铵影响嗜热厌氧反应器污泥丙酸互营氧化产甲烷过程的机理

The anaerobic digestion has been successfully applied in treating the livestock and poultry manure to control the increasing pollution of soil and water environment in China. But, accumulation of ammonium, produced from the degradation of protein-rich materials, is one such detrimental factor in the anaerobic digesters used to treat waste such as livestock and poultry manure. High ammonium concentration would influence the performance of anaerobic digesters, such as the propionate accumulation and inhibition of methane production, and could cause the reactor upset or failure. However, the key microbes involved in syntrophic oxidation of propionate and methanogenesis in thermophilic anaerobic bioreactor sludge, and the mechanisms of ammonium effects on the process have remained poorly understood. In the present study, we collected sludge samples from full-scale manure digester and designed two laboratory microcosm experiments. First, RNA stable isotope probing (RNA-SIP) combined pyrosequencing technologies were used to detect the key microbes involved in syntrophic oxidation of propionate and methanogenesis in thermophilic anaerobic digester sludge. Then, the response of syntrophic propionate oxidizing methanogenesis and microbial populations to ammonium addition were determined by analyzing the rate of propionate degradation, the copy number and transcripts of bacterial and archaeal 16S rRNA gene in the sludge incubations. Finally, the mechanisms of ammonium effects on syntrophic oxidation of propionate and methanogenesis in thermophic anaerobic digester sludge would be clarified.

我国畜禽粪便对土壤和水体环境的污染日趋严重，厌氧消化技术是消除畜禽粪便污染的有效手段。然而，畜禽粪便的厌氧降解容易导致毒性化合物铵的积累，且高温会强化铵的毒性，最终导致厌氧消化过程的停滞。研究发现，铵对丙酸互营氧化产甲烷过程的抑制是厌氧消化停滞的主要原因。但是，目前对参与丙酸互营氧化产甲烷过程的关键微生物及铵影响丙酸互营氧化产甲烷过程的机理并不清楚。基于上述科学问题，本项目设计两部分试验：首先，将采用RNA稳定同位素探针技术结合高通量测序技术，探测嗜热厌氧生物反应器污泥中，参与丙酸互营氧化产甲烷过程的关键互营细菌和产甲烷古菌；其次，采用系列理化分析和基于细菌和古菌16S rRNA基因的克隆、T-RFLP、定量PCR分析和高通量测序等技术，阐明铵对丙酸互营氧化产甲烷过程及相应丙酸互营菌和产甲烷古菌的影响。最终，揭示铵影响嗜热厌氧反应器污泥丙酸互营氧化产甲烷的机理。

在厌氧生物反应器中高铵浓度常会抑制丙酸互营氧化产甲烷过程。然而，该抑制机制并不清楚。我们采用猪粪为底物的厌氧反应器污泥进行了两个独立的试验。在第一部分试验中，采用RNA-SIP技术，探测不同铵浓度下（0, 3 and 7 g NH4+-N L-1）参与丙酸互营氧化产甲烷的有活性的细菌和古菌。在第二部分试验中，采用与第一部分试验同样的培养条件但是不添加外源丙酸，并通过细菌16S rRNA焦磷酸测序和定量PCR技术，检测了再不同铵浓度胁迫条件下细菌群落的动态变化。在第二部分试验中设置了更高的铵浓度处理（10 g NH4+-N L-1）以扩大铵胁迫范围。我们发现细菌 Smithella 和古菌 Methanosaetaceae 和 Methanospirillaceae 是丙酸互营氧化产甲烷过程的主要活性参与者。我们揭示了Smithella, Methanosaetaceae 和 Methanospirillaceae在3 g NH4+-N L-1铵浓度下被相对温和的抑制，而在7-10 g NH4+-N L-1的铵浓度处理中，被相对严重的抑制。然而，发酵细菌展现出对铵胁迫很好的耐受性。此外，在高铵浓度条件下，微生物的响应与挥发性脂肪酸的积累和产甲烷过程的抑制表现是一致的。总之，我们的研究结果阐明铵影响厌氧反应器污泥丙酸互营氧化产甲烷过程的机理。