Two novel anaerobic, moderately thermophilic and cellulose-/cellobiose-digesting bacteria, EBR45 and EBR596, were isolated from anaerobic sludge of a cellulose-degrading methanogenic bioreactor. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belonged to cluster III within the low-G+C-content Gram-positive bacteria. The close relatives of EBR45 were DSM 16021 (sequence identity, 94.6 %) and DSM 1237 (93.4 %). The closest relative of EBR596 was DSM 8532 (95.9 %). Both isolates were rod-shaped sporulators, growing optimally at 60 °C. EBR45 was Gram-staining-reaction-variable and non-motile, formed bright-yellow colonies on solid media, and grew on a relatively narrow range of carbohydrates including cellulose and cellobiose. EBR596 was Gram-staining-reaction-negative and motile, formed glossy white colonies and grew on cellobiose and various carbohydrates except cellulose. Major fatty acid compositions were 16 : 0 iso, 16 : 0 and 16 : 0 dimethylacetal (strain EBR45) and 15 : 0 iso, 16 : 0 iso, 15 : 0 anteiso and 17 : 0 anteiso (strain EBR596). The DNA G+C contents were 36.9 mol% (EBR45) and 51.1 mol% (EBR596). Based on the phenotypic and phylogenetic data and genomic distinctiveness, strains EBR45 and EBR596 represent two novel species, for which the names sp. nov. (type strain EBR45 =DSM 19732 =NBRC 101661) and sp. nov. (type strain EBR596 =DSM 19027 =NBRC 102590) are proposed.

Keyword(s): DMA, dimethylacetal

Article metrics loading...

Loading full text...

Full text loading...



  1. Chan, M., Himes, R. H. & Akagi, J. M.(1971). Fatty acid composition of thermophilic, mesophilic, and psychrophilic clostridia. J Bacteriol 106, 876–881. [Google Scholar]
  2. Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E.(1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.[CrossRef] [Google Scholar]
  3. Felsenstein, J.(1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef] [Google Scholar]
  4. Fitch, W. M.(1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[CrossRef] [Google Scholar]
  5. Galtier, N., Gouy, M. & Gautier, C.(1996).seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12, 543–548. [Google Scholar]
  6. Holdeman, L. V., Cato, E. P. & Moore, W. E. C.(1977).Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Anaerobe Laboratory, Virginia Polytechnic Institute and State University.
  7. Jukes, T. H. & Cantor, C. R.(1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
  8. Kaneda, T.(1991). Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev 55, 288–302. [Google Scholar]
  9. Kato, S., Haruta, S., Cui, Z. J., Ishii, M., Yokota, A. & Igarashi, Y.(2004).Clostridium straminisolvens sp nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int J Syst Evol Microbiol 54, 2043–2047.[CrossRef] [Google Scholar]
  10. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  11. Le Ruyet, P., Dubourguier, H. C., Albagnac, G. & Prensier, G.(1985). Characterization of Clostridium thermolacticum sp. nov., a hydrolytic thermophilic anaerobe producing high amounts of lactate. Syst Appl Microbiol 6, 196–202.[CrossRef] [Google Scholar]
  12. Madden, R. H.(1983). Isolation and characterization of Clostridium stercorarium sp. nov., cellulolytic thermophile. Int J Syst Bacteriol 33, 837–840.[CrossRef] [Google Scholar]
  13. Marmur, J.(1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.[CrossRef] [Google Scholar]
  14. McBee, R. H.(1954). The characteristics of Clostridium thermocellum. J Bacteriol 67, 505–506. [Google Scholar]
  15. Mesbah, M. & Whitman, W. B.(1989). Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. J Chromatogr 479, 297–306.[CrossRef] [Google Scholar]
  16. Ng, T. K., Weimer, T. K. & Zeikus, J. G.(1977). Cellulolytic and physiological properties of Clostridium thermocellum. Arch Microbiol 114, 1–7.[CrossRef] [Google Scholar]
  17. Saitou, N. & Nei, M.(1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. [Google Scholar]
  18. Shiratori, H., Ikeno, H., Ayame, S., Kataoka, N., Miya, A., Hosono, K., Beppu, T. & Ueda, K.(2006). Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor. Appl Environ Microbiol 72, 3702–3709.[CrossRef] [Google Scholar]
  19. Stackebrandt, E. & Goebel, B. M.(1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[CrossRef] [Google Scholar]
  20. Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994).clustalw: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef] [Google Scholar]
  21. Toda, Y., Saiki, T., Uozumi, T. & Beppu, T.(1988). Isolation and characterization of a protease-producing, thermophilic, anaerobic bacterium, Thermobacteroides leptospartum sp. nov. Agric Biol Chem 52, 1339–1344.[CrossRef] [Google Scholar]
  22. Wang, X., Hoefel, D., Saint, C. P., Monis, P. T. & Jin, B.(2007). The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge. J Appl Microbiol 103, 1415–1423.[CrossRef] [Google Scholar]
  23. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef] [Google Scholar]
  24. Wiegel, J.(1980). Formation of ethanol by bacteria. A pledge for the use of extreme thermophilic anaerobic bacteria in industrial ethanol fermentation processes. Cell Mol Life Sci 36, 1434–1446.[CrossRef] [Google Scholar]
  25. Wiegel, J., Tanner, R. & Rainey, F. A.(2006). An introduction to the family Clostridiaceae. In The Prokaryotes. A Handbook on the Biology of Bacteria, 3rd edn, vol. 4, pp. 654–678. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer.

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error