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Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan
FEMS Microbiology Letters 208 (2002) 239^243
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Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy ¢eld soil in Japan Akio Tonouchi
Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki 036-8224, Japan Received 27 October 2001; received in revised form 3 January 2002; accepted 7 January 2002 First published online 6 February 2002
Abstract A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H2 ^CO2 at 20‡C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H2 ^CO2 , 2-propanol-CO2 , or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Hydrogenotrophic methanogen; Rice paddy soil; Methane; Methanospirillum
1. Introduction Flooded rice paddy ¢elds are major sources of atmospheric methane [1]. In rice ¢eld soil, as in other anaerobic environments, acetate and H2 ^CO2 are the major substrates for methanogenesis [2]. Acetotrophic methanogens, such as Methanosarcina mazei [3], Methanosarcina barkeri [4] and hydrogenotrophic methanogens, such as Methanobacterium bryantii [4], Methanobacterium formicicum [4], Methanobrevibacter arboriphilicus [5] and Methanoculleus marisnigri [4] were also isolated from paddy ¢elds. Moreover, a recent study using molecular methods, independent of cultivation, revealed an unidenti¢ed putative methanogen in rice paddy soil, in addition to known methanogenic species [6]. Amongst many factors a¡ecting microbial and methanogenic activities in anaerobic environments, temperature is also important. Methane production rates from rice paddy soil signi¢cantly decreased when the temperature fell from 30 to 15‡C [7]. Such a temperature shift also led to a decrease in the ratio of H2 ^CO2 thus contributing total methane production from ca. 30% (30‡C) to 10^15%
* Tel. : +81 (172) 39 3781; Fax: +81 (172) 39 3781. E-mail address : [email protected] (A. Tonouchi).
(10‡C), consequently, methane was mainly produced from acetate in a low-temperature environment [8]. Moreover, anaerobic incubation of paddy soil with H2 ^CO2 at a low temperature (17‡C) resulted in a dominant enrichment of homoacetogens except for hydrogenotrophic methanogens [9]. Although temperatures of normal £ooded paddy ¢eld soils are relatively low (below 30‡C), enrichment and isolation of methanogens from rice paddies have commonly been done at 30‡C or higher. Hence, the isolation of hydrogenotrophic methanogen(s) apparently active at relatively low temperatures in a natural rice paddy soil environment was attempted. Here the isolation and characterization of a hydrogenotrophic motile methanogen predominantly enriched at a relatively low temperature (20‡C), from a rice paddy ¢eld in Japan, is reported.
2. Materials and methods 2.1. Soil sample Soil samples were collected from a £ooded rice paddy at the Kanagi Farm of Teaching and Research Center for Bio-coexistence at Hirosaki University, Aomori, Japan in August 2000. The soil temperature of the sampling plot
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 6 1 - 5
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below 8 cm of the soil surface was measured every hour using a Thermo Recorder RT11 (ESPEC). 2.2. Strain The M. bryantii IHR1 strain used in this study was formerly isolated from an H2 ^CO2 enrichment culture at 30‡C from rice paddy soil collected from the above mentioned paddy (unpublished).
scribed elsewhere [11], then ampli¢ed from positions 109 to 934 corresponding to Escherichia coli 16S r RNA numbering. The amplicons were cloned in E. coli JM109, using the pGEM-T Easy Vector System and according to the manufacturer’s instructions (Promega). The cloned amplicons were sequenced using the Amersham-Pharmacia Biotech Gene Rapid sequencer. The sequence was deposited in GenBank, EMBL, and DDBJ database under accession number AB062404.
2.3. Media for isolation and culture of methanogens 3. Results and discussion Enrichment cultures were grown in bicarbonate-bu¡ered de¢ned basal medium prepared as described elsewhere [10]. The pH of the medium was adjusted to 7.3 with 2.0 N HCl. For pure cultures, 2 mM acetate was added to the medium as a carbon source or growth-stimulating reagent. Yeast extract, peptone, 2-propanol and other reagents used for anaerobic culture were prepared anaerobically and sterilized by autoclaving. 2.4. Enrichment and isolation of a hydrogenotrophic methanogen All procedures were carried out anaerobically. The soil sample (0.1 g) was added to 10 ml of the basal medium in 25-ml tubes (Sanshin Kogyo). The tubes were sealed with butyl rubber and stoppered with plastic screw caps, and then pressurized with 2.0 kg cm32 H2 ^CO2 (80:20, v/v). The tubes were incubated at 20‡C without shaking. Cultures showing methane production and complete consumption of added hydrogen were then transferred to fresh medium, pressurized with H2 ^CO2 as before. After three more transfers, a methanogen was isolated from the enrichment culture by repeated application of an anaerobic deep agar dilution series (agar shake) using the same medium except that 0.1% (¢nal concentration) of yeast extract and 2 mM (¢nal concentration) of acetate were added. The purity of the isolate was checked microscopically, then inoculated into the heterotrophic medium under an N2 ^CO2 (80:20) atmosphere.
