Combined influence of rumen microorganisms pre-incubation time and dosage on methanogenic performance and microbial community from wheat straw anaerobic digestion.

Rumen microorganisms (RMs) are reported for their strong lignocellulose-degrading capacity, but the influence of their pre-incubation time and dosage on anaerobic digestion (AD) remains underexplored. This study systematically evaluated the effects of RM pre-incubation times (0, 24, 72, 120, and 144 h) and dosage (25 %, 40 %, 50 %, and 60 % of the reactor working volume) on AD performance of wheat straw (WS). The optimal condition (C-120, 50 % dosage and 120 h pre-incubation) resulted in the highest methane yield of 19.7  mL/g VS, which was 32.8 times greater than the control group (C-0, 50 % dosage and 0 h pre-incubation). Additionally, C-120 led to 1.2- and 1.3-times higher reductions in cellulose and hemicellulose compared to C-0, and improved system stability and thermodynamic favorability. Microbial community analysis revealed that Methanobrevibacter (17 % relative abundance) was the dominant methanogen in C-120, producing methane via hydrogen/formate pathways. The bacterial community was dominated by Rikenellaceae_RC9_gut_group (29 %), which facilitated lignocellulose degradation and acetate production. These genera showed positive correlation with acetate and formate utilization and methane production, which enhanced AD system resilience. These findings underscore the critical role of RMs pre-incubation and dosage in lignocellulose deconstruction and methane generation, offering strategies to improve biomass energy recovery. This study provides new insights into microbial syntrophy and metabolic interactions, offering effective strategies to enhance methane production in AD systems treating agricultural residues.