Effect of extreme pH conditions on methanogenesis: Methanogen metabolism and community structure

Abstract

The control of pH is effective for inhibiting methanogenesis in the chain elongation fermentation (CEF) system. However, obscure conclusions exist especially with regard to the underlying mechanism. This study comprehensively explored the responses of methanogenesis in granular sludge at various pH levels, ranging from 4.0 to 10.0, from multiple aspects including methane production, methanogenesis pathway, microbial community structure, energy metabolism and electron transport. Results demonstrated that compared with that at pH 7.0, pH at 4.0, 5.5, 8.5 and 10.0 triggered a 100%, 71.7%, 23.8% and 92.1% suppression on methanogenesis by the end of 3 cycles lasting 21 days. This might be explained by the remarkably inhibited metabolic pathways and intracellular regulations. To be more specific, extreme pH conditions decreased the abundance of the acetoclastic methanogens. However, obligate hydrogenotrophic and facultative acetolactic/hydrogenotrophic methanogens were significantly enriched by 16.9%-19.5 fold. pH stress reduced the gene abundance and/or activity of most enzymes involved in methanogenesis such as acetate kinase (by 81.1%-93.1%), formylmethanofuran dehydrogenase (by 10.9%-54.0%) and tetrahydromethanopterin S-methyltransferase (by 9.3%-41.5%). Additionally, pH stress suppressed electron transport via improper electron carriers and decreased electron amount as evidenced by 46.3%-70.4% reduced coenzyme F₄₂₀ content and diminished abundance of CO dehydrogenase (by 15.5%-70.5%) and NADH:ubiquinone reductase (by 20.2%-94.5%). pH stress also regulated energy metabolism with inhibited ATP synthesis (e.g., ATP citrate synthase level reduced by 20.1%-95.3%). Interestingly, the protein and carbohydrate content secreted in EPS failed to show consistent responses to acidic and alkaline conditions. Specifically, when compared with pH 7.0, the acidic condition remarkably reduced the levels of total EPS and EPS protein while both levels were enhanced in the alkaline condition. However, the EPS carbohydrate content at pH 4.0 and 10.0 both decreased. This study is expected to promote the understanding of the pH control-induced methanogenesis inhibition in the CEF system.

Keywords: Acidic/alkaline conditions; Anaerobic digestion; Chain elongation; Enzyme; Methanogenesis.