【目的】采取人工构建复合菌系的方法探索微生物协同降解纤维素的机理及菌间关系。【方法】从一组高温发酵木质纤维素原料产沼气的菌群中分离获得若干菌株，其中一株细菌经16S rRNA基因全序列测序比对后鉴定为地衣芽孢杆菌(Bacillus licheniformis)，将该菌株与厌氧纤维素分解菌Clostridium thermocellum CTL-6进行共培养，菌株组合表现出很强的滤纸纤维素分解能力。【结果】两菌共培养9 d，累计滤纸分解量为484.6 mg，滤纸相对分解率高达93.2%；pH变化呈先下降后逐步回升，培养3 d后pH由初始时的7.00降到最低值6.57，第9天升至7.73；菌株组合能同时产生纤维素酶和半纤维素酶，培养过程中两种酶活性大小均呈不断上升趋势，最大值分别为0.32 U/mL和0.57 U/mL。利用HPLC监测了乳酸、甲酸、乙酸、丙酸和丁酸5种有机酸含量的变化，其中丁酸、丙酸代谢量最高，分别为1 477.3 mg/L和1 068.8 mg/L；除丙酸外，其他4种有机酸含量变化趋势与滤纸降解的变化均无明显相关性。5种有机酸总含量的变化与pH的变化趋势一致，表明对pH变化起决定性作用的很可能是某种未检测的酸性较强的物质含量变化。【结论】Bacillus licheniformis能有效促进Clostridium thermocellum CTL-6的纤维素分解活性，且该菌株组合可作为后期进一步构建纤维素甲烷转化复合菌系的基础。
[Objective] To explore synergy mechanism of cellulose biodegradation and relationships among the bacterial consortium, we took an approach of artificial constructing composite consortia. [Methods] Some strains were isolated from a microbial community which could ferment lignocellulose to produce biogas at high temperature. One of strains was identified as Bacillus licheniformis by sequencing nearly complete 16S rRNA gene. The strains’ combination, which consisted of the Bacillus licheniformis and Clostridium thermocellum strain CTL-6, had strong filter paper cellulose degradation ability. [Results] Throughout the 9-day co-cultivation, the cumulative degradation amount of filter paper was 484.6 mg and relative degradation ratio was as high as 93.2%. Overall, the variation of pH decreased firstly and then gradually increased. The initial pH of the culture solution was 7.00. The pH dropped to the lowest value (about 6.57) in 3 d. At the end of the culture period (9 days), the pH was 7.73. The combination could produce cellulase and hemicellulase, and two kinds of enzyme activity all represented the rising trend. The maximum of cellulase activity and hemicellulase activity were 0.32 and 0.57 U/mL, respectively, on the day 9. Lactic acid, formic acid, acetic acid, propionic acid and butyric acid were detected by HPLC during the co-culture. Among the five organic acids, the propionic acid and butyric acid had higher metabolism yield and the maximum concentrations were 1 068.8 and 1 477.3 mg/L, respectively. In addition to propionic acid, the concentration change trends of other 4 organic acids had no significant correlation with the change of the filter paper degradation. The total concentration variation of the five organic acids was in accordance with the variation of pH. This result indicated that it probably existed some not-detected acidic substances, the concentration variation of which played a decisive role in the pH variation of the co-culture system. [Conclusion] Bacillus licheniformis could effectively promote the cellulolytic activity of Clostridium thermocellum CTL-6, and the strains’ combination could also be used to artificially construct composite microbial which was able to convert cellulose to produce methane.
国家自然科学基金项目(No. 31370506，31500422)；湖北省自然科学基金项目(No. 2014CFB666)