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Microbial Ecology and Functional Diversity of Natural Habitats

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Abstract

Methane (CH4) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH4 budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH4 that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska’s interior. The water column CH4 oxidation potential for these shallow (2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH4, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH4-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.

Introduction

Methane (CH4) is a potent greenhouse gas responsible for about 20% of the direct radiative forcing from all long-lived greenhouse gases (IPCC, 2001). The concentration of CH4 in the atmosphere has increased from 0.7 to 1.8 p.p.m.v. since the industrial revolution because of increased anthropogenic inputs (Etheridge et al., 1998). Natural aquatic systems, in particular wetlands and lakes, also contribute substantial quantities of CH4 to the atmosphere and are an important component of the continental greenhouse gas budget (IPCC, 2007; Reeburgh, 2007; Bastviken et al., 2011). Presently, lakes north of 45° N emit an estimated 24.2±10.5 Tg CH4 per year directly to the atmosphere (Walter et al., 2007), which is more than twice that from the oceans (Reeburgh, 2007). Moreover, with additional climate warming, CH4 emissions from high-latitude lakes are expected to increase during this century, particularly in arctic regions with continuous, organic-rich permafrost (Zimov et al., 2006).

Aerobic CH4 oxidation in the sediment and water column has an important role in mitigating CH4 release from mid and low latitude freshwater ecosystems to the atmosphere (Frenzel et al., 1990; Bender and Conrad, 1994; Hanson and Hanson, 1996; Carini et al., 2005; Duc et al., 2010). The availability of CH4 and oxygen (O2), the substrates for aerobic methanotrophy, are the most significant factors determining the location and rates of CH4 oxidation in sediments (Hanson and Hanson, 1996). CH4 oxidation is generally most active at the oxic-anoxic transition in sediments near the sediment-water interface (Kuivila et al., 1988; Frenzel et al., 1990; Bender and Conrad, 1994). In addition, CH4 is also consumed in oxygenated portions of the water column in lakes (Carini et al., 2005; Bastviken et al., 2008).

Aerobic methanotrophic diversity in lakes is greater than that of peat or marine environments (Costello and Lidstrom, 1999). Diverse methanotrophs have been reported in low and mid-latitude freshwater ecosystems and varied with environmental conditions (Auman, 2001; Jugnia et al., 2006; Rahalkar and Schink, 2007). Arctic lakes differ substantially from low and mid-latitude lakes in terms of the seasonal shifts in temperature, dissolved oxygen (DO) and CH4 concentration associated with the dynamics of ice cover and thaw (Miller et al., 1980; Phelps et al., 1998; Clilverd et al., 2009). These environmental conditions could favor an aerobic methanotrophic community distinct from warmer climates. However, to date, no study has addressed the identity or ecological ramifications of arctic lake CH4 oxidation. In this study, we characterize the microbial community associated with aerobic CH4 oxidation in the water column and sediments (0–25 cm) from two thermokarst lakes in Alaska, an arctic tundra lake on the North Slope of the Brooks Range and a subarctic taiga lake in Alaska’s interior. In addition to the cultivation of aerobic methanotrophs from the water column, we applied DNA-based stable isotope probing (DNA-SIP) coupled with terminal restriction fragment length polymorphism (T-RFLP), quantitative PCR (Q-PCR) and pyrosequencing to determine the identity and diversity of active aerobic CH4-oxidizing microorganisms along sediment depth profiles.

Materials and methods

Study site and sampling

Water and sediment samples were collected in July 2009 from two lakes: (a) Lake Qalluuraq, a tundra lake with a persistent CH4 seep located near Atqasuk on the north slope of the Brooks Range in Alaska; and (b) Lake Killarney, a subarctic taiga lake with moderate CH4 ebullition in Fairbanks, Alaska (Brosius, 2010) (Figure 1 and Table 1). Two sites, LQ-BGC and LQ-WEST, were sampled at Lake Qalluuraq. Site LQ-BGC is the location with high CH4 ebullition (Wooller et al., 2009), while CH4 ebullition was not observed in the LQ-WEST site. A single site (LK-NW) was sampled in the northwest portion of Lake Killarney.

