera) (1, 2). The n-damo process constitutes a unique link between the two major global nutrient cycles of carbon and nitrogen (1). Furthermore, it may act as an important and overlooked sink of the greenhouse gas methane (3). Methane is greater than 25-fold more effective at trapping heat than is carbon dioxide (CO 2 ) on a per-molecule basis and is responsible for 20% of global warming (4, 5). The n-damo process may alleviate the greenhouse effect by converting methane to CO 2 . Although several enrichment cultures of M. oxyfera-like bac- teria have been obtained from freshwater sediments (1, 68) and peatlands (9), the distribution of these bacteria in environments is not well understood. Two recent studies reported the presence of M. oxyfera-like bacteria in two freshwater lakes, Lake Con- stance (10) in Germany and Lake Biwa in Japan (11). Wang et al. (12) and Zhu et al. (9) reported the distribution of M. oxyfera-like bacteria in a paddy field and in a minerotrophic peatland, respectively. In addition, Shen et al. (13, 14) recently reported the distribution of M. oxyfera-like bacteria in the sedi- ments of Qiantang River (China) and Jiaojiang Estuary (China). However, molecular evidence for the presence of M. oxyfera-like bacteria is not a definitive indication of the occurrence of n-damo process. To date, direct evidence to support the occur- rence of n-damo in the environment was lacking. Deutzmann and Schink (10) used radiotracer experiments ( 14 CH 4 ) to detect n-damo activity in Lake Constance. The lake sediments were evaluated by tracking 14 CO 2 formation in the presence of different electron acceptors, including nitrate, nitrite, sulfate, and oxygen. It was found that the addition of nitrate led to a significant increase in 14 CO 2 formation, suggesting the occurrence of n-damo in this lake (10). Wetlands are the worlds largest natural source of methane, accounting for 2040% of total methane emissions (5, 15). The n-damo is predicted to occur near the oxicanoxic interface, having low concentrations of oxidizable substrates other than methane, as well as low level of sulfate and high level of nitrate (16). Such conditions prevail in anoxic wetlands (17). To date, however, there have been no reports of direct evidence for the occurrence of n-damo in wetlands. The primary objective of this study was to determine whether n-damo acts as a previously overlooked methane sink in wet- lands. To achieve this objective, three different types of fresh- water wetlands, including the Xiazhuhu wetland (natural wetland), the Xixi wetland (urban wetland), and a paddy field (man-made wetland), were studied in southeastern China. The potential n-damo rates in these three wetlands were determined using 13 CH 4 isotope-labeling experiments. The distribution and diversity of M. oxyfera-like bacteria were studied based on 16S rRNA and particulate methane monooxygenase (pmoA) gene clone library analyses, and the abundance of these bacteria was quantified by quantitative (q) PCR. Significance Given the current pressing need to more fully understand the methane cycle on Earth, in particular, unidentified sinks for methane, identifying and quantifying novel sinks for methane is fundamental importance. Here, we provide previously un- identified direct evidence for the nitrite-dependent anaerobic methane oxidation (n-damo) process as a previously over- looked microbial methane sink in wetlands by stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library anal- yses. It is estimated that n-damo could consume 4.16.1 Tg of CH 4 m 2 per year in wetlands under anaerobic conditions, which is roughly 26% of current worldwide CH 4 flux estimates for wetlands. Given the worldwide increase in nitrogen pollu- tion, this methane sink may become more important in the future. Author contributions: X.-y.X., P.Z., and Y.-f.H. designed research; B.-l.H., L.-d.S., X.L., Q.Z., S.L., Q.H., Z.-f.H., and S.G. performed research; B.-l.H., L.-d.S., X.L., Q.Z., S.L., Q.H., D.-q.C., and L.-p.L. analyzed data; and B.-l.H., L.-d.S., and Y.-f.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The sequences reported in this paper have been deposited in the Gen- Bank database [accession nos. KC905781KC905856 (Candidatus Methylomirabilis oxyfera 16S rRNA) and KC905857KC905908 (Candidatus Methylomirabilis oxyfera pmoA)]. 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1318393111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1318393111 PNAS | March 25, 2014 | vol. 111 | no. 12 | 44954500 ECOLOGY Downloaded by guest on April 17, 2020

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Page 1: Evidence for nitrite-dependent anaerobic methane oxidation ... · is fundamental importance. Here, we provide previously un-identified direct evidence for the nitrite-dependent anaerobic

Evidence for nitrite-dependent anaerobic methaneoxidation as a previously overlooked microbialmethane sink in wetlandsBao-lan Hua, Li-dong Shena, Xu Liana, Qun Zhub, Shuai Liua, Qian Huanga, Zhan-fei Hea, Sha Genga, Dong-qing Chengb,Li-ping Loua, Xiang-yang Xua, Ping Zhenga, and Yun-feng Hea,1

aDepartment of Environmental Engineering, Zhejiang University, Hangzhou 310058, China; and bDepartment of Life Science, Zhejiang Chinese MedicalUniversity, Hangzhou 310053, China

Edited by Donald E. Canfield, University of Southern Denmark, Odense, Denmark, and approved February 11, 2014 (received for review October 2, 2013)

