耐低温木质纤维素降解菌群的富集培养及其种群结构分析  

Enrichment Culturing and Bacterial Community Structures Analysis of a Cold-adapted Lignocellulose Degrading Microflora

作　　者：罗立津[1,2,3] 万立[2] 陈宏[1] 温翠莲[4] 徐福乐 贾纬[1] 聂毅磊[1] 袁红莉[2] 

机构地区：[1]福建省微生物研究所福建省新药(微生物)筛选重点实验室,福州350007 [2]中国农业大学生物学院/农业生物技术国家重点实验室,北京100193 [3]福建超大现代农业科技研究所,福州350003 [4]福州大学材料科学与工程学院,福州350108

出　　处：《农业生物技术学报》2015年第6期727-737,共11页Journal of Agricultural Biotechnology

基　　金：福州市科技计划项目(No.2013-G-97);福州市仓山区科技计划项目(仓科[2014]14号);教育部留学回国人员科研启动基金(LXKQ201305)

摘　　要：针对农业生物质废弃物在冬季低温环境下生物转化效率低的问题,本研究通过采集高寒地区长期堆积牛粪的土壤,经低温继代富集培养,获得一组耐低温的具有较高降解纤维素活性的菌群A25-3。分析表明其在15℃能迅速生长,在72 h内使牛粪的失重率达48.28%,15℃培养菌液的羧甲基纤维素酶(carboxymethyl cellulose,CMCase)酶活高于25℃培养的菌液,在120 h时达到42.37 U高峰。提取两种温度下培养的细菌总DNA,分别构建基于通用引物PCR扩增的16S r RNA基因克隆文库,通过HinfⅠ和Csp6Ι限制性内切酶进行限制性片段长度多态性分析(amplified ribosomal DNA restriction analysis,ARDRA),将所有阳性克隆分为若干个可操作分类单元(operational taxonomic unit,OTU),通过构建两种温度下细菌克隆文库的系统发育树,并分析种群结构表明,在25℃培养条件下,A25-3菌群中优势种群主要有短波单胞菌属(Brevundimonas)、假单胞菌属(Pseudomonas)、金黄杆菌属(Chryseobacterium)、不动杆菌属(Acinetobacter)、鞘氨醇杆菌属(Sphingobacterium)、芽胞杆菌属(Bacillus)等,但在15℃低温下,优势菌种只有不动杆菌属、Psychrobacillus和芽胞杆菌属。由此推断,菌群A25-3在15℃中具有更高的纤维素酶活性是由于耐低温降解纤维素细菌重要种属(特别是不动杆菌)更占优势。本研究为今后菌群活性菌株分离及纤维素酶合成条件优化提供科学依据。In the face of the inefficiency of bioconversion of agricultural biomass waste in low temperature environment in the winter, this study was aimed at enrichment culture and community structure analysis of a cold-adapted cellulose degrading microflora A25-3, which was isolated from the soil in alpine region. The microflora A25-3 could grow rapidly by enrichment culture at 15℃, with 10% cow dung and 10% LB sodium salt, and the weight loss ratio of cow dung was 48.28% in 72 h. The carboxymethyl cellulose （CMCase） activity of the supernatant of microflora culture at 15 ℃, with a peak of 42.37 U in 120 h was higher than that at 25 ℃. Total microbial DNA was directly extracted from the microflora cultured at different temperatures of 15 and 25 ℃. The clone library of 16S rRNA genes was amplified using PCR with universal bacterial primer sets. Each unique restriction fragment polymorphism pattern, which was created with 2 restriction endonucleases （Hinf I and Csp6 I ）, was designated as an operational taxonomic unit （OTU）. Amplified DNA was used for diversity analysis. The microflora phylogenetic trees of bacterial clone library at different temperatures of 15 and 25 ℃ was constructed. Community structure analysis revealed that the genera Brevundimonus, Pseudomonus, Chryseobacterium, Acinetobacte, Sphingobacterium and Bacillus were dominant in the clone library from microflora cultured at 25 ℃, while the genera Acinetobacte, Psychrobacillus and Bacillus were dominant at 15 ℃. The results suggested that the dominant population of cold-adapted cellulose degrading bacteria important species, especially Acinetobacte, provided the effect of increasing the CMCase activity at 15 ℃. The result will provide a scientific basis for the next study to separate active bacteria strains from the bacterial community and to optimize cellulase synthesis conditions.

关 键 词：耐低温 纤维素降解菌 16S rRNA基因克隆文库 种群结构 不动杆菌 

分 类 号：S646[农业科学—蔬菜学]