Chromate bioreduction is a promising bioremediation strategy, but its application in wastewater treatment is limited by the Cr(VI) biotoxicity, particularly due to the generation of Cr(VI)-induced reactive oxygen species (ROS). To mitigate these adverse effect, this study synthesized bio-Pd 0 in Citrobacter freundii to modulate distinct Cr(VI) aerobic/anaerobic reduction pathways. With the incorporation of bio-Pd 0 , aerobic and anaerobic Cr(VI) specific removal rate increased by 18.1–21.2 and 51.5–87.9 times, respectively. The genomics, RT-qPCR, respiratory inhibition experiments, ROS levels and antioxidant enzyme activity assays demonstrated that bio-Pd 0 enhanced intracellular Cr(VI) aerobic bioreduction mainly via upregulating the chromate multi-electron reductase (Cr-MER) (related with YieF, NfsA and NemA), together with the energy-dependent resistance system (antioxidant enzyme superoxide dismutase and catalase, efflux pump Emr, DNA repair protein RecG). Cr-MER and resistance system effectively reduced the generation and accumulation of ROS, respectively. For anaerobic conditions, bio-Pd 0 facilitated hydrogenase-mediated short respiratory chain (S-chain) to reduce Cr(VI) extracellularly (related with HypE, RutF and OmcG), avoiding ROS generation and the energy consumption of the intracellular detoxification system, and maximized the energy utilization for Cr(VI) extracellular reduction. This study provides a new strategy for Cr(VI) bioreduction and elucidates the underlying mechanism.
Microbially synthesized palladium nanoparticles (bio-Pd 0 ) hold great promise for catalysis, but the catalytic activity of bio-Pd 0 relies heavily on its subcellular localization. Electron donors could affect enzymatic reduction efficiency of Pd(II) and control the subcellular location of bio-Pd 0 , yet the underlying mechanisms were unclear. Here, the regulatory mechanism of electron donors on Pd(II) reduction pathways in the non-model bacterium B. megaterium Y-4 was comprehensively investigated by multiple methodologies including cell fractionation, DPV/CV, XPS, FTIR, TEM, proteomics. Key findings revealed that membrane-bound hydrogenases drove periplasmic Pd 0 synthesis via biohydrogen, while the new one-electron extracellular transfer (EET) channel consisting of membrane-attached cytochromes ( ccsB ) and multi-heme cytochromes-bound flavin mediated the extracellular Pd(II) reduction. Notably, electron donors could regulate Pd(II) reduction routes, thereby altering the subcellular distribution of bio-Pd 0 . In lactate-added systems, the higher expression of these proteins related to EET processes (NADH dehydrogenase, membrane-attached cytochrome c, heme-based electron transfer proteins and flavodoxin) promoted extracellular Pd 0 formation (65.4 %). Whereas, in formate-added systems, hydrogenase-driven periplasmic Pd 0 synthesis (64.0 %) was facilitated due to more active formate dehydrogenase, hydrogenase and NADPH-reducing hydrogenase. Moreover, synergistical metabolism of formate and lactate balanced both pathways and maximized the apparent Pd(II) reduction efficiency (98.13 %). Proteomic analysis confirmed formate-specific energy rewiring and compensatory energy via Na⁺-translocating Rnf complex and V-ATPase overexpression. These findings significantly advance our understanding of microbial metal reduction by delineating a true dual-pathway strategy in a Gram-positive bacterium, highlighting the mechanistic diversity beyond model Gram-negative systems and offering new insights for harnessing non-model organisms in biocatalysis and nanoparticle synthesis.
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