玉米秸秆低温高效降解复合菌系GF-20的菌种组成及降解稳定性

【目的】探索不同温度及pH条件对玉米秸秆低温高效降解复合菌系的玉米秸秆分解稳定性及菌群结构稳定性的影响，完善菌系培养方法，促进其应用开发利用。【方法】以低温高效降解复合菌系GF-20为研究对象，在10℃条件下连续继代培养45代、不同温度和pH条件下连续继代培养15代，分别获得多组不同代数（F）、不同温度（T）和pH（P）条件下的菌系。测定各复合菌系发酵液pH、玉米秸秆降解率及纤维素酶活，评价复合菌系的玉米秸秆分解稳定性；利用PCR-DGGE技术结合主成分分析法对菌群组成结构的稳定性进行研究。【结果】在10℃条件下经连续继代培养40代和在温度4-30℃、pH 6.0-9.0条件下继代培养获得的不同复合菌系发酵液pH均随发酵时间的延长趋近中性；玉米秸秆降解率在27.59%-32.53%，除F40显著高于F5外，其余无显著差异；纤维素酶活性呈高代菌系大于低代菌系，温度4-10℃和pH 6.0-9.0条件下，对复合菌系产酶有促进作用，纤维素酶活为1.34-1.84 IU·mL^-1；复合菌系的纤维素酶在较低的温度和较宽的pH范围内具有良好的温度和pH稳定性，酶促反应温度15-30℃和pH 4.0-9.0内仍保持80%以上的纤维素酶活力；复合菌系F5-F45、T4-T30和P6.0-P9.0的DGGE条带差异不显著，表明菌系的菌种组成稳定；而在偏酸（pH=4、5）和偏碱性（pH=10）条件下继代培养，复合菌系秸秆降解率和纤维素酶活均显著降低，菌种组成发生变化，进而影响性质与功能稳定性。PCR-DGGE共检测到18个条带，其中关键菌株分别为Bacillus licheniformis、Azonexus hydrophilusd、Azospira oryzae、Arobacter cloacae、Cellvibrio mixtus、Bacillus tequilensis、Clostridium populeti subsp. Mixtus和Clostridium xylanolyticum。【结论】复合菌系GF-20在温度4-30℃、pH 6.0-9.0条件下经过多代继代培养，仍然保持了较高的玉米秸秆分解活性和菌种组成稳定性，具有良好的应用前景。

Function and Composition Stability of a Composite Microbial System GF-20 with Efficient Corn Stalk Decomposition Under Low Temperature

【Objective】 In order to improve the culturing methodology of corn stalk decomposing microbes and promote its utilization, the present study evaluated the impact of different culturing conditions on the community composition of a composite microbial system GF-20 and on its decomposing activity of corn stalk.【Method】 Composite microbial system GF-20 was continuously sub-cultured to the 45th generation under 10℃ and to 15th generation under different temperatures and pH conditions to obtain different microbial communities (F, T, P). Then the dynamics of fermenting pH, corn stalk decomposing ratio and cellulose enzyme activities were determined, so as to estimate the corn stalk decomposing activity of the composite microbial system. Furthermore, the community composition stability was analyzed by PCR-DGGE technique combined with a principal component analysis. 【Result】 The results showed that the pH value tended to be neutral over the fermentation time, and the straw degradation rate ranged in 27.59%-32.53%, in which no difference was shown among the successive 40-generation sub-cultured communities except F40 and F5, neither among the sub-cultured communities under temperature of 4-30℃ and under pH of 6.0-9.0. Cellulose enzyme activity of high generation was higher than that of low generation, and the enzyme production of the microbial community could be promoted under temperature of 4-10℃ and pH of 6.0-9.0, cellulase activity ranged in 1.34-1.84 IU·mL^-1. Cellulase activity of composite microbes showed good stability under lower temperature and in a wide pH range, enzymatic reaction temperature within 15-30℃ and pH within 4.0-9.0 could keep more than 80% of enzyme activity. Furthermore, DGGE bands of F5-F45, T4-T30 and P6-P9 showed no significant differences indicated that the strains composition maintained good stability. However, under acidic (pH=4, 5) or alkaline (pH=10) subculture conditions, the corn stalk degradation rate and cellulase activity significantly decreased, and community composition also changed remarkably, thereby affected the property and functional stability. According to PCR-DGGE profiles, a total of 18 strains were detected, the key strains of which were Bacillus licheniformis, Azonexus hydrophilusd, Azospira oryzae, Arobacter cloacae, Cellvibrio mixtus subsp. Mixtus, Bacillus tequilensis, Clostridium populeti and Clostridium xylanolyticum, respectively. 【Conclusion】Composite microbial community GF-20 sub-cultured under different conditions (temperature 4-30℃, pH 6.0-9.0) kept efficient corn stalk decomposing activity and stable community composition structure, a good application prospect should be anticipated.