Isolation, Identification Of Cellulolytic Bacteria And Optimization Of Cellulase Production By Paenibacillus Terrae ME27-1

The aerobic bacteria which can hydrolyze cellulose grow fast in the simplest medium only containing cellulosic materials and inorganic salts, especially with enzyme production in a very short time of cultivation. Therefore, production of fermentable sugars from various cellulosic materials by these cellulolytic bacteria is an effective and environment-friendly method for utilization of cellulosic wastes.From different natural reserves in the subtropical region of Guangxi and Yunnan provinces of China, a total of245aerobic bacterial strains were isolated on the agar plates containing sugarcane bagasse pulp as the sole carbon source. Of the245strains,22showed hydrolyzing zones on agar plates containing sodium carboxymethyl cellulose after Congo-red staining. Molecular identification showed that the22strains belonged to10different genera. The genus Burkholderia exhibited the highest strain diversity, accounting for36.36%of all the22strains and was the dominant cellulose-degrading bacteria in these samples collected from different soils.Three isolates among the22strains showed higher carboxymethyl cellulase (CMCase) activity (≥0.01U/mL) by using dinitrosalicylic-acid method, and isolate ME27-1exhibited the highest CMCase production (0.17U/mL) in liquid cultivation. The strain ME27-1was identified as Paenibacillus terrae based on16S rRNA gene sequence analysis, physiological and biochemical identification as well as Biolog Microbial Identification System analysis.The cultivation conditions for CMCase production by P. terrae ME27-1were optimized. The optimal pH and temperature for CMCase production was pH8.0and28℃, respectively; the optimal carbon and nitrogen sources were5%(w/v) wheat bran and0.3%(w/v) NH4Cl, respectively; the highest CMCase activity of P. terrae ME27-1was obtained after incubating for60h with2%(v/v) inoculum size. The optimum pH and temperature for CMCase activity produced by P. terrae ME27-1were pH5.5and50℃, respectively, and the CMCase was stable at a wide pH range of4.0-11.0. Finally, a12-fold improvement in the CMCase activity (2.08U/mL) of P. terrae ME27-1was obtained under the optimal cultivation conditions.With a very short period of CMCase production, high CMCase activity and pH stability, P. terrae ME27-1was a good candidate strain for cloning and characterizing cellulase genes. Meanwhile, this study also provided valuable information about the diversity of cellulose-degrading bacteria in the subtropical region of China.