Diversity of Aerobic Methanotrophic Bacteria in a Permafrost Active Layer Soil of the Lena Delta, Siberia
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With this study, we present first data on the diversity of aerobic methanotrophic bacteria (MOB) in an Arctic permafrost active layer soil of the Lena Delta, Siberia. Applying denaturing gradient gel electrophoresis and cloning of 16S ribosomal ribonucleic acid (rRNA) and pmoA gene fragments of active layer samples, we found a general restriction of the methanotrophic diversity to sequences closely related to the genera Methylobacter and Methylosarcina, both type I MOB. In contrast, we revealed a distinct species-level diversity. Based on phylogenetic analysis of the 16S rRNA gene, two new clusters of MOB specific for the permafrost active layer soil of this study were found. In total, 8 out of 13 operational taxonomic units detected belong to these clusters. Members of these clusters were closely related to Methylobacter psychrophilus and Methylobacter tundripaludum, both isolated from Arctic environments. A dominance of MOB closely related to M. psychrophilus and M. tundripaludum was confirmed by an additional pmoA gene analysis. We used diversity indices such as the Shannon diversity index or the Chao1 richness estimator in order to compare the MOB community near the surface and near the permafrost table. We determined a similar diversity of the MOB community in both depths and suggest that it is not influenced by the extreme physical and geochemical gradients in the active layer.
Aerobic methanotrophic bacteria (MOB) of the phylum Proteobacteria primarily contribute to the consumption of methane in terrestrial environments. The microbial conversion of methane into carbon dioxide was estimated to account for 30% to 90% in flooded rice fields [1, 2], for 13% to 38% of the methane produced in temperate and subarctic peat soils , and for 15% to more than 90% of the diffusive methane flux in wetlands [36, 47]. The group of MOB comprises the three families Methylococcaceae, Methylocystaceae, and Beijerinckiaceae [5, 9, 10, 11, 12]. The only exception is Crenothrix polyspora, a filamentous, sheathed microorganism recently discovered to be methanotrophic . Members of the Methylococcaceae are termed type I MOB and belong to the γ-subdivision of the Proteobacteria phylum. Members of the Methylocystaceae and Beijerinckiaceae are termed type II MOB and belong to the α-subdivision of the Proteobacteria phylum [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]. The diversity and composition of MOB have been investigated in several environments such as freshwater sediments [8, 42], landfill soils , rice field soils [22, 24], habitats with only atmospheric methane concentrations [29, 33, 35], and peat bogs with very low pH values [39, 40].
Northern wetlands and tundra contribute about 20% to the global natural methane emission annually [6, 7, 18]. Given that the global warming potential of methane on a molecular basis and a time horizon of 100 years is 23-fold that of carbon dioxide , the ecology of the MOB community as the major sink for methane in these environments is of importance. Methanotrophic communities were found to be abundant and active also in cold environments such as, for example, northern peat lands, eastern Antarctica, and Fennoscandian deep ground waters . It was reported that MOB are viable in deep Siberian permafrost sediments with ages of 1,000–100,000 years as well . In addition, based on the temperature response of potential methane oxidation rates, it was recently shown that methane oxidizing bacteria are well adapted to the temperature regime in permafrost active layer soils of the Lena Delta . However, our knowledge on MOB from high-latitude environments in terms of diversity and composition remains very poor .
With this study, we present first data on the methanotrophic diversity (richness and evenness) in Arctic tundra soils of the Lena Delta with steep gradients of temperature and methane. In addition to the 16S ribosomal ribonucleic acid (rRNA) gene, we analyzed the phylogeny of the particulate methane monooxygenase gene (pmoA), known as a functional marker for MOB . With this study, we will show to what extent the methanotrophic diversity is influenced by the harsh environmental conditions in Siberian permafrost active layer soils.
