Deciphering the role and mechanism of biochar dosage and inoculum-to-substrate ratio in modulating interspecies electron transfer and alleviating acid inhibition

Biochar is a potential additive that can improve anaerobic digestion (AD) of kitchen waste by facilitating direct interspecies electron transfer (DIET). However, little is known about the relationship between biochar dosage and inoculum-to-substrate ratio (ISR) in promoting AD of kitchen waste. Here, the methanogenic performance was tested under the conditions of different biochar dosages (5, 15, and 45 g/L) and ISRs (0.52, 0.26 and 0.13) using batch AD. The results showed that acidogenesis outperformed methanogenesis when ISR was reduced from 0.52 to 0.13. Although increasing biochar dosage significantly improved the methanogenic performance at each ISR, the regulatory ability of biochar on methanogenesis gradually decreased with the decline of ISR to 0.13. Microbial analysis revealed that altering ISR not only caused selective enrichments of the VFA-producers and DIET participants, but also changed methanogenesis from multiple methanogenic pathways to the DIET-based CO₂ reduction pathway in the biochar-amended AD systems. The positive effects of biochar on methanogenesis at low ISRs might be mainly ascribed to the enhancement of DIET between Petrimonas and Methanosaeta. The findings offered an important guidance for using biochar to treat perishable organic wastes at different ISRs.