Coenzyme M and 2-methylbutyric acid were not required in the presence of rumen fluid. 16S rDNA sequence analysis confirmed the signature sequence of the ...
International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1081–1087
DOI : 10.1099/ijs.0.01903-0
Methanobrevibacter acididurans sp. nov., a novel methanogen from a sour anaerobic digester 1
Agharkar Research Institute, G. G. Agarkar Road, Pune 411 004, India
D. V. Savant,1 Y. S. Shouche,2 S. Prakash2 and D. R. Ranade1
2
National Centre for Cell Science, Pune University Campus, Pune 411 007, India
Author for correspondence : D. R. Ranade. Tel : j91 020 5653680. Fax : j91 020 5651542. e-mail : drr!aripune.ernet.in
A novel acid-tolerant, hydrogenotrophic methanogen, isolate ATMT, was obtained from an enrichment performed at pH 50 using slurry from an acidogenic digester running on alcohol distillery waste. The original pH of the slurry was 57 and the volatile fatty acid concentration was 9000 p.p.m. Cells of isolate ATMT were Gram-positive, non-motile and 03–05 µm in size. They did not form spores. The isolate could grow in the pH range 50–75, with maximum growth at pH 60. The optimum temperature for growth was 35 SC. Formate, acetate, methanol, trimethylamine, 2-propanol and 2-butanol were not utilized as growth substrates. Rumen fluid and acetate were required for growth on H2/CO2. Coenzyme M and 2-methylbutyric acid were not required in the presence of rumen fluid. 16S rDNA sequence analysis confirmed the signature sequence of the genus Methanobrevibacter. Morphological and biochemical characteristics of the isolate, together with the 16S rDNA sequence analysis, clearly revealed that the isolate could not be accommodated within any of the existing species of the genus Methanobrevibacter. Therefore, it is proposed that a novel species of the genus Methanobrevibacter should be created for this isolate, Methanobrevibacter acididurans sp. nov., and the type strain is strain ATMT (l MCM B 613T l OCM 804T).
Keywords : methanogen, Methanobrevibacter, low pH, anaerobic digestion
INTRODUCTION
Anaerobic treatment of industrial wastes is becoming increasingly popular because of the generation of a valuable by-product, methane. The success of anaerobic treatment depends mainly on the optimal activity of methanogens. These bacteria are highly sensitive to changes in environmental conditions such as temperature and pH. Changes in operational parameters, e.g. an increase in the organic loading rate, lead to an increase in the volatile fatty acid concentration and a decrease in the pH of the digesting mass. These changes adversely affect the growth of methanogens (Kugelman & Chin, 1971 ; Pohland, 1992). This kind of effect is commonly seen with .................................................................................................................................................
Published online ahead of print on 22 March 2002 as 10.1099/ijs.0.01903-0. A colour micrograph of the novel isolate is available as supplementary material in IJSEM Online (http ://ijs.sgmjournals.org/). The GenBank accession number for the 16S rDNA sequence of Methanobrevibacter acididurans ATMT is AF242652. 01903 # 2002 IUMS Printed in Great Britain
digesters running on high-strength organic wastewaters such as those arising from alcohol distilleries and food-processing units. To overcome the problems arising from sour digestion, one of the alternatives is to utilize methanogens that tolerate low pH ("5n5–6n0) and high concentrations of volatile fatty acids. Over 65 species of methanogen belonging to 20 different genera are known today (Sowers, 1995). However, most of them grow optimally in the pH range around neutral to slightly alkaline conditions (pH 6n8–8n5). Many groups of scientists have attempted the isolation of acid-tolerant methanogens (Williams & Crawford, 1985 ; Ladapo & Barlaz, 1997). A methanogen identified as Methanobacterium uliginosum (Ko$ nig, 1984) has been reported to grow at pH 6n0–8n0. Patel et al. (1990) obtained Methanobacterium espanolae, which was able to grow at pH 5n5–6n2 ; this can be considered as an example of a truly acidophilic methanogen. Certain strains of Methanosarcina have been shown to grow at low pH, using methanol and H as the substrate # 1081
