Microbial adaptation to H2 improves the conversion of volatile fatty acids to methane during in situ biomethanation even in CO2-depleted conditions.

The in situ biomethanation process aims to increase the CH4 content in biogas by injecting H2 into anaerobic digesters, improving its energetic value. However, H2 injection causes CO2 depletion hampering in situ biomethanation performances. This work investigated the effect of H2 addition and CO2 depletion on the adaptation of two microbial consortia to in situ biomethanation. In the in situ biomethanation reactors under CO2-non-limiting conditions (biogas CO2 concentration ∼ 18 %) CH4 was the most produced metabolite (∼80 % of the produced gCOD) whatever the microbial consortia. However, in the in situ biomethanation reactors under CO2 limitation (biogas CO2 concentration ∼ 3 %), CH4 and Volatile Fatty Acids (VFA) accounted for 60 and 40 % of the produced gCOD, despite the tested microbial consortia. Interestingly, all control reactors (operated without H2 and/or CO2 addition) produced mostly VFA instead of CH4 (∼70 to 30 % COD-based-proportion). Hence, VFA accumulation was alleviated by H2 injections in both in situ biomethanation conditions. This lower VFA accumulation was associated with the adaptation of the microbial consortia to H2, evidenced by the improved growth of hydrogenotrophic methanogens (HM). Moreover, competition between HM clades may play a role in microbial adaptation to H2. However, low CO2 availability hindered HM growth and led to lower VFA conversion to CH4 in the in situ biomethanation conditions under CO2 limitation. Methanobacterium spp. was highly resistant to CO2 depletion, dominating the archaeal community in these conditions. This study demonstrated that the microbial adaptation to H2 addition, characterised by an improved HM activity, boosted methanogenesis and enhanced indirectly acetogenesis, preventing VFA accumulation even under CO2-depleted conditions.