Improved anaerobic digestion of waste activated sludge under ammonia stress by nanoscale zero-valent iron/peracetic acid pretreatment and hydrochar regulation: Insights from multi-omics analyses

This study developed a novel strategy combining a nanoscale zero-valent iron (nZVI)/peracetic acid (PAA) pretreatment and hydrochar regulation to enhance anaerobic digestion of waste activated sludge (WAS) under ammonia-stressed conditions. The strategy significantly enhanced methane production at ammonia concentrations below 3000 mg/L, with the regulation groups (AN3000/REG) achieving a 50.1 % increase in cumulative methane yield. Metagenomic analysis demonstrated a 14.2 % enrichment of key functional microorganisms, including syntrophic fatty acid-oxidizing bacteria and hydrogenotrophic methanogens, in the AN3000/REG groups. Some of them promote the conversion of butyrate and valerate to acetate through the upregulation of key genes in the fatty acid β-oxidation pathway, thereby supplying sufficient substrates for acetoclastic methanogenesis. Beyond enhancing acetoclastic methanogenesis, the AN3000/REG groups exhibited significant upregulation of other metabolic pathways, with a 34.2 % increase in syntrophic acetate oxidation-hydrogenotrophic methanogenesis genes and a 17.1 % increase in methanol/methylotrophic methanogenesis-related genes. These findings were further validated by the metatranscriptomic and metaproteomic combination analyses. Furthermore, the AN3000/REG groups exhibited a significant enhancement in direct interspecies electron transfer, with functional microbes (e.g., Geobacter, Methanosarcina , and Methanobacterium ), pili, and cytochrome c showing significant increases of 1.38-fold, 12.7-fold, and 5.6-fold, respectively. This might be due to the synergistic effects of nZVI and hydrochar in the regulation groups. Additionally, metabolomic analyses revealed that the regulation strategy improved the microbial adaptability to ammonia stress by modulating metabolic products, such as alkaloids. Our study not only provides a promising strategy for alleviating ammonia inhibition during the anaerobic digestion of WAS but also provides a strong basis for understanding the underlying mechanism under ammonia-stressed conditions.