Figure 1
figure 1

Sampling sites in the study site. Lake Qalluuraq, an arctic tundra lake with a persistent CH4 seep located near Atqasuk on the North Slope of the Brooks Range in Alaska; and Lake Killarney, a subarctic taiga lake with moderate CH4 ebullition in Fairbanks, Alaska.

Table 1 Physical and chemical characteristics of sediment and water column in the study sites

Water samples were collected from 20 cm under the water surface (top), 1 m below the water surface (middle) and 10 cm above the sediment-water interface (bottom), respectively, at each sampling location. Water temperature, DO and pH at the different sampling depths at each sampling location were measured using a YSI 556 MPS meter (YSI Environmental Inc., Yellow Springs, OH, USA). Cores of sediment 25 cm long were collected at each site using polycarbonate tubes (7 cm o.d.). The sediment sub-samples were immediately placed into plastic zipper freezer bags, homogenized and then sub-samples were kept at 4 °C for CH4 oxidation incubations and SIP experiments. Additional sub-samples were freeze-dried and analyzed for their organic carbon and nitrogen content as described by Wang and Wooller (2006). Sediment particle size was analyzed by the Bouyoucos hydrometer method (Gee and Bauder, 1986).

Sediments for determination of dissolved CH4 concentrations were collected as 3 ml plugs at 4–5 cm intervals from the sediment cores. The samples were transferred to 20 ml serum vials, sealed with 1 cm thick septa and stored at −20 °C. Headspace CH4 concentration in the vials was analyzed using a gas chromatograph equipped with a flame ionization detector and converted to aqueous concentrations using the method of Hoehler et al. (2000).

SIP microcosms and CH 4 oxidation potential

Incubations for SIP and CH4 oxidation potential were conducted in microcosms in sterile 60 ml glass serum vials, containing either 5 g (wet weight) of sediment or 10 ml of water, sealed with butyl rubber stoppers. Microcosms were injected with 13CH4 (99 atom % 13C, Sigma-Aldrich, Saint Louis, MO, USA) or CH4 (99.5% pure, as control) to achieve a headspace concentration of 10% CH4 (v/v) and incubated on a rotary platform shaker (100 r.p.m.) at 4, 10 or 21 °C. Calculated by Henry's Law and the van’t Hoff equation to correct for the effect of temperature on CH4 solubility (Lide and Frederikse, 1995), the theoretical saturated CH4 concentration in the pore water is 151 μM at the initial CH4 concentration of 10% (v/v). CH4 consumptions at 4 and 10 °C were both very slow in the sediments from the LQ-BGC site (Supplementary Figure S1). Therefore, we focused on incubations at 21 °C, a temperature that is similar to the maximum water temperatures of 20 °C (July) detected during a 4-year study of arctic lakes (Miller et al., 1980) and the measured temperature of water at Lake Qalluuraq, to investigate the comparative maximum activity and community structure of methanotrophs between and within the lakes. All samples were incubated in triplicate. Autoclaved water and sediment samples were used as abiotic controls.

Gas headspace samples (50 μl) were periodically withdrawn from the headspace of microcosms and analyzed for residual CH4 using gas chromatograph-flame ionization detector. CH4 oxidation potential was assessed from the zero-order decrease in CH4 concentration in the headspace of the serum vials within 12 h (Kightley et al., 1995), and expressed as μmol CH4 per g dry weight per day (μmol g−1 d−1) or nmol cm−3 d−1. CH4 oxidation potential of water samples was measured during 48 h incubations. SIP incubation was performed as described previously (He et al., 2012) and was continued until 0.2 mmol 13CH4/CH4 g−1 (wet weight) was oxidized (38–74 days for the LQ-WEST and LK-NW sediments and 108–212 days for the LQ-BGC sediments), which is sufficient to produce a UV visible 13C-DNA band with ethidium bromide after an equilibrium (isopycnic) density gradient centrifugation (Radajewski et al., 2002). At the end of the incubation, the sediment samples were harvested and frozen immediately at −80 °C.

