Gammaproteobacterial Methanotrophs Dominate Cold Methane Seepsin Floodplains of West Siberian Rivers

Igor Y. Oshkin,a Carl-Eric Wegner,b Claudia Lüke,c Mikhail V. Glagolev,d,e,f,g Illiya V. Filippov,f Nikolay V. Pimenov,a Werner Liesack,b,h

Svetlana N. Dedysha

S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russiaa; Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germanyb;Radboud University Nijmegen, Nijmegen, The Netherlandsc; Moscow State University, Moscow, Russiad; Laboratory of Computational Geophysics, Tomsk State University,Tomsk, Russiae; Yugra State University, Khanty-Mansiysk, Russiaf; Institute of Forest Science, Russian Academy of Sciences, Uspenskoe, Russiag; Center for SyntheticMicrobiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, Germanyh

A complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the riverMukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4 flux from most (90%) of these gas ebullition sitesdid not exceed 1.45 g CH4 h�1, while some seeps emitted up to 5.54 g CH4 h�1. The �13C value of methane released from theseseeps varied between �71.1 and �71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situtemperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4 ml�1 day�1) were measured in mudsamples. Fluorescence in situ hybridization detected 107 methanotrophic bacteria (MB) per g of mud (dry weight), which ac-counted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacte-rial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding partic-ulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved andanalyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosomagroup, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuatingmethane fluxes from these local but strong CH4 sources in floodplains of high-latitude rivers have a large proportion of poten-tially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.

Methane (CH4) is an important greenhouse gas responsiblefor about 20% of the warming induced by long-lived green-

house gases (1, 2). Since the beginning of the Industrial Era, theatmospheric CH4 concentration has increased by a factor of 2.5(2). A sustained increase in atmospheric CH4 levels in the 1980swas followed by a slowdown in the increase in the 1990s and ageneral stabilization from 1999 to 2006. Since 2007, CH4 levelshave been rising again (3), indicating a recent imbalance betweenCH4 sources and sinks that is not yet fully understood (1, 4). Theglobal methane emission is currently estimated between 550 and678 Tg year�1 (1, 2). Natural wetlands are the largest source ofatmospheric methane, contributing 175 to 217 Tg CH4 year�1 (1,2). The second-largest natural source is geologic methane, whichis emitted from coal beds, natural gas deposits, and gas hydrates.Recent estimates appraise the total methane emitted naturallyfrom all geologic sources at more than 50 Tg year�1 and poten-tially approaching 80 Tg year�1 (5). The emission of geologic CH4

occurs from seepage, including microseeps, terrestrial macro-seeps, geothermal/volcanic emissions from the Earth’s crust, andmarine seeps. Microseepage is the slow, diffuse loss of CH4 overwide areas on the order of 100 to 102 mg m2 day�1. Macroseeps arelocalized flows and gas vents, both on land and on the seafloor,which emit up to 102 tons CH4 year�1, while mud volcanoes rep-resent the largest visible expression of geologic methane emission,producing flows of up to 103 tons CH4 year�1 (6, 7, 8). Majoremissions are related to natural fossil methane formed in sedi-mentary basins (both microbial and thermogenic methane) and,subordinately, to geothermal areas (5).

The global seepage area and the number of seep sites on differ-ent continents remain uncertain. Thus far, mud volcanoes andmicroseepage in low-latitude countries, such as Italy, Greece, Ro-

mania, Azerbaijan, and Taiwan, have received the most researchattention (7, 9, 10). Recently, however, 77 previously undocu-mented fossil seep sites, comprising more than 150,000 vents tothe atmosphere, were mapped in Alaska (11). These seeps werecharacterized by anomalously high fluxes of methane (up to 8 tonsCH4 site�1 day�1) and were located in regions of permafrost thawand receding glaciers. Gases emanating from these seeps weremainly thermogenic in origin. In Greenland, Walter Anthony andcolleagues (11) documented a number of seep sites associated withyounger methane, originating from the decomposition of organicmatter and located in zones of ice sheet retreat. These findingsindicate that geologically sourced methane emissions may wellincrease as ice sheets, glaciers, and permafrost melt (12, 13).

Our knowledge of the microbiology of terrestrial methanevents is by far less advanced than that of marine methane seeps.Most of the currently available information was obtained for mudvolcanoes in countries with warm climates (14–16). The only ex-ception is a study on microbial community composition in a hy-persaline methane seep in the Canadian High Arctic (17). Themolecular analysis revealed the presence of the anaerobic methane

Received 10 May 2014 Accepted 10 July 2014

Published ahead of print 25 July 2014

Editor: J. E. Kostka

Address correspondence to Svetlana N. Dedysh, dedysh@mail.ru.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01539-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01539-14

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group 1a (ANME-1a) clade of anaerobic methane-oxidizing ar-chaea, while bacterial methane oxidizers were absent from thisextreme (�24% salinity) habitat. Methanotrophs that attenuatemethane fluxes from terrestrial methane vents require special at-tention. Although these microorganisms are unlikely to signifi-cantly affect CH4 fluxes from macroseeps, their role in reducingmethane emissions from microseepage and small vents is morefeasible.

