Interspecies electron transfer regulation in a multistage anaerobic hythane reactor: The role of inoculum, activated carbon, and organic loading rate

Understanding and regulation of interspecies electron transfer, includinghydrogen/formate mediated interspecies electron transfer (MIET) and directinterspecies electron transfer (DIET), is crucial for enhancing the efficiency and stabilityof anaerobic digestion (AD). In this study, interspecies electron transfer characteristicswere investigated in a newly developed stage-separated AD system, multistageanaerobic hythane reactor (MAHR), via inoculum management, activated carbon (AC)addition, and organic loading rate (OLR) regulation. The results showed that theoperational stability, organic removal, as well as methane production were obviouslyenhanced in MAHRs with AC addition in methane production zone (Mm) (RpCMm andRnpCMm). The reactors RpCMm and RnpCMm achieved start-up within 20 days,showing a 20% and 104% increase in maximum methane production rates,respectively. In contrast, MAHRs with AC addition in hydrogen/hythane productionzone (Mh) (RpCMh and RnpCMh) did not exhibit a marked enhancement of methaneproduction and organics removal. Microbial community structure and biofilmcharacteristics confirmed that AC in Mm facilitated DIET establishment, but its additionin Mh did not. This assertion is supported by the observed tighter and more positivecorrelation between microbial aggregate conductivity and potential DIET-relatedmicrobes in RpCMm and RnpCMm, as opposed to RpCMh and RnpCMh. Further, adual drive of DIET and MIET was revealed through comprehensive analysis, includingoperational performances, microbial community, Spearman correlation and structureequation model. Additionally, the study highlighted a dynamic shift of DIET and MIETwith OLR variation via electron fluxes analysis. The DIET was proved more efficientthan MIET in Mm with AC addition and OLR below 40 g COD/L/d, while high OLR (80 gCOD/L/d) favored MIET, albeit with lower methane production efficiency. This studysheds light on the nuanced characteristics of interspecies electron transfer, offeringvaluable insights for the further optimization of stage-separated AD systems.