SIP DNA extraction and 13 C-DNA separation

DNA was extracted from 0.5–1 g (wet weight) of the SIP sediment sub-samples using the Bio101 Fast DNA Spin Kit for soil (MP Biomedicals, Solon, OH, USA). Equilibrium (isopycnic) density gradient centrifugation and fractionation were performed using cesium trifluoroacetate (CsTFA, GE Healthcare, Chalfont St Giles, UK) solution as described by He et al. (2012). Gradients were fractionated into 20 250-μl fractions, buoyant density (BD) was measured and DNA was precipitated and re-suspended as previously reported (Leigh et al., 2007).

The relative abundance of bacterial DNA in gradient fractions ranging in BD from 1.559 to 1.663 g ml−1 was determined by Q-PCR as described by Leigh et al. (2007). After the range of fractions containing 13C-labeled DNA or unlabeled DNA was identified, they were combined to compose compiled ‘heavy’ fractions from each sample for subsequent molecular analyses.

Planktonic methanotroph enrichment cultures

Fifty milliliters each of the triplicate water samples were centrifuged at 5000 r.p.m. for 15 min. The cell pellets were transferred into a sterile 60 ml glass serum vial with 10 ml methanotroph medium prepared as described by Graham et al. (1992). The vials were sealed with a butyl rubber stopper, injected with CH4 to 10% (v/v) and incubated at room temperature (21 °C), 10 or 4 °C, on a shaker (100 r.p.m.). CH4 consumption was very slow at low temperatures (4 and 10 °C) in the enrichment cultures from Lake Qalluuraq (that is, LQ-BGC and LQ-WEST sites) even if they were incubated for 1 month (Supplementary Figure S2). Considering this, we chose 21 °C incubations to investigate and compare culturable planktonic methanotrophs between and within the lakes. When the cultures became turbid, 1 ml was transferred into fresh methanotroph medium, and incubated again. After the enrichment, the culture was centrifuged at 14 000 r.p.m. for 10 min and the pellet was harvested and frozen at −80 °C. The DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) was used to extract DNA from enrichment cultures.

Methanotroph Q-PCR analyses

Q-PCR of 16S rRNA genes of type I and type II methanotrophs and pmoA in the heavy fractions from 13C-labeled DNA and unlabeled control DNA was performed using the primer sets described by Martineau et al. (2010) and conducted in four replicates of 15 μl reactions containing SYBR green master mix (Applied Biosystems, Foster City, CA, USA), 4.5 pmol each primer and 1 μl template. Thermal cycler conditions were as follows: an initial stage at 50 °C for 2 min; denaturation at 95 °C for 5 min; 60 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s for pmoA or 72 °C for 30 s for 16S rRNA genes of type I and type II methanotrophs. Standards were made from 10-fold dilutions of linearized plasmids containing the same fragment of 16S rRNA genes of type I and type II methanotrophs and pmoA as Q-PCR product that was cloned from amplified pure culture DNA. The limit of detection by Q-PCR was about 102 copies per reaction for pmoA and about 103 copies per reaction for 16S rRNA genes of type I and type II methanotrophs.

T-RFLP analysis and multivariate analyses

Bacterial community profiling was performed using T-RFLP of 16S rRNA gene amplicons as described previously (He et al., 2012). T-RFLPs were analyzed using GeneMapper software version 3.7 (Applied Biosystems). The peak height threshold for T-RFLPs was set at 300 FU (fluorescence units). Terminal restriction fragments less than 50 and above 1000 bp were eliminated from all data sets, and then normalized such that peak heights represented a percentage of the total peak height in each sample. The different bacterial communities were then subjected to a multivariate cluster analysis using Euclidean distance with the open source statistical application PAST (Hammer et al., 2001).

Sequencing and phylogenetic analyses

Sequence analysis was performed on 16S rRNA gene amplicons from 13C-DNA and DNA from the enrichment cultures of water using pyrosequencing as described by He et al. (2012). Sequences were first trimmed of the primer region and low-quality sequences were removed by Ribosomal Database Project’s (RDP) pyrosequencing pipeline. From 41 to 66% of the total sequences (2631–4794) except for 31% of the total sequences of 2931 for LQ-WEST (0–1 cm) passed this filter (average length of 330–331 bp) and were assigned to taxonomic groups by RDP’s Naïve Bayesian Classifier (80% confidence threshold). The nucleotide sequences have been deposited to NCBI Short Read Archive under the accession number of SRP005485.