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(Maestrojua! n & Boone, 1991). However, methanol is not commonly available in anaerobic digesters. Many hyperthermophiles have been reported to grow at low pH (Jones et al., 1983 ; Burggraf et al., 1990 ; Jeanthon et al., 1999). However, these methanogens have limited potential as far as anaerobic digesters are concerned. In our laboratory, a two-stage anaerobic treatment process of alcohol distillery waste was developed. Although the fermenting slurry of the acidogenic digester had a pH around 5n6–5n8 and a volatile fatty acid concentration of 8000–10 000 p.p.m., methanogens were regularly observed in this digester. For this reason, methanogens were isolated that might have potential application in the treatment of acidic wastewaters and the recovery of sour anaerobic digesters. METHODS Source of inoculum. A two-stage anaerobic treatment pro-
cess for distillery waste was developed in the laboratory in 25 l continuously stirred tank reactors. The sludge of a fieldscale anaerobic digester (initiated with cattle dung as the inoculum and running on distillery waste) was used as the starter culture for establishing this process. The acidogenic digester was operated initially at a hydraulic retention time of 10 days on undiluted distillery waste neutralized to pH 7n0 with Ca(OH) . The loading rate was increased to reach an # kg COD l−" day−" with a hydraulic retention optimum at 18 time of 4 days. Under these conditions and at ambient temperature, the biogas produced contained 15 % (v\v) CH , the remaining gas being CO . The pH of the digesting % was 5n7p0n1. The volatile# fatty acid content was slurry 8000–10 000 p.p.m. The slurry of this digester was used as the inoculum for the isolation of methanogens. Before sample collection, the digester was in operation for 60 days. Growth medium. In the light of the composition of distillery waste and the nutritional requirements of methanogens in general, DVS medium was formulated. It had the following composition (l−" distilled water) : 0n3 g KH PO , 0n3 g # ;% 2H O, K HPO , 1n5 g (NH ) SO , 0n6 g NaCl, 0n08 g CaCl # % % # % # # g 0n1 g MgCl ; 6H O, 2n0 g yeast extract, 1n0 g peptone, 1n0 # # tryptone, 2n5 g sodium acetate, 2n5 g sodium formate, 1 ml trace-element solution (Touzel & Albagnac, 1983), 1 ml trace-vitamin solution (Wolin et al., 1963), 20 ml fatty acid mixture (Sowers & Schreier, 1995), 0n5 g cysteine hydrochloride, 0n5 g Na S ; 9H O and 1 mg resazurin. Coenzyme M was added at #a final #concentration of 50 ng ml−". The medium was prepared under a H \CO (80 : 20, v\v) gas # # using 10 M HCl phase. The pH of the medium was adjusted or NaHCO . The medium was sterilized by autoclaving at $ min. 121 mC for 30 Clarified rumen fluid (Ranade & Gadre, 1988b) was acidified, using 5 M HCl, to pH 5n0 under N and was sterilized # (10 %, v\v) as a by autoclaving. Rumen fluid was added source of unknown growth factors. DVS medium supplemented with rumen fluid was termed DVSR medium. Anaerobic techniques originally developed by Hungate (1969), modified by Miller & Wolin (1974) and further described by Ranade & Gadre (1988a) were followed. All experiments were carried out in duplicate under static conditions at 35 mC unless mentioned otherwise. The vials with H \CO (80 : 20, v\v) as the substrate were repressurized # # to 101n3 kPa unless mentioned otherwise. every other day 1082
Enrichment. Enrichments were performed in DVS medium adjusted to different pH values (4n0, 5n0 and 6n0) under the H \CO (80 : 20, v\v) gas phase. Vials containing 9 ml # # were inoculated with 1 ml inoculum and incubated medium at 35 mC. Isolation. Methanogens were isolated in roll tubes from the fifth transfer of the enrichment at pH 5n0 after 7 days incubation under static conditions. DVSR medium at pH 5n0 in H \CO (80 : 20, v\v) and solidified with 3 g % agar # was# used. (Difco) Purity of the culture. Colonies obtained in the roll tubes showing methane production were transferred to liquid DVSR medium in an anaerobic glove box (Forma Scientific). The gas phase in these vials was replaced with H \CO (80 : # medium # 20, v\v). The organism was re-isolated on solid from the liquid cultures. To check whether the culture was free from aerobic contaminants, it was inoculated on nutrient agar plates. The presence of anaerobic contaminants was checked using liquid PYG medium (Holdeman & Moore, 1975) under N . # Maintenance. The culture was maintained by serial transfers in liquid DVSR medium (pH 5n0) every 3 weeks at 10 % (v\v) inoculum. Microscopy. Cells were routinely observed under a microscope equipped with phase-contrast and a fluorescence attachment with a UV excitation filter in the range 380–420 nm (Nikon). The Gram character was determined using a standard Gram-stain kit (Hi Media). Motility