The enrichment culture incubated under an H2 ^CO2 atmosphere at 20‡C showed methane production in the headspace and acetate production in the aqueous phase, which correlated with consumption of H2 ^CO2 . After the fourth transfer to fresh medium, the enrichment culture was microscopically observed (Fig. 1). The enrichment culture contained predominantly auto£uorescent motile rods morphologically resembling Methanospirillum and non-£uorescent rods with pointed ends morphologically similar to the homoacetogenic Acetobacterium. Methanobacterium-like rods, which are considered a dominant methanogen in rice paddies, were rare in the culture. Nevertheless, when enrichment was applied at 30‡C, growth of Methanobacterium-like rods occurred in addition to Methanospirillum- and Acetobacterium-like cells. The same result was obtained using other paddy soil samples, collected from a rice paddy in Shiga Pref., Japan, indicating that the existence of Methanospirillum-like methanogens is not limited to the paddy in the Kanagi farm. However, predominant growth of Methanospirillum-like cells in the enrichment culture was unexpected, because almost all previous studies on enrichment and isolation of hydrogenotrophic methanogens from rice pad-
2.5. Determination of the DNA G+C content Cells of the isolated strain were lysed with sodium dodecyl sulfate (SDS) and proteinase K treatment. Genomic DNA was puri¢ed with phenol^chloroform treatment and subsequent ethanol precipitation. The G+C content of the DNA was determined by high-performance liquid chromatography. 2.6. 16S rRNA gene sequence determination and analysis A partial region of the 16S rRNA gene of the isolated strain was polymerase chain reaction-ampli¢ed with archaeal group-speci¢c primers (109f and 934r), as de-
Fig. 1. Phase-contrast photomicrograph of the enrichment culture. Cells indicated by arrowheads are representative ones predominantly grown in the culture. (M) Blue-auto£uorescent curved motile rods, (A) non£uorescent rods with pointed ends. Bar, 10 Wm.
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Table 1 Characteristics of strains TM20-1 and M. hungateii
Source Gram-staining Size (Wm) Motility Catalase Temperature range (‡C) Optimum temperature (‡C) pH range Optimum pH NaCl concentration range (mM) Optimum NaCl concentration (mM) G+C content (mol%) Acetate requirement Substrate used a
TM20-1
M. hungateii JF1a
rice ¢eld negative 0.4U6^13 + 3 15^40 30 6.2^8.6 7.2^7.6 0^350 6 17 40.7 + H2 ^CO2 , 2-propanol-CO2 , formate
sewage sludge negative 0.5U7.4 + ND ND 30^37 ND 6.6^7.4 ND ND 45 3 H2 ^CO2 , formate
Data from [16].
dies led to the isolation of Methanobacterium spp. [4,12^ 14]. Members of Methanobacteriaceae are therefore regarded as the predominant group of hydrogenotrophic methanogens in rice paddy soils [15]. However, the presence of Methanospirillum-like methanogen in a paddy soil in France was reported despite unsuccessful isolation [12]. To isolate a Methanospirillum-like methanogen, a deep agar dilution series (agar shake) was prepared using H2 ^ CO2 as a methanogenic substrate. The isolate was named TM20-1. Colonies of TM20-1 formed in deep agar cultures were white and had irregular shapes. TM20-1 was Gram-negative with symmetrically curved cells (Fig. 2), which resembled that of Methanospirillum [16]. TM20-1 occasionally formed wavy ¢laments over 100 Wm. TM20-1 could utilize H2 ^CO2 , 2-propanol-CO2; or formate as methanogenic and growth substrates. Other general substrates for methanogenesis such as acetate and methanol were not used by TM20-1. Acetate was required for growth. TM20-1 was halotolerant and grew at NaCl concentrations of 0^350 mM, with an optimum below 17 mM.
