INTERNATIONAL JOURNAL SYSTEMATIC OF BACTERIOLOGY, 1988, p. 119-121 Jan. 0020-7713/88/010119-03$02.

00/0 Copyright 0 1988, International Union of Microbiological Societies

Vol. 38, No. 1

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 often been used for the simultaneous saccharification and fermentation of lignocellulosic biomasses (18, 27). Cellulolytic bacteria have been isolated from hot springs (21, 24) and decomposing biomasses (22). Strains with broad substrate ranges that are of potential biotechnological use have been described (7, 13, 15, 23). C. thermocellum cultures are often associated with contaminants (26), and identification of the species is hampered by differences in the descriptions, even its Gram reaction. Lignocellulosic substrates (wood pulp, filter paper, cotton, eucalyptus wood, and heat-treated pinewood) in perforated polyethylene pots were buried in the sediment of nine hot spring outfalls in Hveragerthi, Iceland (14). After 2 to 4 weeks, the enrichment pots were recovered, and six strains of C. thermocellum were isolated by liquid culture in medium CM3 (25) containing filter paper strips. The isolates were then cultured on plates under hydrogen-carbon dioxide and in roll tubes under oxygen-free nitrogen (lo), using CM3 medium supplemented with 5% cellobiose or 5% ball-milled cellulose at 60C. Liquid cultures up to 5 liters were incubated for 4 to 5 days to harvest cells. Media and conditions for the growth of other clostridia were as follows: C. thermocellum NCIMB 10682T, DSM 1313, J1, and YS were grown by the method of Weimer and Zeikus (25); C. cellobioparum NCIMB 10669T and C. formicaceticum DSM 92T were cultured as described above except that the atmosphere was 100% carbon dioxide; C. thermohydrosulfuricum DSM 571T and C. thermosaccharolyticum DSM 571T were grown as described by Hollaus and Sleytr (9); C. thermosulfurigenes DSM 2229* was grown as described by Schink and Zeikus (20); C. formicaceticum DSM92T was grown as described by Gottwald et al. (5); C. cellobioparum NCIMB 10669Twas grown as described by Chung (3); C. pupyrosolvens NCIMB 11756 was grown as described by Madden et al. (16); and C. stercorarium NCIMB 11754 was grown as described by Madden (15). Six strains of C. thermocellum were purified from four of the hot springs, which had the following conditions: B2 and M2, pH 7.8 to 8.0 and 68 to 70C; A2, pH 8.0 to 8.5 and 61 to 65C; Rt and H20, pH 7.8 to 8.0 and 60 to 65C; G2, pH 9.0 to 9.1 and 68C. Cells examined by phase-contrast microscopy were single or paired rods (0.4 to 0.8 by 2.5 to 6 km), with filaments only on agar surfaces. Colonies were light yellow to tan and caused yellowing of the adjacent cellulose.

* Corresponding author.
119

All strains were gram variable throughout growth and were lysed by 3% potassium hydroxide (6). Zones of cellulose clearing were distinct in 1to 2 days at 60C. Terminal spores (1.2 to 2.0 by 2.0 to 2.2 pm) were round to oval. All strains were obligate anaerobes and required yeast extract. All strains fermented cellulose, D-cellobiose, D-glucose, and sorbitol. Strains B2, A2, H20, and G2 fermented D-fructose, and strains B2 and H20 also fermented raffinose and lactose weakly. No strains fermented L-arabinose, D-xylose, Dribose, D-galactose, D-mannose, L-rhamnose, L-sorbose, maltose, sucrose, trehalose, D-melibiose, dextrin, glycogen, inulin, pectin, xylan, amygdalin, esculin, salicin, adonitol, dulcitol, erythritol, meso-inositol, mannitol, glycerol, or xylitol. All of the strains produced the following (per mole of anhydroglucose in cellulose): acetic acid (0.23 to 1.12 mol), hydrogen (0.49 to 1.12 mol), carbon dioxide (0.78 to 1.42 mol), and also free glucose (0.19 to 0.49 mol) (17). No strain produced formic acid (12) or the D isomer of lactic acid (8), but strain B2 formed butyric acid (2). Strain B2 extensively hydrolyzed filter paper and bleached wood pulp (95%), cotton yarn (Sl%), milled wheat straw (63%), milled corn stover, and eucalyptus wood (42%). Hydrolysis of heattreated pinewood was much less complete (3 to 10%). The optimum growth temperature (50 to 55C) was determined by an increase in cell protein (1) in a temperature gradient incubator (Scientific Industries Inc., Minola, N:Y .) over 10 days. Growth was negligible at 40 and at 60C. Peptidoglycan was prepared from cells grown on CM3 medium with cellobiose and analyzed as described previously (19). In all strains, meso-diaminopimelic acid, alanine, and glutamate were qualitatively detected. Mid-exponential-phase cells were harvested, and deoxyribonucleic acid (DNA) purification was done as described previously (4). After density gradient centrifugation, the standard saline citrate (SSC; DNA was denatured in 0 . 1 ~ l x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for the determination of mean base composition and also labeled by nick translation for nitrocellulose filter DNA-DNA hybridization (Table 1). C. thermocellum NCIMB 10682T showed high homology (>76%) with four other non-Icelandic strains of the species and 73 to 97% homology with the Icelandic isolates. Homology between C. thermocellum and other thermophilic and mesophilic clostridia was less than 11%. These results were confirmed by spectrophotometric reassociation (11)between solutions of sheared DNA of selected strains.

