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Isolation and Characterization of New Strains of Methanogens from Cold Terrestrial Habitats
System. Appl. Microbiol. 26, 312–318 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam
Isolation and Characterization of New Strains of Methanogens from Cold Terrestrial Habitats Maria V. Simankova1,2, Oleg R. Kotsyurbenko1, Tillmann Lueders2, Alla N. Nozhevnikova1, Bianca Wagner2, Ralf Conrad2, and Michael W. Friedrich2 1 2
Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany Received: April 2, 2003
Summary Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase α-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1–5 °C, all of them have their growth optima in the range of moderate temperatures (25–35 °C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas. Key words: cold habitats – archaea – methanogens – psychrotolerant
Introduction Methanogenesis is a widespread microbial process that occurs in various habitats, such as gastrointestinal tracts of many animals, wetland soils, and lake sediments, as well as in various extreme habitats, including hot springs, hydrotherms, and lakes of high-salinity. Most methanogenic archaea isolated from the above-mentioned ecosystems are unable to grow at low temperatures (5–15 ºC). However, psychrophilic and psychrotolerant methanogens seem to be widely distributed in natural environments. Most habitats of the Earth have a low average temperature (15 ºC and lower), and more than 80% of the biosphere is permanently cold. Low-temperature ecosystems, such as soils of boreal forests, northern wetlands, anoxic freshwater sediments contribute up to 34% to the total global methane flux from wetlands [3]. Decomposition of organic matter with methane production
in cold habits has been studied in detail over the last 10–15 years [5, 7, 16, 17, 18, 19, 25, 26, 27, 31, 32]. However, up to the beginning of the 1990s, methanogenic archaea able to grow and produce methane at low temperatures (1–5 °C) had not been isolated and described. In the review by Jones et al. [14], three species of methanogens were mentioned that had growth temperatures below 30 °C, but none of the species could grow below 10 °C. The first pure culture of a psychrophilic methanogenic archaeon was isolated and described by Zhilina and Zavarzin [36]. This organism, which belongs to the genus Methanosarcina, grew at temperatures of 5–28 °C. Later, two new species of methanogenic archaea, Methanococcoides burtonii and Methanogenium frigidum, were isolated from the anoxic hypolimnion of Ace Lake in Antarctica [8, 10]. These organisms are rep0723-2020/03/26/02-312 $ 15.00/0
Isolation and Characterization of new Strains of Methanogenes from Cold Terrestrial Habitats
resentatives of the moderately halophilic methanogenic microflora of an extremely cold habitat. Recently, a psychrotolerant methanogen, Methanosarcina lacustris, was isolated from a cold freshwater sediment [33]. However, up to now our knowledge about methanogens from lowtemperature terrestrial ecosystems is scarce. Here, we report the isolation and characterization of five new strains of methanogenic archaea from different cold terrestrial ecosystems. The studied microorganisms are able to grow at temperatures as low as 1–5 °C.
Materials and Methods Isolation sources Samples were taken from the following low-temperature environments: tundra wetland soil [18]; pond polluted with papermill waste water [17]; anoxic sediments of Baldegger Lake (Switzerland) [26]; and cattle manure digested at low temperature [16]. Characteristics of habitats from which samples were taken are given in Table 1. Reference strains Methanosarcina lacustris was from our own culture collection. Methanocorpusculum parvum DSM 3823 and Methanocorpusculum labreanum DSM4855 were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). DNA of Methanomethylovorans hollandica was kindly provided by Dr. H. J. M. Op den Camp, University of Nijmengen, Nijmegen, The Netherlands. Medium and cultivation The medium used for enrichment and further cultivation contained 10 ml/l of a mineral solution [28], 0.05% (w/v) Na2S · 9H2O, 0.15% NaHCO3, 0.002% resazurin, 0.02% yeast extract. 2 ml of microelement and vitamin solutions were added per l of medium. The vitamin solution was prepared as described by Pfennig and Lippert [29]. The trace element solution contained (g/l): FeSO4 · 7H2O, 0.3; Na2SeO4, 0.14; CoCl2 · 6H2O, 0.18; MnCl2 · 4H2O, 0.1; Na2MoO4 · 2H2O, 0.06;
Table 1. Characteristics of habitats from which the samples for the isolation of methanogens were taken. Characteristics of habitat
Name of isolate
Tundra wetland soil (Russia, Polar Ural, 68 N, 65 E). Samples were taken at a depth of 30–40 cm. The soil temperature at this depth was 5–6 ºC, and the pH was 6.1.
MT
Anoxic sediments of a pond polluted with paper-mill waste water of the Syktyvkar Forest Industry Complex (Russia, 62 N, 51 E). Samples were taken at a depth of 5–10 cm. The temperature of the upper layer of the sediments was permanently 4–6 ºC.
MS, MSP
Anoxic sediments of Baldegger Lake (Switzerland). The temperature of sediment was 5–6 ºC throughout the year.
