Engineering Bacillus biofilm and its cellulase to enhance cellulose degradation

In nature, most bacteria live within a surface-associated matrix known as biofilm. Biofilm is an extracellular matrix composed mainly of polysaccharides and proteins.  We studied whether biofilm enhances the efficiency of cellulose degradation by bacteria living within, which could provide a useful approach to improve biomass conversion. To increase cellulase production of Bacillus subtilis, we first transformed the bacteria with a recombinant cellulase plasmid that contained a strong promoter, a signal peptide, optimized codons and a synthetic ribosomal binding site.  Transformation of B. subtilis with the recombinant plasmid increased cellulase production by more than 66 folds.  To study the effects of biofilm on cellulase degradation, we introduced the cellulase plasmid into B. subtilis strains that produce different biofilm components. All engineered B. subtilis strains were grown with filter paper as the substrate for biofilm formation and cellulose degradation. We found that bacteria expressing both TasA, an extracellular protein, and extracellular polysaccharides (EPS) degraded cellulose much faster than bacteria without either.  EPS contribute more to the enhancement of cellulose degradation.  We found that biofilm is able to capture exogenous cellulases and form a bacteria-enzyme-substrate complex that promotes synergistic cellulose degradation. Our study shows that biofilm enhances cellulose degradation by concentrating bacteria and enzymes in close proximity to the substrate. This study brings new insight to the importance of biofilm in cellulose degradation and potentiates the development of biofilm-based technology to enhance biomass degradation/conversion for biofuel production.