Inoculum microbiota diversity determines anaerobic digester resilience to acidic shock

Anaerobic digestion (AD) is pivotal for decarbonizing organic waste streams. However, systematic criteria for evaluating inocula and understanding the mechanistic links between inoculum microbiota and digester performance under shock loading remain unclear. In this study, three AD model systems inoculated with sewage sludge (SLAD), pig manure (PMAD), and water hyacinth residue (WHRAD) were exposed to two consecutive acidic shock stages (35 d and 25 d) using tofu wastewater (pH = 3.6) under identical conditions. The maximum methane production rates in stage II were 2,466 ± 52, 1,457 ± 20, and 1,804 ± 49 mL/d, respectively. The SLAD system showed higher performance and stability than the WHRAD system, followed by the PMAD system. In the SLAD system, phylum Chloroflexi, class Anaerolineae, genus Methanosarcina , and family Methanosarcinaceae were identified as biomarkers. In the PMAD system, order Syntrophomonadales and genus Methanosphaerula served as biomarkers, whereas in the WHRAD system, phylum Firmicutes, class Clostridia, genus Methanobacterium , and class Methanobacteria were dominant biomarkers. Community assembly in all systems was initially governed by stochastic processes, gradually shifted toward deterministic processes, and finally reached a stochastic–deterministic equilibrium. Significantly positive correlations were observed between the performance and stability indicators with biodiversity. The SLAD system had multiple functional modules and accomplished parallel processing, which led to better performance and stability than those of the WHRAD and PMAD systems. These results provide mechanistic insights into how inoculum microbiota shape AD performance and stability, guiding rational inoculant selection and process optimization.