Direct interspecies electron transfer was not the sole mechanism contributing to enhanced methanogenesis in biochar-mediated high-solid anaerobic digestion of kitchen waste.

Biochar has been demonstrated to improve methanogenesis in high-solid anaerobic digestion (HSAD) systems. However, the key underlying mechanisms governing biochar's impact on methanogenesis in HSAD systems remain poorly understood under low inoculum-to-substrate ratio (ISR) conditions. Here, the efficacy of biochar prepared at 300 °C (B300), 500 °C (B500) and 800 °C (B800) was investigated in mesophilic HSAD of kitchen waste under ISR of 0.79. Compared to the biochar-unamended control, three types of biochar can effectively address over-acidification and accelerate methanogenesis. B500 showed better performance in promoting the maximum methane production rate (Rm) than B300 and B800. Rm in the B500 group was 15-fold higher than that in the control group during the first HSAD process. In the second HSAD process, the enhancement of Rm for the B500 group was even more pronounced, with an increase of 5952-fold. Further analysis showed that biochar, especially B500, could primarily enhance the buffering capacity against acidification at the start-up stage of HSAD, which created a favorable environment for the subsequently enhanced HSAD performance. Analysis of succession and metabolic prediction of the enriched microbial consortia indicated that, apart from primarily stimulating methanogenesis via direct interspecies electron transfer, biochar (especially B500) may also stimulate methane production from acetate, methanol, and methylamine. This study provides insight into the mechanisms of regulating methanogenesis via biochar under low ISR conditions.