particularly Methanomicrobiales populations, throughout the treatment period. This is contrary to the general
understanding that aceticlastic Methanosaetaceae species dominate in stable AD systems [8]. Two
hydrogenotrophic orders (Methanomicrobiales and Methanobacteriales) together constituted 67.5 90.1% of
the total methanogen population (TMP; sum of the 16S rRNA gene concentrations of target methanogen
groups), with Methanomicrobiales being the most abundant group (up to 78% of TMP), during the operation
of our reactor. This marked dominance of hydrogenotrophic methanogens indicates that hydrogen-utilizing
pathway, possibly coupled with the conversion of acetate into H2 and CO2, is likely the major route of
methanogenesis in the reactor [9]. Given that the maximum specific growth rate ( m) is generally higher in
hydrogenotrophic methanogens (0.3 4.1 d-1) than in aceticlastic methanogens (0.1 0.7 d-1) [10], our finding
might suggest the possibility of improving the process performance by a good understanding and control of
hydrogenotrophic methanogens, particularly Methanomicrobiales, although it will require substantial further
research. Methanosaetaceae constituted the second dominant group after Methanomicrobiales at most time
points (including time zero). The starting population size of each methanogen group increased every cycle,
leading to a steady increase in the starting TMP over three cycles from 5.4 × 107 to 2.1 × 109 copies/mL (40-
fold). Consequently, similar to bacteria, a new cycle always started with more methanogens compared to the
preceding cycle, which likely contributed to the increase in methanogenesis rate over cycles (Fig. 1B).