Pyrite-enhanced sludge digestion via stimulation of direct interspecies electron transfer between syntrophic Propionate- and Sulfur-oxidizing bacteria and Methanogens: Genome-centric metagenomics evidence

The supplementation of conductive materials (CMs) is a promising strategy to improve the methane (CH₄) yield from anaerobic digestion (AD) of sewage sludge. However, most CMs are relatively costly, and the mechanisms by which they influence microbial functional traits (e.g., direct interspecies electron transfer (DIET) and extracellular polymeric substances (EPS)) remain poorly understood, which limits the application of CMs for enhancing CH₄ production. Here, we investigated the effects of micro- and milli-scale particles of pyrite (an iron-based (semi)conductive waste, supplemented at doses of 5–40 g/L) on sludge digestion, and the underlying mechanisms. The results indicated that CH₄ production rate and yield were improved mostly (by 20.8 % and 37.8 %, respectively) by the addition of 5 g/L micro-scale pyrite. The hydrolysis, acidification, and acetoclastic methanogenesis steps were accordingly promoted by 17.0–98.2 %, and the corresponding activities of key enzymes were elevated by 27.2–33.4 %. Pyrite enriched the populations of Anaerolineae and Burkholderiales, which are putative EPS producers according to genome-centric metagenomics, thus boosting the secretion of redox-active components in EPS and facilitating extracellular electron transport. Interestingly, pyrite may mediate “dual-drive” DIET, in which bin 116 (Syntrophobacteraceae sp.) and bin 2 (Thiobacillaceae sp.) directly transfer the electrons released by the oxidation of propionate and sulfide, respectively, to methanogen bin 28 (Methanothrix soehngenii). These results provide deep insights into the highly favorable performance of pyrite-supplemented AD systems and the microbial mechanisms. The proposed pyrite-mediated dual-drive DIET could expand the diversity of electron-donating bacteria and provide a novel microbial strategy for the simultaneous recovery of CH₄ and elemental sulfur from waste organic streams in CM-supplemented AD systems.