Numerous small gas vents (0.5 to 5 cm in diameter) and natu-ral structures similar to mud microvolcanoes (10 to 100 cm indiameter) were found during our field studies in floodplains ofsmall rivers near Khanty-Mansiysk, West Siberia, Russia. This re-gion in the subzone of Middle Taiga is relatively well characterizedwith regard to methane emissions from natural wetlands and an-thropogenic sources related to gas field explorations (18–21).Mud microvolcanoes, however, are still missing on the list of CH4

sources studied in these cold regions. In addition, our knowledgeof methanotrophic bacteria (MB) that thrive in low-temperatureenvironments remains limited. At present, only two representa-tives of obligately psychrophilic aerobic methanotrophs areknown. These are the Antarctic methanotroph Methylosphaerahansonii (22) and the tundra methanotroph Methylobacter psy-chrophilus (23), both of which belong to type I, or gammapro-teobacterial, methanotrophs. Several psychrotolerant metha-notrophs have also been described. These include both type IMB (Methylobacter tundripaludum, Methylomonas scandi-navica) and type II, or alphaproteobacterial, MB (Methylocystisrosea, Methylocella tundrae), but these organisms have growthoptima at 15 to 20°C (24).

Our study, therefore, was initiated in order to map and toassess methane fluxes from mud microvolcanoes in floodplains ofhigh-latitude rivers. In particular, we aimed to get insight into theaerobic methanotrophic community that develops around thispreviously unrecognized natural CH4 source and mitigates meth-ane emission to the atmosphere.

MATERIALS AND METHODSStudy site and methane flux measurements. Our studies were performedin the Mukhrinskaya River Valley (60°53=N, 68°42=E), Khanty-Mansi Au-tonomous Okrug, West Siberia, Russia, where the mean annual temper-ature is �1.1°C and the mean annual precipitation is 596 mm. Othercharacteristics of the study site have been described previously (25–27).This is one of the poorly accessible and virtually unpopulated taiga re-gions. Due to the high water level in the Irtysh River in spring and earlysummer, this area is largely flooded and represents a complex network offloodplains and forested wetlands. In early August, however, the waterlevel begins to decline, and numerous gas ebullition sites become clearlyvisible in the floodplain as well as in the riverbed (see Fig. S1 in the sup-plemental material). In 2012, this time period was used to perform meth-ane flux measurements at 28 bubbling pools, including both larger andsmaller seeps (Fig. 1). Output of the bubbling gases was measured bymeans of a funnel connected to a graduated cylinder filled with water andby counting the time required to displace water out. This method is widelyused for assessing methane emissions by bubbling (28–30). The methaneconcentration in a gas released from seeps was measured using a Crystal5000 gas chromatograph (Chromatek, Russia) equipped with a flame ion-ization detector (detector, 250°C, 3-m glass column filled with HayeSep N[Supelco, USA]; column temperature, 40°C). The stable isotopic compo-sition of methane carbon (�13C) was determined on a gas chromatograph(Thermo Electron, Karlsruhe, Germany), coupled with a Delta Plus massspectrometer, by following the procedure described by Ivanov et al. (31).

The isotopic analyses were carried out in triplicate; measurement errorwas �0.1‰.

Water sampling and analysis. We sampled surface water (0 to 5 cm)from 5 bubbling pools, which were located at a distance of 100 to 300 mapart from each other. Sampling glass containers were rinsed with distilledwater and then with the water from bubbling pools before sampling. Thetemperatures of muddy fluids were measured using temperature sensors(Termochron iButton DS 1921 and 1922 [Dallas Semiconductor, USA]).pH was determined using an SG-8 pH meter (Mettler Toledo, Switzer-land); measurement error was �0.002. Water conductivity was deter-mined using an SG-7 electrical conductivity (EC) meter (Mettler Toledo,Switzerland); measurement error was �0.5%. Concentrations of Li�,Na�, NH4

�, K�, Mg2�, Mn2�, Ca2�, F�, Cl�, NO2�, NO3

�, SO42�, and

Br� in upwelling water were determined by means of ion chromatogra-phy (Metrohn, Switzerland). Concentrations of CO3