Results

Physical and chemical characteristics

The water in Lake Qalluuraq (LQ-BGC and LQ-WEST sites) was weakly acidic and DO was between 9.89 mg l−1 and 10.11 mg l−1 at all depths (Table 1). At 17 °C, the temperature of the lake during sampling, the DO concentrations correspond to 102–105% saturation (the theoretically saturated concentration of DO is 9.65 mg l−1). Water in Lake Killarney (LK-NW site) had apparently high dissolved organic carbon (dark in color) and a sharp decrease of DO and temperature with increasing depth. The sediment from the seep site (LQ-BGC site) was mostly composed of sand-sized particles of 0.1–2 mm in size, accounting for 85.5%. However, the sediments from the LQ-WEST and LK-NW sites were mainly composed of fine-grained particles of 0.05–0.1 mm (45.0–68.2%) and 0.002–0.05 mm (20.4–41.5%), respectively. The sediments from LQ-WEST site had the highest carbon (11.14%) and nitrogen (0.69%) contents, which are about 5 and 70 times higher than those from the LK-NW and LQ-BGC sites, respectively.

At the LQ-BGC site, dissolved CH4 concentrations were less than 15 μM from 0–10 cm sediment depth, and increased to 1334 μM at 25 cm sediment depth (Figure 2). At the LQ-WEST site, dissolved CH4 concentrations from near the sediment-water interface to 25.5 cm sediment depth varied from 483 μM to 2062 μM. Lower dissolved CH4 concentrations (25–68 μM) were found in sandy intervals from 30–36 cm sediment depth. Compared with Lake Qalluuraq (LQ-BGC and LQ-WEST sites), dissolved CH4 concentration in the pore water from LK-NW site was lower (about 16–390 μM).

Figure 2
figure 2

Sediment depth profile for dissolved CH4 concentration in the sampling sites. (a) LQ-BGC site; (b) LQ-WEST site; and (c) LK-NW site.

CH 4 oxidation potential

The highest water column CH4 oxidation potential occurred in the subarctic lake site (LK-NW) samples, followed by the arctic lake seep location (LQ-BGC site) (Figure 3). At all locations the CH4 oxidation potential increased with depth, and the highest rates were obtained from water samples collected from near the bottom of the lakes. The CH4 oxidation potential in water from the near-bottom of the LK-NW site was 1450 nmol cm−3 d−1, which is approximately seven times higher than that from the LQ-BGC site. Near zero CH4 consumption was observed in water from the LQ-WEST site, which is located 583 m from the Lake Qalluuraq seep (LQ-BGC site).

Figure 3
figure 3

CH4 oxidation potential of water samples from indicated depths: top (20 cm under the water surface); middle (1 m below the water surface); bottom (10 cm above the sediment-water interface). Different letters within the graph refer to significant difference at 5% level based on least significant difference (LSD) method.

The CH4 oxidation potential of surface sediment (0–1 cm) from the LQ-WEST site (77–80 μmol g−1 d−1) is over an order of magnitude higher than the LQ-BGC seep site and 1.8–13.0 times higher than surface sediment from the subarctic lake site, LK-NW (Figure 4). After 2 days of incubation, measurable CH4 consumption occurred at the 0–20 cm sediment depths from LQ-BGC and LQ-WEST sites, and at the 0–5 cm depths from the LK-NW site. After 10 days of incubation, CH4 consumption was measured in sediments from every depth (0–25 cm), with CH4 oxidation potentials of 0.7–76.8 μmol g−1 d−1.

Figure 4
figure 4

CH4 oxidation potential of sediment from (a) LQ-BGC site; (b) LQ-WEST site; and (c) LK-NW site. □ incubated for 2 days; incubated for 5 days; ▪ incubated for 10 days. Note different scales on y axes (CH4 oxidation potential).

