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Clostridium Thermocellum from Icelandic Hot Springs ANDREW C INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 119-121 Vol. 38, No. 1 0020-7713/88/010119-03$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Isolation and Properties of Clostridium thermocellum from Icelandic Hot Springs ANDREW C. STAINTHORPE AND RALPH A. D. WILLIAMS* Biochemistry Department, The London Hospital Medical College, London El 2AD, United Kingdom Six strains of Clostridium thermocellum were isolated from hot springs near Hveragerthi, Iceland, and compared with strains of this and other species of clostridia. Deoxyribonucleic acid homology showed that the strains of this species formed a genetic species distinct from all others. The anaerobic thermophile Clostridium thermocellum has All strains were gram variable throughout growth and were often been used for the simultaneous saccharification and lysed by 3% potassium hydroxide (6). Zones of cellulose fermentation of lignocellulosic biomasses (18, 27). Cellulo- clearing were distinct in 1 to 2 days at 60°C. Terminal spores lytic bacteria have been isolated from hot springs (21, 24) (1.2 to 2.0 by 2.0 to 2.2 pm) were round to oval. All strains and decomposing biomasses (22). Strains with broad sub- were obligate anaerobes and required yeast extract. All strate ranges that are of potential biotechnological use have strains fermented cellulose, D-cellobiose, D-glucose, and been described (7, 13, 15, 23). C. thermocellum cultures are sorbitol. Strains B2, A2, H20, and G2 fermented D-fructose, often associated with contaminants (26), and identification of and strains B2 and H20 also fermented raffinose and lactose the species is hampered by differences in the descriptions, weakly. No strains fermented L-arabinose, D-xylose, D- even its Gram reaction. ribose, D-galactose, D-mannose, L-rhamnose, L-sorbose, Lignocellulosic substrates (wood pulp, filter paper, cot- maltose, sucrose, trehalose, D-melibiose, dextrin, glycogen, ton, eucalyptus wood, and heat-treated pinewood) in perfo- rated polyethylene pots were buried in the sediment of nine inulin, pectin, xylan, amygdalin, esculin, salicin, adonitol, hot spring outfalls in Hveragerthi, Iceland (14). After 2 to 4 dulcitol, erythritol, meso-inositol, mannitol, glycerol, or weeks, the enrichment pots were recovered, and six strains xylitol. All of the strains produced the following (per mole of of C. thermocellum were isolated by liquid culture in me- anhydroglucose in cellulose): acetic acid (0.23 to 1.12 mol), dium CM3 (25) containing filter paper strips. The isolates hydrogen (0.49 to 1.12 mol), carbon dioxide (0.78 to 1.42 were then cultured on plates under hydrogen-carbon dioxide mol), and also free glucose (0.19 to 0.49 mol) (17). No strain and in roll tubes under oxygen-free nitrogen (lo), using CM3 produced formic acid (12) or the D isomer of lactic acid (8), medium supplemented with 5% cellobiose or 5% ball-milled but strain B2 formed butyric acid (2). Strain B2 extensively cellulose at 60°C. Liquid cultures up to 5 liters were incu- hydrolyzed filter paper and bleached wood pulp (95%), bated for 4 to 5 days to harvest cells. Media and conditions cotton yarn (Sl%), milled wheat straw (63%), milled corn for the growth of other clostridia were as follows: C. stover, and eucalyptus wood (42%). Hydrolysis of heat- thermocellum NCIMB 10682T, DSM 1313, J1, and YS were treated pinewood was much less complete (3 to 10%). grown by the method of Weimer and Zeikus (25); C. cello- The optimum growth temperature (50 to 55°C) was deter- bioparum NCIMB 10669T and C. formicaceticum DSM 92T mined by an increase in cell protein (1) in a temperature were cultured as described above except that the atmo- gradient incubator (Scientific Industries Inc., Minola, N:Y .) sphere was 100% carbon dioxide; C. thermohydrosulfuricum over 10 days. Growth was negligible at 40 and at 60°C. DSM 571T and C. thermosaccharolyticum DSM 571T were Peptidoglycan was prepared from cells grown on CM3 grown as described by Hollaus and Sleytr (9); C. thermosul- medium with cellobiose and analyzed as described previ- furigenes DSM 2229* was grown as described by Schink and ously (19). In all strains, meso-diaminopimelic acid, alanine, Zeikus (20); C. formicaceticum DSM92T was grown as and glutamate were qualitatively detected. described by Gottwald et al. (5); C. cellobioparum NCIMB Mid-exponential-phase cells were harvested, and deoxy- 10669T was grown as described by Chung (3); C. pupyrosol- ribonucleic