2� and HCO3� were

determined by titration with 0.05 M HCl.Potential methane oxidation. For radioisotope and molecular analy-

ses, the samples of muddy fluids were collected from the surfaces of twobubbling pools located at a distance of 300 m from each other (indicatedby black stars in Fig. 1). Methane concentration in the samples was deter-mined by the headspace method (32). Methane oxidation rates were de-termined by the radioisotope method with [14C]methane dissolved indegassed distilled water. Mud samples collected from the methane seepwere subsampled (3 ml) by means of 5-ml-cutoff plastic syringes; the openend was then sealed with a gas-tight butyl rubber stopper. Labeled sub-strates (0.2 ml of [14C]methane solution, 1 �Ci per sample) were injectedthrough the stopper. The samples were incubated in the dark at the in situtemperature (4°C) for 24 h, fixed with 0.5 ml 2 M KOH, and transportedto an isotope facility. For the analysis, the samples were adjusted to pH 2with HCl, and released CO2 was trapped in a scintillation liquid contain-ing 2-phenylethylamine. Organic matter was also completely oxidized bythe persulfate method (33), and released CO2 was trapped again. BothCO2 portions trapped in the scintillation liquid were combined, and ra-dioactivity was measured on a RackBeta scintillation counter (LKB, Swe-den). The rates of methane oxidation were calculated using the equation I(rate) � r C/R T, where r is the radioactivity of the formed product,C is dissolved methane concentration in the sample, R is the total radio-activity of the added 14C-CH4, and T is the incubation time. Samples fixedwith KOH and stored for 2 h prior to the injection of labeled substrateswere used as controls.

FISH. The fixation procedure was performed at the sampling site byadding ethanol (1:1, vol/vol) to the mud samples. The samples were thendelivered to the laboratory and stored at �20°C prior to the analysis. Inorder to overcome the problem of high autofluorescence of clay particles,the protocol developed for fluorescence in situ hybridization (FISH) insoil (34) was employed. Briefly, 7 mg of polyvinylpolypyrrolidone (PVPP)was added to each sample, which consisted of 1.5 ml of a fixed mudsuspension collected in a 2-ml Eppendorf tube. Following the addition of5 mM Na2-EDTA, the tubes were shaken horizontally at 250 rpm for 20min, after which large soil particles were allowed to settle for 5 min. Sub-sequently, 1 ml of the supernatant was transferred to new 2-ml Eppendorftubes on top of a 1-ml Nycodenz cushion (1.3 g ml�1 in sterile water)(Gentaur, Belgium). The samples were centrifuged at 16,438 g for 30min at 18°C, and 1.8 ml was collected from the supernatant in a new tube.Samples were vortexed and immobilized on black membrane filters (poresize, 0.2 �m; diameter, 47 mm; Millipore, Germany) and washed with 4ml PBS (in g/liter, NaCl, 8.0; KCl, 0.2; Na2HPO4, 1.44; NaH2PO4, 0.2; pH7.0). To prevent pressure damage, black filters were placed onto nitrocel-lulose membrane filters (pore size, 0.45 �m; diameter, 47 mm; Millipore,Germany). These were further transferred into dishes containing absor-bent paper soaked with ethanol and successively dehydrated in 50%, 80%,and 96% ethanol for 6 min each time. Filters were then air dried, cut intopieces, and placed on gelatin-coated (0.1%, wt/vol) and dried Teflon-laminated slides with eight wells for independent positioning of the sam-ples (Magv, Germany). FISH was carried out using the Cy3-labeled oligo-

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nucleotide probes M84/M705 and M450. These have reported groupspecificities for type I and type II MB, respectively (35). Enumeration ofMethylocella-like methanotrophs that are not targeted by the above-men-tioned assays was carried out using the combination of probes Mcell-1026, Mcellt-143, and Mcells-1024 (36). In addition, the Bacteria-specificprobe EUB338-mix was applied as described by Dedysh et al. (37). Fol-lowing hybridization, the slides were stained with the universal DNA stain4=,6-diamidino-2-phenylindole (DAPI; 1 �M) for 7 min in the dark,rinsed again with distilled water, and finally air dried. Cell counting wascarried out with a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany)equipped with Zeiss filter 20 for Cy3-labeled probes and Zeiss filter 02 forDAPI staining.