Enrichment cultures of planktonic methanotrophs

Microorganisms harvested from discrete samples from different water and then incubated in methanotroph medium showed rapid CH4 consumption (0.1 mmol CH4 was first completely consumed within 2 days in the cultures from LK-NW water samples, and 7 days from LQ-BGC and LQ-WEST water samples). Although near zero CH4 consumption was measured in water from the LQ-WEST site, the T-RFLP profiles of 16S rRNA gene amplicons from the LQ-WEST enrichments were similar to those from enrichment cultures from the seep site (LQ-BGC) within the same lake (Supplementary Figures S3a, b and S4a, b). Some variation in T-RFs with water depth was visible within the LK-NW site (Supplementary Figures S3c and S4c).

As T-RFLP analyses indicated that the enrichment cultures from various depths were generally similar in community structure (Supplementary Figure S4), we combined DNA extracts obtained from the top, middle and bottom depths together at each sampling location for pyrosequencing analysis of 16S rRNA gene amplicons. RDP classification of pyrosequencing reads showed that the type II methanotroph, Methylocystis, was dominant in the water sample enrichment cultures (Table 2). The type I methanotrophs, Methylomonas, Methylobacter and unclassified Methylococcaceae, were detected as a small proportion (1.7%) of the enrichment culture from water from the LK-NW site, while they were not evident in the sites from Lake Qalluuraq.

Table 2 Relative abundance (% of total reads) and phylogenetic affiliations of pyrosequencing reads from DNA of enrichment cultures in methanotroph medium cultivated from lake water samples

Interestingly, Methylophilus, an obligate methylotroph that uses methanol as the sole source of carbon and energy (Vries et al., 1990; Bratina et al., 1992), was present in all the enrichment cultures and was especially abundant in the LK-NW water culture, where 25.2% of pyrosequencing reads for the enrichment culture were affiliated with Methylophilus. In addition to the Proteobacteria, members of the Actinobacteria, Bacteroidetes and Verrucomicrobia (note there are three previously known methanotrophs in the phylum Verrucomicrobia; Dunfield et al., 2007; Pol et al., 2007; Islam et al., 2008) were also found in the enrichment cultures from all the study locations.

Active CH 4 oxidizers in sediments

To characterize the active sedimentary methanotrophic community, we employed SIP to track the incorporation of 13C-labeled CH4 carbon into microbial DNA (Radajewski et al., 2003; Dumont and Murrell, 2005). Q-PCR data indicate that significant quantities of 13C-DNA were present in SIP sediment incubated with 13CH4 (Supplementary Figure S5). Unlabeled control DNA had a peak of abundant DNA at BD ranging from 1.589 to 1.601 g ml−1. Negligible bacterial DNA was detected when the BD was >1.620 g ml−1 for unlabeled control DNA fractions, while a second DNA peak in high density fractions was present in the 13CH4 incubated samples. Based on the Q-PCR results, we combined the fractions with BD ranging from 1.620 to 1.644 g ml−1 into a compiled heavy fraction for each sample for subsequent molecular analyses.

The 16S rRNA gene of type I methanotrophs and pmoA were detected using Q-PCR at 7.0 × 105–5.7 × 107 and 1.4 × 106–7.1 × 107 copies μl−1, respectively, in all 13C-DNA samples (Figure 5). The 16S rRNA gene of type II methanotrophs was present at levels of 3.0 × 105–1.0 × 107 copies μl−1 in the 13C-DNA from the LK-NW sediment ranging in depth from 1–25 cm, but was only detected in 13C-DNA from deeper LQ-WEST sediment (20–25 cm) at 1.4 × 107 copies μl−1 as well as in LQ-BGC sediments (15–25 cm), which had 2.3 × 106–8.1 × 106 copies μl−1. Type II methanotroph sequences were below the limit of Q-PCR detection of 103 copies per reaction in all other 13C-DNA samples. For all the heavy fractions from unlabeled control DNA, the 16S rRNA genes of type I and type II methanotrophs and pmoA were below detection limits.

Figure 5
figure 5

Quantitative real-time PCR (Q-PCR) of pmoA (▪) and 16S rRNA genes of type I () and type II (□) methanotrophs in 13C-DNA from SIP sediments. The limit of detection by Q-PCR was about 102 copies per reaction for pmoA and about 103 copies per reaction for 16S rRNA genes of type I and type II methanotrophs. (a) LQ-BGC site; (b) LQ-WEST site; and (c) LK-NW site.

