Screening, Identification, Optimization of Its Enzyme Production and Research on Bioaugmentation of Cellulose-degrading Bacteria C-19
LI Hao1,2, YANG Heng1, ZHANG Min-Qi1, XU Xue-Ming1, ZHANG Yuan-Hao1, ZHANG Ran1,2, ZHANG Zhen-Dong1,2, ZHANG Hui-Wei1, GUI Zhong-Zheng1,2,*
1 School of Biotechnology, Jiangsu University of Science and Technology, Zhengjiang 212100, China; 2 Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhengjiang 212100, China
Abstract：Rich in refractory cellulose is the key limiting factor restricting the resource utilization efficiency of silkworm excrement by composting. Screening the cellulose-degrading bacteria will provide high-quality strains for harmless and rapid decomposition treatment of silkworm excrement. In this study, 16 cellulose-degrading bacteria were screened from silkworm (Bombyx mori) excrement substrates and intestinal samples of housefly larvae (Musca domestica) in the system of high-efficiency conversion of silkworm excrement waste via housefly larvae. Firstly, the strains were preliminarily screened by CMC-Na culturing method and Congo red dyeing and re-screened with filter paper enzyme activity test. A strain C-19 with the highest cellulose-degrading ability from the intestinal of housefly larvae was identified by morphological observation, physiological and biochemical tests, and phylogenetic analysis of the 16S rRNA gene sequence. The result showed that the C-19 strain was short rod-shaped, Gram-negative bacteria, its colony was milky white, mucus-like, smooth surface, and opaque shape. Physiological and biochemical assays showed positive for the Voges-Proskauer test (V-P test), indole production, salt tolerance, and starch hydrolysis test; negative for methyl red test and gelatin liquefaction test. Molecular biological identification results showed that the length of the 16S rRNA gene sequence from the strain C-19 was about 1 500 bp, and it was 99% similar to Klebsiella pneumoniae (GenBank No. MH111429), clustered on one branch in the phylogenetic tree. Combining morphological and physiological biochemical features, the strain C-19 was identified as K. pneumoniae. Then, the enzyme production conditions of C-19 were optimized by a single-factor experiment, and the cellulase activity of strain C-19 was studied by the DNS (3,5-dinitrosalicylic acid) method. The result demonstrated that the optimum temperature and pH of strain C-19 were 60 ℃ and 5, respectively. Under the optimum conditions, the filter paper enzyme activity (FPase), carboxymethyl cellulase (CMCase) activity, and exonuclease (Avicelase) activity reached up to 16.89、13.86, and 14.30 U/mL, respectively. Finally, in order to evaluate the practical application effect of this strain in a complex environment, the bioaugmentation effect of C-19 on silkworm excrement cellulose in housefly larvae vermicomposting systems was also investigated. The degradation rate of cellulose in silkworm excrement was 61.2% after strain C-19 supplemented housefly larvae vermicomposting system (6 d), which was significantly higher than that of the control group (54.6%, P<0.05). In summary, the strain C-19 was an acid-resistant and heat-resistant cellulose-degrading strain, and its cellulase activity was high, which had a bioaugmentation effect on silkworm excrement cellulose in housefly larvae vermicomposting systems. It had the potential to be prepared as microbial inoculum, which can be applied to harmless quick composting of silkworm excrement. This study has a guiding role in the development and application of microbial agents for the harmless treatment of silkworm excrement and provides a new choice for the application of strains for rapid ripening and decomposition of silkworm excrement and resource treatment.
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