PmoA pyrosequencing. Six subsamples of mud material (3 samplesfrom each of the two seeps studied), each of 0.5 g (wet weight), were takenfor DNA extraction and processed separately. The extraction was per-formed using a FastDNA Spin kit for soil (Bio101, Carlsbad, CA, USA)according to the manufacturer’s instructions. pmoA gene amplicons weregenerated using the two primer sets A189r/A682r (38) and A189f/mb661r(39). The A189f primer included a 454-A pyrosequencing adaptor and asample-specific 6-bp barcode sequence, while both reverse primers incor-porated 454-B pyrosequencing adaptors. PCR amplifications were per-formed in a thermal cycler (model Mastercycler gradient; Eppendorf,Hamburg, Germany) under the following conditions: an initial denatur-ation (5 min at 96°C) and 30 cycles consisting of denaturation (30 s at

94°C), primer annealing (1 min at 56°C), and elongation (1 min at 72°C),with a final elongation step for 5 min at 72°C. pmoA amplicons weregenerated in triplicate from each DNA extract, pooled in equal amounts,and purified using Wizard SV gel and a PCR clean-up system (Promega,Madison, WI, USA). Quantification of the PCR products was performedusing a Quant-iT double-stranded DNA (dsDNA) BR assay kit and Qubitfluorometer (Invitrogen GmbH, Karlsruhe, Germany). The purifiedproducts were subjected to pyrosequencing at the Max Planck GenomeCentre, Cologne, Germany.

Since the primer sets A189r/A682r and A189f/mb661r do not detectmethanotrophs from the phylum Verrucomicrobia (40), an additionalPCR assay was performed in order to assess the presence of these bacteriain our samples using the “Methylacidiphilum”-specific pmoA primersV170f/V631b designed by Sharp et al. (41).

Bioinformatic analyses. Raw sequence data were preprocessed priorto downstream analysis using PRINSEQ (lite version 0.20.3) (42). Thesettings were as follows: a minimum sequence length of 200 bp, a mini-mum mean quality score of 20, a maximum allowed rate of N=s of 1%,removal of characters other than ATGC, ends trimmed (from the 3= end)by a quality score of less than 10, and a low-complexity threshold (entropy7). Quality-controlled sequence data were analyzed using QIIME. Oper-ational taxonomic units (OTUs) were picked using a sequence similaritythreshold of 87%, which was found to be roughly equivalent to a 16SrRNA gene similarity of 97% (43). Using the default naive Bayesian clas-

FIG 1 Methane fluxes and seep distribution in the Mukhrinskaya River Valley. Circles represent the relative magnitudes of individual fluxes from single seeps(g CH4 h�1) as follows: red, 0.9 to 5.5; yellow, 0.4 to 0.5; and green, 0.05 to 0.16. Each white circle represents a single seep, while the sites with numerous seepsare indicated by triangles; a big triangle represents 1,800 to 5,000 seeps, a medium triangle represents 150 to 500 seeps, and a small triangle represents 40 to 100seeps. The two bubbling pools where the samples of muddy fluids were collected for 14CH4 oxidation rate measurements and molecular analyses are indicated byblack stars. Scale bar, 800 m. (Inset) Location of the study site (black circle) on a map of West Siberia (adapted from 2GIS-City Expert [www.2gis.ru]). The basemap is adapted from Bing. Microsoft product screen shot reprinted with permission from Microsoft Corporation.

Oshkin et al.

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sifier (44) of mothur (45) implemented in QIIME and a customized pmoAdatabase (provided in the supplementary material of Deng et al. [46]),taxonomic assignments were done based on representative sequences ofthe individual OTUs. Alpha diversity and evenness were assessed by cal-culating Chao1 (47), Shannon (48), and Simpson (49) metrics. Sequencesrelated to amoA were extracted and further analyzed by OTU clusteringusing UPARSE (50) and USEARCH (51) and a threshold of divergence of0.05. Representative sequences from OTUs were randomly sampled andtaxonomically assigned by BLASTX (52) searches against sequences in theNCBI nr database (53). Hits with more than 95% amino acid sequenceidentity over the complete read length to AmoA sequences of culturedrepresentatives were considered to be valid taxonomic assignments.

For further quality check, alignment, and phylogenetic tree construc-tion, the sequences were imported into the ARB software package (54).Sequences containing errors resulting in reading frame shifts and se-quences shorter than 139 amino acids were manually excluded from fur-ther analysis. For a first overview, sequences were added to an existingneighbor-joining tree of 5,010 published pmoA and amoA sequences. Asubset of 184 sequences covering the diversity in the study sites was se-lected for thorough tree construction, together with the reference data setof 5,010 published pmoA/amoA sequences. This neighbor-joining tree wasconstructed based on 126 amino acid positions using the Kimura correc-tion.

Nucleotide sequence accession numbers. The pyrosequencing reads(raw data) have been deposited under the study number SRP037754 inthe NCBI Sequence Read Archive with the following accession num-bers: SRR1168458 and SRR1169858.

RESULTSDistribution of seeps and methane emission rates. Numerousgas ebullition sites represented by either bubbling water pools orcone-shaped, muddy-fluid-discharging craters exposed to thesurface were revealed in a valley of the river Mukhrinskaya (seeFig. S1 in the supplemental material). Most (99%) of these ventsare relatively small (0.5 to 5 cm in diameter), while some of thesestructures achieve the size of 1 m. Overall, we were able to mapover 25,000 seeps, with some of them occurring as single gas ventsand others as groups composed of up to several hundred vents(Fig. 1).