The process of nitrite-dependent anaerobic methane oxidation(n-damo) was recently discovered and shown to be mediated by“Candidatus Methylomirabilis oxyfera” (M. oxyfera). Here, evi-dence for n-damo in three different freshwater wetlands locatedin southeastern China was obtained using stable isotope measure-ments, quantitative PCR assays, and 16S rRNA and particulatemethane monooxygenase gene clone library analyses. Stable iso-tope experiments confirmed the occurrence of n-damo in the ex-amined wetlands, and the potential n-damo rates ranged from0.31 to 5.43 nmol CO2 per gram of dry soil per day at differentdepths of soil cores. A combined analysis of 16S rRNA and partic-ulate methane monooxygenase genes demonstrated that M. oxy-fera-like bacteria were mainly present in the deep soil witha maximum abundance of 3.2 × 107 gene copies per gram of drysoil. It is estimated that ∼0.51 g of CH4 m−2 per year could belinked to the n-damo process in the examined wetlands basedon the measured potential n-damo rates. This study presents pre-viously unidentified confirmation that the n-damo process is a pre-viously overlooked microbial methane sink in wetlands, and n-damohas the potential to be a globally important methane sink due toincreasing nitrogen pollution.

activity | methane cycle | overlooked methane sink

Nitrite-dependent anaerobic methane oxidation (n-damo) isa recently discovered process that was reported to be per-

formed by “Candidatus Methylomirabilis oxyfera” (M. oxyfera)(1, 2). The n-damo process constitutes a unique link between thetwo major global nutrient cycles of carbon and nitrogen (1).Furthermore, it may act as an important and overlooked sink ofthe greenhouse gas methane (3). Methane is greater than 25-foldmore effective at trapping heat than is carbon dioxide (CO2) on aper-molecule basis and is responsible for 20% of global warming(4, 5). The n-damo process may alleviate the greenhouse effectby converting methane to CO2.Although several enrichment cultures of M. oxyfera-like bac-

teria have been obtained from freshwater sediments (1, 6–8) andpeatlands (9), the distribution of these bacteria in environmentsis not well understood. Two recent studies reported the presenceof M. oxyfera-like bacteria in two freshwater lakes, Lake Con-stance (10) in Germany and Lake Biwa in Japan (11). Wanget al. (12) and Zhu et al. (9) reported the distribution ofM. oxyfera-like bacteria in a paddy field and in a minerotrophicpeatland, respectively. In addition, Shen et al. (13, 14) recentlyreported the distribution of M. oxyfera-like bacteria in the sedi-ments of Qiantang River (China) and Jiaojiang Estuary (China).However, molecular evidence for the presence of M. oxyfera-likebacteria is not a definitive indication of the occurrence ofn-damo process. To date, direct evidence to support the occur-rence of n-damo in the environment was lacking. Deutzmannand Schink (10) used radiotracer experiments (14CH4) to detectn-damo activity in Lake Constance. The lake sediments wereevaluated by tracking 14CO2 formation in the presence of

different electron acceptors, including nitrate, nitrite, sulfate, andoxygen. It was found that the addition of nitrate led to a significantincrease in 14CO2 formation, suggesting the occurrence of n-damoin this lake (10).Wetlands are the world’s largest natural source of methane,

accounting for ∼20–40% of total methane emissions (5, 15). Then-damo is predicted to occur near the oxic–anoxic interface,having low concentrations of oxidizable substrates other thanmethane, as well as low level of sulfate and high level of nitrate(16). Such conditions prevail in anoxic wetlands (17). To date,however, there have been no reports of direct evidence for theoccurrence of n-damo in wetlands.The primary objective of this study was to determine whether

n-damo acts as a previously overlooked methane sink in wet-lands. To achieve this objective, three different types of fresh-water wetlands, including the Xiazhuhu wetland (natural wetland),the Xixi wetland (urban wetland), and a paddy field (man-madewetland), were studied in southeastern China. The potentialn-damo rates in these three wetlands were determined using13CH4 isotope-labeling experiments. The distribution and diversityof M. oxyfera-like bacteria were studied based on 16S rRNA andparticulate methane monooxygenase (pmoA) gene clone libraryanalyses, and the abundance of these bacteria was quantified byquantitative (q) PCR.

Significance

Given the current pressing need to more fully understand themethane cycle on Earth, in particular, unidentified sinks formethane, identifying and quantifying novel sinks for methaneis fundamental importance. Here, we provide previously un-identified direct evidence for the nitrite-dependent anaerobicmethane oxidation (n-damo) process as a previously over-looked microbial methane sink in wetlands by stable isotopemeasurements, quantitative PCR assays, and 16S rRNA andparticulate methane monooxygenase gene clone library anal-yses. It is estimated that n-damo could consume 4.1–6.1 Tg ofCH4 m−2 per year in wetlands under anaerobic conditions,which is roughly 2–6% of current worldwide CH4 flux estimatesfor wetlands. Given the worldwide increase in nitrogen pollu-tion, this methane sink may become more important inthe future.

Author contributions: X.-y.X., P.Z., and Y.-f.H. designed research; B.-l.H., L.-d.S., X.L., Q.Z.,S.L., Q.H., Z.-f.H., and S.G. performed research; B.-l.H., L.-d.S., X.L., Q.Z., S.L., Q.H., D.-q.C.,and L.-p.L. analyzed data; and B.-l.H., L.-d.S., and Y.-f.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database [accession nos. KC905781–KC905856 (CandidatusMethylomirabilis oxyfera16S rRNA) and KC905857–KC905908 (Candidatus Methylomirabilis oxyfera pmoA)].1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1318393111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1318393111 PNAS | March 25, 2014 | vol. 111 | no. 12 | 4495–4500

