Screening Of High-Yield Cellulase Strains And Its Application In The Production Of Feed By Spent Mushroom Substrate

In recent years, duo to the rapid development of the edible fungus growth, the production of spent mushroom substrate is greatly increased. So it caused the problem how to use the spent mushroom substrate resource. The main components of spent mushroom substrate are crude fiber, crude protein, crude fat. Because fiber is difficult to be degradated, it has caused reuse limitation. In this study, the soil-samples were collected under the accumulated withered leafage, and cellulose decomposing microorganisms were separated from soil-samples. Three strains enzyme-producing conditions were respectively optimized and they were induced by physical and chemical methods to improve CMCase activities. As a result, High producing cellulase strains degraded spent mushroom substrate, instead of parttraditional feed. The specific experiment results are showed as follows:(1) CMC-Na medium was used in preliminary medium to obtain strains from Jinhua North mountain, and congo red staining method was used to screening strains..Morphological characteristics of bacterial and spore were observed by electron microscope. Two bacterial strains were isolated and identified as Aspergillus Nigera the other one was Penicillium. The CMCase activity of strain 1, strain2 and strain3 determined by DNS method reached to 0.214IU/mL,0.188 IU/mL and 0.137 IU/mL separately.(2) The experiment was conducted to determinate nutrition composition of Pleurotus ostreatus mushroom bran and Pleurotus eryngii bran. Results showed that the content of crude fiber in Pleurotus ostreatus mushroom bran and Pleurotus eryngii bran was 40.0% and 25.4%, crude protein content was 9.07% and 7.41%, crude fat content was 0.38% and 1.00%, Ca content was 1.17% and 1.12%, P content was 0.71% and 0.98%, respectly. Moreover, the two spent mushroom substrates contained little amount of microminerals such as Mg, Fe, Mn, Zn. Pleurotus ostreatus mushroom bran and Pleurotus eryngii bran were used as the only carbon source of medium cultured. The degradation rate of Pleurotus ostreatus mushroom bran and Pleurotus eryngii bran was 26.7% and 23.6%, the degradation rate is higher than the latter.(3) This experiment is aimed to increase the CMCase production and CMCase activity, the optimal environmental conditions including carbon source, nitrogen source, pH, cultivation time and cultivation temperature were further investigated. The optimal conditions of cellulose production for strain 1 and strain 2 were 1% 10 g/L CMC-Na,3g/L yeast extract,0.3g/L MgSO4, 0.3g/LCaCl2, 4g/LKH2PO4, pH 4 and 40℃, cultivation for 7 days, the crude CMCase activities of strain1 and strain2 reached the maximum at 0.504IU/mL and 0.277IU/mL. The optimal conditions for strain 3 is lOg/L glucose and 3g/L ammonium nitrate, pH 8 and 50℃, cultivation for 7 days. The crude CMCase activities reached 0.692 IU/mL.(4) In order to further increased cellulase-producting activities, the original three strains were mutated by UV, NaNO2, hot and compound mutagenesis. After UV mutagenesis, the CMCase activities of strain 1 and strain2 enhanced by 7.79% and 6.77%, respectively, and enzyme activity of strain3 was1.43 times than CK. After NaNO2 mutation, CMCase activities of strain 1 and strain 2 increased by 9.19% and 7.35% than the original strains, CMCase activity of strain3was 1.33 times than the original strain. Through hot mutagenicity, CMCase activities of strain 1 and strain 2 improved by 9.19% and 7.35%, while CMCase activity of strain3 decreased. Finally, after compound mutagenesis, CMCase activities of strain1, strain 2and strain 3 were 0.239 IU/mL,0.210 IU/mL and 0.178 IU/mL, CMCase activities of strain 1, strain 2 and strain 3 increased by 11.4%,12.2% and 30.0% than original strains.(5) The degradation effect of 3 original strains on Pleurotus ostreatus mushroom bran and Pleurotus eryngii bran was studid, and the results were compared with the special bacteria crude fibre or straw inoculants. The degradation of Pleurotus ostreatus mushroom bran, Pleurotus eryngii bran was largest, When strain2 was used of degrading spent mushroom substrate. Pleurotus ostreatus mushroom bran, Pleurotus eryngii bran degradation rate achieved 26.7% and 23.6%.(6) In order to improve the degradation of spent mushroom substrate, the fermentation conditions of strain 2 were optimized. The optimal conditions of cellulose production for Pleurotus ostreatus mushroom bran degradated by strain 2 were inoculation amount 9%, pH 4 and 30℃, the degradation rate of Pleurotus ostreatus mushroom bran reached the maximum at 34.5%, which increased by 29.2%. For Pleurotus eryngii bran, the optimal conditions of cellulose production was inoculation amount was 9%, pH 6 and 20℃, the degradation rate achieved 29.8% which is 26.3% higher than the control.