ZB
Digested cattle manure from an anaerobic laboratory reactor operated at 6 ºC.
MM
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ZnSO4 · 7H2O, 0.15; KAl(SO4) · 12H2O, 0.025; NiCl2 · 6H2O, 0.18; H3BO3, 0.01; CuCl2 · 2H2O, 0.025. The medium was anaerobically dispensed into vials, the head space was filled with an N2/CO2 mixture (80:20). Methanol (30 mM), trimethylamine (20 mM), or H2/CO2 were used as substrates for enrichment cultures. When H2/CO2 (80:20) was used as substrate, the gas mixture was added to the head space. Inoculated vials were incubated at 10–15 °C. For the isolation of monocultures and pure cultures, repeated transfers to medium containing 200 mg/l of vancomycin were carried out using 1:10 serial dilutions. Physiological studies To determine the range of substrates utilized, the following compounds were tested: monomethylamine chloride, 20 mM; dimethylamine chloride, 20 mM; trimethylamine chloride, 20 mM; sodium acetate, 20 mM; sodium formate, 20 mM; methanol 30 mM; dimethyl sulfide (DMS), 10 mM; methanethiol, 5 mM; 2-propanol, 20 mM; 2-butanol, 20 mM; H2+CO2, 80:20. Growth was monitored by phase-contrast microscopy and by measurements of methane production. The growth rates were calculated for exponentially growing cultures by a linear regression of the logarithm of the total amount of methane that accumulated versus time. Methane was measured with a gas chromatograph equipped with a thermal conductivity detector and a column packed with activated coal; argon was used as carrier gas (40 ml/min). DNA extraction The procedure used for DNA extraction and purification has been described previously [12]. To verify DNA qualities, aliquots of DNA extracts were analyzed by agarose (1%) gel electrophoresis. PCR amplification of 16S rRNA genes To amplify the nearly complete 16S rRNA gene, the following primers were used: Ar12f (5′ GTT GAT CCT GCC AGA GGY YA-3′) [22] and Ar1542r (5′-GTC CCT GCT CCT TGC ACA CA-3′) [15]. The total volume (50 µl) of the PCR reaction mixture contained 1× PCR buffer II, 50 µM of each deoxyribonucleoside triphosphate, 1.5 µM MgCl2, 0.5 µM of each primer, 1.25 U of AmpliTaq DNA polymerase, and 1 µl of a 1:10 dilution of the DNA template. The PCR reaction was performed by using a GeneAmp PCR System 9700 (Applied Biosystems) with the following thermal program: an initial denaturation step (94 °C, 4 min) was followed by 28 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 90s). After a final extension step (72 °C, 6 min) samples were kept at 4 °C until further analysis. PCR amplification of mcrA genes The following primer combinations were used to amplify mcrA fragments: ME1 (5′- GCM ATG CAR ATH GGW ATG TC –3′) and ME2 (5′- TCA TKG CRT AGT TDG GRT AGT –3′) [11] yielding ~740 bp amplicons; and additionally a new forward primer, MR1 (5′- GAC CTC CAC TWC GTV AAC AAC GC –3′) in combination with ME2, yielding ~1100 bp amplicons. The PCR reaction mixtures and thermal cycling were as previously described [21]. Also, partial mcrA amplicons from strain MSP were cloned and sequenced as published before [21]. Sequence analysis PCR products were purified with the QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Sequencing of PCR products was performed using the BigDye terminator cycle sequencing kit on an ABI 377A DNA sequencer (Applied Biosystems, Forster City, CA). Sequences were assembled using the Laser-
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gene software package (DNASTAR, Madison, Wis.). Alignment and reconstruction of phylogenetic trees were performed using the ARB software package [34] as previously described [21]. 16S rDNA and mcrA sequences obtained in these analyses have been deposited at GenBank under the accession numbers AY260430 to AY260436 and AY260437 to AY260448, respectively.
Results Enrichments for psychrotolerant methanogens were inoculated with samples from permanently cold habitats such as tundra wetland soil, anaerobic laboratory reactor, and anoxic lake and waste-water pond sediments (Table 1) using methanol, trimethylamine, or H2/CO2 as substrates. Pure cultures were obtained after repeated
transfers in the presence of vancomycin for at least half a year and up to two years. Morphology The isolated strains of methanogens exhibited different morphologies. Three strains (MM, MS, and MT) had a characteristic sarcina-like cell arrangement and morphology (Fig. 1). The cells of all these strains formed aggregates of varying size. The cell aggregates of strain MT were large, reaching up to 1 mm in diameter. The aggregates formed by cells of other strains were smaller with a diameter of 0.1–0.5 mm. The aggregates were visible by the unaided eye. The cell morphology of strain ZB differed from that of the three above strains. Cells of this strain never formed aggregates. Only packets of 3–4 cells
Fig. 1. Phase-contrast micrographs of (a) strain MT, (b) strain MM (c) strain ZB, (d) strain MSP. Strains MM and MS (not shown) have the same morphology.