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ResultsPhysicochemical Characteristics of the Wetlands. The vertical pro-files of the redox potential, pH, temperature, ammonium, nitrite,nitrate, total nitrogen, organic carbon, and methane of the soilcores at 10-cm intervals are shown in Fig. S1. The redox potentialshowed a decreasing trend from the surface layer to the deeplayer, which decreased from 62.3 to 19.0, 43.9–23.8, and 69.2–20.4 mV, respectively, in soil cores collected form the Xiazhuhuwetland, the Xixi wetland, and the paddy field, suggesting theanoxic conditions of the soil cores. The soil nitrite concentrationof soil cores collected from the Xiazhuhu wetland peaked at 10-to 20-cm depth and then decreased with depth from 2.18 to 0.12mg·kg−1 N and rapid decrease of nitrite concentrations wereobserved at depths of 20–30, 50–60, and 90–100 cm (Fig. S1).The soil nitrite concentration of soil cores collected from thepaddy field also peaked at 10- to 20-cm depth and then de-creased with depth from 1.12 to 0.16 mg·kg−1 N, and rapiddecreases of nitrite concentrations were observed at depths of10–30, 50–60, and 90–100 cm (Fig. S1). The soil nitrite concen-tration of soil cores collected from the Xixi wetland decreasedwith depth from 1.72 to 0.15 mg·kg−1 N, and rapid decreases ofnitrite concentrations were observed at depths of 20–40, 50–60,and 90–100 cm (Fig. S1). For the methane, the electron donor ofn-damo, showed increasing trends with depth (Fig. S1). Theconcentration of methane in soil gas collected from the Xiaz-huhu wetland increased with depth from 3.1 × 103 to 5.5 × 104mg·m−3, and rapid increases of methane concentrations wereobserved at depths of 50–60 and 90–100 cm. The methaneconcentration of soil cores collected from the Xixi wetland in-creased with depth from 8.9 to 3.2 × 104 mg·m−3, and rapidincreases of methane concentrations were also observed atdepths of 50–60 and 90–100 cm. The methane concentration ofsoil cores collected from the paddy field increased with depthfrom 8.7 × 102 to 1.0 × 105 mg·m−3, and rapid increases ofmethane concentrations were observed at depths of 10–30, 50–60, and 90–100 cm. It was hypothesized that the methane at thelayers of 20–30, 50–60, and 90–100 cm has the potential to beoxidized anaerobically via nitrite because of the observed op-posing gradients of methane and nitrite. The sulfate, which is themost common electron acceptor for anaerobic methane oxida-tion (AOM) in marine environments (4), was below the de-tection limit (∼2 mg·kg−1 S) in all core samples, whereas theturbidimetric method used for determination of soil sulfate is notvery sensitive. Therefore, the possibility that the below-groundmethane could also be oxidized via sulfate cannot be excluded.Based on the above observations, a total of nine core samplescollected from depths of 20–30, 50–60, and 90–100 cm of eachwetland were selected for further molecular analyses and ac-tivity tests.

Phylogenetic Analysis of M. oxyfera-Like 16S rRNA Genes. Phyloge-netic analyses showed that the recovered 16S rRNA genesequences were grouped into eight separate clusters, which wereassigned to two groups of M. oxyfera-like bacteria, groups A andB (Fig. 1), according to Ettwig et al. (6). Sequences of cluster I,which were recovered from the Xixi wetland, showed a highsimilarity to the 16S rRNA gene of M. oxyfera at 97.2–97.8%identity. Sequences of cluster II, which were recovered from thepaddy field, were less similar to the 16S rRNA gene of M. oxyfera,showing 95.6–96.7% identity. Sequences of cluster III, which wererecovered from the Xiazhuhu wetland, showed 94.7–95.4% simi-larity to the 16S rRNA gene of M. oxyfera. Sequences of clustersIV and V, which were recovered from the Xixi wetland, showed92.6–92.8% and 91.8–92.4% identities to the 16S rRNA gene ofM. oxyfera, respectively. Sequences of clusters VI and VII, whichwere recovered from the paddy field, showed 91.2–91.6% and90.4–90.8% identities to the 16S rRNA gene of M. oxyfera, re-spectively. Sequences of cluster VIII, which were recovered fromthe Xiazhuhu wetland, showed only 88.2–90.1% identity to the16S rRNA gene of M. oxyfera. Furthermore, the most abundantclone sequences at depths of 50–60 and 90–100 cm were affiliated

with group A, whereas the majority of sequences recovered fromdepth of 20–30 cm were affiliated with group B (Fig. 1).

Phylogenetic Analysis of M. oxyfera-Like pmoA Genes. Phylogeneticanalyses showed that the retrieved pmoA gene sequences weregrouped into three distinct clusters (Fig. 2), which was consistentwith the detection of three distinct clusters of 16S rRNA genes ingroup A (Fig. 1). Sequences of cluster I that were recovered fromthe Xixi wetland showed 90.5–91.3% similarity to the pmoA geneofM. oxyfera. Sequences of cluster II, which were recovered from

Fig. 1. Neighbor-joining phylogenetic tree showing the phylogeneticaffiliations ofM. oxyfera-like 16S rRNA gene sequences in soil cores collectedfrom the three wetlands. Bootstrap values were 1,000 replicates, and thescale bar represents 2% of the sequence divergence. The identifiers XZ30,XZ60, and XZ100 represent samples collected from depths of 20–30, 50–60,and 90–100 cm, respectively, at the Xiazhuhu wetland. The identifiers XX30,XX60, and XX100 correspond to samples collected from depths of 20–30, 50–60, and 90–100 cm, respectively, at the Xixi wetland. The identifiers PF30,PF60, and PF100 indicate samples collected from depths of 20–30, 50–60, and90–100 cm, respectively, at the paddy field.

Fig. 2. Neighbor-joining phylogenetic tree showing the phylogenetic affili-ations ofM. oxyfera-like pmoA gene sequences in soil cores collected from thethree wetlands. Bootstrap values were 1,000 replicates, and the scale barrepresents 5% of the sequence divergence.

