Multi-omics insights into choline chloride-enhanced methane production from waste activated sludge anaerobic digestion: linking rheological regulation with microbial metabolic responses

Higher solids contents of waste activated sludge (WAS) improve the volumetric efficiency of anaerobic digestion but simultaneously reduce sludge flowability and substrate accessibility, leading to slower hydrolysis and methane production. Here, choline chloride (ChCl) was employed to overcome these constraints across 2-6% TSS, and its mechanism was resolved from sludge interfaces to microbial gene expression. ChCl increased cumulative methane production by 87.0-201.5%. This enhancement arose from a coordinated cascade rather than a single effect: ChCl first improved sludge flowability, reducing viscosity by 5.2-47.0%, and lowered the interfacial energy barrier by 42.3-55.3%, indicating interfacial reconfiguration and destabilization of the compact floc network. These physicochemical changes expanded the soluble substrate pool, increasing soluble proteins, carbohydrates, and VFAs by 101.5-274.6%, 87.2-132.8%, and 8.4-18.0 folds, respectively. Genome-centric metatranscriptomics revealed a fermentative-methanogenic relay: the fermentative MAG112 (Promineofilum) was enriched from 10.4% to 17.9% and transcriptionally activated in carbohydrate, amino acid, and energy metabolism, whereas MAG2 (Methanosarcina) simultaneously expressed hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis pathways, enabling flexible utilization of diversified intermediates. Network analysis further showed that ChCl strengthened bacterial-archaeal cooperation most strongly under 6% TSS, where positive interactions increased from 53.1% to 64.4%. By coupling rheological regulation to microbial metabolic reprogramming, ChCl linked sludge-structure relaxation with methanogenic carbon conversion, offering a potential cross-scale strategy for energy recovery from WAS anaerobic digestion.