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NOTES

INT. J. SYST. BACTERIOL.

TABLE 1. Mean base composition and DNA-DNA homology of C. thermocellum and other clostridia
Strain TmU("C) in 0.1x ssc DNA hybridization with [3H] DNA from strain:
% G+Cb

NCIMB 10682

B2

NCIMB 11756

C. thermocellum NCIMB 10682= DSM 2360 DSM 1313 JI YS Icelandic C . thermocellum B2 M2 A2 Rt H20

69.0 69.2 68.9 68.6 68.8 67.8 68.6 68.4 69.3 68.2 68.1 64.8 65.9 67.1 66.6 66.3 65.7 66.5 67.9 69.7

38.0 38.4 37.8 37.1 37.6 35.8 37.1 36.7 38.6 36.3 36.1 29.2 32.0 34.4 33.4 33.0 31.1 32.8 36.1 39.4

100.0 104.0 92.0 88.0 76.0 96.7 85.0 75 .O 73 .O 82.5 85.0 5.1 5.9 4.9 5.8 5.0 5.2 6.6 6.3 ND'

84.0 105.7 86.6 86.9 64.6 100.0 91.4 83.8 76.2 87.6 83.O 5.8 6.1 5.1 6.5 4.8 5.1 3.6 7.9 11.4

8.9 6.0 5.0 5.7 4.6 12.4 6.0 5.3 4.7 5.5 5.5 3.5 4.0 4.7 5.1 100.0 58.7 3.6 4.5 ND

c1 1

Other clostridia C . thermosaccharolyticum DSM 571 C. thermohydrosulfuricum DSM 567 C. formicaceticum DSM 92 C. thermosulfurigenes DSM 2229 Clostridium sp. strain NCIMB 11756 Clostridium sp. strain NCIMB 11755 C. cellobioparum NCIMB 10669 C. papyrosolvens NCIMB 11394 C . stercorarium NCIMB 11754 lhermus thermophilus HB8

IT.coli B
T,,,, Melting temperature. G+C, Guanine-plus-cytosine; % G+C 'ND, Not determined.
=

75.2
50.9

50.9

1.7

2.9

1.3

+ 2.08 (T,,, - T,,, of E . coli DNA [Sigma Chemical Co., St. Louis, Mo.]).
structure of some hyperthermophilic saccharolytic clostridia. Arch. Microbiol. 86:129-146. 10. Hungate, R. E. 1969. A roll-tube method for cultivation of strict anaerobes, p. 117-132. I n J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. B. Academic Press (London) Ltd., London. 11. HUSS,V. A. R., H. Festl, and K. H. Schleifer. 1983. Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4:184-192. 12. Lang, E., and H. Lang. 1972. Spezifische Farbreaktion zum direkten Nachweis der Ameisensaure. Z. Anal. Chem. 260:&10. 13. Le Ruyet, P., H. C. Dubourguier, and G. Albagnac. 1984. Thermophilic fermentation of cellulose and xylan by methanogenic enrichment cultures. Syst. Appl. Microbiol. 5247-253. 14. Locher, P., and A. Binder. 1971. Investigations in the hot springs of Iceland. Natturufraedingurinn 41:129-143. 15. Madden, R. H. 1983. Isolation and characterization of Clostridium stercorariurn sp. nov. , a cellulolytic thermophile. Int. J. Syst. Bacteriol. 33:837-840. 16. Madden, R. H., M. J. Bryder, and N. J. Poole. 1982. Isolation and characterization of an anaerobic cellulolytic bacterium, Clostridium papyrosolvens sp. nov. Int. J. Syst. Bacteriol. 32: 87-91. 17. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153:375-380. 18. Ng, T. K., A. Ben-Bassat, and J. G. Zeikus. 1981. Ethanol production by thermophilic bacteria: fermentation of cellulosic substrates by cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum. Appl. Environ. Microbiol. 41: 1337-1343, 19. Pask-Hughes, R. A., and R. A. D. Williams. 1978. Cell envelope

We are grateful for the interest and support of Biogen Inc., Eloston, Mass. LITERATURE CITED 1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 2. Carlsson, J. 1973. Simplified gas chromatographic procedure for identification of bacterial metabolic products. Appl. Microbiol. 25~287-289. 3. Chung, K. T. 1976. Inhibitory effects of hydrogen on growth of Clostridium cellobioparum. Appl. Environ. Microbiol. 31:342348. 4. Dent, V. E., and R. A. D. Williams. 1984. Actinomyces howellii, a new species from the dental plaque of dairy cattle. Int. J. Syst. Bacteriol. 34:316-320. 5. Gottwald, M., J. R. Andreesen, J. LeGall, and L. G. Ljungdahl. 1975. Presence of cytochrome and menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum. J. Bacteriol. 122:325-328. 6. Halebian, S., B. Harris, S. M. Finegold, and R. D. Rolfe. 1981. Rapid method that aids in distinguishing gram-positive from gram-negative anaerobic bacteria. J. Clin. Microbiol. 13:444448. 7. Hayashida, S., B. K. Ahn, S. Yoshino, and S. Ogata. 1983. Fermentation of cellulose by a newly isolated thermophilic Clostridium sp. J . Fac. Agric. Kyushu Univ. 27:99-107. 8. Hohorst, H. J. 1963. L( +)Lactate. Determination with lactic hydrogenase and DPN, p. 266-270. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Verlag Chemie, Weinheim, Federal Republic of Germany. 9. Hollaus, F., and U. Sleytr. 1972. On the taxonomy and fine

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