Humin (HM) has been reported to function as an external electron mediator (EEM) in various microbial reducing reactions. In this study, the effect of isolation methods on EEM functionality and the chemical/electrochemical structures of HM were examined based on the correlation between dechlorination rates in the anaerobic HM-dependent pentachlorophenol (PCP)-dechlorinating consortium and the chemical/electrochemical structures of HM. A lack of PCP dechlorination activity suggested no EEM function in the HM samples prepared as a soluble fraction in dimethyl sulfoxide and sulfuric acid (which did not contain any electric capacitance). Other HM samples exhibited EEM functionality as shown by the dechlorination activity ranging from 0.55 to 3.48 (µmol Cl⁻) L⁻¹d⁻¹. The comparison of dechlorination activity with chemical structural characteristics suggested that HM with EEM functionalities had predominantly aliphatic and carbohydrate carbons with the partial structures C=O, O=C–N, and O=C–O. EEM functionality positively correlated with the proportion of O=C–N and O=C–O, suggesting an association between peptidoglycan structure and EEM functionality. The lack of detection of a quinone structure in one HM sample with EEM functionality and a negative correlation with aromatic or C=C carbon suggested that the mechanism containing quinone structures is a minor component for the functionality of EEM.

Acetogenesis and methanogenesis have attracted attention as CO₂-fixing reactions. Humin, a humic substance insoluble at any pH, has been found to assist CO₂-fixing acetogenesis as the sole electron donor. Here, using two CO₂-fixing consortia with acetogenic and methanogenic activities, the effect of various parameters on these activities was examined. One consortium utilized humin and hydrogen (H₂) as electron donors for acetogenesis, either separately or simultaneously, but with a preference for the electron use from humin. The acetogenic activity was accelerated 14 times by FeS at 0.2 g/L as the optimal concentration, while being inhibited by MgSO₄ at concentration above 0.02 g/L and by NaCl at concentrations higher than 6 g/L. Another consortium did not utilize humin but H₂ as electron donor, suggesting that humin was not a universal electron donor for acetogenesis. For methanogenesis, both consortia did not utilize extracellular electrons from humin unless H₂ was present. The methanogenesis was promoted by FeS at 0.2 g/L or higher concentrations, especially without humin, and with NaCl at 2 g/L or higher concentrations regardless of the presence of humin, while no significant effect was observed with MgSO₄. Comparative sequence analysis of partial 16S rRNA genes suggested that minor groups were the humin-utilizing acetogens in the consortium dominated by Clostridia, while Methanobacterium was the methanogen utilizing humin with H₂.

This study aimed to elucidate the origin of extracellular electron mediating (EEM) functionality and redox-active center(s) in humic substances, where they are ubiquitously distributed. Here, we show the emergence of EEM functionality during the humification of rice straw in artificial soil (kaolin and sand) with a matric potential of −100 cm at 20 °C for one year. We used the dechlorination activity of an EEM material-dependent pentachlorophenol-dechlorinating anaerobic microbial consortium as an index of the EEM functionality. Although rice straw and its mixture with artificial soil did not initially have EEM functionality, it emerged after one month of humification and increased until six months after which the functionality was maintained for one year. Chemical and electrochemical characterizations demonstrated that the emergence and increase in EEM functionality were correlated with the degradation of rice straw, formation of quinone structures, a decrease in aromatic structures, an increase in nitrogenous and aliphatic structures, and specific electric capacitance during humification. The newly formed quinone structure was suggested as a potential redox-active center for the EEM functionality. These findings provide novel insights into the dynamic changes in EEM functionality during the humification of organic materials.