4496 | www.pnas.org/cgi/doi/10.1073/pnas.1318393111 Hu et al.

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the paddy field, showed 88.4–89.5% identity to the pmoA geneof M. oxyfera. Sequences of cluster III that were recovered fromthe Xiazhuhu wetland showed 85.6–86.4% identity to the pmoAgene of M. oxyfera. Moreover, the pmoA gene sequences wereonly detected within the deeper layers (50–60 and 90–100 cm) ofthe soil cores, and pmoA gene sequences were not detected inthe upper layer (20–30 cm) (Fig. 2).

Diversity of M. oxyfera-Like 16S rRNA and pmoA Genes. Similarlevels of M. oxyfera-like 16S rRNA gene diversity were observedin the Xiazhuhu and Xixi wetlands (Table S1 and Fig. S2).DOTUR analysis indicated that a total of five operational tax-onomic units (OTUs) were observed in the Xiazhuhu and Xixiwetlands, respectively, based on a threshold of 3% difference inthe recovered 16S rRNA gene sequences. A higher diversity ofM. oxyfera-like 16S rRNA genes was observed in the paddy fieldhaving nine OTUs. The diversity of M. oxyfera-like pmoA geneswas lower than the 16S rRNA gene diversity. DOTUR analysisindicated that only one OTU was detected in the three wetlandsbased on a threshold of 7% difference in the recovered pmoAgene sequences (Table S1 and Fig. S2).

Abundance of M. oxyfera-Like Bacteria. The copy numbers of M.oxyfera-like 16S rRNA genes in the nine core samples were de-termined using qPCR, as previously described (6). The copy num-bers of M. oxyfera-like 16S rRNA genes varied from 3.0 ± 0.5 × 106

to 3.2 ± 0.7 × 107, 1.7 ± 0.2 × 106 to 1.0 ± 0.1 × 107, and 1.5 ± 0.2 ×106 to 4.5 ± 0.3 × 106 copies per gram of dry soil in the Xiazhuhuwetland, the Xixi wetland, and the paddy field, respectively. Thecopy numbers of M. oxyfera-like 16S rRNA genes varied at dif-ferent depths of soil cores collected from the three wetlands,with the highest copy number detected at a depth of 50–60 cm(Fig. 3A).

Potential Rates of n-damo Process. Stable isotope tracer experi-ments were conducted on the nine samples to determine thepotential rates of n-damo in the examined wetlands. The resultsshowed that for the slurries amended with only 13CH4, no sig-nificant accumulation of 13CO2 was observed at any soil coredepth (except for the 20- to 30-cm soil core depth of the Xixiwetland and the paddy field) (Fig. 4), indicating that the levels ofambient dissolved oxygen and NOx

− in most incubations were solow after preincubation that can’t be used by methane-oxidizingbacteria. When both 13CH4 and NO2

− were present, 13CO2 ac-cumulated at each analyzed depth for the majority of soil cores(Fig. 4), suggesting the occurrence of n-damo. For those slurries

amended with 13CH4 and SO42−, however, 13CO2 values remained

near background levels in each of soil incubation (Fig. 4), in-dicating a lack of discernible activity of sulfate-dependent AOM inthe examined soil samples. The potential n-damo activities rangedfrom 0.31 ± 0.06–5.43 ± 0.19, 0.68 ± 0.13–4.92 ± 0.04, and 1.68 ±0.03–2.04 ± 0.06 nmol of CO2 per gram of dry soil per day in theXiazhuhu wetland, the Xixi wetland, and the paddy field, re-spectively. It was found that the potential n-damo activity in thedeeper layers (50–60 and 90–100 cm) was much higher than in theupper layer (20–30 cm) (Fig. 3B). The highest potential n-damorates were detected at a depth of 50–60 cm for the Xiazhuhu andXixi wetlands and at 90–100 cm for the paddy field. In contrast,the lowest potential n-damo activity was observed at 20- to 30-cmdepth for the Xiazhuhu and Xixi wetlands, and the potential n-damo activity was below the detection limit at a depth of 20–30 cmfor the paddy field.

DiscussionIn this study, the potential role of n-damo as a previously over-looked methane sink was studied in three different types offreshwater wetlands. The presence ofM. oxyfera-like bacteria wasconfirmed by 16S rRNA and pmoA gene clone library analyses.qPCR results further confirmed the presence of M. oxyfera-likebacteria throughout the examined soil cores, with abundancevarying from 1.5 × 106 to 3.2 × 107 16S rRNA gene copies pergram of dry soil. Incubations using 13CH4 tracer indicated theoccurrence of n-damo in the majority of the examined soil cores,and the potential n-damo rates ranged between 0.31 and 5.43nmol CO2 per gram of dry soil per day. Taken together, theseresults demonstrate that the n-damo process represents a pre-viously overlooked microbial methane sink in wetlands.The distribution and diversity of M. oxyfera-like bacteria have

