高温秸秆降解菌的筛选及其纤维素酶活性研究

Screening of thermophilic cellulolytic bacteria and investigation of cellulase thermostability

筛选能在高温(55~75℃)好氧堆肥中高效降解纤维素的菌株,并评估其降解秸秆的能力与产纤维素酶的热稳定性。从高温期堆肥中采样,以水稻秸秆粉为唯一碳源,通过55、65℃和75℃连续高温传代驯化并分离筛选耐高温菌,结合水解圈和水稻秸秆崩解试验筛选不同高温下高效降解秸秆的目标菌株,采用16S rRNA测序和系统发育分析鉴定目标菌株分类地位,通过分析目标菌株纤维素降解相关酶在50~90℃之间的热稳定性,解析其高温适应性机制,评价其在实际生产中的应用潜力。高温驯化分离得到13株耐高温降解菌,其中B-5、B-6、B-7和B-11的纤维素和秸秆降解能力较强,而只有B-7和B-11在55~65℃和75℃具有高效降解水稻秸秆的能力,将其认定为目标菌株。系统发育分析表明B-7和B-11菌株与芽孢杆菌科高度相似,分别命名为短小芽孢杆菌B-7和嗜热脂肪芽孢杆菌B-11。酶活热稳定性分析发现B-7和B-11各纤维素酶活性在50~90℃之间先升高后降低,其最适温度范围不同,分别为55~65℃和70~80℃,其中B-11在85℃时的相对酶活仍高于60%。研究表明,菌株B-7和B-11是耐高温高效秸秆降解菌,其具有不同高温偏好性,纤维素酶热稳定性强,在秸秆高温好氧堆肥中具有潜在的应用前景。

The aim of this study was to screen efficient cellulolytic bacteria at high temperatures(55~75℃)and evaluate the straw degradation and thermostability of cellulase.Thermophilic cellulolytic bacteria were isolated from composts enriched by a serial subcultivation in rice straw at 55,65℃,and 75℃.Targeted strains were screened based on hydrolytic activity and degradation efficiency.The resulting bacteria were subjected to 16S rRNA sequencing and phylogenic analyses,and enzyme thermostability investigation.Bacterial strain B-7 and B-11 were screened from 13 thermophilic cellulolytic bacterial isolates owing to their high cellulolytic efficiency and the carboxymethyl cellulose activity.The 16S rRNA analysis revealed that B-7 and B-11 belong to Geobacillus stearothermophilus and Bacillus pumilus,respectively.Cellulase thermostability results showed that the cellulase activity of B-7 and B-11 first increased and then decreased at 50~90℃with optimal enzyme activity at 55~65℃and 70~80℃,respectively.B-7 and B-11 are efficient thermophilic cellulolytic bacteria with different temperature preferences.These two thermophilic bacteria show great potential for a potential for broad application in high temperature aerobic composting.