一株分离自绿化废弃物堆肥枯草芽孢杆菌的常压室温等离子诱变及其发酵条件

【背景】为了提高堆肥降解有机废弃物的效率,高效堆肥菌剂成为了研究热点,其中以真菌应用的研究为多,但真菌也有对氧气和底物敏感等缺点,细菌对堆肥的作用开始被研究。本实验室以羧甲基纤维素钠(CMC-Na)为底物,从绿化废弃物堆肥中筛选得到枯草芽孢杆菌(Bacillussubtilis,B.subtilis) BL03,它具有较好的纤维素分解能力,能提高绿化废弃物堆肥中纤维素降解和腐殖质合成的速度。【目的】进一步提高B.subtilisBL03的纤维素酶生产能力。【方法】利用常压室温等离子(Atmospheric and room temperature plasma,ARTP)诱变BL03菌,通过CMC-刚果红固体培养基观察水解透明圈,以及液体发酵后检测酶活力的方法进行3轮筛选;通过连续多代培养观察突变株的遗传稳定性;通过梯度温度、p H培养研究突变株发酵的最适生长温度、培养基初始pH;利用正交设计方法研究适合突变株发酵培养的工业级原料配方。【结果】筛选到2株正突变株,酶活力分别提高了69%和72%;连续10代培养稳定,验证了突变株的遗传稳定性;其中酶活力最高的突变株BLA3890最适培养温度为37°C、培养基初始pH为5.0-6.5,研究得到较经济的发酵培养基配方。【结论】ARTP诱变B. subtilis BL03后得到的突变株BLA1973和BLA3890在绿化废弃物堆肥或其他纤维素降解行业具有进一步研究和应用的价值。

Mutagenesis of Bacillus subtilis isolated from green waste compost by atmospheric and room temperature plasma treatment and its fermentation conditions

Background] High-efficiency composting agents, especially fungi, have become a research hotspot for improving the efficiency of green waste compost. Due to the disadvantaged of fungi application such as its sensitivity to oxygen and substrate, the role of bacteria in composting process has gradually been studied. Bacillus subtilis (B. subtilis) BL03 was screened from the composting process of green waste using CMC-Na as substrate in our laboratory. The BL03 strain with good cellulose decomposition ability could improve the speed of cellulose degradation and humus synthesis in the green waste composting. Objective] To further improve the cellulase activity of BL03. Methods] The atmospheric pressure room temperature plasma (ARTP) was used to mutate the bacteria. Three rounds of screening were carried out via measuring the diameter of the hydrolyzed transparent circle on CMC-Congo red solid medium and detecting the activity of cellulase after liquid fermentation. The genetic stability of the mutant was measured after 10-generations culturing. The optimum growth temperature and initial pH of medium for mutant fermentation were determined by setting gradient temperature and pH culture. Orthogonal design was adopted to determine the industrial raw materials for the fermentation of the mutants. Results] Two mutant strains, of which the enzyme activity increased by 69% and 72% respectively, were obtained. Also, the cellulase activity of the two mutants were stable after 10 generations culturing. The optimum culture temperature for the highest cellulase activity mutant, BLA3890, was 37 °C, and the initial pH of the medium was 5.0?6.5. An economical fermentation medium was obtained from this study. Conclusion] BLA1973 and BLA3890, which obtained from Bacillus subtilis BL03 mutated by ARTP, are valuable in composting process of green waste or other cellulose degradation applications and need further research.