Materials and Methods
The study site is located in the eastern part of Samoylov Island (N 72°22′, E 126°28′) in the Lena Delta, Siberia. For a detailed description of the geomorphology of the Lena Delta, refer to Schwamborn et al. . During the expedition LENA 2005 (7th of July to 1st of September), active layer cores (ø 56 mm) of a low-centered polygon were sampled. The cores were frozen immediately after sampling and were kept frozen until further processing. For this study, we used core number 33, sampled on the 25th of August, 2005, at the rim of the polygon. In the laboratory, the core was sectioned under sterile conditions, and samples of depths 6–11 (near surface) and 20–28 cm (near permafrost table) were selected for molecular analysis. The selection of these samples was according to a previously observed shift in the temperature optima of potential methane oxidation rates between the near surface and the near permafrost table within the active layer of the polygon rim .
Soil and Pore Water Analysis
Vertical profiles of soil CH4 concentrations were obtained from the elevated rim of the polygon through extraction of CH4 from soil pore water by injection of 5 ml water into saturated NaCl solution, shaking the solution and subsequently analyzing the CH4 headspace concentration with gas chromatography. Soil temperature measurements (Greisinger GTH 100/2 equipped with Ni–Cr–Ni temperature sensor) were carried out in 5-cm increments from 0 to 38 cm soil depth before core sampling. The total carbon and total nitrogen (TN) contents were determined with an automatic element analyzer (Elementar VARIO EL III). The total organic carbon (TOC) content was measured on corresponding samples after HCl (10%) acid digestion to remove the carbonate on the same analyzer (Elementar VARIO EL III). The pH values were determined with the MultiLab 540 (WTW, Germany) in pore water, which was extracted by applying Rhizons into undisturbed active layer samples according to the method of Seeberg-Elverfeldt et al. . Cell counts of MOB were obtained based on fluorescence in situ hybridization with the MOB-specific probes Mg705/Mg84 (type I MOB) and Ma450 (type II MOB)  according to a protocol described recently .
Extraction of Total DNA
Prior to the deoxyribonucleic acid (DNA) extraction, the core sections were thoroughly homogenized. Afterwards, four subsamples of each depth were used for extraction. Total genomic DNA was extracted with the UltraClean™ Soil and the PowerSoil™ DNA Isolation Kit (Mo Bio Laboratories, USA) following to the manufacture’s protocol. Both kits combine heat, detergents, and mechanical force against beads to lyse microbial cells. The released DNA is then bound to a silica spin filter and finally purified. The PowerSoil™ DNA Isolation Kit is intended for use with environmental samples containing a high humic acid content. The size of the genomic DNA was checked by electrophoresis on a 1% agarose gel against a Lamda EcoRI/HindIII marker (ABgene, UK) with SYBR gold staining.
Methanotroph specific primers used in this study
E. coli positiona fragment length (bp)b
5′-CCT TCG GGM GCY GAC GAGT-3′
5′-GAT TCY MTG SAT GTC AAGG-3′
5′-GAG TTT GAT CMT GGC TCA G-3′
5′-CAT CTC TGR CSA YCA TAC CGG-3′
5′-GGN GAC TGG GAC TTC TGG-3′
5′-CCG GMG CAA CGT CYTTACCC-3′
5′-ACG TCC TTA CCG AAG GT-3′
Denaturing Gradient Gel Electrophoresis
PCR products were separated on an 8% polyacrylamide gel in 1×Tris–acetate–ethylenediamine tetraacetic acid buffer using a D-Code System (Bio-Rad). The denaturing gradient (100% denaturant consisted of 7 M urea and of 40%, v/v, deionized formamide) ranged from 30% to 60% and from 40% to 70%, respectively. Gradient gel electrophoresis ran constantly at 100 V for 14 h at 60°C. The gels were stained for 30 min with SYBR gold and visualized with a GeneFlash system (Syngene, UK). Distinct bands were excised with a sterile scalpel, eluted overnight at 4°C, and reamplified with primers without GC-clamp in 20–25 cycles. Products were purified as described and sequenced by MWG (Germany).
