草鱼肠道纤维素降解细菌的分离与鉴定

摘要

The microbiota in the animal digestive tracts plays an important role in the nutrition and metabolism of their host. Many cellulose-ingesting animals need the microorganisms in their digestive tracts for degrading cellulose and therefore providing the energy. For example, cellulose-decomposing bacterial population has been frequently found in the intestine of grass carp (Ctenopharyngodon idella), an herbivore fish species. However, knowledge about this type of bacteria remains unclear. In this study, we characterized the species of the cellulose-decomposing microbiota in the content and mucosa in the intestine of grass carp. We isolated and cultured the cellulolytic bacteria in the foregut, mid-gut and hindgut of the intestine using the medium with carboxymethycellulose (1%) as its sole carbon source. Entero-bacterial repetitive intergenic consensus (ERIC) PCR fingerprint was utilized to identify different genotypes of the iso-lated bacteria. The cellulose-degrading activity of each strain was indicated by the ratio of the diameter of the clear zone to the diameter of the colony. We identified the genotypes of these bacteria by sequencing the 16S rRNA gene. Forty strains of cellulose-decomposing bacteria from intestinal content and mucosa of grass carp were identified. Two geno-types of the cellulose-decomposing bacteria were discovered in the content of foregut and hindgut, and in the mucosa of midgut and hindgut. Only one genotype of the cellulose-decomposing bacteria was discovered in the mucosa of foregut and midgut. The 16S rRNA sequencing analysis showed that the majority of the cellulolytic bacteria was Aeromonas which accounted for 85%of the total bacterial isolates. Enterobacter accounted for 7.5%and the“uncultured bacterium”accounted for 5%. The ability of degrading cellulose varied remarkably among different bacterial strains. High enzy-matic activity was observed in some isolates of the cellulose-degrading bacteria. The bacterial isolates with a ratio (the diameter of clearing zone to the diameter of the colony) greater than 5.0 had significantly higher cellulolytic ability than the others. These bacterial isolates were identified as Aeromonas veronii (MC2, BC6),“uncultured bacterium”(BM3) in Enterobacteriaceae, and A. jandaei (HC9). Here we reported that A. jandaei, Plesiomonas shigelloides, Enterobacter cloacae and E. aerogenes were cellulose-decomposing bacteria in grass carp intestine for the first time. The results of the present study indicated that there were a variety of cellulose-degrading bacteria in the intestine of grass carp. Our study contributed to the knowledge about cellulolytic bacterial species in the intestine of grass carp, and will provide the guidance for the selection of cellulose-degrading probiotics.%为更好地弄清草鱼(Ctenopharyngodon idella)肠道纤维素降解细菌的种类，采用羧甲基纤维素(CMC)作为唯一碳源的选择性培养基，分别从草鱼肠道内容物和肠道黏膜中分离到了40株产纤维素酶细菌。16S rRNA基因序列的分析结果显示，大多数产纤维素酶细菌为气单胞菌属(Aeromonas)的种类，其次为肠杆菌属(Enterobacter)的细菌以及未经分离纯培养的细菌(Uncultured bacterium)。进一步研究细菌产纤维素酶能力发现，纤维素酶活性显著性高于其他菌株的分别是A. veronii MC2、A. veronii BC6、肠杆菌科(Enterobacteriaceae)中一种未经分离纯培养的细菌BM3(Uncultured bacterium BM3)和A. jandaei HC9。草鱼肠道中简答气单胞菌(A. jandaei)、类志贺邻单胞菌(Plesiomonas shigelloides)、阴沟肠杆菌(E. cloacae)以及产气肠杆菌(E. aerogenes)是被作为产纤维素酶细菌的首次报道。