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Fig. 2. Phylogenetic tree reconstructed according to 16S rRNA gene sequences indicating the relationship of the new isolates (marked in bold) to known methanogens. The dendrogram was calculated from almost fulllength sequences using distance matrix based neighbor-joining (shown) and maximum likelihood analyses. The scale bar represents 10% sequence difference. Type strains are as indicated.
were observed (Fig. 1). The cells in the packets were nonmotile irregular cocci with a diameter of 1.0–1.7 µm. Strain MSP was represented by motile irregular cocci with a diameter of 0.3–0.6 µm (Fig. 1). Substrate specificity Strains MM, MS, and MT metabolized a wide variety of substrates, such as mono-, di- and trimethylamines, methanol, and H2/CO2 mixture (Table 2). Strains MT and MS also used acetate, whereas strain MM did not utilize this substrate. All above-mentioned isolates did not grow on formate, methanethiol, and DMS. Strain ZB used only
methylated compounds, such as mono-, di- and trimethylamines, methanol, methanethiol, and DMS. An H2/CO2 mixture and acetate were not utilized. The only substrates for strain MSP were H2/CO2 mixture and formate but growth and methane production during formate consumption were negligible. Phylogenetic analysis of 16S rRNA gene and mcrA gene sequences Almost full-length 16S rRNA gene sequences (~1440 nucleotides) of the new isolates were obtained. According to the sequences data, three strains were affiliated with
Table 2. Some characteristics of new strains of methanogenic archaea. MT
MM
MS
ZB
MSP
Cell diameter (µm) Motility Temperature range (°C) Temperature optimum (°C)
1.2–2.2 – 5–40 35
0.8–1.8 – 1–32 25
0.9–1.8 – 1–32 25
1.0–1.7 – 1–38 30
0.3–0.6 + 5–35 25
Substrate range: H2/CO2 Formate 2-Propanol/CO2 2-Butanol/CO2 Methanol Monomethylamine Dimethylamine Trimethylamine Methanethiol Dimethyl sulfide Acetate
+ – – – + + + + – – +
+ – – – + + + + – – –
+ – – – + + + + – – +
– – – – + + + + + + –
+ + – – – – – – – – –
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Fig. 3. Phylogenetic tree of partial McrA sequences indicating the relationship of the new isolates (marked in bold) to known methanogens. From the isolate MSP and a pure culture of Methanocorpusculum parvum, mcrA-amplicons were cloned prior to sequencing and revealed the presence of more than one gene copy in these strains. The dendrogram was reconstructed from deduced amino acid sequences of partial mcrA gene sequences using Fitch distance matrix analysis. The scale bar represents 10% sequence divergence.
the genus Methanosarcina (Fig. 2). Strain MT was closely related to Methanosarcina mazei (99.8% of sequence similarity). Two strains (MM and MS) were identical in sequence to each other and exhibited 98.5% similarity with Methanosarcina lacustris. Strain ZB was 99.9% similar in 16S rRNA gene sequence to Methanomethylovorans hollandica and strain MSP was classified as a member of the genus Methanocorpusculum. It was closely related to Methanocorpusculum parvum (99.6% sequence similarity) and Methanocorpusculum labreanum (99.4% sequence similarity). Partial sequences of mcrA genes (1100 nucleotides for strains MM and MS, and about 740 nucleotides for the other studied strains) were obtained. Analysis of the mcrA gene sequence fragments showed that strain MT is identical in mcrA gene sequence to Methanosarcina mazei (100% similarity). Strains MM and MS were identical in sequence to each other (Fig. 3) and to Methanosarcina lacustris (100% each) (Fig. 3). The partial mcrA gene of strain ZB was highly related to Methanomethylovorans hollandica (97.7% for DNA and 99.1% for deduced amino acids). Surprisingly, although strain MSP contained only one 16S rRNA gene copy according to direct sequencing, it was impossible to directly obtain an unambiguous mcrA sequence from this culture. Therefore, amplicons were cloned and a small number of clones (n = 10) was sequenced. All clones were definitely affiliated with the genus Methanocorpusculum, but sequencing revealed the presence of at least 3 different mcrA gene copies in this culture, represented by three groups of MSP mcrA clone sequences (1, 2, and 7 clones, each; Fig. 3), which were all related to each other between 95 and 97.3% on the DNA and between 95.3 and 97.5% on the amino acid level. To test, whether more than one mcrA gene copy oc-
curs in one Methanocorpusculum species, mcrA amplicons were generated and cloned also from Methanocorpusculum parvum DSM 3823. Here, sequencing of clones suggested the presence of two distinct gene copies, however they were more than 99% identical.