T-RFLP profiles of bacterial 16S rRNA gene amplicons for 13C-DNA from sediment showed that a diverse array of bacteria derived carbon from 13CH4 in all sediment incubations, while few obvious peaks were found in the heavy fractions from the control DNA (Supplementary Figures S6, S7). The bacteria active in CH4 utilization varied in composition among different lakes, sampling sites and sediment depths. A multivariate cluster analysis of T-RFLP profiles using Euclidean distance showed that the composition and structure of active methanotrophic communities clustered by depth and site (Figure 6).

Figure 6
figure 6

Cluster analysis of T-RFLP profiles of 16S rRNA genes amplicons digested with HhaI for 13C-DNA from SIP of sediment collected after about 0.2 mmol 13CH4 g−1 (wet weight) consumed. The hierarchical tree was produced by PAST software (Hammer et al., 2001) based on Euclidean distance. The sign (*) in the figure shows the sediment samples used for pyrosequencing analysis.

On the basis of the T-RFLP-based multivariate cluster analysis for 13C-DNA from the SIP sediments of different depths and sites, we selected 13C-DNA from the uppermost (0–1 cm) sediment and the deeper (15–20 cm) sediment at each sampling location for comparative pyrosequencing analysis (Figure 6, pyrosequencing samples noted with asterisks). Although numbers of pyrosequencing reads were low in the LQ-WEST 0–1 cm sediment (903) (Table 3), rarefaction curves showed a higher diversity in the sediment than the deep sediments and LK-NW uppermost sediment (Supplementary Figure S8). Phylogenetic affiliations of pyrosequencing reads from 13C-DNA by RDP classifier showed a diverse bacterial community was involved directly or indirectly in acquiring CH4-derived carbon. Members of the phylum Proteobacteria predominated CH4 carbon assimilation, especially in the sediments from the LK-NW site and the deep (15–20 cm) sediment from the LQ-WEST site, where Proteobacteria accounted for more than 91% of the total pyrosequencing reads. The phyla Actinobacteria, Firmicutes, Bacteroidetes, Acidobacteria, Verrucomicrobia, Planctomycetes and Chloroflexi were all found in the deep (15–20 cm) sediment from the LQ-BGC site and the uppermost (0–1 cm) sediment from the LQ-WEST site.

Table 3 Relative abundance (% of total reads) and phylogenetic affiliations of pyrosequencing reads from 13C-DNA obtained from SIP of lake sediments with 13CH4

Both type I (Methylomonas, Methylobacter, Methylosoma and unclassified Methylococcaceae) and type II methanotrophs (Methylocystis, Methylosinus and Methylocella) were detected in the 13C-DNA from sediments. Compared with type II methanotrophs, type I methanotrophs were more dominant in 13C-DNA from the uppermost (0–1 cm) sediment, especially at the LK-NW site, where Methylobacter accounted for 60.3% of the pyrosequencing reads (Table 3). Type I (Methylobacter) and type II methanotrophs (Methylocystis, Methylosinus and Methylocella) were all detected in the deeper (15–20 cm) sediment from the LK-NW site. The type II methanotroph, Methylocystis, was the most abundant methanotroph in the deeper sediment from the seep site (LQ-BGC) at Lake Qalluuraq, while the type I methanotroph, Methylobacter, was the most abundant type in deeper sediments from LQ-WEST site in the same lake.

Discussion

Differences in CH4 oxidation potential in the water column were observed both between and within our study lakes, Lake Qalluuraq (a tundra arctic lake) and Lake Killarney (a subarctic taiga lake) (Figure 1). A 15 to 60-fold greater CH4 oxidation potential was observed in the water column above the active CH4 seep (LQ-BGC) site compared with the non-seep (LQ-WEST) site within the same arctic lake, which was likely due to the seep providing a constant input of carbon and energy sources that caused increased microbial biomass in the water column (Reay et al., 1993; Treude and Ziebis, 2010) (Figure 3). Compared with the arctic lake CH4 seep, a considerably (4 to 7-fold) higher CH4 oxidation potential was exhibited by the subarctic lake (LK-NW site) water. The highest CH4 oxidation potential was present in the deep water from this subarctic lake (LK-NW site), which was a hypoxic zone in situ (Table 1).

