Acid soluble extracellular matrix confers structural stability to marine Bacillus haynesii pellicle biofilms

In natural and engineered settings, bacteria predominantly thrive in biofilms, which are complex microbial communities embedded in a self-produced extracellular polymeric substances (EPSs) matrix. Pellicles are complex macroscopic biofilms floating at air-water interface. Though pellicle formation has been studied in detail in Bacillus subtilis, a soil bacterium, it is not reported in aquatic bacteria, which may use pellicle-growth as survival-strategy. This study shows that Bacillus haynesii isolated from a marine environment forms robust pellicle biofilms at air-water interface. B. haynesii pellicles showed complex architecture, involving dense cell-aggregates with interconnecting thread-like structures in an extracellular matrix. In situ staining by Alcian blue, Concanavalin A and ThioflavinT (ThT), respectively, localized acidic polymers, glycoconjugates and amyloid-like fibers in the pellicle. The pellicle was rigid and not disrupted by common EPS extraction protocols. Hence, a set of reagents and conditions were evaluated for solubilizing the EPS and pellicle. Acetic acid was able to effectively solubilize the structural EPS and pellicle structure. Acid soluble structural EPS contained chemical signatures for both proteins and carbohydrates, as revealed by elemental analysis, Fourier Transform Infrared Spectroscopy and Raman Spectroscopy. Ex situ staining of acid soluble EPS by ThT showed recovery of amyloid-forming proteins from pellicle. Results show that structural stability of the pellicle is mainly conferred by amyloid-like fibers of the EPS matrix. The robust pellicle-growth reported here may represent a survival-strategy in the aquatic bacterium. The findings reported here can support future research on biofilm structure, EPS matrix and its formation, which are critical for understanding how microbes thrive in natural and engineered settings.