Influence of substrate freshness on metabolite dynamics and methane emissions from pig slurry during laboratory...

Freeze/thaw pretreatment is commonly applied to animal slurry samples in laboratory studies, but its impact on organic matter transformation and methane (CH₄) emissions remains uncertain. In this study, slurry incubations were performed at 20°C with three treatments differing in freshness of feces, urine, and inoculum. CH₄ production and metabolite dynamics were monitored over 30 days. Freeze/thaw of feces and urine combined with fresh inoculum yielded the highest and most stable CH₄ production (20 g kg^-1 volatile solids), supported by the gradual transformation of protein-derived metabolites and no inhibition. In contrast, fully fresh material led to rapid hydrolysis and volatile fatty acids accumulation and slight inhibition of methanogenesis, whereas freezing the inoculum caused a pronounced delay likely due to impairment of methanogens. Metabolomics identified protein-related compounds (e.g., L-phenylalanine, 5-aminovaleric acid, and L-norleucine) as early substrates correlated to CH₄ formation. In contrast, lignin/carboxyl-rich aliphatic molecules and lipid compounds persisted as residual fractions. These results demonstrate that inoculum freshness is the decisive factor controlling freeze/thaw effects on CH₄ production and should be prioritized when preparing substrates for laboratory incubations. When fresh materials are not practical, freeze/thaw feces and urine can substitute it when combined with fresh inoculum.