was determined by the hanging-drop method, using a glass cavity slide. Spore formation was checked by using the pasteurization test and by observation of the treated cell suspension by phase-contrast microscopy. For scanning electron microscopy, cells from the exponential phase of growth were fixed using Trumps 4F : 1G fixative on a glass coverslip at 4 mC overnight. Cells were washed with distilled water and dehydrated by gradual washing with an increasing gradient of ethanol. The sample was air-dried and mounted on a stub. Gold coating (20 nm thick) of the sample was done using an E 5200 Auto Sputter Coater (Bio-Rad). Observations were made on a stereoscan S120 scanning electron microscope (Cambridge Instruments). Growth substrates. DVSR medium without acetate and formate, prepared under N , was used for substrate-utiliz# of filter-sterilized substrates ation studies. Anaerobic stocks (acetate, methanol, formate, trimethylamine, 2-propanol and 2-butanol) were prepared and added separately at final concentrations of 50 mM. A freshly grown culture was inoculated at 10 % (v\v) and vials were incubated at 35 mC for 28 days. Medium under H \CO served as the control. # substrate was also The ability to use H \acetate# as the # checked. Growth was determined on the basis of the methane produced. Susceptibility to lysis. Cells from the exponentially growing culture were used to check susceptibility to lysis by 1 % SDS and distilled water as a hypotonic solution (Boone & Whitman, 1988). Lysis was determined by visual and microscopic observation of the culture. Unexposed culture served as the control. Bile sensitivity. The sensitivity of the culture to different bile salts was examined in DVSR medium. Anaerobic stocks of sodium taurocholate, sodium deoxycholate and ox bile were prepared by dissolving the required amount of salt in a known quantity of distilled water that had been pre-boiled and cooled under a flow of N . The pH of the solution was # under N . The stocks were adjusted and the vials were sealed # International Journal of Systematic and Evolutionary Microbiology 52
Methanobrevibacter acididurans sp. nov. sterilized by autoclaving and then added separately to the medium to achieve final concentrations of 0n1, 0n1 and 2n0 % (w\v), respectively. The culture was inoculated at 10 % (v\v) and incubated at 35 mC for 28 days. Methane production in each vial was then checked. Growth experiments. The culture was grown in DVSR medium at 35 mC with shaking at 150 r.p.m. It was observed that growth, in terms of OD at 660 nm, had a linear relationship with methanogenesis during the exponential phase and for most of the subsequent linear phase. Hence, for quantitative studies, growth was monitored by measuring the amount of methane produced. The specific growth rate was calculated from the exponential parts of the methane-formation curve. Maximum methane, 17n67 µmol (ml headspace)−", was produced in 120 h when the OD was 0n640. Optimum temperature. The optimum temperature was determined in DVSR medium at pH 5n0. Vials inoculated with 10 % (v\v) culture were incubated at temperatures in the range 25–40 mC, using 5 mC intervals. The vials were pressurized daily with H \CO to ensure an adequate supply # of substrate. Growth was# measured every other day in terms of methane produced (Boone & Whitman, 1988). Optimum pH. The pH requirement was determined at the
optimum temperature in DVSR medium adjusted to pH values ranging from 5n0 to 7n5 using different concentrations of NaHCO or 10 M HCl. The proportion of CO was kept constant at$ 20 % (v\v) as H \CO (80 : 20, v\v) #in the gas # # was maintained after phase. The pH of the medium autoclaving. The vials were inoculated with freshly grown culture (10 %, v\v). The headspace was pressurized to 101n3 kPa every day and methane production was measured. The vial headspace was then reflushed for 3 min and then repressurized to maintain the pH of the broth (Patel et al., 1990). After incubation, the pH remained within 0n1 unit of the desired value. Specific growth rates were calculated on the basis of the cumulative methane production. Nutritional requirements. Cells from a freshly grown culture
were harvested by centrifugation at 10 000 g for 10 min at 4 mC and then washed twice with anaerobic saline to prevent carry-over of nutrients. The pellet was resuspended in anaerobic saline and used as the inoculum. DVS and DVSR media were used to study the requirement for rumen fluid, acetate, coenzyme M and 2-methylbutyric acid. Growth was determined by measuring methane production. 16S rDNA sequencing. The DNA was extracted from 3 ml
