ISSN 0003-6838, Applied Biochemistry and Microbiology, 2008, Vol. 44, No. 1, pp. 42–47. © Pleiades Publishing, Inc., 2008. Original Russian Text © O.V. Berezina, S.P. Sineoky, G.A. Velikodvorskaya, W. Schwarz, V.V. Zverlov, 2008, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 1, pp. 49–55. 42 Bacteria of the genus Clostridium are gram-positive spore-forming bacillary anaerobes, very diverse in both their physiology and genetics. For example, the optimal growth temperature for clostridia varies from 34–37°C for C. beijerinckii to 75–78°C for C. thermohydrosulfu- ricum. The content of guanosine and cytidine (GC com- position) in DNA of different species also varies in a wide range from 24% in C. pasteurianum to 55% in C. barkeri. However, all members of the genus Clostridium have a common ancestor, which diverged from the gram-positive bacteria rather early in the evo- lution. Some clostridia produce acetone, ethanol, butanol, isopropanol, butyric acid, and acetic acid as metabolic side products. Such bacteria are named solven- togenic bacteria. Five species of solventogenic clostridia are known so far, namely, C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum, C. saccharobutylicum, and C. ljungdahlii. They differ considerably in their abil- ities to produce alcohols and short-chain fatty acids and to utilize nutrient substrates [1, 2]. The common feature of all solventogenic clostridia is that they all are meso- philes with a low GC composition of DNA [3]. Acetone, butanol, and ethanol production with the help of anaerobic bacteria C. acetobutylicum is the old- est industrial-scale bacterial solvent-fermentation. It was first described by Pasteur as early as at the end of the XIX century [4] and transformed into large-scale commercial production in the first half of the XX cen- tury by Weizmann [5]. By the mid-XX century, acetone-butanol manufacture became second micribial fermentation process after the yeast-based ethanol pro- duction. However, development of the petrochemical industry and elevation in the prices for traditional sub- strates (flour, corn starch, and glucose) led to a decrease in the solvent production to its complete cessation [3]. Acetone-butanol plants successfully worked in the Soviet Union to the late 1980s. The nowadays rise in prices of oil and the need to decrease the carbon dioxide discharge into the atmosphere stimulate the search for and development of alternative energy sources. Interest in butanol production using clostridia is therefore renewed. In addition to active use of butanol in several fields of chemical synthesis, it can with time partially substitute gasoline and diesel fuel due to its high energy content, low volatility, good miscibility, and high octane number. Because substrates have a considerable share in pro- duction costs for butanol, the production process can again become profitable when inexpensive substrates, become accessible, in particular, plant biomass with cellulose and hemicellulose as its main components. The goal of this work was to study the cellulase and hemicellulase activities of various butanol-producing Clostridium strains and the yield of solvents produced by the strains grown on the substrates containing plant biomass. MATERIALS AND METHODS Bacterial strains and cultivation media. The strains Clostridium sp. 6 and Clostridium sp. 7 were purchased from the collection of Moscow State Univer- sity (according to the records, these strains were used at the Efremov acetobutyl plant), and the strain Clostri- Extracellular Glycosyl Hydrolase Activity of the Clostridium Strains Producing Acetone, Butanol, and Ethanol O. V. Berezina a , S. P. Sineoky b , G. A. Velikodvorskaya a , W. Schwarz c , and V. V. Zverlov a a Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia e-mail: mashchenko@yandex.ru b Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545 Russia c Institute of Microbiology, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany Received March 22, 2006 Abstract—Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were stud- ied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. ace- tobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be par- tially substituted with plant biomass. DOI: 10.1134/S0003683808010079