been studied in a limited number of freshwater lakes, rivers, andwetlands. For example, based on the detection of M. oxyfera-like16S rRNA genes, three and five OTUs have been observed inprofundal and littoral sediments of Lake Constance (10), re-spectively, and six OTUs have been found in profundal sedi-ments of Lake Biwa (11). A relatively higher diversity of M.oxyfera-like 16S rRNA genes has been reported in freshwaterriver sediments, with a total of 15 OTUs (13). M. oxyfera-likepmoA gene sequences have been detected in Lake Constance(10), Lake Biwa (11), Qiantang River (13), Jiaojiang Estuary(14), and the deep layer of paddy soil (12) and peatland (7).DOTUR analysis of pmoA genes deposited in GenBank showedthat pmoA genes recovered from these habitats were very closelyrelated to one another (with only one OTU being observed foreach habitat), except for the Qiantang River (13) and JiaojiangEstuary (14), for which a total of 13 and 16 OTUs were observed,respectively. In this study, a total of five, five, and nine OTUs of16S rRNA genes from M. oxyfera-like bacteria were observed inthe Xiazhuhu wetland, the Xixi wetland, and the paddy field,respectively, and only one OTU of pmoA genes was observed inthe three wetlands (Table S1 and Fig. S2). Thus, the diversity ofM. oxyfera-like 16S rRNA genes and pmoA genes in the exam-ined wetlands was similar to the diversity observed in the re-ported lake sediments and wetlands but lower than the diversityobserved in river sediments and estuarine sediments.Previous studies demonstrated that group A of M. oxyfera-like

bacteria was the dominant bacteria responsible for carrying outthe n-damo process (1, 6–10, 18). The most abundant clonesequences of the 16S rRNA genes obtained from the deeperlayers (50–60 and 90–100 cm) of the examined soil cores wereaffiliated with group A, whereas most sequences detected in theupper layer (20–30 cm) were affiliated with group B (Fig. 1).Furthermore, pmoA genes of M. oxyfera-like bacteria could onlybe recovered from the deeper layers (50–60 and 90–100 cm) ofthe examined soil cores, whereas they could not be recoveredfrom the upper layer (20–30 cm; Fig. 2). These results are inagreement with those obtained from other studies, in whichgroup A M. oxyfera-like bacteria and pmoA genes were bothpresent in deep profundal sediment of lakes (10, 11) and the

A B

Fig. 3. The abundance of M. oxyfera-like bacteria (A) and the potential n-damo rates (B) of soil cores collected from the three wetlands.

Hu et al. PNAS | March 25, 2014 | vol. 111 | no. 12 | 4497

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deep layer of paddy fields (12) and minerotrophic peatlands (9),all of which are characterized by stable environmental conditionsand the coexistence of methane and nitrate/nitrite.qPCR showed that M. oxyfera-like bacteria were present

throughout the examined soil cores, and their 16S rRNA geneabundance varied from 1.5 × 106 to 3.2 × 107 copies per gram ofdry soil, which is similar to values reported for minerotrophicpeatland and river sediments (106–107 copies per gram of dry soil)(9, 13). In the present study, a higher abundance ofM. oxyfera-likebacteria was observed in the deeper layers (50–60 and 90–100 cm)(Fig. 3A). These results are similar to those observed in peatland,where a higher abundance ofM. oxyfera-like bacteria was found inthe deep layer (80–85 cm) relative to shallow layers (9).In addition to the presence of M. oxyfera-like bacteria, stable

isotope experiments confirmed the occurrence of n-damo in the

examined wetlands. In incubations amended with only 13CH4,it was observed that a very small amount of 13CO2 seemed to beaccumulated at a depth of 20–30 cm of the Xixi wetland (0–20 h)and the paddy field (0–10 h) (Fig. 4). This may indicate that thepreincubation did not remove all of the ambient nitrite at adepth of 20–30 cm of these two wetlands, which a relativelyhigher nitrite concentration than was observed in situ (Fig. S1).Therefore, it is likely that the residue nitrite after preincubationstimulates the production of 13CO2 in incubations amended withonly 13CH4 at a depth of 20–30 cm of the Xixi wetland and thepaddy filed. In addition, it was found that significant amountof 13CO2 accumulated at each analyzed depth (especially atdepths of 50–60 and 90–100 cm) for the majority of soil cores inincubations amended with 13CH4 and NO2

− compared with theincubations amended with only 13CH4 or

13CH4 and SO42−. This

Fig. 4. The formation of 13CO2 from 13CH4 in incubations of soils collected from different depths of the three wetlands.

4498 | www.pnas.org/cgi/doi/10.1073/pnas.1318393111 Hu et al.

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suggests that the oxidation of methane in the examined soil coresis driven by nitrite (not sulfate) under anoxic conditions, and then-damo is limited by nitrite under in situ conditions. Previously,direct evidence for the occurrence of n-damo in natural envi-ronments had been lacking. Recently, Deutzmann and Schink(10) reported the occurrence of n-damo in Lake Constance usingradiotracer experiments, with a potential activity of 1.8–3.6 nmolof CO2 per milliliter of sediment per day. However, the slurrieswere incubated for weeks to months, indicating that microbialgrowth should be taken into consideration. Besides, becausea high concentration of nitrate (2 mM) was added for deter-mining the potential n-damo activity in this reported study, theoccurrence of the recently reported AOM coupled to nitratereduction cannot be ruled out (19). In addition, Zhu et al. (9)investigated the potential importance of n-damo in a minero-trophic peatland. After 3 mo of incubation using 13CH4 andNO2

− amendments, the deep soil core layer (80–100 cm) clearlyshowed n-damo activity, producing 9.0 nmol of CO2 per gram ofsoil per day. However, methane oxidation was not detectedwithin the first 2 wk of incubation, indicating that the increase ofn-damo activity was attributable to the enrichment and growth ofM. oxyfera-like bacteria. In the present study, n-damo activity wasconfirmed in the majority of the examined soil core sampleswithin 20 h of incubation (Fig. 4). Higher potential n-damo rateswere observed in the deep layers of examined wetlands (Fig. 3B),where a high abundance of group A M. oxyfera-like bacteria wasobserved relative to the surface layer (Fig. 3A). A recent studyhas demonstrated that the addition of oxygen to M. oxyfera-likeenrichment cultures results in an instant decrease in the methaneand nitrite conversion rates (20). Therefore, the low abundanceof M. oxyfera-like bacteria and the low potential n-damo ratesobserved in the upper soil layer may be caused by the possiblepenetration of oxygen into this layer, which has a negative effecton the abundance and activity of these anaerobes.The incubation experiments that mimicked the in situ soil

nitrite and methane concentrations were conducted in the cur-rent study. The in situ concentrations of NO2

