Optimization of scalable biofilm bioreactors with genetically improved bacteria for microbial biosurfactant production

The Gram-positive soil bacterium Bacillus subtilis is a potential producer of a very powerful biosurfactant called surfactin. It offers many environmental advantages because surfactin is biodegradable and less toxic than chemical compounds. In the phytosanitary field, surfactin represents a very promising biocontrol agent as it is able to induce systemic resistance in plants. Furthermore, due to its exceptional foaming and emulsifying properties, surfactin has many applications in different industrial sectors.
Industrial surfactin production in conventional stirred tank reactors necessitates the control of excessive foam formation. Biofilm bioreactors are more efficient alternative bubbleless systems for biosurfactant production since foam formation can be avoided while a high air/liquid mass transfer can be obtained. This is a very important parameter because a high aeration rate is necessary for a good surfactin productivity. However, the control of the cell colonization on the reactor support is challenging as biofilm formation is a heterogeneous phenomenon. The laboratory strain B. subtilis 168 is very easy to cultivate and manipulate genetically. Yet, this strain has very low cell adhesion capacities making it difficult to fix the cells by natural cell immobilization through biofilm formation on the bioreactor support. Here, genetically modified B. subtilis 168 mutants have been tested for improved colonization capacities on the bioreactor support to achieve a more robust and stable process.
The cell adhesion capacities of the selected B. subtilis 168 mutants have been improved through the restoration of the biofilm matrix production and the induction of cell filamentation by genetic engineering. Through the additional change of cell shape, the aim was to promote the initial cell adhesion step followed by the support colonization and to reduce the cell detachment under stress conditions. The biofilm adhesion capacities were analyzed by means of a drip flow biofilm reactor. The mutants with the best performance were then selected for the cultivation in a lab-scale trickle-bed biofilm bioreactor. This device contains a structured metal packing providing a very high specific surface area for cell immobilization.
For the mutant with functional biofilm matrix production the cell attachment capacities were significantly improved compared to the control strain resultant in an improved surfactin productivity. Cell filamentation seemed to decrease cell detachment under stress conditions, leading to a more stable colonization and process.