Abstract:Anaerobic digestion is an important approach for reducing the volume of urban sludge and achieving resource recovery. However, conventional anaerobic digestion is limited by the diffusion of hydrogen and formic acid, which can easily lead to metabolic blockage, eventual acidification, and system collapse. Direct interspecies electron transfer (DIET) has been proven to effectively mitigate these issues and enhance the efficiency of anaerobic digestion. Previous studies have shown that the addition of a small amount of glycerol can enrich electroactive microorganisms and promote DIET. However, the use of exogenous glycerol increases operational costs. To address this, the present study combined alkali pretreatment and yeast fermentation to produce glycerol in situ from urban sludge, thereby promoting DIET and supporting efficient methane production. The results showed that the optimal duration for alkaline pretreatment was 10 hours, which increased the soluble sugar content by 43.4% compared to untreated sludge. The optimal inoculation ratio for yeast fermentation was 10%, and the optimal fermentation duration was 9 hours, during which glycerol accounted for 2.43% of the total chemical oxygen demand (COD) of the sludge. Anaerobic digestion performance was evaluated based on indicators such as methane yield, volatile solids (VS) removal rate, and organic matter conversion efficiency. Compared with the control group, the yeast fermentation group and the combined alkaline pretreatment and yeast fermentation group achieved increases in methane production of 11.8% and 15.4%, respectively. The VS removal rate was similar across all groups (approximately 45%), while the organic matter conversion efficiency for the yeast fermentation group and the combined group increased by 5.80% and 9.30%, respectively. Furthermore, analysis of the electron transfer coefficient (ETC) revealed the intrinsic mechanism of system enhancement. Compared to the control group, the discharging ETC in the yeast fermentation group and the combined pretreatment group increased by 11.1% and 16.8%, respectively. Similarly, the charging ETC in these groups increased by 11.1% and 17.3%, respectively. Finally, microbial community analysis indicated that Methanothrix soehnenii GP6 and Fastidiosipila sanguinis were enriched in the combined pretreatment group, suggesting their potential involvement in DIET. Close-