Four hundred and sixteen strains of aerobic, endospore-forming bacteria belonging to the genus Bacillus were physiologically characterized using a total of 329 miniaturized tests. Overall similarities of all strains for 295 unit characters were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and the SJ coefficients as measures of similarity. The test error and cophenetic correlation were within acceptable limits. Comparison of photometric and visual test reading revealed overall differences of 12.3%. A total of 30 clusters (2 or more strains) and 20 single member clusters were defined at the 86.7 to 89.4% levels (SSM). Generally similar groupings were obtained with the Jaccard coefficient with some changes in the definition of clusters. Heterogeneity was found within the species Bacillus brevis, B. circulans, B. macerans, B. coagulans, B. megaterium, B. sphaericus and B. stearothermophilus, thus confirming the results of previously published data. For the clusters and for some subclusters containing two or more strains the minimum number of differentiating characters was selected by computer programs (CHARSEP, DIACHAR, MOSTTYP) and a frequency matrix for probabilistic identification was constructed consisting of 40 tests versus 36 clusters and subclusters. This matrix was theoretically evaluated using a computer program (MATIDEN), and out of 391 strains, a total of 287 (73.40%) were correctly identified with a Willcox probability p>0.99. Another 34 strains (8.69%) achieved Willcox probabilities above 0.90, for 19 strains (4.85%) p exceeded 0.8, and 27 strains (6.90%) achieved Willcox probabilities >0.5. For 24 strains (6.13%) no correct identification result was obtained.
A microcrystalline cellulose (Avicel)-hydrolyzing cellulase (an endocellulase) was isolated from a preparation of Trichoderma reesei CDU-11 and purified successively by DEAE-Sepharose CL 6B and Concanavalin A-Sepharose column chromatography, and preparative electrophoresis and on Sephadex G-100 gel filtration. The cellulase preparation showed a single protein band on SDS-PAGE slab and gel disc electrophoresis. Its molecular weight was estimated to be 64, 000 by Sephadex G-100 gel filtration and 67, 000 by SDS slab electrophoresis. The isoelectric point was at pH 4.75 on polyacrylamide gel electric focusing. This cellulase hydrolyzed more specifically water insoluble microcrystalline cellulose such as Avicel rather than water soluble cellulose such as carboxymethyl-cellulose. It produced glucose, cellobiose and cellotriose from cellotetraose and cellohexaose, and it produced a significant amount of glucose and cellobiose from Avicel. Based on these results, it appears that the cellulase should be regarded as an endo-type cellulase, although it hydrolyzes Avicel relatively easier than CM-cellulose. We obtained no evidence that this cellulase is an exo-type one named cellobiohydrolase (CBH) in the systematic nomenclature.
The mode of submerged spore (SS) formation in Kitasatosporia setae was studied. A time course study of SS formation with pulse-labeling and radioautography demonstrated that at the tips of the hyphal branches, premature spore chains were produced and grew into short spore chains. Then the short spore chains were fragmented to yield SS. This was supported by the morphological changes observed by Nomarski differential interference microscopy, which showed that K. setae differs from Streptomyces griseus in the way SSs are formed.
The enzymatic hydrolysis of native and pretreated chitin by the culture filtrate of Myrothecium verrucaria NCIM 903 was studied. Also, factors which influence the enzymatic hydrolysis such as pH, temperature, enzyme and substrate concentration and enzyme adsorption characteristics (n-value) were studied. Chitin hydrolysate was also used as a substrate for Single Cell Protein (SCP) production using Saccharomycescerevisiae NCIM 3052. With M. verrucaria chitinase complex, native and acid swollen chitin were hydrolyzed at pH 5.0 and 40°C, 12.7% and 39.6% respectively. The n values which is a measure of adsorption characteristics of an enzyme were 0.4 and 0.63 for native and acid swollen chitin, respectively. This indicates that the swelling of the ordered structure results in efficient hydrolysis. As the end-product of hydrolysis of chitin was mainly N-acetyl-D-glucosamine, its further utilization as a substrate for SCP production was investigated. A biomass of 9.5g/l with a growth yield of 0.27g/g of substrate utilized was obtained. The total protein and nucleic acid content of the biomass was 61% and 3.1%, respectively.
The partial base sequences of 18S and 26S rRNAs were examined in eighteen strains of Debaryomyces, Torulaspora, and Yamadazyma species including two strains of D. udenii. All of the strains of Debaryomyces species constituted a single group (cluster) phylogenetically. In the partial base sequence (positions 493 through 622, 130 bases) of 26S rRNA, the maximum homologies were 79-99% among Debaryomyces species. T. globosa and Y. philogaea had 71-78% and 81-87% maximum homologies, respectively, with Debaryomyces species. In the partial base sequence (positions 1611 through 1835, 225 bases) of 26S rRNA, the base differences numbered 5-0 among Debaryomyces species. T. globosa and Y. philogaea had 15-13 and 10-8 base differences, respectively, with Debaryomyces species. In the partial base sequence (positions 1451 through 1618, 168 bases) of 18S rRNA, Debaryomyces species were divided into two subgroups (subclusters). The first subgroup was comprised of D. hansenii, D. melissophilus, D. udenii, and so on, and the second subgroup comprised of D. castellii, D. polymorphus, D. yamadae, and so on. The base difference numbered 1 between the two subgroups. T. globosa and Y. philogaea had 5-4 and 1-0 base differences, respectively, with Debaryomyces species. Between T. globosa and S. cerevisiae, there was 1 base difference. D. tamarii occupied a distant position (maximum homologies, 63-71%; base differences, 50-48 and 20-19, respectively).
Partial sequence comparisons of 18S ribosomal RNA comprising 558 nucleotides in the positions 384-562, 942-1119, and 1419-1623 were used to determine the evolutionary affinities among eleven species of Aspergillus and associated teleomorphs, including five species of the most diverse sect. Ornati, and two species of Penicillium with teleomorphs. The species compared were Eurotium repens, Aspergillus fumigatus, Sclerocleista ornata, Hemicarpenteles paradoxus, H. acanthosporus, Warcupiella spinulosa, A. raperi, Emericella nidulans, A: flavus, A. oryzae, Chaetosartorya cremea, Eupenicillium crustaceum, and Talaromyces flavus. Phylogenetic trees were constructed by the unweighted pair-group method using arithmetic averages (UPGMA) and the neighbor-joining (NJ) method from evolutionary distances (Knuc). The sequence differences between the eleven species of Aspergillus and associated teleomorphs were slight, indicating only recent diversification. These observations contrast with the diversity of teleomorphs and the heterogeneity of ubiquinone systems. Both UPGMA and NJ trees based on the above sequence data, and the present known data of 18S rRNA partial sequences comprising a total of 188 nucleotides in the positions 1419-1607 from Aspergillus, Penicillium and associated teleomorphs showed that the anamorphic genera Aspergillus and Penicillium are well separated from each other. The distinguished character of "aspergillum" for Aspergillus and of "penicillus" for Penicillium appears to be a phylogenetic indicator.