TM20-1 grew at temperatures between 15 and 40‡C, with an optimum around 30‡C. No growth was observed at 45 and 10‡C. Growth was observed between pH 6.2 and 8.6, with an optimum between pH 7.2 and 7.6. Yeast extract and peptone did not stimulate growth. Cell lysis did not occur with 2% SDS or 4 M urea treatment. The G+C content of TM20-1 genome DNA was 40.7mol%. Major features of TM20-1 are listed in Table 1. The 16S rRNA gene sequence of TM20-1 was compared with the 16S rRNA gene sequences of known methanogens (Table 2). The 16S rRNA gene sequence of TM20-1 was more related to that of SAGMA-J2 (similarity of 98,5%), an uncultured archaeon, than to known Methanospirillum hungateii (similarity of 94.8%). The phylogenetic position of TM20-1 between closely related taxa is shown in Fig. 3. Sequence similarity between TM20-1 and M. hungateii is less than 97%, thereby indicating that TM20-1 and M. hungateii are to be regarded as separate species [17]. Thus, TM20-1 can be regarded as a new species of the genus Methanospirillum. From May 12 to September 2 in 2001, when rice ¢elds are £ooded, the temperature of rice ¢eld soil at a sampling Table 2 16S rRNA gene sequence similarities of TM20-1 with the related taxa
Fig. 2. Phase-contrast photomicrograph of the strain TM20-1. Bar, 10 Wm.
Organism
Homology (%)
TM20-1 SAGMA-J2a Soyang 1AF-1100Arb Methanospirillum hungateii Soyang 1AF-1100Arb Methanoculleus olentangyi Methanoculleus palmolei Methanoculleus marisnigri
^ 98.5 95.8 94.8 89.1 86.4 84.5 84.3
a An environmental 16S rDNA sequence of an uncultured archaeon retrieved from the deep subsurface of a South African gold mine (Takai et al., 2000, unpublished). b Environmental 16S rDNA sequences of uncultured archaea retrieved from the sediments of Lake Soyang (Go and Ahn, 1998, unpublished).
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plot, 8 cm below the soil surface, shifted from 11.8 to 27.6‡C and the mean temperature through the period was 21‡C. Hence, selective growth of Methanospirillumlike cells in the enrichment culture at 20‡C suggested that Methanospirillum-like methanogens are more active at relatively low temperatures in a natural paddy environment than all known hydrogenotrophic methanogens, family Methanobacteriaceae, which were considered to be dominant in rice paddies [15]. In view of this, speci¢c growth rates of TM20-1 and M. bryantii IHR1 strain, which was formerly isolated from an enrichment culture on H2 ^CO2 at 30‡C using rice paddy soil (unpublished), were determined at di¡erent temperatures (Fig. 4). As expected, the speci¢c growth rate of TM20-1 below 30‡C was greater than that of IHR1, and TM20-1 was less sensitive to the low temperature: TM20-1 but not IHR1 could grow at 15‡C. The enrichment of Methanospirillum-like cells at 20‡C and faster growth of TM20-1 below 30‡C suggested that there are hydrogenotrophic methanogens like TM20-1 which have an important role in consuming hydrogen at relatively low temperatures and are suitable for inhabiting natural environments of rice paddy soil. A newly identi¢ed hydrogenotrophic methanogen belonging to the genus Methanospirillum in rice paddy soil in Japan has been described. It appeared that the family Methanospirillaceae is also a member of a methanogenic archaeal community in paddy soil. However, no such study has been published except for a single report [12]. Methanospirillum-like cells may be sensitive to change in environment, i.e. exposure to oxygen or starvation, so that it could be di⁄cult to handle the soil samples for isolation of such methanogens. This suggestion is supported by the fact that the anaerobic incubation of an air-dried soil sample under an H2 ^CO2 atmosphere led to no growth of Methanospirillum-like cells. However, a physiological analysis, the tolerance of isolate TM20-1 to oxygen, has not
Fig. 3. Denderogram showing the phylogenetic position of the strain TM20-1 amongst the related taxa. Bar indicates the genetic distance. The accession numbers are given in parentheses.
Fig. 4. E¡ect of temperature on the growth rate of TM20-1 (b) and M. bryantii IHR1 (a). Growth was monitored by measuring the optical density at 420 nm.
yet been determined, and, further studies are needed on the environmental adaptations of this organism.
