Studies On Isolation And Identification Of Cellulose-degrading Strain From Tibetan Pigs And The Enzymatic Characteristics

Tibetan pigs are grass-type oriented local species of China, which are roughagetolerant, disease resistant et.al. To research the mechanisms that lead to roughagetolerant and isolate highly cellulose-degrading bacteria, carboxymethylcellulose(CMC)-amended agar plates and shake-flask fermentation were used for isolation ofhighly cellulose-degrading strains from the cecal contents of eight-month healthyTibetan and Taihu pigs, and a thermophilic bacterial strain, marked as BY-3, wasnewly isolated; The “single-factor” design and response surface methodology (RSM)was then employed to optimize fermentation condition and medium composition ofstrain BY-3, characteristics of the crude cellulase produced by the strain were alsodescribed. This study could lay experimental foundation for industrial utilization ofcellulolytic microbes and cellulases. The main conclusions are as follows:1, A thermophilic bacterial strain BY-3was isolated by carboxymethylcellulose(CMC)-amended agar plates and shake-flask fermentation. Based on the results ofbiochemical, morphological characterizations, and the phylogenetic analysis of strainBY-3, the strain was designated as B. subtilis BY-3.2, The growth curve of strain BY-3indicated that the strain was grown tologarithmic phase after6h cultivation, followed by stationary phase after21h; themaximum enzyme activity was up to1.603±0.039U/mL when cultivated in the basalfermentation medium for24h.3, The “single-factor” design was applied to determine the optimal fermentationconditions of strain BY-3: inoculums3%, fermentation temperature42℃, rotationspeed220rpm and loading dose50mL/250mL.4, A combination of “single-factor” experiments and RSM design was used foroptimizing a medium for production of cellulase bystrain BY-3. Corn stover andsoybean meal were screened as the optimal carbon source and nitrogen source,respectively. NaCl (X3) and corn stover (X6) were identified as the critical variablesfor cellulase production and the optimized medium composition (g/L): MgSO4·7H2O 0.3, K2HPO4·3H2O1.5, NaCl5.772, Yeast extract0.5, Soybean meal20, Corn stover28.499, CaCl2·2H2O0.1and Tween800.1%(v/v), for maximum cellulase productionby B. subtilis BY-3was obtained by RSM. Under the optimal medium, an over all3.15-fold increase in cellulase yield with an average maximum carboxymethylcellulase (CMCase) activity of5.305±0.073U/mL was achieved in shake flasks after24h submerged fermentation, which showed good agreement with the predictedresponse (5.263U/mL) by RSM.5, Maximum carboxymethyl cellulase (CMCase) activity of the crude cellulase inassays was observed at pH5.5and60°C. The enzyme was active on rice straw, wheatstraw, wheat bran, corn stover and posseed good stability at60°C for at least1h.Surprisingly, the cellulase exhibited strong antibacterial activity againstStaphylococcus aureus.The results of this study were helpful to reveal the mechanisms of roughage ofTibetan pigs; this study will also provide a potential solution to utilize straws anddevelop cellulase preparation.