Upwelling water and muddy fluids were characterized by near-neutral pH values of 6.8 to 6.9 and conductivity values of 543 to627 �S cm�1 and had relatively high contents of Ca2�, Mg2�, andCO3

2�/HCO3� ions (Table 1). Water temperature ranged be-

tween 3.5 and 5°C. The gases released from these bubbling poolsconsisted primarily of methane (70 to 99%), with �13C-CH4 val-ues of �71.1 to �71.3 ‰, which are indicative of biogenic meth-ane. CH4 emission was measured for 28 randomly chosen seeps(Fig. 1). While methane flux from most (90%) of these sites didnot exceed 1.45 g CH4 h�1, some of the seeps emitted up to 5.54 gCH4 h�1 (see Fig. S2 in the supplemental material).

Potential methane oxidation. The samples of muddy fluidscollected from the two bubbling pools (mapped by black stars inFig. 1) were used for measuring in situ methane concentrationsand potential methane oxidation rates. Methane concentration inmuddy fluids was in the range 116 to 233 nmol CH4 ml�1. Poten-tial methane oxidation rates determined at the in situ temperatureof 4°C were highly similar for the two sets of samples and rangedbetween 15.5 and 15.9 nmol CH4 ml�1 day�1.

FISH-based analysis. To determine the in situ abundance ofmethanotrophic bacteria, the probes M84/M705 and M450 withreported group specificity for type I and type II MB, respectively,were applied to mud slurries. This direct approach, however,

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turned out to be inefficient because we faced the problem of thehigh autofluorescence of soil particles masking the signal of theprobes. To overcome this problem, we tested an earlier developedprotocol for FISH in soil. It is based on separating microorganismsfrom soil particles by buoyant density centrifugation on Nycodenzand concentrating cells on membranes (34). This protocol workedwell and allowed us to visualize numerous cells of various sizes andshapes that occurred singly or formed long cell chains or aggre-gates (Fig. 2).

In situ hybridization with probes M84 and M705 revealed thattype I methanotrophs were present at a high abundance in the twoseeps examined in our study. The number of cells targeted by theseprobes ranged between 2.74 107 and 4.29 107 per g of mud(dry weight) (Table 2). In contrast, only a very few randomlyoccurring cells were detected with the probe M450, which targetsthe Methylosinus/Methylocystis group of alphaproteobacterial typeII MB. The number of cells revealed by this probe was only 2.84 105 to 11.70 105 cells per g of mud (dry weight). The attempt todetect Methylocella-like methanotrophs by using the probesMcell-1026, Mcellt-143, and Mcells-1024 was unsuccessful. Theabundance of these bacteria in mud slurries was below the detec-tion limit (103 cells per g of dry mud). Therefore, 95.8 to 99.3% ofall aerobic methanotrophs detected in mud by FISH were repre-sented by type I MB, while type II MB were numerically insignif-icant.

As determined by FISH, the total number of aerobic metha-

notrophs in mud samples taken from the seeps was in the range of2.86 107 to 4.32 107 cells per g of mud (dry weight), whichaccounted for 20.2 to 20.5% of all bacterial cells detected byEUB338-mix or 10.9 to 12.2% of all DAPI-stained cells (Table 2).The high abundance and unusually high proportion of metha-notrophs among total bacteria indicate that methane is one of themajor substrates that shape the microbial community structure inthese unique habitats.

Methanotrophic community analysis via pmoA pyrotag se-quencing. To obtain a more detailed view of methanotroph diver-sity in cold Siberian seeps, we applied amplicon pyrosequencing ofpmoA genes. These encode the -subunit of particulate methanemonooxygenase (pMMO), the key enzyme of nearly all extantmethanotrophs. Since Methylocella-like organisms, which lackthis enzyme, were virtually absent from this habitat, a pmoA-basedapproach had a good potential to identify all numerically impor-tant methanotroph populations. A total of 53,828 quality-checked, partial (average length, �400 bp) pmoA gene sequenceswere retrieved from the mud samples. The read numbers obtainedfor each seep were nearly equal (27,398 and 26,430 reads for seeps1 and 2, respectively).

