NADH-dependent LDHs are conserved among diverse organisms and contribute to NAD ⁺ regeneration in lactic acid fermentation. However, this type of LDH is also present in nonfermentative bacteria, including members of the genus Shewanella , while their physiological roles in these bacteria remain unknown. Here, we show that LdhA (an NADH-dependent d -LDH) works in concert with Dld (a quinone-dependent d -LDH) to transfer electrons from NADH to quinones during sugar catabolism in S. oneidensis MR-1. Our results indicate that d -lactate acts as an intracellular electron mediator to transfer electrons from NADH to membrane quinones. In addition, d -lactate serves as a temporal electron sink when respiratory electron acceptors are not available. Our study suggests novel physiological roles for d -LDHs in providing nonfermentative bacteria with catabolic flexibility under electron acceptor-limited conditions.

Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H₂) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H₂ directly, but rather through a reduced state of humin—a solid-phase humic substance—as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H₂ was used for carbon-dioxide fixation to acetate; the acetate produced was used for the reduction of humin; and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H₂ dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination.

The utilization of extracellular electron transfer by microorganism is highly engaging for remediation of toxic pollutants under “energy-starved” conditions. Humin, an organo-mineral complex of soil, has been instrumental as an external electron mediator for suitable electron donors in the remediative works of reductive dehalogenation, denitrification, and so forth. Here, we report, for the first time, that humin assists microbial acetogenesis as the extracellular electron donor using the electron acceptor ${\mathrm{CO}}_2$ . Humin was obtained from Kamajima paddy soil, Japan. The anaerobic acetogenic consortium in mineral medium containing ${\mathrm{CO}}_2/{\mathrm{HCO}}_3^{-}$ as the inorganic carbon source used suspended humin as the energy source under mesophilic dark conditions. Retardation of acetogenesis under the ${\mathrm{CO}}_2$ -deficient conditions demonstrated that humin did not function as the organic carbon source but as electron donor in the ${\mathrm{CO}}_2$ -reducing acetogenesis. The consortium with humin also achieved anaerobic dechlorination with limited methanogenic activity. Total electron-donating capacity of humin was estimated at about 87 µeeq/g-humin. The metagenomic sequencing of 16S rRNA genes showed the predominance of Firmicutes (71.8 ± 2.5%) in the consortium, and Lachnospiraceae and Ruminococcaceae were considered as the ${\mathrm{CO}}_2$ -reducing acetogens in the consortium. Thus, microbial fixation of ${\mathrm{CO}}_2$ using humin introduces new insight to the holistic approach for sustainable treatment of contaminants in environment.

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.