Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70¢XC. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group £L in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group £L£L), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

Three thermophilic aerobic bacterial strain, Geobacillus thermoleovorans T4, Bacillus subtilis b5 and Bacillus licheniformis b6 were isolated from the wastewater of a Taiwan Sugar Company sugar refinery in Kaohsiung. All strains were capable of growth in cellulose containing medium. We found out that G. thermoleovorans T4 possessed both of the activity of cellulses and xylanase. This was the first report ever in this bacterial spices. Its endoglucanase was active at temperatures ranging from 30¢J to 100¢J, and pH ranging from 4.0 and 8.0. Its £]-glucosidase activities could be measured at 40¢J - 90¢J and pH 5.0 - 9.0. Its xylanase was active from 30¢J to 90¢J and pH from 3.0 to 10.0, while the exoglucanase was active at temperatures from 30¢J to 90¢J. The pH values of xylasnase was better than G. thermoleovorans K-3d. This enzyme was worth of possible in industry application. Both of B. subtilis b5 and B. licheniformis b6 contained a thermoactive endoglucanase. The endoglucanase of B. subtilis b5 was active at pH values between 4.0 and 7.5, and was thermostable at 100¢J for at least 1 hour ( retaining 60% of the original activity ). The stability of this enzyme was better than all known enzymes extracted from Bacillus spices. The endoglucanase of B. licheniformis b6 was active at pH values between 4.0 and 9.0, and thermostable at 60¢J for least 1 hour.