freshly grown culture (Erb & Wagner-Dobler, 1993). The 16S rRNA gene was amplified using a PCR (Perkin Elmer) as described previously (Dighe et al., 1998). The primer pair used here was MbUr\MbUf (Lin & Miller, 1998). The resulting 1n4 kb fragment was cloned directly in the vector pGEM-T Easy (Promega). The complete 1n4 kb sequence was obtained using universal sequencing and internal primers. Phylogenetic analysis. The sequence was analysed by using
and at the Ribosomal Database Project (RDP II ; Maidak et al., 1999). Sequence similarities were determined at RDP II using I (Maidak et al., 1999). Sequences were aligned using (Thompson et al., 1994) at the EBI site. Phylogenetic analyses were done in (Kumar et al., 1994). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) by calculating the genetic distance with Kimura’s two-parameter model (Kimura, 1980) and using the Jukes–
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Cantor model (Jukes & Cantor, 1969). Bootstrap values were obtained from 1000 random samplings. Gas analysis. Methane was analysed on a gas chromatograph (Chemito 3800 ; Toshniwal) equipped with a thermal conductivity detector. The column used was Porapak Q (80\100 mesh, 3n2i2 mm, SS). Hydrogen (30 ml min−") was the carrier gas. The temperature settings were as follows : oven, 50 mC ; injector, 60 mC ; detector, 80 mC. Pure methane (Span Gas) was used as the external standard. Data integration and analysis were done using software.
RESULTS Enrichment
Within 10 days of initiation of the enrichments, methane was detected in the headspace gas. When the methane concentration in the headspace gas reached 50–60 % (v\v), the enrichments were transferred (10 %, v\v) to fresh medium. Upon subsequent transfers, it was observed that the enrichment at pH 4n0 grew poorly and little methane was produced. The enrichment at pH 5n0 was selected for further studies as it showed better methane production than the enrichment at pH 6n0. Microscopic observations revealed cocci with uniform morphology that occurred singly, in pairs and in chains. The cells consistently possessed a characteristic blue-green fluorescence when exposed to UV radiation (a colour micrograph of cells of the novel isolate is available as supplementary material in IJSEM Online ; http :\\ijs.sgmjournals.org\). Addition of vancomycin at 100 µg ml−" during the third transfer reduced the number of non-fluorescent bacteria. Sterile rumen fluid was also added at this stage of enrichment and was found to stimulate the growth of the fluorescent bacteria. Both additions were continued in subsequent transfers. After two more transfers, isolation was undertaken. Isolation
In the roll tubes, methane was produced after 12 days incubation. Well-isolated colonies were selected and transferred to liquid medium in an anaerobic glove box. After the production of methane during growth in the broth, the purity of the isolate was checked by means of re-isolation in a roll tube. All colonies were brown and pinpoint with irregular margins. The absence of organotrophic contaminants was confirmed by failure to grow in anaerobic PYG medium and aerobic nutrient agar plates. The isolate was designated strain ATMT. Morphology
Cells were more or less round, 0n3–0n5 µm in size and occurred singly, in pairs or in chains of 8–12 cells (Fig. 1). Cells tended to form loose aggregates upon prolonged incubation. Cells were Gram-positive and nonmotile. They did not form spores, as indicated by phase-contrast microscopy. Cells showed the bluegreen fluorescence typical of methanogens, and this 1083
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Fig. 2. Effect of pH on the growth of strain ATMT in DVSR medium.
Cell lysis
(a)
No decrease in turbidity was observed after exposure to 1 % SDS or distilled water for 10 min. Microscopic observations also revealed intact cells. Thus, the cells were found to be resistant to lysis by SDS and hypotonic solution. Optimum temperature
The isolate grew at temperatures in the range 25–37 mC. The optimum temperature was 35 mC. No growth was obtained at or above 40 mC. Optimum pH
The isolate grew and produced methane over a wide range of pH (5n0–7n5). On the basis of specific growth rates, the optimum pH was found to be 6n0 (Fig. 2). No growth was obtained at pH 4n5. Bile tolerance (b) .................................................................................................................................................
