Revealing an unrecognized role of free ammonia in sulfur transformation during sludge anaerobic treatment

Free ammonia (FA), the unionized form of ammonium, is presented in anaerobic fermentation of waste activated sludge (WAS) at high levels. However, its potential role in sulfur transformation, especially H2S production, during WAS anaerobic fermentation process was unrecognized previously. This work aims to unveil how FA affects anaerobic sulfur transformation in WAS anaerobic fermentation. It was found that FA significantly inhibited H2S production. With an increase of FA from 0.04 to 159 mg/L, H2S production reduced by 69.9%. FA firstly attacked tyrosine-like proteins and aromatic-like proteins in sludge EPSs, with C--O groups being responded first, which decreased the percentage of alpha-helix/(beta-sheet + random coil) and destroyed hydrogen bonding networks. Cell membrane potential and physiological status analysis showed that FA destroyed mem-brane integrity and increased the ratio of apoptotic and necrotic cells. These destroyed sludge EPSs structure and caused cell lysis, thus strongly inhibited the activities of hydrolytic microorganisms and sulfate reducing bac-teria. Microbial analysis showed that FA reduced the abundance of functional microbes (e.g., Desulfobulbus and Desulfovibrio) and genes (e.g., MPST, CysP, and CysN) involved in organic sulfur hydrolysis and inorganic sulfate reduction. These findings unveil an actually existed but previously overlooked contributor to H2S inhibition in WAS anaerobic fermentation.