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 as protein-rich, lipid-rich, and carbohydrate-rich substrates, affects 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 a 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 the 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 being Chemical Oxygen Demand), lipid-rich and carbohydrate-rich substrates, respectively). These findings underscore that substrate biochemical composition does not significantly affect hydrogen consumption kinetics during the start-up period of batch in-situ biomethanation., providing valuable insights for advancing biogas upgrading technologies.