Distinctive roles of crayfish shell-based biochar on sludge anaerobic digestion: Insights of microbial communities and metabolic pathways

Waste activated sludge (WAS) and crayfish shell waste represent significant organic fractions of municipal solid waste that demand sustainable disposal and resource recovery. This study explored a novel approach using crayfish shell-based biochar to enhance sludge anaerobic digestion, a well-established method for waste resource recovery. Results revealed the complex effects of the biochar on WAS anaerobic digestion. While the biochar accelerated sludge solubilization (improved by 11.0-33.3 %), hydrolysis (improved by 10.6-34.0 %), and acidification (improved by 16.3-22.4 %), it showed minimal impact on methane yield (141.0-144.9 mL/g VSS compared to 144.4 mL/g VSS in control). Microbial community analysis revealed that biochar addition enriched hydrolytic Firmicutes (from 11.0 % to 23.8 %), providing more substrates for acidification, but significantly reduced archaeal diversity (Shannon index decreased from 2.91 to 1.14). The relative abundance of key methanogen Methanobacterium declined from 4.49 % to 1.77 %. Notably, genes associated with stress response showed marked upregulation, including quorum sensing (mqsR: 2.07-fold increase) and two-component systems (Vick increased from 0.06 to 0.14 parts per thousand, vanRC increased by 1.54-fold), indicating microbial adaptation to potential toxicity from the crayfish shell biochar. Unlike previous studies, these findings demonstrate that biochar's properties can differentially affect various stages of anaerobic digestion, emphasizing the importance of tailored biochar selection for specific treatment objectives.