Microbial Transformation of Humic Acid Components Modulates Reduction of Structurally Bound As(V) via Electron Shuttling

Humic acid (HA) facilitates pentavalent arsenic (As(V)) reduction via electron shuttling, but the impact of microbial HA transformation on this process remains unclear. We investigated As(V) reduction in scorodite (FeAsO4·2H2O) mediated by Suwannee River humic acid (SRHA) serving as an electron shuttle. Experiments were conducted in microcosms inoculated with an As(V)-reducing bacterium (RA-2, isolated from high-As groundwater) or a model Fe(III)-reducing bacterium (Shewanella oneidensis MR-1). The MR-1 system exhibited significantly higher reduction levels of As(V) and Fe(III) on scorodite than the RA-2 system. Optical characterization, molecular network analysis, and electrochemical measurements revealed that the MR-1 system preserved the aromatic conjugated networks and quinone moieties of SRHA, maintaining the more negative Gibbs free energy change (ΔGr) that thermodynamically favored spontaneous electron shuttling. In contrast, RA-2 partially degraded these redox-active structures of SRHA into phenolic and protein-like components. This transformation weakened the electron-shuttling capacity of SRHA since these produced components increased the electron-donating capacity (EDC) but decreased the redox reversibility. Consequently, the MR-1 system achieved a relatively higher electron-shuttling efficiency of 23.5%, compared to the RA-2 system (13.7%). More electrons were stored within the transferred SRHA in the RA-2 system, relative to the MR-1 system. This study highlights the critical control of microbial metabolism over HA components and their electron-shuttling capacity for As(V) reduction and provides new insights into As enrichment in groundwater.