Deeper insights into the effects of substrate to inoculum ratio selection on the relationship of kinetic parameters, microbial communities, and key metabolic pathways during the anaerobic digestion of food waste

•Microbial interactions and metabolic pathways at optimal S/I ratio were analyzed.•Methane production at S/I = 1:3 was significantly higher than at other S/I ratios.•The abundance of key bacteria at S/I = 1:3 was higher than 1:4.•Cooperation among microbes promotes methane production.•Key enzymes related to the methanogenesis pathway were most abundant at S/I = 1:3. The substrate to inoculum ratio (S/I) is a crucial factor that affects not only the stability of the anaerobic digestion (AD) of food waste (FW) but also the methanogenic capacity of the substrate. This is of great significance for the start-up of small-scale batch reactors and the directional regulation of methanogenesi and organic acid production. Most studies have merely clarified the optimal S/I ratio for methane production and revealed the basic composition of microbial communities. However, the mechanism of microbial interactions and the metabolic pathways behind the optimal S/I ratio still remain unclear. Herein, the effects of different S/I ratios (VS basis) on the relationship of kinetic parameters, microbial communities, and metabolic pathways during the AD process of FW were holistically explored. The results revealed that high S/I ratios (4:1, 3:1, 2:1, and 1:1) were prone to irreversible acidification, while low S/I ratios (1:2, 1:3, and 1:4) were favorable for methanogenesis. Moreover, a kinetic analysis demonstrated that the methane yield of S/I = 1:3 were the highest. A bioinformatics analysis found that the diversity of bacteria and archaea of S/I = 1:3 were the most abundant, and the enrichment of Bacteroides and Synergistetes could help to establish a syntrophic relationship with hydrogenotrophic methanogens, which could aid in the fulfillment of a unique niche in the system. In contrast to the findings with the other S/I ratios, the cooperation among microbes in S/I = 1:3 was more apparent. Notably, the abundances of genes encoding key enzymes involved in the methanogenesis pathway under S/I = 1:3 were all the highest. This knowledge will be helpful for revealing the influence mechanism of the ratio relationship between microorganisms and substrates on the biochemical metabolic process of anaerobic digestion, thereby providing effective guidance for the directional regulation of FW batch anaerobic reactors. [Display omitted]