− were 0.12–2.18,0.15–1.72, and 0.16–1.12 mg·kg−1 N in soil cores collected fromthe Xiazhuhu wetland, the Xixi wetland, and the paddy field,respectively (Fig. S1). The final concentration of NO2

− in ourslurries was ∼0.67–2.16 mg·kg−1 N. This concentration was in thesame range to the in situ concentration of NO2

− measured in theexamined wetlands. On the other hand, the methane concen-tration in the headspace of our slurries was 6.5 × 104 mg·m−3.The in situ methane concentrations in soil gas ranged from 3.1 ×103 to 5.5 × 104, 8.9–3.2 × 104 and 8.7 × 102 to 1.0 × 105 mg·m−3

at different depths of soil cores collected from the Xiazhuhuwetland, the Xixi wetland, and the paddy field, respectively (Fig.S1). Thus, the methane concentration used in incubation experi-ments was similar to the in situ methane concentration measuredat different depths of soil cores. Coexistence of high concen-trations of soil nitrite (nitrate) and methane were observed in theexamined soil cores (especially at the upper 60-cm depth; Fig. S1).However, the redox potential (19.4–60.2 mV) of the examined soilcores indicated the anoxic conditions of the soil cores. Under theanoxic conditions, the soil nitrite (nitrate) could be reduced viavarious biological processes (such as denitrification and anaerobicammonium oxidation). Thus, the observed profiles of soil nitrite(nitrate) and methane in the current study may suggest a transientsituation of the examined soil cores. Furthermore, the soil am-monium was extracted from the soil using 2 M KCl, and this maylead to the oxidation of ammonium to nitrite (nitrate) after 1-hextraction. In addition, it was found that the potential n-damorates mainly peaked at a depth of 50–60 cm of the three wetlands.However, the in situ nitrite concentration at this layer (0.16–0.70mg·kg−1 N) was substantially lower than the nitrite concentrationin incubations (0.67–2.16 mg·kg−1 N). Therefore, the potential n-damo rates obtained from the incubation experiments may stilloverestimate the in situ n-damo rates.It is estimated that ∼50% of the methane produced in wet-

lands is consumed before it reaches the atmosphere, and this

important microbial methane sink is often exclusively attributedto aerobic methane oxidation (21, 22). In the present study, theoccurrence of n-damo was confirmed in three wetlands, therebyaltering our understanding of the mechanisms for reducingmethane emissions from wetlands. Considering that the nitriteconcentration at a depth of 20–30 cm (0.49–2.18 mg·kg−1 N) wassimilar to the nitrite concentration in incubations (0.67–2.16mg·kg−1 N), the average potential n-damo rate (0.33 nmol ofCO2 per gram of dry soil per day) obtained from depth of 20–30cm of the three wetlands was used to estimate the potentialimportance of n-damo process as a methane sink in wetlands.Based on the average n-damo rate (0.33 nmol CO2 per gram ofdry soil per day) and the average density of soil [2.65 g·cm−3

(23)], we estimate a total of 0.51 g·m−2·y−1 CH4 could be oxi-dized to CO2 via n-damo in wetlands. According to this rate,n-damo has the potential to consume ∼4.1–6.1 Tg of CH4 onaverage each year, assuming that the total area of the world’swetlands is 8–12 million square kilometers (24, 25). This isroughly 2–6% of total current CH4 flux estimates for wetlands[100–200 Tg·m−2·y−1 CH4 (26)]. However, the estimate of theenvironmental importance of n-damo process as a methane sinkin wetlands still appears to be associated with a very large un-certainty because this process is likely to be highly variable inspace and time owing to spatial and temporal heterogeneity insubstrate availability and redox potential in wetlands. Thus, morestudies that mimic the in situ conditions as closely as possible arerequired to determine the quantitative importance of n-damo asa methane sink in wetlands.Worldwide, anthropogenic nitrogen inputs (e.g., inorganic

nitrogenous fertilizer additions) are increasing rapidly in fresh-water habitats, and thus nitrite and nitrate are becoming themajor electron acceptors under anoxic conditions. Therefore,AOM coupled to nitrite reduction could be more likely to occurthan AOM coupled to sulfate reduction in freshwater habitats.Moreover, nitrogen input to marine ecosystems via river runoffhas been increasing, providing electron acceptors (nitrite/nitrate)for AOM other than sulfate which is thought to be the mostcommon electron acceptor for AOM in anoxic marine environ-ments (27, 28). Therefore, the n-damo process may be globallyimportant and has the potential to be an important methane sinkin natural ecosystems due to increasing nitrogen pollution.

ConclusionsIn this study, we provide previously unidentified direct evidencefor the n-damo process as a previously overlooked methane sinkin wetlands. Based on the measured potential n-damo rates, it isestimated that n-damo has the potential to consume 4.1–6.1 Tgof CH4 on average each year in wetlands under anaerobic con-ditions, which is roughly 2–6% of total current CH4 flux esti-mates for wetlands. Given the worldwide increase in nitrogenpollution, this methane sink may become more important inthe future.

