Biofilm-based bioprocess optimization by morphology engineering of Bacillus subtilis

B. subtilis is able to produce naturally different classes of lipopeptides. Lipopeptides have a wide range of application in different industrial sectors since they combine interesting physicochemical properties and biological activities. Yet, lipopeptide production is still challenging, considering complexity associated to secondary metabolic pathways or more basically due to excessive foam formation. Biofilm bioreactors represent a promising alternative production strategy as they provide improved productivity through high cell densities, avoid foam formation and facilitate further metabolite purification. However, biofilm formation implies strong cell phenotypic diversification impairing process reproducibility. Here, we show that morphology engineering permits to improve the surface attachment of the laboratory strain B. subtilis 168 by rendering the cells filamentous due to an induced change in bacterial cell shape. Additionally, the impact of the exopolysaccharide (EPS) production on the support colonization is analyzed. Biofilm growth characteristics of a B. subtilis 168 mutant, exhibiting filamentous growth due to the deletion of the cell division protein SepF, were studied by means of a drip flow biofilm reactor. This device allowed the direct visualization and quantification of biofilm formation under low shear stress conditions. Besides, the biofilm development of filamentous and non-filamentous B. subtilis 168 strains with restored EPS production was investigated. The results have shown that EPS production was a key factor for the support attachment and colonization. The attachment of the strain deficient in EPS production could be increased through genetically-induced filamentous growth. The presence of lipopeptides also seemed to have an impact on the support conditioning through the bulk medium and on the biofilm distribution. The performed experiments pointed out a promising strategy for the design of Bacillus biofilm-based processes. Screening of morphologically engineered B. subtilis strains permitted to gain important insights into their biofilm growth dynamics. Based on these results, an optimized strain combining optimal biofilm formation and lipopeptide production has been identified and will be used in scalable biofilm bioreactors.