Generation of hyperfilamentous B. subtilis mutants to optimize the surfactin production in biofilm bioreators

The Gram-positive bacterium Bacillus subtilis produces surfactin, a powerful biosurfactant with various applications in different industrial sectors . The advantage of surfactin over chemically synthesized surfactants is its biodegradability and its low critical micelle concentration. However, the surfactin production yield has not yet reached an industrial level. The production is challenging due to excessive foam formation and complex downstream process operations. Researchers from the University of Liège – Gembloux Agro-Biotech and the University of Lille1 – Institut Charles Viollette have developed promising continuous production processes based on: (1) a trickle-bed biofilm reactor containing a structured packing as support for the biomass; (2) a bubbleless membrane bioreactor with an air/liquid contactor. The aeration in these two processes is realized without air sparging to avoid foam formation. The objective of this work is to generate hyperfilamentous B. subtilis mutants with enhanced adhesion capacity on the biofilm reactor support in order to simplify the downstream process of the surfactin molecules. Furthermore, the impact of filamentous growth on surfactin production and on cell attachment will be examined.
The filamentous B. subtilis mutants were generated with a markerless gene deletion strategy. Gene deletions provoking filamentous growth due to a less efficient cell division and a change in membrane composition were realised. The biomass development and surfactin production were analyzed under shaking conditions in flasks and in flow cell chambers under static conditions. The behaviour and the cell morphology were further analyzed by flow cytometry and microscopy. Multiple gene deletions resulted in hyperfilamentous bacterial growth. The filamentous mutants did not show a significant decline in surfactin yield compared to the control strain under shaking conditions. The selective adaption of B. subtilis to the production process by genetic engineering seems to be a promising approach in order to optimize the surfactin production yield.