Acknowledgements I thank M. Ohara for critical reading of the manuscript. I also thank Y. Nishizaki and T. Ooki for providing soil samples. Part of this study was done at the Hirosaki University Gene Research Center.
References [1] Minami, K., Mosier, A. and Sass, R. (1994) CH4 and NO2 : Global Emission and Controls from Rice Fields and other Agricultural and Industrial Sources. NIAES Series 2, Yokendo, Tokyo. [2] Schu«tz, H., Seiler, W. and Conrad, R. (1989) Processes involved in formation and emission of methane in rice paddies. Biogeochemistry 7, 33^53. [3] Asakawa, S., Akagawa-Matsushita, M., Morii, H., Koga, Y. and Hayano, K. (1995) Characterization of Methanosarcina mazei TMA isolated from a paddy ¢eld soil. Curr. Microbiol. 31, 34^38. [4] Joulian, C., Ollivier, B., Patel, B.K.C. and Roger, P.A. (1998) Phenotypic and phylogenetic characterization of dominant culturable methanogens isolated from rice¢eld soils. FEMS Microbiol. Ecol. 25, 135^145. [5] Asakawa, S., Morii, H., Akagawa-Matsushita, M., Koga, Y. and Hayano, K. (1993) Characterization of Methanobrevibacter arboriphilicus SA isolated from a paddy ¢eld soil and DNA^DNA hybridization among M. arboriphilicus strains. Int. J. Syst. Bacteriol. 43, 683^ 686. [6] Lueders, T. and Friedrich, M. (2000) Archaeal population dynamics during sequential reduction processes in rice ¢eld soil. Appl. Environ. Microbiol. 66, 2732^2742. [7] Chin, K.-J. and Conrad, R. (1995) Intermediary metabolism in methanogenic paddy soil and the in£uence of temperature. FEMS Microbiol. Ecol. 18, 85^102. [8] Fey, A. and Conrad, R. (2000) E¡ect of temperature on carbon and electron £ow and on the archaeal community in methanogenic rice ¢eld soil. Appl. Environ. Microbiol. 66, 4790^4797.
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A. Tonouchi / FEMS Microbiology Letters 208 (2002) 239^243 [9] Conrad, R., Bak, F., Seitz, H.J., Thebrath, B., Mayer, H.P. and Schu«tz, H. (1989) Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment. FEMS Microbiol. Ecol. 62, 285^294. [10] Tsurumi, R., Takeda, K. and Tonouchi, A. (2000) Characteristics and propionate production of Propionibacterium isolated from a methane fermentation digester. Microb. Environ. 15, 151^159. [11] GroMkopf, R., Janssen, P.H. and Liesack, W. (1998) Diversity and structure of the methanogenic community in anoxic paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl. Environ. Microbiol. 64, 960^969. [12] Joulian, C., Ollivier, B., Neue, H.-U. and Roger, P.A. (1996) Microbiological aspects of methane emission by a rice¢eld soil from the Camargue (France): 1. Methanogenesis and related micro£ora. Eur. J. Soil Biol. 32, 61^70.
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[13] Adachi, K. (1999) Isolation of hydrogenotrophic methanogenic archaea from a subtropical paddy ¢eld. FEMS Microbiol. Ecol. 30, 77^ 85. [14] Rajagopal, B.S., Belay, N. and Daniels, L. (1988) Isolation and characterization of methanogenic bacteria from rice paddies. FEMS Microbiol. Ecol. 53, 153^158. [15] Liesack, W., Schnell, S. and Revsbech, N.P. (2000) Microbiology of £ooded rice paddies. FEMS Microbiol. Rev. 24, 625^645. [16] Ferry, J.G., Smith, P.H. and Wolfe, R.S. (1974) Methanospirillum, a new genus of methanogenic bacteria, and characterization of Methanospirillum hungateii sp.nov.. Int. J. Syst. Bacteriol. 465^469. [17] Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note: a place for DNA^DNA reassociation and 16S rRNA sequence analysis in the present species de¢nition in bacteriology. Int. J. Syst. Bacteriol. 44, 846^849.