Discussion Although methanogenesis is operative at low temperature, only very few psychrophilic and psychrotolerant methanogens have been cultivated up to now [9], in spite of intensive efforts of several laboratories to isolate such organisms. We have isolated five new strains of methanogens from enrichment cultures growing at low temperature. The samples for isolation were taken from different natural and man-made environments being under either periodically or permanently cold conditions. According to their phenotypic characteristics, such as morphology and substrate specificity, the new strains of methanogens can be divided into three groups. The first group includes strains MM, MS, and MT, which have sarcina-like cell morphology and use substrates characteristic of the genus Methanosarcina. The second group, represented by strain ZB, forms packets of 3–4 cells and uses only methylated compounds. The third group is represented by strain MSP, cells of which are small cocci and metabolize only H2+CO2 and formate. Phylogenetic analysis confirmed this division and allowed us to affiliate strains MM, MS, and MT to the genus Methanosarcina and strain MSP to the genus Methanocorpusculum [4]. Strain ZB could be classified into the new genus Methanomethylovorans [20], a genus that has not been validated yet (http://www.dsmz.de/bactname.htm).
Isolation and Characterization of new Strains of Methanogenes from Cold Terrestrial Habitats
Strain MT shows morphological and physiological properties which are quite similar to those of Methanosarcina mazei but differs from this mesophilic species in the temperature range of growth. Phylogenetic analyses of 16S rRNA gene and mcrA gene sequences show that strain MT is almost identical in sequence to Methanosarcina mazei. Based on these data and on some phenotypic characteristics, strain MT can be classified as a new ecotype of Methanosarcina mazei, which is able to grow at lower temperature than the type strain. Strains MM and MS differ from all mesophilic species of the genus Methanosarcina in their temperature characteristics (Table 2). Phylogenetic analyses show that these new strains are identical in both, 16S rRNA and mcrA gene sequence, to each other and exhibit a high 16S rRNA (98.5 %) and mcrA gene (100%) sequence similarity to the recently described psychrotolerant species Methanosarcina lacustris (Fig. 2, 3). Based on these data, we were able to affiliate strains MM and MS to Methanosarcina lacustris. Strain ZB exhibits very low divergence from Methanomethylovorans hollandica in its 16S rRNA and mcrA gene sequences (> 99% similarity, Fig. 2, 3) and can be assigned to Methanomethylovorans hollandica as a new ecotype. This organisms uses methanol and methylamines, as well as DMS and methanethiol. Strain ZB is the first methanogen which is able to degrade these compounds in cold fresh water ecosystems at ambient temperature (4–5 °C). Strain MSP, belonging to the genus Methanocorpusculum, demonstrates high level of 16S rRNA gene sequence similarity (> 99%) to Methanocorpusculum parvum and Methanocorpusculum labreanum and is probably also a new ecotype of one of the species mentioned above. Thus, all new strains exhibit close phylogenetic affinity with known mesophilic and psychrotolerant methanogens. Although the affiliation of strain MSP to the genus Methanocorpusculum was unequivocal according to mcrA gene and McrA sequence comparison, the detection of more than one mcrA gene copy in this culture was a surprise. A second mcrA gene copy was also detected in Methanocorpusculum parvum DSM 3823, although with much higher identities (> 99%) than the genes detected in strain MSP (> 95%). These findings are intriguing, since more than a single copy of the mcrA gene has not been detected in any methanogen to date. Members of the orders Methanobacteriales and Methanococcales are known to contain an isoenzyme of the MCR encoded by the mrt-operon (35), but mrtA genes are phylogenetically distinct and the additional mcrA gene copies detected within the Methanocorpusculum strains were clearly not mrtA-like. All five methanogenic strains, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. Rather, they belong to the psychrotolerant microorganisms having a wide temperature range for growth [2, 24, 30]. Our findings corroborate the observation that the major part of the microflora of permanently cold environments is psychrotolerant, but not psychrophilic [1, 6, 13, 23]. Apparently, psychrotolerant
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rather than psychrophilic methanogenic communities are functioning in different terrestrial ecosystems both those that are under permanently cold conditions and those undergoing temperature fluctuations. Three of five isolates have growth limits 10–15 °C lower than those of known mesophilic species, and demonstrate, at the same time, only 0.1–0.4% of 16S rDNA divergence from these species. None of the new isolates is a member of a new genus of methanogenic archaea, but belong to known genera of methanogens as either new species or new ecotypes. This points out the importance of adaptive capacities of methanogens thriving in various cold terrestrial environments. Future research has to show, whether the isolates we obtained by enrichment are in fact numerically abundant in the environments they were retrieved from compared to other methanogens, preferably by using cultivation-independent molecular techniques. Acknowledgments This study was supported in part by the INTAS (grant 962045) and by the Max-Planck-Society, Munich.
