Mediated electron transfer in the bio-reduction of graphene oxide by Lysinibacillus sphaericus: Mechanisms and facilitation

The microbial reduction of graphene oxide (GO) represents a sustainable approach for producing functional nanomaterials, yet the mechanisms in Gram-positive bacteria remain poorly understood. In this study, Lysinibacillus sphaericus HJ-3 was shown to effectively reduce GO through both direct cytochrome-mediated electron transfer and flavin-dependent shuttling. Whole-genome sequencing revealed complete respiratory chain and flavin biosynthesis pathways, while metabolomic profiling indicated enrichment of riboflavin and aromatic amino acid metabolism under GO stress. qRT-PCR analysis further confirmed the upregulation of key electron transport genes, particularly c-type cytochromes, during GO reduction. Control experiments, including abiotic, heat-killed, and non-reducing Gram-positive strains, verified that the process is a genuine biotic activity requiring metabolic viability. Together, these findings provide multi-omics evidence for the extracellular electron transfer mechanism of GO reduction in L. sphaericus and highlight its adaptive responses to redox stress. Importantly, the study demonstrates that endogenous flavins act as efficient electron shuttles to enhance electron transfer, thereby facilitating the microbial reduction of GO. This work not only deepens the understanding of extracellular electron transfer in Gram-positive bacteria but also underscores the potential of microbial strategies as green and scalable approaches for advanced graphene-based materials.