Study On The Cellulase Activity In The Gut Of Holotrichia Parallela And The Construction Of Microbial Community

Holotrichia parallela larvae is fed on the root and tuber of plants which is closely related to the wide diversity of the microbes in the intestinal tract. The cellulase and xylanase produced by the microbes help to degrade the lignocelluloses and digest the food. The major constituents of lignocelluloses are cellulose, hemicellulose and lignin. The cellulase and xylanase are the main enzymes of decomposing lignocelluloses and play an important role in the biodegradation and the development of biofuels.In this study, the enzyme activities of cellulase and xylanase in the gut of H. parallela and the pH and temperature adaptability were detected. The diversity difference of the microbial community enriched from the gut of H. parallela before and after the subculture was compared and the straw degradation was researched. The enzyme activities of the mixed-culture bacteria and single strains fermentation were compared. These studies provide a new idea and theoretical basis for the research of the cellulase activities in the insect gut and the straw biodegradation.1. The cellulase activities in the gut of H. parallelaThe enzyme activities of endoglucanase, P-glucosidase and xylanase of the protein extraction from the head and gut of H. parallela were researched and compared. These enzyme activities in the gut of H. parallela larvae are higher than in the adult’s. These enzyme activities of protein extraction of the third instar larve gut are higher than that of the first instar larvae. The enzyme in the gut has a higher activity than in the head. The optimum temperature of the cellulase in the adult is40℃while in the larvae it is50℃. The xylanase has a wide temperature adaptability. The cellulase can adapt to the acid and alkaline conditions. The active bands of endoglucanase and β-glucosidase were detected through zymogram. The partial sequences of cellulase proteins were identified through tandem mass spectrometry analysis:it’s xylanase screened form Bacteroide ovatus, glycoside hydrolase family43protein screened form Schizophyllum commune H4-8, PE-PGRS family protein from Sorangium cellulosum So ce56.2. The enrichment and subculture of the cellulolytic microbial community in the gut of H.parallelaThe microbial community enriched from the gut of H. parallela larvae was cultured and subcultured with rice straw powder as the sole carbon source. A microbial community was obtained which can utilize rice straw and stably degrade filter paper. The medium inoculated the microbial community enriched from the gut of H.parallela adult could degrade the filter paper in13days while9days were needed by the larvae. Then the composition of microbial community was analyzed. In the subcultured bacteria community enriched from the gut of larvae, the top three genus are Dysgonomonas, Azospirillum and Ruminococcaceae_Incertae_Sedis. While in the community enriched from the gut of adult, the top three genus are Lysinibacillus, Dysgonomonas and Kerstersia. The microbial community enriched from the gut of H. parallela larvae could degrade0.3437g rice straw at35℃in15days which has no significant difference with that of the adult.3. Construction of the cellulolytic mixed-cultured bacteria screened from the gut of H.parallela larvaeThe effect of fermentation time and temperature on the enzyme activities of both mixed-cultured bacteria and single bacteria strains was studied. The results show that enzyme production of mixed-cultured bacteria fermentation is three days in advance than the single strains. The cellulase and hemicellulase activities of mixed-cultured bacteria fermentation is higher than the single strains. The highest enzyme activities was observed at30℃-35℃by the mixed-cultured fermentation. The mixed-cultured bacteria have a milder fermentation temperature which is more suitable to the biodegradation application. About0.5487g rice straw was degraded at35℃in15days by mixed-cultured bacteria.