Cr(VI) bioreduction enhanced by the electron transfer between flavin reductase and persistent free radicals

Persistent free radicals (PFRs) in biochar are an important electron shuttle for mediating electron transfer, which has significant impact on the biogeochemical redox reactions. Although the influence of biochar on the extracellular electron transfer (EET) for redox cycle has been extensively studied, the molecular mechanism for promoting the EET with PFRs remains poorly understood. This study investigated the oxygen-centered PFRs-mediated Cr(VI) reduction by Shewanella oneidensis MR-1 (MR-1) and exhibited the molecular mechanism of electron transfer between flavin substances and PFRs. Results showed that the Cr(VI) bioreduction rate by MR-1 increased from 31% to 70% with the addition of biochar. Electrochemical results illustrated that biochar increased biocurrent generation in the Cr(VI) bioreduction process. 3D-EEM and LC/MS spectra indicated that MR-1 secreted the flavin mononucleotide (FMN) reductase that relied on the [H] to provide the electrons. Electron paramagnetic resonance spectra illustrated that PFRs in biochar accepted the electrons from FMN reductase and stored those bioelectrons. Because of the oxidation of FMN, the electron transfer from FMN reductase to PFRs would increase the intracellular reactive oxygen species, which further produced the extracellular ·O₂^-. The reduced PFRs released the bioelectrons, accelerating the Cr(VI) reduction by ·O₂^-. Together with the results of the mutant strains experiment, it was found that the EET by c-cytochrome and free radicals contributed to the Cr(VI) bioreduction by 7.1% and 92.9%, respectively. These findings revealed that the PFRs could participate in the EET process and promote the redox reactions, providing a new approach for enhancing the remediation of heavy metal pollution by microorganisms and suggesting the important role of PFRs in the electron transfer process.