acid (DNA) purification was done as described vens NCIMB 11756 was grown as described by Madden et previously (4). After density gradient centrifugation, the al. (16); and C. stercorarium NCIMB 11754 was grown as DNA was denatured in 0.1~standard saline citrate (SSC; described by Madden (15). lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for the Six strains of C. thermocellum were purified from four of determination of mean base composition and also labeled by the hot springs, which had the following conditions: B2 and nick translation for nitrocellulose filter DNA-DNA hybrid- M2, pH 7.8 to 8.0 and 68 to 70°C; A2, pH 8.0 to 8.5 and 61 ization (Table 1). C. thermocellum NCIMB 10682T showed to 65°C; Rt and H20, pH 7.8 to 8.0 and 60 to 65°C; G2, pH 9.0 high homology (>76%) with four other non-Icelandic strains to 9.1 and 68°C. Cells examined by phase-contrast micros- of the species and 73 to 97% homology with the Icelandic copy were single or paired rods (0.4 to 0.8 by 2.5 to 6 km), isolates. Homology between C. thermocellum and other with filaments only on agar surfaces. Colonies were light thermophilic and mesophilic clostridia was less than 11%. yellow to tan and caused yellowing of the adjacent cellulose. These results were confirmed by spectrophotometric reas- sociation (11) between solutions of sheared DNA of selected * Corresponding author. strains. 119 120 NOTES INT. J. SYST.BACTERIOL. TABLE 1. Mean base composition and DNA-DNA homology of C. thermocellum and other clostridia - DNA hybridization with [3H] DNA from strain: TmU("C)in Strain % G+Cb NCIMB 0.1x ssc NCIMB B2 10682 11756 C. thermocellum NCIMB 10682= 69.0 38.0 100.0 84.0 8.9 DSM 2360 69.2 38.4 104.0 105.7 6.0 DSM 1313 68.9 37.8 92.0 86.6 5.0 JI 68.6 37.1 88.0 86.9 5.7 YS 68.8 37.6 76.0 64.6 4.6 Icelandic C. thermocellum B2 67.8 35.8 96.7 100.0 12.4 M2 68.6 37.1 85.0 91.4 6.0 A2 68.4 36.7 75 .O 83.8 5.3 Rt 69.3 38.6 73 .O 76.2 4.7 H20 68.2 36.3 82.5 87.6 5.5 c11 68.1 36.1 85.0 83 .O 5.5 Other clostridia C. thermosaccharolyticum DSM 571 64.8 29.2 5.1 5.8 3.5 C. thermohydrosulfuricum DSM 567 65.9 32.0 5.9 6.1 4.0 C. formicaceticum DSM 92 67.1 34.4 4.9 5.1 4.7 C. thermosulfurigenes DSM 2229 66.6 33.4 5.8 6.5 5.1 Clostridium sp. strain NCIMB 11756 66.3 33.0 5.0 4.8 100.0 Clostridium sp. strain NCIMB 11755 65.7 31.1 5.2 5.1 58.7 C. cellobioparum NCIMB 10669 66.5 32.8 6.6 3.6 3.6 C. papyrosolvens NCIMB 11394 67.9 36.1 6.3 7.9 4.5 C. stercorarium NCIMB 11754 69.7 39.4 ND' 11.4 ND lhermus thermophilus HB8 IT.coli B 75.2 50.9 1.7 2.9 1.3 T,,,, Melting temperature. G+C, Guanine-plus-cytosine; % G+C = 50.9 + 2.08 (T,,, - T,,, of E. coli DNA [Sigma Chemical Co., St. Louis, Mo.]). ' ND, Not determined. We are grateful for the interest and support of Biogen Inc., structure of some hyperthermophilic saccharolytic clostridia. Eloston, Mass. Arch. Microbiol. 86:129-146. 10. Hungate, R. E. 1969. A roll-tube method for cultivation of strict LITERATURE CITED anaerobes, p. 117-132. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. B. Academic Press (London) 1. Bradford, M. M. 1976. A rapid and sensitive method for the Ltd., London. quantitation of microgram quantities of protein utilizing the 11. HUSS,V. A. R., H. Festl, and K. H. Schleifer. 1983. Studies on principle of protein-dye binding. Anal. Biochem. 72:248-254. the spectrophotometric determination of DNA hybridization 2. Carlsson, J. 1973. Simplified gas chromatographic procedure for from renaturation rates. Syst. Appl. Microbiol. 4:184-192. identification of bacterial metabolic products. Appl. Microbiol. 12. Lang, E., and H. Lang. 1972. Spezifische Farbreaktion zum 25~287-289. direkten Nachweis der Ameisensaure. Z. Anal. Chem. 3. Chung, K. T. 1976. Inhibitory effects of hydrogen on growth of 260:&10. Clostridium cellobioparum. Appl. Environ. Microbiol. 31:342- 13. Le Ruyet, P., H. C. Dubourguier, and G. Albagnac. 1984. 348. Thermophilic fermentation of cellulose and xylan by methano- 4. Dent, V. E., and R. A. D. Williams. 1984. Actinomyces howellii, genic enrichment cultures. Syst. Appl. Microbiol. 5247-253. a new species from the dental plaque of dairy cattle. Int. J. Syst. 14. Locher, P., and A. Binder. 1971. Investigations in the hot Bacteriol. 34:316-320. springs of Iceland. Natturufraedingurinn 41:129-143. 5. Gottwald, M., J. R. Andreesen, J. LeGall, and L. G. Ljungdahl. 15. Madden, R. H. 1983. Isolation and characterization of Clostrid- 1975. Presence of cytochrome and menaquinone in Clostridium ium stercorariurn sp. nov.
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