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Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy ¢eld soil in Japan Akio Tonouchi
Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki 036-8224, Japan Received 27 October 2001; received in revised form 3 January 2002; accepted 7 January 2002 First published online 6 February 2002
Abstract A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H2 ^CO2 at 20‡C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H2 ^CO2 , 2-propanol-CO2 , or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Hydrogenotrophic methanogen; Rice paddy soil; Methane; Methanospirillum
1. Introduction Flooded rice paddy ¢elds are major sources of atmospheric methane [1]. In rice ¢eld soil, as in other anaerobic environments, acetate and H2 ^CO2 are the major substrates for methanogenesis [2]. Acetotrophic methanogens, such as Methanosarcina mazei [3], Methanosarcina barkeri [4] and hydrogenotrophic methanogens, such as Methanobacterium bryantii [4], Methanobacterium formicicum [4], Methanobrevibacter arboriphilicus [5] and Methanoculleus marisnigri [4] were also isolated from paddy ¢elds. Moreover, a recent study using molecular methods, independent of cultivation, revealed an unidenti¢ed putative methanogen in rice paddy soil, in addition to known methanogenic species [6]. Amongst many factors a¡ecting microbial and methanogenic activities in anaerobic environments, temperature is also important. Methane production rates from rice paddy soil signi¢cantly decreased when the temperature fell from 30 to 15‡C [7]. Such a temperature shift also led to a decrease in the ratio of H2 ^CO2 thus contributing total methane production from ca. 30% (30‡C) to 10^15%
* Tel. : +81 (172) 39 3781; Fax: +81 (172) 39 3781. E-mail address : [email protected] (A. Tonouchi).
(10‡C), consequently, methane was mainly produced from acetate in a low-temperature environment [8]. Moreover, anaerobic incubation of paddy soil with H2 ^CO2 at a low temperature (17‡C) resulted in a dominant enrichment of homoacetogens except for hydrogenotrophic methanogens [9]. Although temperatures of normal £ooded paddy ¢eld soils are relatively low (below 30‡C), enrichment and isolation of methanogens from rice paddies have commonly been done at 30‡C or higher. Hence, the isolation of hydrogenotrophic methanogen(s) apparently active at relatively low temperatures in a natural rice paddy soil environment was attempted. Here the isolation and characterization of a hydrogenotrophic motile methanogen predominantly enriched at a relatively low temperature (20‡C), from a rice paddy ¢eld in Japan, is reported.
2. Materials and methods 2.1. Soil sample Soil samples were collected from a £ooded rice paddy at the Kanagi Farm of Teaching and Research Center for Bio-coexistence at Hirosaki University, Aomori, Japan in August 2000. The soil temperature of the sampling plot
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 6 1 - 5
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below 8 cm of the soil surface was measured every hour using a Thermo Recorder RT11 (ESPEC). 2.2. Strain The M. bryantii IHR1 strain used in this study was formerly isolated from an H2 ^CO2 enrichment culture at 30‡C from rice paddy soil collected from the above mentioned paddy (unpublished).
scribed elsewhere [11], then ampli¢ed from positions 109 to 934 corresponding to Escherichia coli 16S r RNA numbering. The amplicons were cloned in E. coli JM109, using the pGEM-T Easy Vector System and according to the manufacturer’s instructions (Promega). The cloned amplicons were sequenced using the Amersham-Pharmacia Biotech Gene Rapid sequencer. The sequence was deposited in GenBank, EMBL, and DDBJ database under accession number AB062404.
2.3. Media for isolation and culture of methanogens 3. Results and discussion Enrichment cultures were grown in bicarbonate-bu¡ered de¢ned basal medium prepared as described elsewhere [10]. The pH of the medium was adjusted to 7.3 with 2.0 N HCl. For pure cultures, 2 mM acetate was added to the medium as a carbon source or growth-stimulating reagent. Yeast extract, peptone, 2-propanol and other reagents used for anaerobic culture were prepared anaerobically and sterilized by autoclaving. 2.4. Enrichment and isolation of a hydrogenotrophic methanogen All procedures were carried out anaerobically. The soil sample (0.1 g) was added to 10 ml of the basal medium in 25-ml tubes (Sanshin Kogyo). The tubes were sealed with butyl rubber and stoppered with plastic screw caps, and then pressurized with 2.0 kg cm32 H2 ^CO2 (80:20, v/v). The tubes were incubated at 20‡C without shaking. Cultures showing methane production and complete consumption of added hydrogen were then transferred to fresh medium, pressurized with H2 ^CO2 as before. After three more transfers, a methanogen was isolated from the enrichment culture by repeated application of an anaerobic deep agar dilution series (agar shake) using the same medium except that 0.1% (¢nal concentration) of yeast extract and 2 mM (¢nal concentration) of acetate were added. The purity of the isolate was checked microscopically, then inoculated into the heterotrophic medium under an N2 ^CO2 (80:20) atmosphere.
