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Isolation of Acidophilic Methane-Oxidizing Bacteria from Northern Peat Wetlands

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Science  09 Oct 1998:
Vol. 282, Issue 5387, pp. 281-284
DOI: 10.1126/science.282.5387.281

Abstract

Acidic northern wetlands are an important source of methane, one of the gases that contributes to global warming. Methane oxidation in the surface of these acidic wetlands can reduce the methane flux to the atmosphere up to 90 percent. Here the isolation of three methanotrophic microorganisms from three boreal forest sites is reported. They are moderately acidophilic organisms and have a soluble methane monooxygenase. In contrast to the known groups of methanotrophs, 16S ribosomal DNA sequence analysis shows that they are affiliated with the acidophilic heterotrophic bacteriumBeijerinckia indica subsp. indica.

The methane (CH4) concentration in the atmosphere has more than doubled over the last 300 years (1) and is currently increasing at an annual rate of 0.8 to 1.0% per year (2). About half of the total annual flux of CH4 to the atmosphere is contributed by wetlands (3). The massive northern wetlands account for 50% of the global wetland area (4), and their most extensive type, found in northern Europe, West Siberia, the United States, and Canada, is the acidic Sphagnum peat bogs, which have pH values ranging from 3.5 to 5.

The primary barrier that limits the release of CH4 from methanogenic peatlands is its in situ consumption by indigenous methane-oxidizing bacteria (MOB). MOB inhabit a spectrum of diverse environments and have the unique ability to use CH4 as a sole carbon and energy source (5). The colonization of acidic bogs by MOB has been established by measurement of methanotrophic activity (6), MOB signatures in phospholipids (7), and DNA (8, 9) extracted from peat. Nevertheless, virtually all MOB available in pure culture are neutrophiles, and there are no reports of methanotrophs that grow at pH values below 5.0 (10).

We recently reported on the enrichment of methanotrophic communities from acidic peat bogs of four boreal forest sites in West Siberia and European North Russia (11). The key to successful enrichment was the use of a medium of very low ionic strength and low pH (3 to 6), and incubation under CH4-air mixture at moderate temperature (20°C). These communities were moderately acidophilic with growth and activity optima at pH 4.5 to 5.5. We have now isolated in pure culture MOB from three of these four enrichments (12). The colonies of MOB developed after 4 to 5 weeks of incubation. We selected three strains (strains K, M131, and S6), each representing one enriched community, and confirmed their purity (13). The cells of these three strains were Gram-negative, nonmotile, polymorphic, straight or curved rods with a diameter of 0.7 to 1.0 μm and length of 1.0 to 2.0 μm. They shared an identical morphotype, that is, a specific flattened shape with a concave center and round, bent ends (14) (Fig. 1). The same morphotype was observed as one of the dominant components of the primary communities (11).

Figure 1

Cell morphology of acidophilic methanotrophic isolate (strain K). (A) Polymorphic cell appearance, (B) cell bipolarity, and (C) formation of shapeless aggregates.

Strains S6, K, and M131 grew on minimal mineral medium with the addition of a vitamin mixture and CH4 as a sole source of carbon and energy (15). Growth did not occur in control experiments on the same mineral medium containing vitamins and no CH4. The isolates were slow growing with a specific growth rate ≤ 0.8 to 1.0 day−1, which is consistent with the in situ growth rate for MOB of 0.02 day−1(16). The temperature range for growth of isolates was from 10° to 25°C with the optimum at ∼20°C. The same optimum was found for CH4 consumption by native peat samples (17). No growth of isolates occurred at 30°C. Clearly these bacteria are adapted to conditions of their natural habitat where the temperature never exceeds 25°C, even during summer.

Methane consumption peaked at pH 5.1 for all three strains (Fig. 2). The same pattern of pH dependence was also found for the original peat samples and the methanotrophic enrichments (11, 17–19). The acidophilic nature of the isolated bacteria was confirmed by observing exponential growth without a lag phase and the highest specific growth rate at pH 4.8, whereas no growth was recorded in a medium at initial pH 7.4 (Fig. 2). Furthermore, growth of isolates was sustained in serial transfers in pH 5.0 to 5.5 medium.

Figure 2

Effect of medium pH on the growth and methanotrophic activity of isolated strains. Growth at initial pH 4.8 (•) and pH 7.4 (○). (Inset) The dependence of instant methanotrophic activity on medium pH.

Polymerase chain reaction (PCR) assays for the mmoXand mmoY genes, encoding the α and β subunits of the soluble methane monooxygenase (sMMO) hydroxylase component, yielded products of the predicted size of 524 and 602 bp, respectively, with genomic DNA of the strains K, M131, and S6 (20). The nucleotide sequences of the PCR-amplified mmoX gene fragments from the three strains were identical to each other and to the sequence of mmoX clones obtained previously from the methanotrophic communities (21). The amino acid sequence deduced from these mmoX gene fragments corresponds to positions 300 to 458 of the homologous sequence ofMethylococcus capsulatus (Bath), Methylosinus trichosporium OB3b, and Methylocystis sp. strain M (22) (Fig. 3). Although the level of homology is high, the mmoX gene fragment in bog methanotrophs diverged significantly from these gene fragments in known methanotrophs. The level of sequence divergence indicates that themmoX gene of the acidophilic isolates forms a branch distinct from the mmoX sequences of theMethylocystis-Methylosinus and theMethylococcus groups.