Construction of Clone Libraries and Sequencing
The purified PCR products were ligated into the linear Plasmid Vector pCR4 supplied with the TOPO TA CloningR Kit for Sequencing (Invitrogen, Germany) and subsequently transformed into chemically competent E. coli cells via heat shock following the manufacture’s protocol. Cells were incubated overnight at 37°C on agar plates containing 0.05% Ampicillin. Only cells containing a Vector with the insert were competent to grow with ampicillin. Colonies of these cells were screened for correct size of the insert and directly sequenced. Sequencing was performed with the BigDye Terminator cycle sequencing kit, version 3.1 (Applied Biosystems, Germany) with vector primers.
Sequences of the 16S rRNA gene were automatically and manually edited with Sequencing Analysis 5.2 (Applied Biosystems) and checked for chimeras with Bellerophon  and with the Chimera-Check of the Ribosomal Database Project (Michigan State University). Contigs were assembled with Sequencher 4.7 (Gene Codes, USA). Sequences were imported into the ARB 16S rRNA database (www.arb-home.de) and phylogenetically analyzed. Rarefaction analysis and estimation of diversity indices were performed with DOTUR .
Previous to an analysis of the achieved pmoA gene sequences, a database of pmoA gene sequences was created using CLC Free workbench software (http://www.clcbio.com). This database contains all translated pmoA gene sequences of good quality (approximately 1,000 sequences) available in GenBank (http://www.ncbi.nlm.nih.gov/). The translated pmoA gene sequences were aligned using the MUSCLE algorithm . Database and alignment were imported into the ARB program. The pmoA sequences obtained in this study were translated with CLC Free workbench, imported into the newly created pmoA gene database, manually aligned, and phylogenetically analyzed.
Nucleotide and Protein Sequence Accession Numbers
The 16S rRNA and pmoA gene sequences recovered in this study and used for phylogenetic presentation have been deposited in the European Molecular Biology Laboratory, GenBank, and DNA Data Bank of Japan nucleotide sequence databases under the accession numbers EU124838–56 (16S rRNA gene sequences) and EU124857–EU124864 and EU135968 (pmoA gene sequences).
Soil and Pore Water Characteristics
Selected soil properties of the elevated polygonal rim on Samoylov Island, Lena Delta (values of methane concentrations and temperatures were obtained in the field in July, 2005)
CH4 (nmol g−1)
Diversity of Aerobic Methanotrophic Bacteria Based on the 16S rRNA Gene
Phylogenetic characterization of 16S rRNA gene sequences of aerobic methanotrophic bacteria of active layer samples from Samoylov Island, Lena Delta, obtained through DGGE
Sequence IDa (sequence length)
Next cultured relative according to NCBI
Accession number, GenBank
Similarity according to NCBI BLASTb (%)
Similarity according to NJc (%)
LD_DGGE_MOB_T1 (826 bp)
LD_DGGE_MOB_T2 (829 bp)
LD_DGGE_MOB_B1 (855 bp)
LD_DGGE_MOB_B2 (846 bp)
LD_DGGE_MOB_B3 (753 bp)
Diversity indices of 16S rRNA gene sequences of aerobic methanotrophic bacteria in active layer samples from Samoloy Island, Lena Delta
At the genus level (≥95% sequence similarity) , the overall diversity decreased significantly. Based on the Shannon index, it was only half of that at the species level. In addition, the estimated overall richness at the genus level was only 30% that of the species level.
Phylogenetic Analysis of the pmoA Gene
In addition to the diversity of MOB in active layer samples of two depths of a Siberian permafrost active layer soil based on the 16S rRNA gene, we investigated the distribution and phylogenetic relatedness of pmoA gene sequences in the same samples.
DGGE profiles of the pmoA gene could only be obtained for the near-surface samples. We reproducibly detected four distinct bands (Fig. 1b) that were excised and sequenced. DGGE bands of the near-permafrost table samples could not be excised and sequenced successfully as a result of very low amounts of template. For this reason and in order to confirm the DGGE results of the near-surface samples, we constructed small clone libraries of the pmoA gene for the near-surface and the near-permafrost table samples. We obtained 18 clones that gave five additional pmoA gene sequences different from the previous ones.