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Corresponding author: Michael W. Friedrich, Max-Planck-Institute für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, 5043 Marburg, Germany Tel.: ++49-6421-178 830; Fax: ++49-6421-178 809; e-mail: [email protected]
Isolation and Characterization of New Strains of Methanogens from Cold Terrestrial Habitats Maria V. Simankova1,2, Oleg R. Kotsyurbenko1, Tillmann Lueders2, Alla N. Nozhevnikova1, Bianca Wagner2, Ralf Conrad2, and Michael W. Friedrich2 1 2
Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany Received: April 2, 2003
Summary Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase α-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1–5 °C, all of them have their growth optima in the range of moderate temperatures (25–35 °C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas. Key words: cold habitats – archaea – methanogens – psychrotolerant
Introduction Methanogenesis is a widespread microbial process that occurs in various habitats, such as gastrointestinal tracts of many animals, wetland soils, and lake sediments, as well as in various extreme habitats, including hot springs, hydrotherms, and lakes of high-salinity. Most methanogenic archaea isolated from the above-mentioned ecosystems are unable to grow at low temperatures (5–15 ºC). However, psychrophilic and psychrotolerant methanogens seem to be widely distributed in natural environments. Most habitats of the Earth have a low average temperature (15 ºC and lower), and more than 80% of the biosphere is permanently cold. Low-temperature ecosystems, such as soils of boreal forests, northern wetlands, anoxic freshwater sediments contribute up to 34% to the total global methane flux from wetlands [3]. Decomposition of organic matter with methane production
in cold habits has been studied in detail over the last 10–15 years [5, 7, 16, 17, 18, 19, 25, 26, 27, 31, 32]. However, up to the beginning of the 1990s, methanogenic archaea able to grow and produce methane at low temperatures (1–5 °C) had not been isolated and described. In the review by Jones et al. [14], three species of methanogens were mentioned that had growth temperatures below 30 °C, but none of the species could grow below 10 °C. The first pure culture of a psychrophilic methanogenic archaeon was isolated and described by Zhilina and Zavarzin [36]. This organism, which belongs to the genus Methanosarcina, grew at temperatures of 5–28 °C. Later, two new species of methanogenic archaea, Methanococcoides burtonii and Methanogenium frigidum, were isolated from the anoxic hypolimnion of Ace Lake in Antarctica [8, 10]. These organisms are rep0723-2020/03/26/02-312 $ 15.00/0
Isolation and Characterization of new Strains of Methanogenes from Cold Terrestrial Habitats
resentatives of the moderately halophilic methanogenic microflora of an extremely cold habitat. Recently, a psychrotolerant methanogen, Methanosarcina lacustris, was isolated from a cold freshwater sediment [33]. However, up to now our knowledge about methanogens from lowtemperature terrestrial ecosystems is scarce. Here, we report the isolation and characterization of five new strains of methanogenic archaea from different cold terrestrial ecosystems. The studied microorganisms are able to grow at temperatures as low as 1–5 °C.
Materials and Methods Isolation sources Samples were taken from the following low-temperature environments: tundra wetland soil [18]; pond polluted with papermill waste water [17]; anoxic sediments of Baldegger Lake (Switzerland) [26]; and cattle manure digested at low temperature [16]. Characteristics of habitats from which samples were taken are given in Table 1. Reference strains Methanosarcina lacustris was from our own culture collection. Methanocorpusculum parvum DSM 3823 and Methanocorpusculum labreanum DSM4855 were obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). DNA of Methanomethylovorans hollandica was kindly provided by Dr. H. J. M. Op den Camp, University of Nijmengen, Nijmegen, The Netherlands. Medium and cultivation The medium used for enrichment and further cultivation contained 10 ml/l of a mineral solution [28], 0.05% (w/v) Na2S · 9H2O, 0.15% NaHCO3, 0.002% resazurin, 0.02% yeast extract. 2 ml of microelement and vitamin solutions were added per l of medium. The vitamin solution was prepared as described by Pfennig and Lippert [29]. The trace element solution contained (g/l): FeSO4 · 7H2O, 0.3; Na2SeO4, 0.14; CoCl2 · 6H2O, 0.18; MnCl2 · 4H2O, 0.1; Na2MoO4 · 2H2O, 0.06;
Table 1. Characteristics of habitats from which the samples for the isolation of methanogens were taken. Characteristics of habitat
Name of isolate
Tundra wetland soil (Russia, Polar Ural, 68 N, 65 E). Samples were taken at a depth of 30–40 cm. The soil temperature at this depth was 5–6 ºC, and the pH was 6.1.
MT
Anoxic sediments of a pond polluted with paper-mill waste water of the Syktyvkar Forest Industry Complex (Russia, 62 N, 51 E). Samples were taken at a depth of 5–10 cm. The temperature of the upper layer of the sediments was permanently 4–6 ºC.
MS, MSP
Anoxic sediments of Baldegger Lake (Switzerland). The temperature of sediment was 5–6 ºC throughout the year.