The two reverse pmoA-specific primers used in our study, i.e.,A682r and mb661r, are known to display some differences in theirtarget specificities. This was reflected in the diversity patterns gen-erated with each of the two primer sets (Fig. 3). The overall diver-sity recovered with A189f/A682r was higher than that revealed byusing A189f/mb661r (Chao1 indices of 4,602 and 1,523, respec-tively). This is due to the fact that primer A682r has a broaderdetection range and targets not only pmoA but also the phyloge-netically related amoA gene (38). The proportions of amoA se-quences among all 454 reads recovered with A189f/A682r fromseeps 1 and 2 were 27.3 and 24.9%, respectively. These amoA-related reads were removed from further methanotroph analysis,but the information regarding their phylogenetic affiliation islisted in Table S1 in the supplemental material. Other majorgroups of reads retrieved from the seeps with primer set A189f/A682r were Methylobacter-related pmoA sequences (16.4 to 25.6%of total reads), type Ib methanotroph sequences affiliated withlake cluster 2 (7.7 to 16.0%), and pmoA-like sequences from sev-eral as-yet-uncultured bacterial groups (24.1 to 30.4%), includingthose from Crenothrix-like organisms (Fig. 3). The pmoA librariesobtained with primer set A189f/mb661r were dominated byMethylobacter-related sequences (64.8 to 69.5%) and pmoA se-quences from other type Ia methanotrophs, such as Methylomo-nas, Methylovulum, and Methylosoma (19.3 to 22.4%). Commonto both primer sets, the proportion of pmoA sequences from typeII MB did not exceed 5.5% of total reads, and most of them were

FIG 2 Specific detection of type I aerobic methanotrophs in seep mud byFISH. Epifluorescence micrographs of in situ hybridizations with Cy3-labeledprobes M84 and M705 (A) and DAPI staining (B). Panels 1 and 2 display twodifferent microscopic fields of view. Bars, 5 �m.

TABLE 2 Numbers of cells detected by FISH with methanotroph-specific probes in muddy fluids sampled from the seeps

Seep

No. of cells detected with the following oligonucleotide probe(s)(g�1 of dry mud):

No. of DAPI-stained cells(108 g�1 of dry mud)

% methanotrophs of DAPI-stainedcells/% methanotrophs ofEUB338-mix-hybridized cellsM84/M705a (107) M450b (105) EUB338-mix (108)

1 4.29 � 0.10 2.84 � 1.92 2.1 � 1.4 3.98 � 0.57 10.9/20.22 2.74 � 0.18 11.70 � 11.1 1.4 � 3.4 2.33 � 0.18 12.2/20.5a The target specificity of probe M84 includes members of the genera Methylobacter, Methylomonas, Methylococcus, Methylomicrobium, Methylosarcina, and Crenothrix polyspora.Probe M705 targets Methylobacter, Methylomonas, Methylococcus, Methylosarcina, Methylosphaera, and Methylovulum. These probes do not target members of the familyMethylothermaceae (i.e., thermophilic and halophilic methanotrophs of the genera Methylothermus, Methylohalobius, and Methylomarinovum).b Probe M450 targets all currently described members of the genera Methylosinus and Methylocystis.

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affiliated with the genus Methylocystis. pmoA sequences assignedto Methylosinus trichosporium- and Methylocapsa-like MB and tocluster MO3 were detected at lower levels. Only a very few readsrepresented pmoA2 sequences, which were most similar to thoseof Methylocystis bryophila.

Because the pmoA diversity patterns recovered from seeps 1and 2 were similar for each of the two PCR assays, the 454 readsobtained with either A189f/A682r or A189f/mb661r were pooledfor further analysis. In total, 1,850 and 771 species-level OTUs(defined at a nucleotide sequence identity of 87%) were detectedin the seeps with primer sets A189f/A682r and A189f/mb661r,respectively. The most abundant OTUs detected with both prim-ers sets affiliated with the genus Methylobacter, displaying 87 to96% nucleotide sequence identity to pmoA sequences from M.psychrophilus and M. tundripaludum. Several abundant OTUsshowed equal levels of sequence divergence (15 to 18%) frompmoA gene sequences of known representatives of the generaMethylobacter, Methylovulum, and Methylosoma. One of the mostabundant OTUs represented lake cluster 2, displaying 83 to 84%sequence identity to pmoA gene fragments from members of thegenera Methylococcus and Methylocaldum. Abundant OTUs de-tected exclusively with primer set A189f/A682r included Creno-thrix-like and LW (Lake Washington) cluster pmoA sequences.Abundant OTUs detected exclusively with primer set A189f/

mb661r were represented by Methylovulum- and Methylomonas-like pmoA sequences and the sequences from deep-sea cluster 1.Among type II MB, sequences of the most abundant OTU dis-played 97 to 99% identity to pmoA gene fragments from Methylo-cystis hirsuta and Methylocystis rosea.

Sequence reads were manually checked for insertions or dele-tions (indels), in addition to their classification based on k-mers.Good-quality sequences without indels were used for phyloge-netic tree construction. The overall pattern of the methanotrophdiversity detected in the seeps is depicted in Fig. 4.

The tests for the presence of Verrucomicrobia-like metha-notrophs in cold Siberian seeps gave negative results, which ap-pears logical, since these bacteria have so far been detected only ingeothermal acidic environments.

