Screening of Cellulase-producing Bacteria, Whole-gene Analysis, and Optimization of the Enzyme Production Process for the Functional Bacterium NF-101
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Abstract:
Northern China is a major agricultural region, primarily producing cereal crops, which are associated with large amounts of residual straw. To efficiently utilize this natural agricultural resource and identify efficient cellulose degrading bacteria, 100 samples were collected from 20 sites in three northeastern provinces. After preliminary screening, 2 052 strains capable of decomposing cellulose were isolated, which were provisionally identified as 1 207 strains of bacteria, 598 strains of mold, and 195 strains of actinomycetes. These strains were stored in an ultra-low temperature freezer at -80 ℃ for subsequent establishment of a functional cellulase-degrading strain library. After re-screening, 16 functional bacteria with high cellulase production ability were obtained, which were identified based on 16S rDNA analysis and physiological and biochemical identification as Serratia marcescens, Bacillus licheniformis, and halotolerant Bacillus, among which B. licheniformis NF-101 was found to be characterized by the most efficient enzyme production. Following single-factor and response surface optimization, the optimal enzyme-producing medium for cultivating NF-101 was identified as a medium comprising sodium carboxymethylcellulose 0.75 wt.%, peptone 0.6 wt.%, potassium dihydrogen phosphate 0.2 wt.%, and magnesium sulfate 0.05 wt.%. The optimal process conditions were a 4% inoculation amount, initial pH of 7.0, temperature of 37 ℃, and incubation time of 72 h. Optimized filter enzyme activity reached 181.22 U/mL, which represented an approximate 3.63-fold increase compared with that prior to optimization. Wholegene analysis of NF-101 proved that it was a dominant strain for efficient cellulase-mediated degradation. It also provided information on a dominant strain and an experimental basis for cellulase degradation experiments.