ZB
Digested cattle manure from an anaerobic laboratory reactor operated at 6 ºC.
MM
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ZnSO4 · 7H2O, 0.15; KAl(SO4) · 12H2O, 0.025; NiCl2 · 6H2O, 0.18; H3BO3, 0.01; CuCl2 · 2H2O, 0.025. The medium was anaerobically dispensed into vials, the head space was filled with an N2/CO2 mixture (80:20). Methanol (30 mM), trimethylamine (20 mM), or H2/CO2 were used as substrates for enrichment cultures. When H2/CO2 (80:20) was used as substrate, the gas mixture was added to the head space. Inoculated vials were incubated at 10–15 °C. For the isolation of monocultures and pure cultures, repeated transfers to medium containing 200 mg/l of vancomycin were carried out using 1:10 serial dilutions. Physiological studies To determine the range of substrates utilized, the following compounds were tested: monomethylamine chloride, 20 mM; dimethylamine chloride, 20 mM; trimethylamine chloride, 20 mM; sodium acetate, 20 mM; sodium formate, 20 mM; methanol 30 mM; dimethyl sulfide (DMS), 10 mM; methanethiol, 5 mM; 2-propanol, 20 mM; 2-butanol, 20 mM; H2+CO2, 80:20. Growth was monitored by phase-contrast microscopy and by measurements of methane production. The growth rates were calculated for exponentially growing cultures by a linear regression of the logarithm of the total amount of methane that accumulated versus time. Methane was measured with a gas chromatograph equipped with a thermal conductivity detector and a column packed with activated coal; argon was used as carrier gas (40 ml/min). DNA extraction The procedure used for DNA extraction and purification has been described previously [12]. To verify DNA qualities, aliquots of DNA extracts were analyzed by agarose (1%) gel electrophoresis. PCR amplification of 16S rRNA genes To amplify the nearly complete 16S rRNA gene, the following primers were used: Ar12f (5′ GTT GAT CCT GCC AGA GGY YA-3′) [22] and Ar1542r (5′-GTC CCT GCT CCT TGC ACA CA-3′) [15]. The total volume (50 µl) of the PCR reaction mixture contained 1× PCR buffer II, 50 µM of each deoxyribonucleoside triphosphate, 1.5 µM MgCl2, 0.5 µM of each primer, 1.25 U of AmpliTaq DNA polymerase, and 1 µl of a 1:10 dilution of the DNA template. The PCR reaction was performed by using a GeneAmp PCR System 9700 (Applied Biosystems) with the following thermal program: an initial denaturation step (94 °C, 4 min) was followed by 28 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 90s). After a final extension step (72 °C, 6 min) samples were kept at 4 °C until further analysis. PCR amplification of mcrA genes The following primer combinations were used to amplify mcrA fragments: ME1 (5′- GCM ATG CAR ATH GGW ATG TC –3′) and ME2 (5′- TCA TKG CRT AGT TDG GRT AGT –3′) [11] yielding ~740 bp amplicons; and additionally a new forward primer, MR1 (5′- GAC CTC CAC TWC GTV AAC AAC GC –3′) in combination with ME2, yielding ~1100 bp amplicons. The PCR reaction mixtures and thermal cycling were as previously described [21]. Also, partial mcrA amplicons from strain MSP were cloned and sequenced as published before [21]. Sequence analysis PCR products were purified with the QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Sequencing of PCR products was performed using the BigDye terminator cycle sequencing kit on an ABI 377A DNA sequencer (Applied Biosystems, Forster City, CA). Sequences were assembled using the Laser-
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gene software package (DNASTAR, Madison, Wis.). Alignment and reconstruction of phylogenetic trees were performed using the ARB software package [34] as previously described [21]. 16S rDNA and mcrA sequences obtained in these analyses have been deposited at GenBank under the accession numbers AY260430 to AY260436 and AY260437 to AY260448, respectively.
Results Enrichments for psychrotolerant methanogens were inoculated with samples from permanently cold habitats such as tundra wetland soil, anaerobic laboratory reactor, and anoxic lake and waste-water pond sediments (Table 1) using methanol, trimethylamine, or H2/CO2 as substrates. Pure cultures were obtained after repeated
transfers in the presence of vancomycin for at least half a year and up to two years. Morphology The isolated strains of methanogens exhibited different morphologies. Three strains (MM, MS, and MT) had a characteristic sarcina-like cell arrangement and morphology (Fig. 1). The cells of all these strains formed aggregates of varying size. The cell aggregates of strain MT were large, reaching up to 1 mm in diameter. The aggregates formed by cells of other strains were smaller with a diameter of 0.1–0.5 mm. The aggregates were visible by the unaided eye. The cell morphology of strain ZB differed from that of the three above strains. Cells of this strain never formed aggregates. Only packets of 3–4 cells
Fig. 1. Phase-contrast micrographs of (a) strain MT, (b) strain MM (c) strain ZB, (d) strain MSP. Strains MM and MS (not shown) have the same morphology.