DISCUSSION

The type of methane-emitting vents reported here has not beenstudied before. The bubbling pools in a floodplain of the riverMukhrinskaya cannot be referred to as a microseepage, given thata microseepage does not form visible manifestations and is char-acterized by much lower emission rates (10 mg m�2 day�1 onaverage and 1 g m�2 day�1 at a maximum) (6, 7). They also do notfulfill the criteria used to define macroseeps, including mud vol-canoes, which emit between several hundred kilograms and sev-

FIG 3 Relative abundances of different methanotroph groups in methane seeps based on pyrosequencing of pmoA gene fragments. AOB, ammonia-oxidizingbacteria.

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eral thousand tons of methane per year. The average vent of thosemapped in our study emits about 16 g CH4 per day, but thesestructures seem to be widely distributed in a valley of the riverMukhrinskaya. Most of these vents are difficult or even impossibleto map during “high-water” seasons. Although our field studieswere performed after the water level dropped down, the real num-ber of gas vents in the riverbed may have remained underesti-mated. Further research will show whether these methane ventsare characteristic of other rivers in poorly accessible regions of theWest Siberian taiga.

Methane emanating from these seeps was biogenic (or micro-bial) in origin. This is not very common for CH4 emitted fromgeologic sources and suggests that Siberian seeps release methanethat originated from decomposition of organic matter in sedi-mentary basins which are located in zones of permafrost retreat.

The upwelling water had relatively high contents of Ca2�,Mg2�, and CO3

2�/HCO3� ions but low or undetectable concen-

trations of NO2�, NO3

�, and SO42�. This water composition ex-

cludes the occurrence of NO2�/NO3

�- or SO42�-dependent an-

aerobic methane oxidation (55, 56) and suggests the leading roleof aerobic processes in attenuating methane emissions from theseeps. Indeed, the bubbling water pools examined in our studywere abundantly colonized by aerobic methanotrophic bacteriathat actively oxidized CH4 released from the subsurface. This wasevidenced by relatively high rates of methane oxidation measuredin muddy fluids at the in situ temperature of 4°C and also by thehigh number of methanotroph cells detected by FISH. These ac-counted for up to 20.5% of total bacterial cell counts. The probe-conferred fluorescence of the cells targeted with methanotroph-specific probes was very bright (Fig. 2), suggesting high ribosomecontents in these bacteria. According to the results of our FISHanalysis, muddy fluids collected from the seeps were dominated bytype I MB, which accounted for 96 to 99% of all methanotrophcells detected by the probes. These estimations were confirmed bythe pyrosequencing-based analysis of pmoA gene amplicons, be-cause only 0.5 to 5% of total pmoA sequences retrieved from thesamples affiliated with type II MB. The predominance of type Iover type II MB has rarely been observed and occurs mainly incold freshwater ecosystems (57–59) and in permafrost environ-ments (60, 61). Generally, type I MB are very responsive to sub-strate availability and are believed to be indicative of environ-ments with a high methane source strength (62, 63). This is exactlythe situation in Siberian river seeps.

A large proportion of pmoA sequences retrieved in our studybelonged to type Ia methanotrophs of the genera Methylobacter,Methylovulum, and Methylosoma. These sequences formed a phy-logenetic continuum with poorly defined cutoffs between the ge-nus-level groups. The psychrophilic nature of Methylobacter-likemethanotrophs is well documented. Two described species of thisgenus, Methylobacter psychrophilus (23) and Methylobacter tundi-paludum (64), were isolated from permanently cold, arctic tundra

environments. Using stable isotope probing, Methylobacter spe-cies were identified as a major group of metabolically activemethanotrophs in arctic wetlands (65) and arctic lakes (58). No-tably, members of the genera Methylovulum and Methylosomawere also detected among the active methanotrophs in arctic lakesediments (58), a habitat similar to that of cold river seeps asdescribed here. Apparently, these methanotroph genera accom-modate a number of as-yet-undescribed psychrotolerant species.The only currently described species of the genus Methylosoma,Methylosoma difficile, was isolated from littoral sediment of LakeConstance and does not grow below 10°C (66). Methylovulummiyakonense, a methanotroph from a forest soil, does grow at 5°C,but its growth optimum is at 24 to 32°C (67). None of the pmoAsequences retrieved from the cold Siberian seeps could be unam-biguously assigned to either Methylosoma difficile or Methylovu-lum miyakonense. Apparently, they represent novel species withinthese genera.

