牛源纤维素降解菌的分离筛选与鉴定

Isolation,Screening and Identification of Cellulose Degrading Bacteria from Cattle

为了提高牛粪中的纤维素降解效率,筛选牛粪发酵菌剂的优良菌种,采用连续稀释平板法,以羧甲基纤维素纳(CMC-Na)为碳源,从新鲜牛粪和腐熟牛粪中对纤维素降解菌株进行初步分离。对所得菌株进行纤维素水解圈测定,并进一步采用3,5-二硝基水杨酸(DNS)法测定纤维素酶活,再结合菌落形态、生化反应特性和16S rDNA序列分析进行菌种鉴定。研究结果表明:从牛粪样品中初步分离得到了56株纤维素降解菌,进一步水解圈测定筛选出13株菌,其中菌株NA10的水解圈最大,水解圈直径与菌落直径比值(H/C)为5.35,该菌纤维素酶活(CMC)和滤纸酶活(FPA)分别为55.13 U/mL和35.47 U/mL。观察NA10菌落呈乳白色圆盘状,表面光滑不透明。显微镜下,可见NA10经革兰氏染色呈阳性,为短杆状、链状排列,中生芽孢。生化反应显示NA10符合芽孢杆菌属安全芽孢杆菌的特征。经16S rDNA基因序列分析鉴定,该菌株为芽孢杆菌属的安全芽孢杆菌(Bacillus safensis),将其命名为Bacillus safensis NA10。本文分离得到一株具有降解纤维素潜力的菌株,可作为制备发酵菌剂的优势菌种之一。

To improve the efficiency of cellulose degradation in cow dung and screen the excellent strains for fermentation of cow dung,theserial dilution plate method was used to isolate cellulose degrading strains from fresh and decomposed cow dung with sodium carboxymethyl cellulose( CMC-Na) as acarbon source. The activity of cellulase was determined by enzyme digestion( DNS),and then identified by colony morphology observation,biochemical reaction characteristics and 16 S r DNA sequence analysis. The results show that 56 strains of cellulose degrading bacteria preliminarily are isolated from cow dung samples,and 13 strains are screened out by further hydrolysis circle determination.Among them,the NA10 strain has the largest hydrolysis circle,the ratio of hydrolysis circle diameter to colony diameter( H/C) is 5. 35,and its cellulose enzyme activity( CMC) and filter paper enzyme activity( FPA) are 55.13 U/m L and 35.47 U/m L,respectively.The colony of NA10 is milky white,disk-shaped,smooth and opaque. The micro features are gram-positive,short rod-shaped,chain shaped and mesophytic spores.Biochemical reaction shows that the NA10 is consistent with the characteristics of Bacillus. 16 S r DNA gene sequence analysis shows that the strain is Bacillus safensis in Bacillus sp.,and is named Bacillus safensis NA10. The strain with cellulose degradation potential is isolated,which can be used as one of the dominant strains for the preparation of fermentation agents.