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Fig. 2. Phylogenetic tree reconstructed according to 16S rRNA gene sequences indicating the relationship of the new isolates (marked in bold) to known methanogens. The dendrogram was calculated from almost fulllength sequences using distance matrix based neighbor-joining (shown) and maximum likelihood analyses. The scale bar represents 10% sequence difference. Type strains are as indicated.
were observed (Fig. 1). The cells in the packets were nonmotile irregular cocci with a diameter of 1.0–1.7 µm. Strain MSP was represented by motile irregular cocci with a diameter of 0.3–0.6 µm (Fig. 1). Substrate specificity Strains MM, MS, and MT metabolized a wide variety of substrates, such as mono-, di- and trimethylamines, methanol, and H2/CO2 mixture (Table 2). Strains MT and MS also used acetate, whereas strain MM did not utilize this substrate. All above-mentioned isolates did not grow on formate, methanethiol, and DMS. Strain ZB used only
methylated compounds, such as mono-, di- and trimethylamines, methanol, methanethiol, and DMS. An H2/CO2 mixture and acetate were not utilized. The only substrates for strain MSP were H2/CO2 mixture and formate but growth and methane production during formate consumption were negligible. Phylogenetic analysis of 16S rRNA gene and mcrA gene sequences Almost full-length 16S rRNA gene sequences (~1440 nucleotides) of the new isolates were obtained. According to the sequences data, three strains were affiliated with
Table 2. Some characteristics of new strains of methanogenic archaea. MT
MM
MS
ZB
MSP
Cell diameter (µm) Motility Temperature range (°C) Temperature optimum (°C)
1.2–2.2 – 5–40 35
0.8–1.8 – 1–32 25
0.9–1.8 – 1–32 25
1.0–1.7 – 1–38 30
0.3–0.6 + 5–35 25
Substrate range: H2/CO2 Formate 2-Propanol/CO2 2-Butanol/CO2 Methanol Monomethylamine Dimethylamine Trimethylamine Methanethiol Dimethyl sulfide Acetate
+ – – – + + + + – – +
+ – – – + + + + – – –
+ – – – + + + + – – +
– – – – + + + + + + –
+ + – – – – – – – – –
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Fig. 3. Phylogenetic tree of partial McrA sequences indicating the relationship of the new isolates (marked in bold) to known methanogens. From the isolate MSP and a pure culture of Methanocorpusculum parvum, mcrA-amplicons were cloned prior to sequencing and revealed the presence of more than one gene copy in these strains. The dendrogram was reconstructed from deduced amino acid sequences of partial mcrA gene sequences using Fitch distance matrix analysis. The scale bar represents 10% sequence divergence.
the genus Methanosarcina (Fig. 2). Strain MT was closely related to Methanosarcina mazei (99.8% of sequence similarity). Two strains (MM and MS) were identical in sequence to each other and exhibited 98.5% similarity with Methanosarcina lacustris. Strain ZB was 99.9% similar in 16S rRNA gene sequence to Methanomethylovorans hollandica and strain MSP was classified as a member of the genus Methanocorpusculum. It was closely related to Methanocorpusculum parvum (99.6% sequence similarity) and Methanocorpusculum labreanum (99.4% sequence similarity). Partial sequences of mcrA genes (1100 nucleotides for strains MM and MS, and about 740 nucleotides for the other studied strains) were obtained. Analysis of the mcrA gene sequence fragments showed that strain MT is identical in mcrA gene sequence to Methanosarcina mazei (100% similarity). Strains MM and MS were identical in sequence to each other (Fig. 3) and to Methanosarcina lacustris (100% each) (Fig. 3). The partial mcrA gene of strain ZB was highly related to Methanomethylovorans hollandica (97.7% for DNA and 99.1% for deduced amino acids). Surprisingly, although strain MSP contained only one 16S rRNA gene copy according to direct sequencing, it was impossible to directly obtain an unambiguous mcrA sequence from this culture. Therefore, amplicons were cloned and a small number of clones (n = 10) was sequenced. All clones were definitely affiliated with the genus Methanocorpusculum, but sequencing revealed the presence of at least 3 different mcrA gene copies in this culture, represented by three groups of MSP mcrA clone sequences (1, 2, and 7 clones, each; Fig. 3), which were all related to each other between 95 and 97.3% on the DNA and between 95.3 and 97.5% on the amino acid level. To test, whether more than one mcrA gene copy oc-
curs in one Methanocorpusculum species, mcrA amplicons were generated and cloned also from Methanocorpusculum parvum DSM 3823. Here, sequencing of clones suggested the presence of two distinct gene copies, however they were more than 99% identical.
