芦苇秸秆厌氧联产氢气甲烷过程中细菌群落演替规律

Bacterial community structure succession in fermentative biohydrogen with methanogenesis integration from reed straw

基于PCR-DGGE技术研究芦苇秸秆氢气-甲烷厌氧联产过程中,细菌微生物群落结构特征和演替规律。结果表明,厌氧联产过程中细菌群落结构分布存在明显的阶段性差异。产氢阶段初期,细菌群落相似性较小,随着厌氧联产的进行,细菌种类逐渐增多并在产氢高峰期保持稳定,群落相似性较高,戴斯系数(Cs)为83.6%(第12、24小时,泳道H3和H4)。产甲烷高峰期Cs值达到87.4%(第210、258小时,泳道M6,M7),群落结构稳定,产甲烷末期Cs值降低至51.5%(第210、432小时,泳道M6,M9)。序列分析表明,Enterobacter aerogenes产气肠杆菌是具有高效产氢潜力的兼性厌氧细菌,Sedimentibacter产氢产乙酸菌是产氢阶段的优势微生物。Clostridium thermocellum嗜热纤维素菌是厌氧联产过程的优势微生物,具有降解纤维素功能,对芦苇秸秆的能源化利用起到重要作用。

The two-stage coproduction of hydrogen and methane using cellulosic biomass, such as reed straw, is a promising technology for achieving energy saving and emission reduction and developing a circular economy. The enhancement of hydrogen and methane coproduction from reed straw under enzyme pretreatment was evaluated during anaerobic fermentation. The effects of cellulase pretreatment on biogas production performance and intermediate metabolites’ characteristics were investigated in this study. In addition, the combination of polymerase chain reaction （PCR） amplification of 16S rRNA genes with denaturing gradient gel electrophoresis （DGGE） analysis was used to study the composition and succession of bacterial community in fermentative biohydrogen with methanogenesis integration system. The results showed that the maximum accumulative biogas production and hydrogen proportion were 42.5 mL/g and 52.1% respectively in hydrogenogenic stage. And the maximum accumulative biogas production of 137.5 mL/g was 5.36 times higher than the control in methane prodution stage. However, the highest methane proportion of 68.4% in control test was similar to those under cellulase pretreatment. Therefore, the cellulase pretreatment has the benefit of structural damage on refractory organics while improving the hydrogen production potential in this study. Usually, hydrogen and methane formation is accompanied by volatile fatty acids （VFAs） generation during anaerobic digestion process. Hence, the composition and concentration of soluble metabolites produced were useful indicators for monitoring the hydrogenogenic process. The investigation of the soluble metabolites at the end of each stage showed that the main VFAs were distributed under cellulase pretreatment compared with the control. The composition of VFAs in hydrogenogenic stage was butyric acid and acetic acid, indicating that butyric-acid type fermentation was established. During the methanogenic stage, the butyric acid was consumed and the propionic and valeric acid were produced more. These results showed that cellulase pretreatment might be attributed to the diversity of microbial populations in 2 stages after enrichment. The sequences of 16S rDNA DGGE predominant band fragments were determined by comparison with NCBI database. The DGGE patterns showed that the 2 stages experienced different microbial community structure changes during the period. Early in hydrogenogenic stage, the low similarity of bacterial communities was observed. And then the high similarity with Deiss coefficient of 83.6% （lane： H3, H4） and 87.4% （lane： M6, M7） was obtained in peak production period of hydrogen and methane, respectively. The Cs value was reduced to 51.5 at the end of methane production stage （lane： M6, M9）. The majority of the sequences obtained were affiliated withClostridium thermocellum （Band 20）,Enterobacter aerogenes （Band 28） andSedimentibacter （Band 29）. In hydrogenogenic stage, the dominant microorganism wasEnterobacter aerogenes （Band 28）, which can produce hydrogen and dramatically enhance the hydrogen production performance. A hydrogen-producing acetogenic bacterium ofSedimentibacter （Band 29） was also the dominant bacterium, which can produce hydrogen and acetic acid in hydrogen and methane coproduction process. The dominant microorganism ofClostridium thermocellum （Band 20） existed in 2 stages, which can degrade cellulose and play an important role in reed straw utilization process. Hence, the maximum cumulative biogas yield and proportion were increased dramatically under the cellulase pretreatment, which directly impacted the hydrogen and methane production ability. The result provides an important microbiology theoretical basis for the biofortification in biogas coproduction process from cellulosic biomass.