R2 showed a different trend, with total VFAs peaking later at 5.5 g L⁻¹ on day 43, of which 3.9 g L⁻¹ was acetic acid. The concentration gradually dropped to less than 0.7 g L⁻¹ (0.5 g L⁻¹ of acetic acid) by the end of the experiment. In R3, VFA levels peaked within the first 30 days, reaching 9.5 g L⁻¹ on day 22, of which 4.6 g L⁻¹ was acetic acid. Subsequently, VFA levels declined to an average of 6.5 g L⁻¹, with acetic acid stabilizing at 2.5 g L⁻¹ by the end of the experiment. The persistently higher VFA levels in R3 suggest that methanogenic activity was inhibited early in the experiment, delaying the establishment of a stable functional microbial community. This further supports the idea that functional microbial enrichment, rather than overall microbial diversity, plays a crucial role in CO conversion efficiency in TBRs.