Materials and MethodsSite Description and Sample Collection. The Xiazhuhu wetland is located inZhejiang Province and is the largest natural wetland in southeastern China,having a total area of 36.5 km2. The Xixi wetland, located in Hangzhou inZhejiang Province, is a rare urban wetland with a total area of 11.5 km2. It isthe first and only wetland in China combining urban life, farming, andculture. The paddy field selected for this study is also located in Hangzhouand represents a typical agricultural region of subtropical southeasternChina. In each wetland, at least five soil cores were collected in September2012. All soil cores were collected using a stainless steel ring sampler (5 cm indiameter and 100 cm in length).

Analytical Methods. The soil pH, temperature, and redox potential of theintact soil were measured in situ using an IQ150 pH meter (IQ ScientificInstruments). Ammonium, nitrite, and nitrate were extracted from the soilusing 2 M KCl as previously described (29). The soil organic carbon contentwas determined by the K2Cr2O7 oxidation method, and the total nitrogencontent was determined using the FOSS Kjeltec2300 analyzer (FOSS Group).Soil sulfate was extracted using calcium phosphate [Ca(H2PO4)2] and

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determined by barium sulfate (BaSO4) turbidimetric method at 420 nm (30).The below-ground gas samples were gathered through soil gas samplers.The polyvinyl chloride (PVC) tube connected with a rubber tube used as soilgas sampler (Fig. S3). The PVC tubes were placed horizontally at 10-cmintervals. The end of each PVC tube was covered with nylon stocking asseparator to prevent the soil from blocking the holes, and the end of eachrubber tube was sealed with rubber septa. The gas samples were collectedfrom each rubber tube through rubber septa using 100-mL polypropylenesyringes. The first 20 mL of collected gas from the tube was rejected, and theremaining gas was injected into polyethylene-coated aluminum bags forfurther methane concentration analyses; 50-μL gas samples were withdrawnfrom the polyethylene-coated aluminum bags with a gas-tight glass syringe(Agilent) and injected into an Agilent 6890N gas chromatograph (Agilent)equipped with a Porapak Q column and a flame ionization detector. Theoven temperature was set at 100 °C, and the injection and detector tem-peratures were both set at 130 °C.

Isotope Tracer Experiments. Soil samples of knownweight were transferred toHe-flushed, 75-mL glass vials together with He-purged deionized water. Thesoil slurries were preincubated under anaerobic conditions for at least 30 h toremove residual NOx

− and oxygen as closely as possible. The slurries weresubsequently split into three treatment groups receiving different amend-ments: 13CH4 (13C at 99.9%) (treatment 1); 13CH4 + NO2

− (treatment 2); and13CH4 + SO4

2− (treatment 3). The final concentrations of NO2− in treatment 2

and SO42− in treatment 3 were 0.67–2.16 mg·kg−1 N and 1.53–4.80 mg·kg−1 S,

respectively, by injecting 100 μL of He-purged stock solution through thesepta of each vial. Three independent experiments were performed for eachtreatment. Immediately after the preincubation step, 2 mL of headspace gasin each vial was removed and replaced with an equal volume of 13CH4, re-sulting in a final concentration of 6.5 × 104 mg·m−3 methane in the headspaceof each vial. The production of 13CO2 was measured directly from the head-space of each vial using a continuous flow isotope ratio mass spectrometer(Agilent 7890/5975C inert MSD; Agilent) as previously described (6). The po-tential rates of n-damo were calculated by the linear regression of the con-centration of produced 13CO2 in the headspace of the vial over time. The

coefficients of determination (R2) for linear regression of the 13CO2 concen-tration change over time were greater than 0.90 for most datasets.

DNA Extraction and PCR Amplification. Considering the importance of bi-ological replicates as reported by Prosser (31), at least two or three soil coresin each wetland were subject to molecular analyses in the current study. Itwas found that the community structures of M. oxyfera-like bacteria weresimilar at the same depth between different soil cores in each wetland. Thus,the molecular data of one representative soil core in each wetland waspresented. Soil DNA was extracted using a Power Soil DNA kit (Mo BioLaboratories) according to the manufacturer’s instructions. Extracted DNAwas then examined using 1.0% agarose gel electrophoresis. The 16S rRNAand pmoA genes of M. oxyfera-like bacteria were amplified using nestedPCR protocols as previously described (17, 32). Detailed information on theprimers used is provided in Table S2.

Cloning, Sequencing, and Phylogenetic Analysis. The PCR products werecloned using the pMD19-T vector (TaKaRa Bio) according to the manu-facturer’s instructions. Phylogenetic analysis of the sequences was con-ducted using Mega 5 software with the neighbor-joining method, andthe robustness of tree topology was tested by bootstrap analysis (1,000replicates).

qPCR. The copy numbers of the 16S rRNA genes ofM. oxyfera-like bacteria inthe collected samples were determined by qPCR, as previously described (6).The standard curve was constructed from a series of 10-fold dilutions ofa known copy number of plasmid DNA (Fig. S4).

Statistical Analyses. The OTU cutoff values of 3% and 7% were applied todetermine M. oxyfera-like 16S rRNA and pmoA genetic diversity, respectively,using the DOTUR program.

ACKNOWLEDGMENTS. This work was supported by Natural Science Foun-dation Grants 51108408 and 40081198 and the Shanghai Tongji Gao TingyaoEnvironmental Science and Technology Development Foundation.

1. Raghoebarsing AA, et al. (2006) A microbial consortium couples anaerobic methaneoxidation to denitrification. Nature 440(7086):918–921.

2. Ettwig KF, et al. (2010) Nitrite-driven anaerobic methane oxidation by oxygenicbacteria. Nature 464(7288):543–548.

3. Shen LD, et al. (2012) Microbiology, ecology, and application of the nitrite-dependentanaerobic methane oxidation process. Front Microbiol 3:269.