Figure 3

Alignment of partial amino acid sequences deduced from mmoX gene fragments of the acidophilic isolates K, M131, and S6 (stK-M-S) (GenBank accession number AF004554),Methylosinus trichosporium OB3b (Ms.tri),Methylocystis sp. strain M (Mcy.stM), andMethylococcus capsulatus (Bath) (Mc.cap). The sequence stretch corresponds to positions 300 through 458 of the open reading frame published for the mmoX gene of M. capsulatus (Bath) (22). For this stretch, the strains K, M131, and S6 share identical primary structures at the nucleotide and amino acid levels. The identity and similarity values between the amino acid sequence shared by the three strains and these homologous fragments were, for M. capsulatus (Bath), 78.0 and 88.7%, respectively; for Methylosinus trichosporium, OB3b 76.5 and 80.5%, respectively, and for Methylocystis sp. strain M, 78.6 and 83.6%, respectively. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

The 16S ribosomal DNA (rDNA) was sequenced to establish the phylogenetic affiliation of acidophilic strains (23). The 16S rRNA gene sequence was identical for all three strains. The phylogenetic analysis revealed that acidophilic strains are members of the α subclass of Proteobacteria and are most closely related to Beijerinckia indica subsp. indica(Fig. 4); the sequence identity was 96.5%. The identity to the previously known methanotrophic genera,Methylocystis and Methylosinus, was only 90.5 and 92.6%, respectively. Methylocystis spp. andMethylosinus spp. always formed a coherent group in the treeing analyses, which is related to, but clearly distinct from, the branch characterized by the strains K, M131, S6, and B. indica subsp. indica. Apart from B. indicasubsp. indica, Rhodopseudomonas acidophila is the next most closely related organism in the rDNA database, with a similarity value of 93.5%. This organism also is found in acidic, boggy waters and soils and has a pH optimum for growth of 5.5 (24). One of the environmental sequences (clone MPH17) (Fig. 4) included in the phylogenetic analyses was retrieved from a peat core sampled at the Pennine Hills in the north of England (9); it had an identity value of 94.5% to the acidophilic isolates.

Figure 4

16S rDNA-based maximum-likelihood tree constructed for the three acidophilic methanotrophs and 18 reference organisms of the α subclass of Proteobacteriaand three environmental clone sequences [clones MPH14, MPH17 (9), and RB13 (26)] that have been retrieved from acidic environments. Methylococcus capsulatus (Bath) was used as the outgroup. The numbers indicate the statistical significance (percentage of outcome) of the respective interior nodes in 1000 neighbor-joining tests. The scale bar represents the estimated number of base changes per nucleotide sequence position.

Both the mmoX and 16S rRNA gene sequence data indicate that the acidophilic strains represent a lineage of CH4-oxidizing bacteria only moderately related to the known cluster of α-proteobacterial methanotrophs, that is,Methylosinus-Methylocystis spp. Furthermore, the acidophilic strains turned out to be closely affiliated with the heterotrophic bacterium B. indica subsp. indica, contrary to the generally accepted notion that neither known group of methanotrophs has any close relatives that are not methanotrophs (10).Beijerinckia indica is a common inhabitant of acidic soils and has a pH optimum at 5.0 (25). Our isolates and B. indica have many similar features, such as cell morphology, temperature response, pH optimum, and low growth rate. Nevertheless,B. indica is a typical representative of heterotrophic bacteria that uses a large spectrum of sugars and other organic substrates, whereas our strains do not grow on sugars. We attempted to amplify the sMMO gene from B. indica DNA (27), but found no sMMO-like products.

The phylogenetic tree (Fig. 4) is suggestive of a common methanotrophic ancestor for the three acidophilic methanotrophs and theMethylocystis-Methylosinus cluster with a subsequent evolutionary loss of methanotrophic activity in the ancestors of B. indica and R. acidophila because the new strains usually grouped next to theMethylocystis-Methylosinus cluster when the 16SrRNA sequences of B. indica, R. acidophila, and clone MHP17 were excluded from the treeing analyses. However, the bootstrap values in neighbor-joining and maximum parsimony tests were above 80 (percentage of outcome) for the relevant interior node indicating the Methylosinus-Methylocystis group as a monophyletic cluster (82 for the tree shown in Fig. 4), whereas these values were always below 35 for the node shared by the strains K, M131, and S6 and the Methylosinus-Methylocystis group in the same analyses. Thus, considering the known metabolic traits of the acidophilic methanotrophs and of the phylogenetically related bacteria, the hypothesis of a common methanotrophic ancestor for the three acidophilic methanotrophs and the Methylosinus-Methylocystisgroup appears to be very unlikely, but may not be completely discarded. Discussion about the evolutionary history of these two α-proteobacterial methanotrophic groups becomes even more speculative when the amino acid sequence deduced from the partial mmoXgene fragment of the three acidophilic methanotrophs (Fig. 3) is taken into account. This mmoX sequence is clearly distinct from both the Methylocystis-Methylosinus–like mmoXsequences and the M. capsulatus (Bath) mmoXsequence as indicated by the overall identity values between 76.5 and 79% (legend to Fig. 3) to each of the other two mmoXsequence groups.

Most likely, the indigenous methanotrophs of acidic bogs are quite diverse, and our isolates may be the first representatives of a group of previously unculturable MOB. Nevertheless, the ecological fitness of these bacteria to the environmental conditions of Sphagnumbogs suggests that they play a major role in the CH4 cycle in the vast acidic northern wetlands, which are the largest natural source of atmospheric CH4.

  • * To whom correspondence should be addressed. E-mail: tiedjej{at}pilot.msu.edu

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