Our results showed that members within the type I MOB were closely related to only two known genera, Methylobacter and Methylosarcina. This was consistently detected applying two different methods, DGGE and cloning. Given that altogether ten genera belong to the type I MOB [5, 25, 45, 54, 60], this points at a restricted diversity within the studied community of this group. In terms of relative abundance, representatives related to the Methylobacter genus were clearly dominant, whereas the number of representatives of the Methylosarcina genus was only marginal. In a related study on high-arctic sediments based on DGGE profiles, the MOB community was also found to be restricted to only two genera . In that study, members of the genera Methylobacter and Methylosinus and consequently both type I and type II MOB were detected. Similar to our study members of the Methylobacter genus were recovered to be dominant.
A low diversity or a restriction to certain groups of MOB was reported also for nonarctic environments that exhibit extreme environmental conditions. The MOB community of peat bogs or acidic forest soils, for example, was restricted to members of type II MOB [39, 40, 43]. Besides, all known acidophilic isolates belong to the genera Methylocapsa and Methylocella, both type II MOB [9, 10, 11, 12]. The MOB community capable of the consumption of atmospheric methane was studied in different forest and grassland soils and was found to exclude members of the type I group. It was suggested to consist primarily of type II MOB  and also of distinct, uncultivated clades distant from type I and type II MOB [23, 29, 33, 46]. In all studies discussed, the MOB community is exposed to environmental extremes such as freeze–thaw cycles, low pH values, or low substrate concentrations and was found to be restricted to certain taxonomic groups of MOB. In contrast to that, a wide range of MOB genera of both type I and type II MOB was reported for more moderate environments such as landfill soils , rice field soils [22, 24, 28], freshwater sediments [8, 42, 44], and subarctic tundra soils . This points at a trend toward a restricted diversity or a selection for certain groups of MOB in more extreme environments, which is in accordance with the results obtained in the present study.
The absence of type II MOB in our study on the 16S rRNA as well as on the pmoA gene level confirms a previous study indicating a clear dominance of type I over type II MOB in active layer samples from Samoylov Island using phospholipid fatty acid (PLFA) analysis . Our methodological approach is based on primer combinations that were designed to detect both 16S rRNA and pmoA gene sequences of known and unknown type I as well as type II MOB [3, 8, 59]. These primer combinations were used in several studies where type I and type II MOB were detected either based on the 16S rRNA gene , based on the pmoA gene [29, 42], or based on both genes [28, 44]. According to this, we would have detected type II MOB if their abundance was sufficiently high. However, type II MOB were below the detection limit in the studied permafrost active layer soil, while cell counts of type I MOB were in the range of 107 cells g−1 soil. The unsuccessful amplification of type II MOB could therefore be due to a PCR bias against this group of MOB as an artifact caused by a too low abundance of type II MOB in our samples. Previous studies showed that true psychrophilic MOB can be only found within the subgroup of type I MOB [4, 41], which is in accordance with in situ temperatures of permafrost environments. Considering this and the fact that type II MOB outcompete type I MOB only at either extremely low methane concentrations as discussed in the beginning or at distinctly heightened methane concentrations , it is evident that the environmental conditions of the Siberian permafrost lead to the dominance of type I MOB over type II species.
Based on 16S rRNA gene clone libraries, we found a distinct species-level diversity of the type I MOB within the active layer including two new clusters (clusters I and II) of MOB that were specific for the permafrost active layer soil studied here. More than 60% of all OTUs and more than 70% of all sequences detected belong to these clusters. Specific clusters for Siberian permafrost-affected soils based on the 16S rRNA gene could already be detected for other groups of microorganisms such as methanogenic archaea , which was interpreted as clusters formed by methanogens characterized by a specific adaptation to the harsh permafrost conditions. The closest cultured relatives of members of the clusters detected here were M. psychrophilus and M. tundripaludum. M. psychrophilus was isolated from Siberian tundra and was characterized as a true psychrophile with an optimum growth temperature between 3.5°C and 10°C . M. tundripaludum was isolated from an Arctic wetland soil on the island of Svalbard, Norway, and was characterized as a psychrotroph . The sequence similarity of representatives of clusters I and II to M. psychrophilus and M. tundripaludum was less than 95%. Considering the 16S rRNA sequence similarity only, these clusters can be distinguished from known genera .
