Background: This study investigated in situ biomethanation as a biogas upgrading strategy by injecting hydrogen (H₂) into anaerobic fed-batch reactors treating wastewater from the pulp and paper industry.

Methods: Granular sludge was used as inoculum and H₂ was supplied at two pressures (0.6 and 0.9 bar overpressure in CB and CC assays, respectively) to evaluate its impact on treatment efficiency, methane (CH₄) production, and microbial community dynamics compared to control reactors (CA sets) after an adaptation phase with feeding wastewater only.

Results: The CH₄ production increased during the first two feeding cycles with H₂ supplementation accompanied by a reduction in CO₂ emissions. However, this was transient and at the end of cycle 7 acid accumulation (mainly acetic and propionic acids) and reduced CH₄ production was observed in both H₂-supplemented assays. Microbial community structure changed first as a function of new stirred reactor conditions and later according to amount of H₂ addition resulting in three clearly separated groups of communities. The family Syntrophobacteraceae responsible for propionate degradation declined in all reactors due to operational changes and following microbial succession. In control reactors it was out-competed by members of Geobacteraceae and Desulfobulbaceae. In CB and CC assays, Ethanoligenenaceae, Bacillaceae, Kosmotogaceae, Anaerolineaceae, and Anaerolineaceae families were enriched as a result of H₂ supplementation. The most abundant methanogens were affiliated to the acetotrophic Methanothrix and the hydrogenotrophic Methanobacterium in all batch reactors. Upon H₂ addition the relative abundance of Methanobacterium increased and became predominant in later cycles of CB and CC sets. Despite this shift, both genera coexisted throughout the experiments, suggesting that multiple metabolic pathways contributed to CH₄ production under H₂-enriched conditions.

Conclusions: Although the process demonstrated potential for simultaneous biogas upgrade and wastewater treatment, the overall performance was negatively influenced by increased H₂ pressure. This highlights that proper H₂ dosing and microbial monitoring are critical to ensure process stability for in situ biomethanation systems. Considering the fragile balance of the investigated wastewater treatment process an ex situ upgrade of biogas using a separate reactor is recommended.