Technical exploration of the induction of surfactin biosynthesis in Bacillus subtilis by micro-aerobic conditions, modelling, parameterization and evaluation of in-situ product removal by foam fractionation

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Surfactin is a highly potent non-ribosomal lipopeptide biosurfactant produced by Bacillus subtilis. In principle, the production of such lipopeptides is non-trivial and despite extensive efforts, only limited improvements in its bioproduction have been achieved so far. Up to 27 g/L could be achieved in aerobic fed-batch procedures, however, accompanied by severe foam formation, which resulted in excessive addition of antifoam agents as well as an insufficiently convincing specific product yield (YP/X). In contrast to this, we had previously established a promising alternative anaerobic cultivation strategy that resulted in high YP/X values but suffered from other limitations. In the present application, a novel aerobic to micro-aerobic cultivation switch process with integrated product separation will be developed. Model-based bioprocess control of feed and effluent rates via a foam column will be implemented to enable an extended fed-batch process. The objective is to combine the advantages of both, aerobic and anaerobic cultivation while avoiding the respective disadvantages. This should multiply the specific surfactin yield. In addition, foam fractionation and cell recycling will be described in a combined dynamic model. The design used allows the gas flow for foam fractionation to be independent of the bioreactor aeration. Preliminary work has already shown that the promoter of nitrite reductase PnasD is highly expressed under micro-aerobic and anaerobic conditions and can be used for the micro-aerobic induction of surfactin biosynthesis. Therefore, it is to be clarified biologically and technically how the foam formation inside the bioreactor can be minimized by micro-aerobic conditions on the one hand and how maximum induction can be achieved on the other hand, so that the replacement of the native promoter PsrfA by PnasD leads to a micro-aerobic, self-inducible surfactin production process with never before achieved productivities. In summary, this project investigates an unconventional, novel approach to realize a highly efficient surfactin production process combining an efficient aerobic biomass growth phase and a micro-aerobic product formation phase with a highest specific product yield (YP/X). To explore the productivity potential of the cells under micro-aerobic conditions, a model-based process will be used in conjunction with integrated foam fractionation. The fundamental gain in knowledge lies on the one hand in the exploration of the potential of oxygen-limited cells and on the other hand in the understanding of the dynamic behavior of this novel coupled process concept.