Free-standing bacterial films reinforced with 2D layered double hydroxide nanosheets: A biohybrid platform toward engineered living materials

The use of bacteria to fabricate materials has garnered significant attention in both fundamental and applied research. In this study, we introduce a method for producing bacteria-based materials utilizing the bacterial capacity to colonize 2D nanomaterials, which in turn provides additional robustness to the resulting bacterial film. Specifically, we demonstrate the fabrication of self-standing films based on a bacterium Bacillus pumilus reinforced with 2D nanosheets derived from Layered Double Hydroxides. The Live/Dead staining, followed by Confocal Laser Scanning Microscopy (CLSM), revealed an anisotropic distribution of live and dead bacteria within the mature biofilms leading to a stratified biofilm architecture. Scanning Electron Microscopy (SEM) confirmed the presence of endospores in the reinforced biofilm structure. Biological assays further supported this observation. Notably, simulated gastrointestinal passage experiments showed that the presented bacterial films provide significant protection to probiotic bacteria under harsh gastrointestinal conditions, suggesting their potential applicability in advanced delivery systems. The present study offers a useful approach for future innovations in bacteria-based material fabrication. Statement of significance: In this manuscript we present a sustainable approach for producing bacteria-based materials by harnessing the natural ability of bacteria to colonize 2D nanomaterials. Applying 2D LDH nanosheets contributed to endospores formation and structural robustness leading to the reinforced self-standing bacterial films. Moreover, the reinforced biofilms effectively protect probiotic bacteria under harsh gastrointestinal conditions. Our findings can contribute to the development of innovative bacteria-based living materials with potential applications in advanced delivery systems and regenerative medicine.