Culturable methanotrophic communities varied with water depth in the subarctic lake (Supplementary Figures S3c and S4c), but not in the arctic lake (Supplementary Figures S3a, b and S4a, b). The vertical variation of the culturable methantrophic communities is consistent with stratification of the water column in the subarctic Lake Killarney. Cultivation in a methanotroph medium in our study appeared to favor Methylocystis, especially in the enrichment cultures of water from arctic Lake Qalluuraq, whose representatives accounted for 74–75% of the total pyrosequencing reads (Table 2). The prevalence of Methylocystis in the enrichment cultures of water may be an indication of a predominance of these organisms in water and/or their more rapid growth in cultures than other dominant organisms, which has been reported previously in a mid-latitude freshwater system (Bussmann et al., 2004).

In sediments, the communities of active CH4 utilizers varied with depth as well as sampling site (Supplementary Figures S6 and S7), and were likely governed by depth of O2 penetration. The metabolically active community of aerobic methanotrophs in sediment is limited by the in situ availability of O2 and CH4 (Hanson and Hanson, 1996). The O2 penetration depth depends largely on the input of degradable organic matter to the sediment (Brune et al., 2000). In our three study locations, the highest sediment organic matter content was observed in the LQ-WEST site, but the DO was still oversaturated in the water column due to physical mixing of the shallow (<2 m depth) lake (Miller et al., 1980; Burn, 2002; Crump et al., 2003). Compared to the LQ-WEST and LK-NW sediment, the LQ-BGC (active CH4 seep site) sediment was much sandier, potentially allowing a deeper penetration of O2 into the sediment due to higher permeability and lower sediment microbial biomass and metabolic activity, as well as in a faster removal of potentially inhibitory reduced end products (Treude and Ziebis, 2010). Based on microbial community shifts revealed by cluster analysis of T-RFLP profiles (Figure 6), we hypothesize that the position of the oxic-anoxic interface relative to the sediment surface was at about 15 cm depth for the LQ-BGC site, but near the sediment-water interface for the LQ-WEST and LK-NW sites. Compared to the sediment depth profile for dissolved CH4 concentration in the LQ-BGC site (Figure 2a), O2 penetration was deeper based on the T-RFLP-based multivariate cluster analysis for 13C-DNA, most likely due to CH4 seep flow which is similar to hydrothermal vent flow entraining lateral inputs of oxygenated water into the seep. The metabolically active communities of bacteria established in the oxic sediment layers were thus markedly different from those in the deeper, anoxic and anaerobic layers, where methanotrophs are likely dormant, existing as cysts or exospores (Bowman et al., 1993), even when they were exposed to the same SIP incubation conditions. A sharp decrease in dissolved CH4 concentration was observed in the pore water from the LK-NW 15–25 cm sediments (Figure 1), which was likely due to anaerobic CH4 oxidation. Additional studies such as total methanotroph communities (including pmoA) in situ and SIP microcosms incubated under anaerobic conditions need to be conducted to better understand the active methanotrophs in arctic lakes.

Both type I and type II methanotrophs were found to derive carbon from CH4 during the lake sediment microcosm incubations (Table 3), with type I methanotrophs being more active in the uppermost sediment at all sites. In our another study, direct sequencing of 16S rRNA genes of methanotrophs also demonstrated that type I methanotrophs were more abundant than type II methanotrophs in the upper sediment from Lake Qalluuraq (He et al., 2012). However, different genera of type I methanotrophs were dominant in the uppermost sediment at the three study sites: Methylosomas in the LQ-BGC site; Methylomonas in the LQ-WEST site; and Methylobacter in the LK-NW site. In the deep sediment, the type II methanotroph Methylocystis was dominant in the LQ-BGC and LK-NW sites; while type I Methylobacter was abundant in the LQ-WEST and LK-NW sites. Type I methanotrophs grow better in low CH4 and high O2 concentrations whereas type II methanotrophs dominate under high CH4 and low O2 concentrations (Amaral and Knowles, 1995; Henckel et al., 2000). In arctic lakes, the uppermost sediments can be a temporally heterogeneous environment due to the dramatic seasonal variations in the concentrations of O2, CH4 and temperature (Miller et al., 1980; Phelps et al., 1998; Clilverd et al., 2009). Type I methanotrophs generally grow more rapidly than type II methanotrophs, and dominate in variable environments (Henckel et al., 2001), perhaps explaining their dominance in the uppermost sediments in our study lakes.