Type Ib MB were also detected in our study sites. None of these,however, belonged to the currently recognized genera, i.e., Methy-lococcus, Methylocaldum, and Methylogaea. This is not surprisinggiven that all characterized members of these genera are mesoph-ilic or moderately thermophilic bacteria that do not develop at lowtemperatures. The pmoA sequences retrieved in our study affili-ated with two major environmental clades, freshwater lineage 1and freshwater lineage 2 (Fig. 4), which are not represented bycultivated methanotrophs. Freshwater lineage 1 can be separatedinto smaller clusters, such as RPC-1 (rice paddy cluster 1), LWcluster, and OSC (organic soil cluster) (68). Only pmoA sequencesbelonging to the LW cluster were detected in our study. SimilarpmoA sequences are often retrieved from lake sediments but canalso be detected in paddy fields (57, 69, 70). Freshwater lineage 2includes lake cluster 2, FW cluster (freshwater cluster), and anumber of RPCs (rice paddy clusters) (68). A large proportion ofpmoA sequences from seeps 1 and 2 belonged to lake cluster 2,which is composed of sequences retrieved mostly from freshwaterlake sediments (71, 72).

The fact that pmoA sequences from type II MB representedonly a minor proportion of our data sets suggests that these bac-teria play only a secondary role in the oxidation of methane inSiberian microseeps. Although several Methylocystis species, in-cluding M. rosea, M. bryophila, and M. heyeri, were isolated fromcold environments, all currently described members of this genusdisplay a preference for relatively high growth temperatures of 25to 30°C (73). Only a very few pmoA2 sequences from Methylocystisspp. were detected in our study, which appears logical given thehigh CH4 availability in this particular habitat.

Approximately 100 reads displayed 95 to 96% sequence iden-tity to the pmoA gene from Crenothrix polyspora (74). This bacte-rium is commonly found in stagnant water environments and lakesediments. Some filamentous cell forms observed in our FISHexperiments could potentially belong to these methanotrophs

FIG 4 Neighbor-joining tree depicting major pmoA/amoA lineages consistent with those of Lüke and Frenzel (68). Clusters containing sequences fromseep-inhabiting bacteria are indicated by dark gray. Lineages lacking isolates are named according to representative clones or to the environment in which theywere predominantly or initially found (RPC, rice paddy cluster; JRC, Japanese rice cluster; OSC, organic soil cluster; LW, Lake Washington cluster; FW,freshwater cluster; TUSC, tropical upland soil cluster; USC, upland soil cluster; JR, Jasper Ridge, CA; MOB, methane-oxidizing bacteria; AOB, ammonia-oxidizing bacteria). GenBank accession numbers of representative clones are given in parentheses. The RPC-2 and deep-sea 4 lineages cluster within either typeIa or type Ib MB, depending on the subset of sequences or the method used for tree construction. Colored squares next to pmoA lineages depict the number ofsequences retrieved with the A682r reverse primer (dark blue) or the mb661 reverse primer (light blue). The scale bar represents 0.1 change per amino acidposition.

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since they are targeted by the M84 probe. Furthermore, sequencesfrom two new lineages were retrieved (new-1 and new-2 in Fig. 4).Remarkably, sequences related to the alkane monooxygenases ofNocardioides and Mycobacterium were amplified. They are onlydistantly related to known pmoA and amoA sequences, and theprimer sets used in this study did not amplify alkane monooxy-genase genes from pure cultures of Nocardioides and Mycobacte-rium (75, 76). The fact that these sequences were obtained fromthe methane seep samples may be indicative of a high number ofalkane oxidizers in these environments.

In summary, the community composition of aerobic metha-notrophs that attenuate CH4 emissions from the Siberian seepswas quite similar to that found at methane seeps in the littoral andprofundal zones of oligotrophic freshwater Lake Constance, Ger-many (57), and in sediments of various boreal and arctic freshwa-ter lakes (58, 77, 78). All these environments are characterized bythe predominance of Methylobacter-like organisms and type IbMB from as-yet-uncultivated clades. Apparently, these metha-notrophs are the major CH4 oxidizers in cold eco-niches with highmethane source strength. The isolation and characterization ofpotentially novel, psychrotolerant methanotrophs from these en-vironments represent a challenge for future cultivation studies.

ACKNOWLEDGMENTS

I.Y.O. and S.N.D. were supported by the program Molecular and CellBiology of the Russian Academy of Sciences and the Russian Foundationfor Basic Research (project no. 12-04-00768). M.V.G. was supported bythe Tomsk State University Competitiveness Improvement Program.C.-E.W. is a member of the International Max Planck Research School forEnvironmental, Cellular, and Molecular Microbiology (Marburg, Ger-many). W.L. was supported by the LOEWE Center for Synthetic Micro-biology (SYNMIKRO).

We greatly appreciate the help of E. D. Lapshina (Yugra State Univer-sity) and also thank A. F. Sabrekov (Tomsk State University), N. Shnyrev(Yugra State University), and I. E. Kleptsova (Tomsk State University).

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