Fig. 1. Morphology of isolate ATMT. (a) Fluorescence micrograph of cells showing the shape of the cells and the formation of chains ; bar, 25 µm. A colour version of this image showing the characteristic blue-green fluorescence of cells of strain ATMT when exposed to UV radiation is available as supplementary material in IJSEM Online (http ://ijs. sgmjournals.org/). (b) Scanning electron micrograph ; bar, 1 µm.
remained consistent over a longer duration. Neither flagella nor polar fibrils were observed under scanning and transmission electron microscopy. Growth substrates
Isolate ATMT used only H \CO as the substrate for # was# observed with acetmethanogenesis. No growth ate, formate, trimethylamine, methanol, 2-propanol or 2-butanol with N as the gas phase. Acetate plus H # was not utilized. # 1084
At pH 7n0, 0n1 % sodium taurocholate, 0n1 % sodium deoxycholate and 2n0 % (w\v) ox bile were inhibitory. At pH 5n0, sodium taurocholate (0n1 %, w\v) was inhibitory, but ox bile and sodium deoxycholate were precipitated. Thus, the isolate was found to be sensitive to bile salts. Nutritional requirements
When the culture grown in DVSR medium was inoculated into DVS medium (10 %, v\v), growth was greatly diminished. Furthermore, after successive transfers in DVS medium, growth ceased. A culture washed with anaerobic saline failed to grow when inoculated into DVS medium. Thus, the presence of acetate, coenzyme M and 2-methylbutyric acid in DVS medium was not sufficient for growth and maintenance of the strain. However, the addition of rumen fluid restored growth in DVS medium without acetate, coenzyme M and 2-methylbutyric acid. This showed that the addition of rumen fluid was necessary for the growth and maintenance of strain ATMT. Addition of acetate (20 mM) stimulated growth in DVSR medium. International Journal of Systematic and Evolutionary Microbiology 52
Methanobrevibacter acididurans sp. nov. .....................................................................................................
Fig. 3. Phylogenetic position of strain ATMT. The tree was constructed by the neighbourjoining method based on 16S rDNA sequences. The sequence from Methanosphaera stadtmanae was included as an outgroup. Methanobrevibacter sp. strains SH, PG, HO, CW4C, GS and RT are faecal isolates described by Lin & Miller (1998). Methanobrevibacter sp. strains MB9 (Tokura et al., 1999) and NT7 (G. N. Jarvis, C. Stro$ mpl, E. R. B. Moore and K. N. Joblin, unpublished) are unidentified species. Sequences of Methanobrevibacter ruminantium and Methanobrevibacter arboriphilicus were obtained from RDP II. GenBank accession numbers are given in parentheses. Numbers at nodes indicate bootstrap percentages (1000 bootstrap resamplings for each node). The scale bar represents distance measured from nucleotide differences by using the Jukes–Cantor method.
No such effect was seen with the addition of 2methylbutyric acid and\or coenzyme M. Organic nitrogen in the form of amino acids was required. 16S rDNA sequence analysis
Using primers for the plasmid vector and internal primers for the 16S rRNA gene, a sequence of 1359 bp was obtained. It showed the presence of the sequence 5h-TGTGAGC(A\C)AATCGCG-3h, corresponding to Escherichia coli positions 375–388, which is considered as the signature sequence for the genus Methanobrevibacter. When analysed by and at RDP II, the sequence showed a high degree of similarity to many Methanobrevibacter sequences, including many partial sequences, uncharacterized species and clones from environmental samples. For subsequent analyses, only those sequences with more than 1000 bases, excluding uncultured organisms, were compared, and a phylogenetic tree was drawn (Fig. 3). Strain ATMT showed a separate lineage. DISCUSSION
Cells of strain ATMT are Gram-positive, non-motile cocci, 0n3–0n5 µm in diameter, that occur singly, in pairs or in chains. The cells do not form spores and are resistant to lysis by SDS and hypotonic solutions. The isolate is hydrogenotrophic. The optimum temperature for growth is 35 mC. All these characteristics are shared by members of the genus Methanobrevibacter (Holt et al., 1994). The presence of the signature sequence of Methanobrevibacter species in the 16S rDNA of strain ATMT confirmed this assignment. The genus Methanobrevibacter consists of seven wellcharacterized species, namely Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter arboriphilicus (Holt et al., 1994), Methanohttp://ijs.sgmjournals.org