Discussion Although methanogenesis is operative at low temperature, only very few psychrophilic and psychrotolerant methanogens have been cultivated up to now [9], in spite of intensive efforts of several laboratories to isolate such organisms. We have isolated five new strains of methanogens from enrichment cultures growing at low temperature. The samples for isolation were taken from different natural and man-made environments being under either periodically or permanently cold conditions. According to their phenotypic characteristics, such as morphology and substrate specificity, the new strains of methanogens can be divided into three groups. The first group includes strains MM, MS, and MT, which have sarcina-like cell morphology and use substrates characteristic of the genus Methanosarcina. The second group, represented by strain ZB, forms packets of 3–4 cells and uses only methylated compounds. The third group is represented by strain MSP, cells of which are small cocci and metabolize only H2+CO2 and formate. Phylogenetic analysis confirmed this division and allowed us to affiliate strains MM, MS, and MT to the genus Methanosarcina and strain MSP to the genus Methanocorpusculum [4]. Strain ZB could be classified into the new genus Methanomethylovorans [20], a genus that has not been validated yet (http://www.dsmz.de/bactname.htm).
Isolation and Characterization of new Strains of Methanogenes from Cold Terrestrial Habitats
Strain MT shows morphological and physiological properties which are quite similar to those of Methanosarcina mazei but differs from this mesophilic species in the temperature range of growth. Phylogenetic analyses of 16S rRNA gene and mcrA gene sequences show that strain MT is almost identical in sequence to Methanosarcina mazei. Based on these data and on some phenotypic characteristics, strain MT can be classified as a new ecotype of Methanosarcina mazei, which is able to grow at lower temperature than the type strain. Strains MM and MS differ from all mesophilic species of the genus Methanosarcina in their temperature characteristics (Table 2). Phylogenetic analyses show that these new strains are identical in both, 16S rRNA and mcrA gene sequence, to each other and exhibit a high 16S rRNA (98.5 %) and mcrA gene (100%) sequence similarity to the recently described psychrotolerant species Methanosarcina lacustris (Fig. 2, 3). Based on these data, we were able to affiliate strains MM and MS to Methanosarcina lacustris. Strain ZB exhibits very low divergence from Methanomethylovorans hollandica in its 16S rRNA and mcrA gene sequences (> 99% similarity, Fig. 2, 3) and can be assigned to Methanomethylovorans hollandica as a new ecotype. This organisms uses methanol and methylamines, as well as DMS and methanethiol. Strain ZB is the first methanogen which is able to degrade these compounds in cold fresh water ecosystems at ambient temperature (4–5 °C). Strain MSP, belonging to the genus Methanocorpusculum, demonstrates high level of 16S rRNA gene sequence similarity (> 99%) to Methanocorpusculum parvum and Methanocorpusculum labreanum and is probably also a new ecotype of one of the species mentioned above. Thus, all new strains exhibit close phylogenetic affinity with known mesophilic and psychrotolerant methanogens. Although the affiliation of strain MSP to the genus Methanocorpusculum was unequivocal according to mcrA gene and McrA sequence comparison, the detection of more than one mcrA gene copy in this culture was a surprise. A second mcrA gene copy was also detected in Methanocorpusculum parvum DSM 3823, although with much higher identities (> 99%) than the genes detected in strain MSP (> 95%). These findings are intriguing, since more than a single copy of the mcrA gene has not been detected in any methanogen to date. Members of the orders Methanobacteriales and Methanococcales are known to contain an isoenzyme of the MCR encoded by the mrt-operon (35), but mrtA genes are phylogenetically distinct and the additional mcrA gene copies detected within the Methanocorpusculum strains were clearly not mrtA-like. All five methanogenic strains, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. Rather, they belong to the psychrotolerant microorganisms having a wide temperature range for growth [2, 24, 30]. Our findings corroborate the observation that the major part of the microflora of permanently cold environments is psychrotolerant, but not psychrophilic [1, 6, 13, 23]. Apparently, psychrotolerant
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rather than psychrophilic methanogenic communities are functioning in different terrestrial ecosystems both those that are under permanently cold conditions and those undergoing temperature fluctuations. Three of five isolates have growth limits 10–15 °C lower than those of known mesophilic species, and demonstrate, at the same time, only 0.1–0.4% of 16S rDNA divergence from these species. None of the new isolates is a member of a new genus of methanogenic archaea, but belong to known genera of methanogens as either new species or new ecotypes. This points out the importance of adaptive capacities of methanogens thriving in various cold terrestrial environments. Future research has to show, whether the isolates we obtained by enrichment are in fact numerically abundant in the environments they were retrieved from compared to other methanogens, preferably by using cultivation-independent molecular techniques. Acknowledgments This study was supported in part by the INTAS (grant 962045) and by the Max-Planck-Society, Munich.
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Corresponding author: Michael W. Friedrich, Max-Planck-Institute für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, 5043 Marburg, Germany Tel.: ++49-6421-178 830; Fax: ++49-6421-178 809; e-mail: [email protected]