不同pH缓冲液对由乙酸产甲烷菌群结构的影响

Effects of p H on methanogenesis and methanogenic community in the cultures amended with acetate

【目的】研究不同p H缓冲液对乙酸产甲烷过程及对细菌和古菌群落结构的影响。【方法】分别添加磷酸盐(PB)、4-羟乙基哌嗪乙磺酸(HEPES)、哌嗪-1,4-二乙磺酸(PIPES)和Na HCO3/CO2缓冲液到乙酸产甲烷菌系中,定期监测甲烷产生趋势,到稳定期后收集菌体,进行16S rRNA基因的末端限制性片段多态性分析(T-RFLP)。【结果】发现PB组的乙酸产甲烷菌系延滞期约为40d,显著高于其他组的20-24 d(P<0.05);Na HCO3/CO2组乙酸转化为甲烷的比例为(88.3±0.5)%,显著高于其他组的77%-81%(P<0.05);不同缓冲液组的最大甲烷比生长速率为0.46-0.57 d-1(P>0.05);Na HCO3/CO2组的细菌群落变化最明显,主要是未培养细菌(unclassified bacteria)、螺旋菌科细菌(Spirochaetaceae)和未培养WWE1类群的丰度较其他组分别增加到(15.5±9.4)%、(7.3±4.6)%和(17.6±6.3)%,而互养菌科(Synergistaceae)的细菌丰度降低到(8.9±8.1)%。AC+PB组中的古菌类群发生了明显变化,以竹节状甲烷鬃毛菌(Methanosaeta harundinacea)相关的产甲烷古菌占主导(97±2%),而在HEPES、PIPES和Na HCO3/CO2组和不加缓冲液组中同时存在两类乙酸营养型产甲烷古菌M.harundinacea和联合鬃毛甲烷菌(Methanosaeta concilii),以及属于甲烷杆菌目(Methanobacteriales)的氢营养型产甲烷古菌。【结论】在乙酸产甲烷菌系中加入PB增加了甲烷产生的延滞期,加入Na HCO3/CO2增加了甲烷产量,但是添加p H缓冲液不会影响到菌系的最大甲烷比生长速率。加入PB和Na HCO3/CO2都会显著改变微生物的菌群结构。这些研究为设计适宜的产甲烷菌系生长条件提供了参考。

[ Objective ] To evaluate the effects of pH on methane production from acetate and the methanogenic community structures. [ Methods ] Solutions of phosphate （ PB ） , 2-hydroxyethyl （HEPES） , NaHCO3/CO2 or piperazine-1,4- bisethanesulfonic acid （PIPES） were added into the methanogenic cultures, separately. The substrate consumption was determined by monitoring cumulative methane production, the methanogenic community structuresin the stationary-phase cultures were analyzed using terminal restriction fragment length polymorphism （T-RFLP） of 16S rRNA gene fragments. [ Results ] The period of lag phase of methane production in the PB addition culture （ ca. 40 d） was much longer than that in other pH buffer cultures （20 -24 d, P 〈 0.05 ）. Approximate 88.3% of acetate was converted into methane in the NaHCOJCOz addition cuhure, while the value decreased to 77% -81% in other pH buffer cultures （P 〈 0.05）. The maximum specific methane production rate was similar between different pH buffer cultures （P 〉 0.05）. The relative abundance of members of unclassified bacteria, Spirochaetaceae and uncultured WWE1 increased to （ 15.5 ± 9.4） % , （7.3 ± 4.6）% and （17.6 ± 6.3）%, respectively, in the NaHCO3/CO2 addition culture, while synergistaeeae decreased to （8.9 ± 8.1 ） %. In archaeal domain, the acetotrophic methanogen related with Methanosaeta harundinacea became predominant （97 ± 2% ） in the PB buffer culture, on the contrary, the concurrence of M. harundinacea, M. concilii and hydrogenotrophic methanogen related with Mcthanobacteriales were detected in the cultures amended with HEPES, PIPES and NaHCO3/CO2. [ Conclusion ] PB retarded the methane production in the acetatemethanogenic culture,NaHCO3/CO2 addition improve methane production from acetate, the pH buffers had not obvious effects on the maximum specific methane production rate of the cultures, the microbial community structures obviously changed along with PB and NaHCO3/CO2 addition. The research would help us to design suitable condition for the growth of methanogenic culture.