The enrichment culture incubated under an H2 ^CO2 atmosphere at 20‡C showed methane production in the headspace and acetate production in the aqueous phase, which correlated with consumption of H2 ^CO2 . After the fourth transfer to fresh medium, the enrichment culture was microscopically observed (Fig. 1). The enrichment culture contained predominantly auto£uorescent motile rods morphologically resembling Methanospirillum and non-£uorescent rods with pointed ends morphologically similar to the homoacetogenic Acetobacterium. Methanobacterium-like rods, which are considered a dominant methanogen in rice paddies, were rare in the culture. Nevertheless, when enrichment was applied at 30‡C, growth of Methanobacterium-like rods occurred in addition to Methanospirillum- and Acetobacterium-like cells. The same result was obtained using other paddy soil samples, collected from a rice paddy in Shiga Pref., Japan, indicating that the existence of Methanospirillum-like methanogens is not limited to the paddy in the Kanagi farm. However, predominant growth of Methanospirillum-like cells in the enrichment culture was unexpected, because almost all previous studies on enrichment and isolation of hydrogenotrophic methanogens from rice pad-
2.5. Determination of the DNA G+C content Cells of the isolated strain were lysed with sodium dodecyl sulfate (SDS) and proteinase K treatment. Genomic DNA was puri¢ed with phenol^chloroform treatment and subsequent ethanol precipitation. The G+C content of the DNA was determined by high-performance liquid chromatography. 2.6. 16S rRNA gene sequence determination and analysis A partial region of the 16S rRNA gene of the isolated strain was polymerase chain reaction-ampli¢ed with archaeal group-speci¢c primers (109f and 934r), as de-
Fig. 1. Phase-contrast photomicrograph of the enrichment culture. Cells indicated by arrowheads are representative ones predominantly grown in the culture. (M) Blue-auto£uorescent curved motile rods, (A) non£uorescent rods with pointed ends. Bar, 10 Wm.
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Table 1 Characteristics of strains TM20-1 and M. hungateii
Source Gram-staining Size (Wm) Motility Catalase Temperature range (‡C) Optimum temperature (‡C) pH range Optimum pH NaCl concentration range (mM) Optimum NaCl concentration (mM) G+C content (mol%) Acetate requirement Substrate used a
TM20-1
M. hungateii JF1a
rice ¢eld negative 0.4U6^13 + 3 15^40 30 6.2^8.6 7.2^7.6 0^350 6 17 40.7 + H2 ^CO2 , 2-propanol-CO2 , formate
sewage sludge negative 0.5U7.4 + ND ND 30^37 ND 6.6^7.4 ND ND 45 3 H2 ^CO2 , formate
Data from [16].
dies led to the isolation of Methanobacterium spp. [4,12^ 14]. Members of Methanobacteriaceae are therefore regarded as the predominant group of hydrogenotrophic methanogens in rice paddy soils [15]. However, the presence of Methanospirillum-like methanogen in a paddy soil in France was reported despite unsuccessful isolation [12]. To isolate a Methanospirillum-like methanogen, a deep agar dilution series (agar shake) was prepared using H2 ^ CO2 as a methanogenic substrate. The isolate was named TM20-1. Colonies of TM20-1 formed in deep agar cultures were white and had irregular shapes. TM20-1 was Gram-negative with symmetrically curved cells (Fig. 2), which resembled that of Methanospirillum [16]. TM20-1 occasionally formed wavy ¢laments over 100 Wm. TM20-1 could utilize H2 ^CO2 , 2-propanol-CO2; or formate as methanogenic and growth substrates. Other general substrates for methanogenesis such as acetate and methanol were not used by TM20-1. Acetate was required for growth. TM20-1 was halotolerant and grew at NaCl concentrations of 0^350 mM, with an optimum below 17 mM.
