Substrate biochemical composition does not impact hydrogen consumption rate but metabolite production during batch in-situ biomethanation process

Biogas upgrading through in-situ biomethanation offers a sustainable alternative to traditional technologies, yet the impact of key factors such as substrate biochemical composition remains inadequately explored. This study investigates how the substrate composition, such protein-rich, lipid-rich, and carbohydrate-rich substrates, affect batch in-situ biomethanation performance. Using inoculum pre-adapted through parent continuous anaerobic digestion reactors, experiments revealed distinct methane yields and microbial dynamics influenced by substrate composition. Protein-rich substrates resulted in elevated ammonia levels (2 g total ammonia nitrogen-N/L), leading to higher abundance of hydrogenotrophic methanogens. In contrast, carbohydrate-rich substrates demonstrated stable methane production and low ammonium accumulation, facilitating balanced microbial activity. Reactors processing lipid-rich and carbohydrate-rich substrates maintained consistent microbial communities, contrasting with a higher abundance of hydrogenotrophic methanogens in protein-rich waste reactors. Despite these variations, hydrogen consumption rates were comparable across all reactors, regardless of inoculum source (37 ± 1.5, 38 ± 2.0, and 34 ± 4.0 mg COD/L/h for protein-rich (COD = Chemical Oxygen Demand), lipid-rich and carbohydrate-rich substrate, respectively). These findings underscore that the substrate biochemical composition does not significantly affect hydrogen consumption kinetics during the startup period of batch in-situ biomethanation., providing valuable insights for advancing biogas upgrading technologies. • No impact of substrate composition on hydrogen consumption rate • Higher abundance of Methanosarcinae in reactor treating protein-rich waste • Higher VFA accumulation during batch in-situ biomethanation of protein-rich waste