Our additional analysis of the phylogeny and distribution of the pmoA gene revealed a remarkable congruence with the phylogeny of the 16S rRNA gene sequences. The pmoA cluster I coincided with the two specific 16S rRNA gene permafrost clusters (clusters I and II), whereas pmoA cluster II confirmed cluster III in the 16S rRNA tree. The first group of sequences was directly affiliated to the species M. psychrophilus and M. tundripaludum. The second group of sequences was not directly affiliated to any known species, but the closest cultivated relatives were again M. psychrophilus and M. tundripaludum. Based on this, the majority of MOB detected in this study was found to be closely related to phsychrophilic and psychrotrophic MOB isolated from Arctic environments. In addition, there is indication of a vertical shift in MOB population. Based on the 16S rRNA gene sequences, clusters III and IV are exclusively bottom. With respect to the pmoA gene sequences, cluster I is exclusively bottom, too, whereas III and IV are top. Although it is difficult to deduce a straight phylogenetic relationship between the distinct clusters of environmental sequences detected, the similarity in the 16S rRNA and pmoA gene sequence clustering suggest that species of the same type I lineages were detected by both approaches. In addition, the comparison of the diversities obtained by analysis of both genes covered similar ranges.
Based on our data, we were able to compare the diversity of the active layer MOB near the surface and near the permafrost table. This comparison was necessary as we were aiming at investigating whether the gradients of temperature and methane within the active layer influence the diversity of the MOB community. Although, compared to the near surface, the active layer zone close to the permafrost table is characterized by a negative redox potential , by temperatures constantly below 2°C, and significantly lower cell counts of MOB , diversity and estimated richness values were found to be almost equal in both depths. Thus, the gradients within the studied active layer do not seem to influence the diversity of the MOB, although they do influence their abundance and, as discussed before, most likely their community structure. This indicates that, firstly, the gradients within the active layer are too weak to affect the diversity of the MOB community. Secondly, diversity and abundance must be regulated by different parameters. This is in accordance with results obtained from the phospholipid biomarker analysis. The PLFA distribution of near-surface and near-permafrost horizons of the active layer showed that the microbial communities of both horizons do not incorporate significantly more unsaturated fatty acids under cooler conditions (Mangelsdorf and Wagner, personal communication). This indicates a high level of adaptation to low temperatures of permafrost microbial communities in general.
With this study, we present first data on the diversity of the MOB community in an Arctic permafrost affected environment from northeast Siberia at a high phylogenetic resolution. The spatial and temporal “snapshot” of methanotroph diversity presented here revealed a community of MOB that is specialized to the extreme environment it is exposed to. We showed that, in contrast to a distinct species-level diversity, the diversity of the MOB community at a higher taxonomic level is restricted to members closely related to only two known genera. The presented data support a trend reported in other studies toward a restricted diversity of MOB in more extreme environments. In the context of the climatic changes presently affecting the Siberian Arctic and its polygonal tundra landscapes, a restricted diversity could negatively affect the community of MOB and their function as a primary sink for methane in these regions.
The authors acknowledge J. Harder (Max Planck Institute for Marine Microbiology) for helpful discussions and T. Sachs (Alfred Wegener Institute for Polar and Marine Research) for critical reading of the manuscript. In addition, we want to thank the crew of the Expedition LENA 2005, in particular Waldemar Schneider for logistic and Günter “Molo” Stoof for technical support (both Alfred Wegener Institute for Polar and Marine Research). Finally, we thank all our Russian partners, in particular Dimitry Yu. Bolshiyanov (Arctic Antarctic Research Institute), Alexander Yu. Dereviagin (Moscow State University), Mikhail N. Grigoriev (Permafrost Institute Yakutsk), Dmitri V. Melnitschenko (Hydro Base Tiksi), and Alexander Yu. Gukov (Lena Delta Reserve). This research was conducted on the frame of the International Max Planck Research School for Marine Microbiology (MarMic).
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