一株蚕沙纤维素降解菌的鉴定及产酶优化和生物强化效果

Identification of a Cellulose-Degrading Bacteria from Silkworm Excrement and Optimization of Enzyme Production and Its Bioaugmentation Effect

为了提高蚕沙生物转化资源化处理效率,从蚕沙废弃物中筛选出1株高效纤维素降解菌株DC-15,优化产酶条件并评估其对蝇蛆生物转化蚕沙体系的强化作用,以期为蚕沙高效资源化处理提供优质菌株。采用羧甲基纤维素钠(sodium carboxymethyl cellulose, CMC-Na)平板法和刚果红染色法进行筛选,通过形态学观察、生理生化试验和16S rRNA基因序列系统发育分析进行种属鉴定,利用DNS法(3,5-二硝基水杨酸法)测定菌株DC-15的纤维素酶活力,并优化其产纤维素酶条件。结果表明,获得的高效纤维素降解菌DC-15鉴定为特基拉芽孢杆菌(Bacillus tequilensis),该菌株最优生长及产酶碳源和氮源为羧甲基纤维素钠(CMC-Na)和蛋白胨;最适接种量、产酶温度和pH分别为2%、37℃和7。优化后菌株DC-15的纤维素内切酶(CMCase)、滤纸酶(FPase)、纤维素外切酶(CBH)最高酶活力分别可达11.66、11.62、10.01 U/mL;菌株DC-15回补蝇蛆生物转化蚕沙体系后(6 d),蚕沙纤维素降解率为71.2%,显著高于对照组的59.6%。获得的菌株DC-15能够高效降解蚕沙纤维素,其纤维素酶活力较高,对蝇蛆生物转化蚕沙降解纤维素体系有生物强化作用,制成的微生物菌剂具有应用于蚕沙无害化快速腐熟的潜力。

In order to improve the bioconversion and resource utilization efficiency of silkworm excrement, a high-efficiency cellulose-degrading bacteria DC-15 was screened out from silkworm excrement substrates. The conditions of cellulase production were optimized, and its bioaugmentation effect on housefly larvae biotransformation silkworm excrement system was evaluated, so as to provide high-quality strains for efficient resource treatment of silkworm excrement. The strains were screened by sodium carboxymethyl cellulose(CMC-Na) culturing method and Congo red staining method. The strain was identified by morphological observation, physiological and biochemical tests and phylogenetic analysis of the 16S rRNA gene sequence. The cellulase activity of strain DC-15 was studied by the DNS method(3, 5-dinitrosalicylic acid method), and its cellulase production conditions were optimized. The results showed that the highly efficient cellulolytic strain DC-15 was identified as Bacillus tequilensis. The best carbon and nitrogen sources for the growth and enzyme production of the strain were CMC-Na and peptone. The optimum inoculation amount, temperature and pH were 2%, 37 ℃ and 7, respectively. After optimization, the endocellulase(CMCase) activity, the filter paper enzyme(FPase) activity, and exocellulase(CBH) activity reached up to 11.66, 11.62, 10.01 U/mL, respectively. The degradation rate of cellulose in silkworm excrement was 71.2% after strain DC-15 supplemented housefly larvae vermicomposting system(6 d), which was significantly higher than that of the control group(59.6%). The strain DC-15 can efficiently degrade silkworm excrement cellulose, and its cellulase activity is relatively high, which has a bioaugmentation effect on the cellulose degradation system, and has the potential to make microbial inoculum to be applied to harmless quick composting of silkworm excrement.