Green reduction of Graphene Oxide mediated by Sporosarcina pasteurii in harsh conditions, changing the paradigm of rGO production with a non-pathogenic nanomaterial-resistant bacterium

Abstract

Graphene oxide (GO) reduction to reduced GO (rGO) is pivotal for producing graphene materials with significant applications in the fields of electronics, energy storage, sensing, and biomedicine. Traditional reduction methods often involve toxic reagents and high temperatures, which pose significant environmental and safety concerns. Therefore, developing green reduction techniques has become essential. Several studies have demonstrated the ability of certain anaerobic species, particularly Shewanella spp., and a few aerobic bacteria to reduce oxygen functionalities of GO to obtain rGO. Although the reduction has been effective, the process requires controlled growth conditions and/or specific nutrients. In this study, we investigated for the first time the reduction of GO using Sporosarcina pasteurii at controlled temperature in water without nutrients. S. pasteurii is a bacterium known for its ureolytic activity, able to precipitate calcium carbonate, and with a high capacity for survival without nutrients. By harnessing the extracellular signalling of S. pasteurii in starvation conditions, we achieve an efficient GO reduction both at low and high temperatures, 4 °C and 30 °C, respectively. This avoids the use of hazardous chemicals or specific formulations and can occur even at low temperatures, with the possibility to scale up the process thanks to the use of a non-pathogenic bacterium whose viability is not impaired by graphenic materials, offering potential for large-scale production. We also investigated the mechanism responsible for the GO reduction by putting it in contact with several bacterial by-products, i.e. cell membranes, spores and soluble metabolic products. Spectroscopic and microscopic analyses confirm the removal of oxygen-containing groups and the partial restoration of the graphene π-electron conjugated structure with different effects produced by each specific cell byproduct. This approach demonstrates that S. pasteurii can act through both soluble mediators and membrane-associated factors to achieve reduction. Through this biocatalytic approach, we contribute to the development of sustainable and efficient processes for biomedical applications, microbial fuel cells, and bio-batteries based on rGO.