As SIP often requires in vitro incubations that only partially reflect conditions in situ, this method may distort the relative abundance of organisms active in a particular process (Radajewski et al., 2002, 2003). Nonetheless, SIP is valuable for revealing the identity of active methanotrophs in the environment and enables tracking carbon from a specific substrate through the microbial food web by examining the increase in diversity of 13C-labeled organisms over time (Radajewski et al., 2003; McDonald et al., 2005). Methanotrophic community structure and activity varies with temperature (Mor et al., 2006; Liebner and Wagner, 2007; Chanton et al., 2008). Although we conducted SIP microcosms at 21 °C that is similar to the maximum water temperature in arctic lakes (Miller et al., 1980) and harvested SIP samples at the similar CH4 consumption and different incubation time, our some core samples and depths show type I methanotrophs while others show that type II methanotrophs are dominant, which might be due to factors such as depth and geochemistry known to be associated with varying microbial community structure.

In addition to methanotrophs, we found an unexpectedly high abundance of methylotrophs, especially Methylophilus, to be active in utilizing carbon from CH4 in the deep (15–20 cm) sediment from the LQ-WEST site (Table 3). Methanotrophic bacteria usually metabolize CH4 completely to CO2, with methanol, formaldehyde, and formate produced as intermediates (Hanson and Hanson, 1996). This series of reactions reportedly occurs intracellularly and no methanol is released extracellularly (Corder et al., 1986). However, the interruption of the enzymatic reactions by manipulation of the environmental conditions or mutation has been reported to produce excess extracellular methanol (Corder et al., 1986; Lee et al., 2004). Because of the SIP in vitro incubations, these methylotrophs could have derived carbon through cross-feeding, in which non-methanotrophs incorporate 13C into their DNA through the metabolism of by-products derived from methanotrophs such as 13CO2 or organic matter (Radajewski et al., 2003; McDonald et al., 2005). In addition to using carbon derived from CH4 during SIP of sediments, surprisingly, Methylophilus was also detected by pyrosequencing analyses in our enrichment cultures of water. We isolated several methanotrophic strains from the enrichment cultures of water, but the 16S rRNA sequences of isolates were not affiliated with Methylophilus (data not shown). Taken together, our results suggest that methanotrophs and methylotrophs, including Methylophilus, play important roles in the microbial food web that processes carbon from CH4 oxidation in these arctic and subarctic lakes.

In conclusion, SIP and cultivation methods demonstrated that type I methanotrophs, including Methylomonas, Methylobacter and Methylosoma, and type II methanotrophs, including Methylocystis and Methylosinus, are abundant and actively assimilate CH4 in these arctic and subarctic lakes. The community structure and activity of methanotrophs varied alongside the physical-chemical properties of the sediments and water, such as sediment composition, oxygen and CH4 concentrations. Our findings provide new fundamental information regarding the activity and diversity of methanotrophs in cold regions that may aid predicting and modeling future CH4 flux from warming arctic and subarctic environments.

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Acknowledgements

We thank Ben Gaglioti, Nathan Stewart, Doug Whiteman, Monica Heintz, Catharine Catranis for field and laboratory assistance. We also thank Carolyn Ruppel for help with mapping the sampling sites. Any use of trade names is only for descriptive purposes and does not imply endorsement by the U.S. Government. This work was supported by funding from United States Department of Energy National Energy Technology Laboratory (Grant DE-NT000565).

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Correspondence to Mary Beth Leigh.

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He, R., Wooller, M., Pohlman, J. et al. Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments. ISME J 6, 1937–1948 (2012). https://doi.org/10.1038/ismej.2012.34

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Keywords

  • methanotrophs
  • CH4 oxidation potential
  • arctic lakes
  • stable isotope probing

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