Mutual dosing of tungsten, molybdenum and selenium impact anaerobic digestion microbiome.

Metals are critical in anaerobic digestion, but their co-occurrence effects on microbiome structure and function are underexplored. This study hypothesized that exposure of methanogenic granules to a trace element (TE) mixture alongside molybdenum (Mo), tungsten (W) or selenium (Se)-would alter (i) extracellular polymeric substances (EPS) protein and carbohydrate content, (ii) microbial composition and function (iii) methanogenic pathways.To test this, anaerobic batch reactors (n = 35) were set up in a fed batch mode, with sacrificial reactors (n = 14) used to collect biomass for analyses, including DNA: RNA co-extraction, amplicon sequencing, and determination of the concentrations of total and soluble metals, Scanning Electron Microscopy- Energy Dispersive X-ray (SEM-EDX) and EPS extraction over a 24-day period.The results reveal that, Mo and W increased the concentration of soluble Fe in abiotic controls, enhancing Fe and S retention. The presence of W, Mo, W + Se, and Se had a positive effect on methane production, with W + Se and W enhancing acetoclastic methanogenesis. Additionally, Se increased EPS protein and carbohydrate contents in the biomass. Shifts in the microbiome composition were mainly driven by Mo and Se, with typically dominant Anaerolineacaeae, Capriciproducens, Macelibacteroides and Clostridium sensu stricto 5 taxa. Functional potential suggested an enrichment of nucleotide metabolism and, importantly, Vitamin (B12, B6 and B9) metabolic potential.These finding inform Anaerobic digestion (AD) stakeholders about the impacts of Fe, W, Mo, and Se co-dosing on process performance and microbiome structure and function, offering insights to optimize biogas production through tailored metal supplementation combinations, given demonstrations at lab and pilot scales.