Screening and indentification of cellulase-producing bacteria from soil in Nan province

Abstract

In the isolation and screening of cellulose-producing bacteria from soil samples collected in Pua district (80 samples) and Santisuk district (40 samples), NAN provinces. Sixty-five strains were isolated from soil collected in Pua district and 10 strains from soil in Santisuk district. On the basis of their phenotypic and chemotaxonomic characteristics including the phylogenetic analysis using 16S rDNA sequences, 2 strains were identified as Brevibacillus, 20 as Paenibacillus and 53 as Bacillus. The similarity of 16S rDNA sequences revealed that strains P5-5 (Group 1) was closely related to Brevibacillus agri DSM 6348T (97.2%). P2-1 (Group 2) and S10-4 (Group 3 ) showed 96.1 and 99.1% similarity to Paenibacillus cineris 3998T , respectively. P2-3 and P4-7 (Group 4), S9-2 (Group 5) P6-8 (Group 6), P4-8, P5-2, P7-1 and P7-3 (Group 7) P1-5 (Group 8) P6-7 (Group 9) were closely related to Bacillus subtilis KCTC 3135T (95.5, 97.8, 95.6, 94.3, 96.1, 96.8, 94.5, 98.3, 99.3 and 99.9%, respectively). The tested strains of Brevibacillus, Paenibacillus and Bacillus contained meso-diaminopimelic in cell wall peptidoglycan. The DNA G+C contents of Brevibacillus strain was 54.2 mol%. The DNA G+C contents of Paenibacillus strains were 52.7 to 53.5 mol% and Bacillus strains were 35.5-47.8 mol%. Predominanant menaquinones of the tested strains in Brevibacillus, Paenibacillus, and Bacillus were 7 isoprene units (MK-7). In this study, strain P5-5 was the new species in Brevibacillus. Strains P2-3, S9-2, P6-8, P4-8, P5-2, and P7-1 were found to be the novel Bacillus species and P2-1 was the new species in Paenibacillus. Their phenotypic characteristics were differentiated from the tested type strains. The strains P4-7, P7-3, P1-5, P6-7 that showed 97.8-99.9% sequence similarity to Bacillus subtilis KCTC 3135T were required the DNA-DNA hybridization experiments to make clear their taxonomic position. Among 75 strains screened, a maximum cellulolytic activity exhibited to be 8.5 cm in diameter of clear zone. Fifty-two strains could produce cellulase ranged from 0-0.005 U/ml and 20 strains did from 0.005-0.1 U/ml. Furthermore, 3 strains (P4-6, P3-1 and P4-8) could produce cellulase from 0.0127-0.0153 U/ml. The selected strains, P2-1, P2-3, P7-7 that showed the high HC value and P3-1 and P4-6 that exhibited the highest enzyme activity in CMC medium, produced cellulase optimally at 50o C and pH 7.0.