Quantitative Standardization of Preincubation for Reliable Biochemical Methane Potential

This study established a standardized preincubation method for the inoculum prior to the biochemical methane potential test. The conventional approach, which relies on arbitrary incubation periods, suffers from reproducibility issues. Therefore, a quantitative criterion was proposed to address this limitation. Preincubation was conducted in reactors with a working volume of 285 mL, which were subsequently used with the preincubated inoculum and waste activated sludge for the biochemical methane potential tests. The end of preincubation was determined by a performance-based criterion: daily biogas production per unit working volume falling below the threshold of 0.035 mL/day. The ratio of availability to total chemical oxygen demand was reduced by 18%, volatile fatty acids were reduced by 99%, and volatile solids were reduced by 27% compared to the inoculum. Microbial analysis detected decreases in the proportions of bacterial phyla Bacteroidota and Bacillota, and an increase in the proportion of phylum Pseudomonadota, and decreases in the proportions of archaeal family Methanoregulaceae and genus Methanospirillum. Compared to preincubation, the biochemical methane potential resulted in a reduction in relative standard deviation values, with relative standard deviation decreasing by 20.1% (from 20.9% to 0.8%) for daily biogas production and by 16.8% (from 20.7% to 3.9%) for cumulative biogas production. The modified Gompertz and logistic models showed that the maximum biogas production rate in the preincubation had a relative standard deviation of 20.4%. In comparison, the biochemical methane potential had a relative standard deviation of 6%, representing a reduction of 14.4%. Therefore, we propose standard criteria for preincubation in the biochemical methane potential test of <0.035 mLbiogas/mLworking volume/day. This is the first step toward establishing a quantitative criterion for preincubation.