TM20-1 grew at temperatures between 15 and 40‡C, with an optimum around 30‡C. No growth was observed at 45 and 10‡C. Growth was observed between pH 6.2 and 8.6, with an optimum between pH 7.2 and 7.6. Yeast extract and peptone did not stimulate growth. Cell lysis did not occur with 2% SDS or 4 M urea treatment. The G+C content of TM20-1 genome DNA was 40.7mol%. Major features of TM20-1 are listed in Table 1. The 16S rRNA gene sequence of TM20-1 was compared with the 16S rRNA gene sequences of known methanogens (Table 2). The 16S rRNA gene sequence of TM20-1 was more related to that of SAGMA-J2 (similarity of 98,5%), an uncultured archaeon, than to known Methanospirillum hungateii (similarity of 94.8%). The phylogenetic position of TM20-1 between closely related taxa is shown in Fig. 3. Sequence similarity between TM20-1 and M. hungateii is less than 97%, thereby indicating that TM20-1 and M. hungateii are to be regarded as separate species [17]. Thus, TM20-1 can be regarded as a new species of the genus Methanospirillum. From May 12 to September 2 in 2001, when rice ¢elds are £ooded, the temperature of rice ¢eld soil at a sampling Table 2 16S rRNA gene sequence similarities of TM20-1 with the related taxa
Fig. 2. Phase-contrast photomicrograph of the strain TM20-1. Bar, 10 Wm.
Organism
Homology (%)
TM20-1 SAGMA-J2a Soyang 1AF-1100Arb Methanospirillum hungateii Soyang 1AF-1100Arb Methanoculleus olentangyi Methanoculleus palmolei Methanoculleus marisnigri
^ 98.5 95.8 94.8 89.1 86.4 84.5 84.3
a An environmental 16S rDNA sequence of an uncultured archaeon retrieved from the deep subsurface of a South African gold mine (Takai et al., 2000, unpublished). b Environmental 16S rDNA sequences of uncultured archaea retrieved from the sediments of Lake Soyang (Go and Ahn, 1998, unpublished).
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plot, 8 cm below the soil surface, shifted from 11.8 to 27.6‡C and the mean temperature through the period was 21‡C. Hence, selective growth of Methanospirillumlike cells in the enrichment culture at 20‡C suggested that Methanospirillum-like methanogens are more active at relatively low temperatures in a natural paddy environment than all known hydrogenotrophic methanogens, family Methanobacteriaceae, which were considered to be dominant in rice paddies [15]. In view of this, speci¢c growth rates of TM20-1 and M. bryantii IHR1 strain, which was formerly isolated from an enrichment culture on H2 ^CO2 at 30‡C using rice paddy soil (unpublished), were determined at di¡erent temperatures (Fig. 4). As expected, the speci¢c growth rate of TM20-1 below 30‡C was greater than that of IHR1, and TM20-1 was less sensitive to the low temperature: TM20-1 but not IHR1 could grow at 15‡C. The enrichment of Methanospirillum-like cells at 20‡C and faster growth of TM20-1 below 30‡C suggested that there are hydrogenotrophic methanogens like TM20-1 which have an important role in consuming hydrogen at relatively low temperatures and are suitable for inhabiting natural environments of rice paddy soil. A newly identi¢ed hydrogenotrophic methanogen belonging to the genus Methanospirillum in rice paddy soil in Japan has been described. It appeared that the family Methanospirillaceae is also a member of a methanogenic archaeal community in paddy soil. However, no such study has been published except for a single report [12]. Methanospirillum-like cells may be sensitive to change in environment, i.e. exposure to oxygen or starvation, so that it could be di⁄cult to handle the soil samples for isolation of such methanogens. This suggestion is supported by the fact that the anaerobic incubation of an air-dried soil sample under an H2 ^CO2 atmosphere led to no growth of Methanospirillum-like cells. However, a physiological analysis, the tolerance of isolate TM20-1 to oxygen, has not
Fig. 3. Denderogram showing the phylogenetic position of the strain TM20-1 amongst the related taxa. Bar indicates the genetic distance. The accession numbers are given in parentheses.
Fig. 4. E¡ect of temperature on the growth rate of TM20-1 (b) and M. bryantii IHR1 (a). Growth was monitored by measuring the optical density at 420 nm.
yet been determined, and, further studies are needed on the environmental adaptations of this organism.
Acknowledgements I thank M. Ohara for critical reading of the manuscript. I also thank Y. Nishizaki and T. Ooki for providing soil samples. Part of this study was done at the Hirosaki University Gene Research Center.
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