Engineered Bacillus subtilis Biofilm@Biochar living materials for in-situ sensing and bioremediation of heavy metal ions pollution

The simultaneous sensing and remediation of multiple heavy metal ions in wastewater or soil with microor-ganisms is currently a significant challenge. In this study, the microorganism Bacillus subtilis was used as a chassis organism to construct two genetic circuits for sensing and adsorbing heavy-metal ions. The engineered biosensor can sense three heavy metal ions (0.1-75 mu M of Pb2+ and Cu2+, 0.01-3.5 mu M of Hg2+) in situ real-time with high sensitivity. The engineered B. subtilis TasA-metallothionein (TasA-MT) biofilm can specifically adsorb metal ions from the environment, exhibiting remarkable removal efficiencies of 99.5% for Pb2+, 99.9% for Hg2+and 99.5% for Cu2+ in water. Furthermore, this engineered strain (as a biosensor and absorber of Pb2+, Cu2+, and Hg2+) was incubated with biochar to form a hybrid biofilm@biochar (BBC) material that could be applied in the bioremediation of heavy metal ions. The results showed that BBC material not only significantly reduced exchangeable Pb2+ in the soil but also reduced Pb2+ accumulation in maize plants. In addition, it enhanced maize growth and biomass. In conclusion, this study examined the potential applications of biosensors and hybrid living materials constructed using sensing and adsorption circuits in B. subtilis, providing rapid and cost-effective tools for sensing and remediating multiple heavy metal ions (Pb2+, Hg2+, and Cu2+).