Interspecies electron transfer regulation in a stage-separated anaerobic digestion system: The role of inoculum strategies, activated carbon integration, and organic loading rate adjustment

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Understanding and regulation of interspecies electron transfer, including hydrogen-mediated interspecies electron transfer (MIET) and direct interspecies electron transfer (DIET), is crucial for enhancing anaerobic digestion (AD). In this study, MIET and DIET were regulated in a multistage anaerobic hythane reactor (MAHR) via inoculum management, activated carbon (AC) addition, and organic loading rate (OLR) adjustment. The operational stability, organic removal, and methane production can be enhanced, with AC addition in the methane production zone (Mm) (RpCMm and RnpCMm). The reactors of RpCMm and RnpCMm achieved start-up within 20 days and showed a 20 % and 104 % increase in maximum methane production rates, respectively. Microbial community characteristics confirmed that AC in Mm could facilitate DIET establishment, but its addition in Mh could not. This assertion was supported by the observed tighter and more positive correlation between biofilm conductivity and DIET-related microbes in RpCMm and RnpCMm, as opposed to RpCMh and RnpCMh. Further, the dual drive of DIET and MIET was revealed through the comprehensive analysis of operational performances and microbial community with the structure equation model. The electron fluxes analysis highlighted a dynamic shift of DIET and MIET in the methanogenic stage with AC addition and OLR variation. The DIET was proved more efficient than MIET in Mm with AC addition and OLR below 40 g COD/L/d, while high OLR (80 g COD/L/d) favored MIET with lower methane production efficiency. The results of this study shed light on the nuanced characteristics of interspecies electron transfer, providing valuable insights for the further design and operation of stage-separated AD.