4. Knittel K, Boetius A (2009) Anaerobic oxidation of methane: Progress with an un-known process. Annu Rev Microbiol 63:311–334.

5. Denman KL, et al. (2007) Couplings between changes in the climate system andbiogeochemistry. Climate change 2007: The physical Science Basis. Contribution ofWorking Group I to the Fourth Assessment Report of the Intergovernmental Panel onClimate Change, eds Solomon S, et al. (Cambridge Univ Press, Cambridge, UK).

6. Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MS, Strous M (2009) Enrich-ment and molecular detection of denitrifying methanotrophic bacteria of the NC10phylum. Appl Environ Microbiol 75(11):3656–3662.

7. Hu S, et al. (2009) Enrichment of denitrifying anaerobic methane oxidizing micro-organisms. Environ Microbiol Rep 1(5):377–384.

8. Kampman C, et al. (2012) Enrichment of denitrifying methanotrophic bacteria forapplication after direct low-temperature anaerobic sewage treatment. J HazardMater 227-228:164–171.

9. Zhu BL, et al. (2012) Anaerobic oxidization of methane in a minerotrophic peatland:Enrichment of nitrite-dependent methane-oxidizing bacteria. Appl Environ Microbiol78(24):8657–8665.

10. Deutzmann JS, Schink B (2011) Anaerobic oxidation of methane in sediments of LakeConstance, an oligotrophic freshwater lake. Appl Environ Microbiol 77(13):4429–4436.

11. Kojima H, et al. (2012) Distribution of putative denitrifying methane oxidizing bac-teria in sediment of a freshwater lake, Lake Biwa. Syst Appl Microbiol 35(4):233–238.

12. Wang Y, et al. (2012) Co-occurrence and distribution of nitrite-dependent anaerobicammonium and methane-oxidizing bacteria in a paddy soil. FEMS Microbiol Lett336(2):79–88.

13. Shen LD, et al. (2014) Distribution and diversity of nitrite-dependent anaerobicmethane-oxidising bacteria in the sediments of the Qiantang River.Microb Ecol 67(2):341–349.

14. Shen LD, et al. (2014) Molecular evidence for nitrite-dependent anaerobic methane-oxidising bacteria in the Jiaojiang Estuary of the East Sea (China). Appl MicrobiolBiotechnol, 10.1007/s00253-014-5556-3.

15. Bastviken D, Tranvik LJ, Downing JA, Crill PM, Enrich-Prast A (2011) Freshwatermethane emissions offset the continental carbon sink. Science 331(6013):50.

16. Thauer RK, Shima S (2006) Biogeochemistry: Methane and microbes. Nature 440(7086):878–879.

17. Zhu G, Jetten MS, Kuschk P, Ettwig KF, Yin C (2010) Potential roles of anaerobicammonium and methane oxidation in the nitrogen cycle of wetland ecosystems. ApplMicrobiol Biotechnol 86(4):1043–1055.

18. Luesken FA, et al. (2011) Diversity and enrichment of nitrite-dependent anaerobicmethane oxidizing bacteria from wastewater sludge. Appl Microbiol Biotechnol92(4):845–854.

19. Haroon MF, et al. (2013) Anaerobic oxidation of methane coupled to nitrate re-duction in a novel archaeal lineage. Nature 500(7464):567–570.

20. Luesken FA, et al. (2012) Effect of oxygen on the anaerobic methanotroph ‘Candi-datus Methylomirabilis oxyfera’: Kinetic and transcriptional analysis. Environ Micro-biol 14(4):1024–1034.

21. Reeburgh WS (2007) Oceanic methane biogeochemistry. Chem Rev 107(2):486–513.22. Bodelier PLE (2011) Interactions between nitrogenous fertilizers and methane cycling

in wetland and upland soils. Curr Opin Environ Sustainability 3(5):379–388.23. Boyd CE (1995) Bottom Soils, Sediment, and Pond Aquaculture (Chapman & Hall, New

York), p 348.24. Ramsar Secretariat, Climate change and wetlands: Impacts, adaptation, and mitiga-

tion. Wetlands: Water, Life and Culture, Eighth Meeting of the Conference of theContracting Parties to the Convention on Wetlands (Ramsar, Iran, 1971), Valencia,Spain, November 18–26, 2002.

25. Lehner B, Döll P (2004) Development and validation of a global database of lakes,reservoirs and wetlands. J Hydrol (Amst) 296(1):1–22.

26. Dlugokencky EJ, Nisbet EG, Fisher R, Lowry D (2011) Global atmospheric methane:Budget, changes and dangers. Phil Trans R Soc A 369(1943):2058–2072.

27. Orphan VJ, House CH, Hinrichs KU, McKeegan KD, DeLong EF (2002) Multiple ar-chaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc NatlAcad Sci USA 99(11):7663–7668.

28. Holler T, et al. (2011) Carbon and sulfur back flux during anaerobic microbial oxi-dation of methane and coupled sulfate reduction. Proc Natl Acad Sci USA 108(52):E1484–E1490.

29. Shen LD, et al. (2013) Broad distribution of diverse anaerobic ammonium-oxidizingbacteria in chinese agricultural soils. Appl Environ Microbiol 79(19):6167–6172.

30. Chesnin L, Yien CH (1950) Turbidimetric determination of available sulfates. Soil SciSoc Am Proc 5:149–151.

31. Prosser JI (2010) Replicate or lie. Environ Microbiol 12(7):1806–1810.32. Luesken FA, et al. (2011) pmoA primers for detection of anaerobic methanotrophs.

Appl Environ Microbiol 77(11):3877–3880.

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