西南地区高山湖泊中可培养细菌多样性及其所产胞外活性物质的特性

Diversity of culturable bacteria and their extracellular active substance properties in alpine lakes of Southwest China

【目的】研究西南不同地区的高山湖泊中可培养细菌的多样性及其产胞外蛋白酶、纤维素酶和胞外多糖的能力。【方法】以西南4个不同地区的高山湖泊:雷波的马湖(LB)、中缅边境的凯邦亚湖(ZM)、沙德的莲花湖(SD)、腾冲的青海湖(TC)的水样为研究对象,利用稀释涂布平板方法对可培养细菌进行分离筛选,然后通过对可培养细菌的生理生化指标和16S r RNA基因序列进行分析,初步确定细菌属别;对分离得到的菌株进行产胞外蛋白酶和纤维素酶活性测定和产胞外多糖能力检测。【结果】从西南地区4个湖泊中共分离筛选得到41株细菌,其中LB 15株、ZM 13株、SD 7株、TC 6株。根据16S r RNA基因序列的系统进化分析,4个地区可培养细菌的组成和丰度存在明显差异,其中LB和ZM的优势菌属是芽孢杆菌属(Bacillus),其次是气单胞菌属(Aeromonas)和假单胞菌属(Pseudomonas),分离的TC菌株全部属于芽孢杆菌属(Bacillus),分离的SD菌株特异性较强。进一步酶活性和胞外多糖检测表明,分离得到的41株细菌中有28株菌的发酵产物具有蛋白酶活性,6株具有纤维素酶活性,17株可产胞外多糖(Exopolysaccharides,EPS)。其中有2株细菌同时产蛋白酶、纤维素酶和胞外多糖,10株细菌同时产蛋白酶和胞外多糖,2株细菌同时产蛋白酶和纤维素酶,1株细菌同时产纤维素酶和胞外多糖。【结论】西南4个高山湖泊中存在丰富的微生物菌种资源,且4个湖泊中筛选的可培养细菌受所处环境的影响大。其中莲花湖由于高海拔和较偏僻等特点,人为干扰小,分离得到的细菌类群与其他湖泊相比明显不同;而马湖、凯邦亚湖和青海湖3个湖泊的海拔相对较低,受人类活动影响较大,分离得到的细菌均较常见。此外高山湖泊中的可培养细菌具有分泌多种胞外活性物质特性,为工业化应用奠定了资源基础,极具更深入的开发和研究价值。

[Objective] The main aim of this study is to study the diversity of culturable bacteria in alpine lakes of different regions located in Southwest China, and to analyze their capabilities for producing proteases, cellulases and exopolysaccharides. [Methods] Four water samples in different alpine lakes located in Southwest China, including Horse Lake in Leibo （LB）, Kanbon sub Lake in Sino-Burmese border （ZM）, Lotus Lake in Shade （SD） and Qinghai Lake in Tengchong （TC）, were collected to isolate the cultural bacteria based on the spread plate method. The genera of culturable bacteria were identified by analyzing the physiological and biochemical indexes and 16S rRNA gene sequences. The strains were further detected for production of proteases, cellulases and exopolysaccharides. [Results[ A total of 41 strains were isolated from four lakes in southwestern China, of which 15 strains from LB, 13 strains ZM, 7 strains SD and 6 strains TC. According to phylogenetic analysis of 16S rRNA gene sequences, there were obvious differences in composition and abundance of culturable bacteria among four lakes. Aeromonas and Pseudomonas in LB and ZM. All The dominant genus was Bacillus, and followed by isolates were Bacillus in TC, whereas strains were highly specific in SD. Further research on enzyme activities and exopolysaccharides were detected in 41 cultural bacteria, of which 28 strains had protease activity, 6 strains had cellulase activity and 17 strains produced exopolysaccharides （EPS）. In addition, 2 strains produced proteases, cellulases and exopolysaccharides simultaneously, 10 strains produced proteases and exopolysaccharides, 2 strains produced proteases and cellulases, and only 1 strain produced cellulases and exopolysaccharides. [Conclusion] The culturable bacteria in alpine lakes were capable of secreting various extracellular active substances, with potential for further development and research.