低温沼气发酵微生物区系的筛选及其宏基因组文库构建

Screening of Microbial Community in Biogas Fermentation under Low Temperature and Construction of its Metagenome Library

从四川、浙江、山西、北京等地采集冬季产气良好户用沼气池及沼气工程中的沼液样品作为微生物接种源,在实验室条件下进行了500ml反应器的梯度降温模拟沼气发酵试验,筛选出5个在21℃、18℃和15℃等不同温度下均可正常产气的沼液样品Z-2、Z-3、Z-6、Z-8和Z-10,选取其中3个产气量较高的沼液样品Z-3、Z-6和Z-10作为接种源,进一步建立了2500ml反应器实验室模拟15℃低温沼气发酵系统,经过连续传代筛选获得一个在15℃低温下产气量较高且稳定的沼气发酵系统Z-6,产气率达到0.15m3/m3d。提取该系统内沼气发酵微生物宏基因组DNA,以pBluescriptSK(+)质粒为载体成功构建了约10000个克隆的沼气发酵微生物宏基因组质粒文库。随机分析文库中的克隆结果表明,该文库的外源片段平均长度约为5kb,文库总容量为48Mb,插入片段带型多样性较高。该文库的建立为沼气发酵过程中水解酶基因资源的挖掘利用奠定了基础。

Eight biogas slurry samples were collected from some rural household biogas digesters in Sichuan,Zhejiang and Shanxi Provinces or from biogas project in Beijing.The result of biogas fermentation experiment in 500ml reaction system under different temperatures（21℃ 、18℃ and 15℃ respectively）shown that five of the samples Z-2、 Z-3、Z-6、Z-8 and Z-10 could produce biogas under different temperatures.Three of the biogas slurry samples Z-3、Z-6 and Z-10 which produce higher yield of biogas were chose for further biogas fermentation experiment in 2 500 ml reaction system under 15℃ lower temperature,a higher yield biogas fermentation system Z6 with the biogas yield of 0.15m3/m3day was selected from the three samples according to the results of two times continuous biogas fermentation experiments.The metagenomic DNA of microbial community from the Z-6 biogas fermentation system was extracted by direct extraction method,purified through Wizard DNA Clean-Up System,partial digested by Sau3AⅠand then ligated with plasmid vector of pBluescript SK（＋）.A metagenomic library containing 10 000 transformations was constructed,stochastic analysis of the library indicated that the average size of the foreign DNA fragments was 5 kb and the capacity of foreign DNA in the library was 48 Mb.The diversity of the restriction types of the foreign DNA fragments was higher.The results laid a foundation for utilization of gene resources of the key hydrolytic enzymes in biogas fermentation.