brevibacter oralis (Ferrari et al., 1994), Methanobrevibacter curvatus, Methanobrevibacter cuticularis (Leadbetter & Breznak, 1996) and Methanobrevibacter filiformis (Leadbetter et al., 1998). Isolate ATMT is morphologically similar to only two species, Methanobrevibacter smithii and Methanobrevibacter ruminantium. It differs from both of these species with respect to cell size, utilization of formate as a growth substrate and temperature and pH maxima for growth. It also differs from Methanobrevibacter smithii in terms of bile tolerance and amino acid requirements. When analysed at the RDP II, strain ATMT showed a very high degree of similarity to sequences of Methanobrevibacter sp. MB9 and Methanobrevibacter sp. NT7 (which are cultured, but unidentified, methanogens from GenBank). Amongst the well-characterized species of Methanobrevibacter, it shows a maximum similarity value of 95n8 % to Methanobrevibacter ruminantium, followed by a value of 94n8 % for Methanobrevibacter smithii ALI. Comparison of the available 600 bp 16S rRNA gene sequence of Methanobrevibacter oralis with that of strain ATMT also shows 94n8 % sequence similarity. For different species of Methanobrevibacter, the similarity values are in the range 93–97 %. In their analyses of faecal isolates, Lin & Miller (1998) reported similarity values of 97 % and above for the isolates that they proposed as novel species. Compared to these values, the similarity values of ATMT are low. Moreover, phylogenetic analysis with suggests that strain ATMT is in a distinctly different lineage from the known species of Methanobrevibacter. Thus, the results obtained in sequence analysis, along with nutritional and physiological studies, clearly suggest that strain ATMT represents a novel species within the genus Methanobrevibacter. Strain ATMT stands apart from all the reported species of Methanobrevibacter with respect to pH require1085
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ments for growth. It grows at pH values above pH 5n0, the optimum pH for growth being 6n0. Within the genus Methanobrevibacter, only Methanobrevibacter ruminantium has been reported to grow at pH 6n0, other species growing at neutral to slightly alkaline pH values. Even Methanobrevibacter ruminantium does not grow at pH values below 6n0 (Smith & Hungate, 1958). On the basis of the pH behaviour of the new species and the fact that it has been isolated from an environment containing high levels of volatile fatty acids, the name Methanobrevibacter acididurans sp. nov. is proposed. Of all the acid-tolerant and\or acidophilic hydrogenotrophic methanogens growing at ambient temperature, this is the only organism belonging to the genus Methanobrevibacter. The remaining isolates belong to the genus Methanobacterium. The isolation of strain ATMT indicates the likelihood of acidtolerant and acidophilic methanogens in other genera. It also offers an explanation for the continuous production of methane in small amounts in acidogenic digesters. Better understanding of the ecological role of this organism will help to solve the problems of sour digestion and to enhance methanogenesis under acidic conditions. Description of Methanobrevibacter acididurans sp. nov.
Methanobrevibacter acididurans (a.ci.di.duhrans. N.L. neut. n. acidum acid, L. part. adj. durans resisting, N.L. part. adj. acididurans acid-resisting). Cells occur singly, in pairs or in chains. They are cocci, 0n3–0n5 µm in diameter. Cells are Gram-positive, do not form spores and are resistant to lysis by SDS and hypotonic solutions. Hydrogenotrophic. Formate, acetate, methanol, trimethylamine, 2-propanol and 2-butanol are not utilized as growth and energy substrates. Mesophilic. The optimum temperature is 35 mC. No growth occurs at or above 40 mC. The optimum pH for growth is pH 6n0. However, it can also produce methane at pH 5n0. Bile-sensitive. Rumen fluid is required for growth. Acetate and amino acids are required. Coenzyme M and 2-methylbutyric acid are not required in the presence of rumen fluid. The type strain was obtained from an acidogenic digester. The type strain, ATMT, has been deposited as MCM B 613T at the Agharkar Research Institute, Pune, India, and as OCM 804T at the Oregon Collection of Methanogens, USA. ACKNOWLEDGEMENTS We are grateful to Professor H. G. Tru$ per, Germany, for his suggestions regarding nomenclature. We also thank Dr A. S. Dighe, National Centre for Cell Science, Pune, India, for helpful discussions. The authors are grateful to Mr Rajdeep Dongre for help with the scanning electron microscopy. We also acknowledge the Council for Scientific and Industrial 1086
Research, New Delhi, India, for providing a research fellowship and a contingency grant to D. V. S.
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