Propionic acid-related inhibition during anaerobic digestion: Insights into methane production and microbial community responses

Propionic acid (HPr) accumulation is a major indicator of anaerobic digestion (AD) dysfunction, yet the respective contributions of acidity, undissociated HPr and the propionate ion (Pr⁻) to inhibition and microbial community composition remain unclear. Using mesophilic batch microcosms fed with municipal sewage sludge, we compared HPr, sodium propionate (NaPr), NaCl and HCl across two experimental series. Inhibition by HPr was governed by the pH drop rather than by Pr⁻: 20 mM HPr reduced the maximal methane rate by 22%, while 81 mM HPr (pH 5.1) abolished methane production, an effect reproduced by HCl at the same pH. At near-neutral pH, the propionate salt had only a secondary, concentration-dependent effect (81 mM NaPr, −40% on the rate), largely attributable to ionic strength (81 mM NaCl, −23%). 16S rRNA gene amplicon sequencing revealed compound-specific community restructuring: methanogenic archaea fell from 2 to 3% to < 0.2% of relative abundance under 81 mM HPr, and the > 100 differentially abundant ASVs were largely shared with HCl reactors, confirming the dominant role of acidity. The number of differentially abundant ASVs was negatively correlated with the methane rate (R² = 0.97). These results clarify the respective roles of pH, undissociated HPr and Pr⁻ in both methane inhibition and microbial community restructuring.