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

表面活性素是枯草芽孢杆菌产生的一种高效非核糖体脂肽生物表面活性剂。原则上，这种脂肽的生产是不平凡的，尽管进行了广泛的努力，但迄今为止在其生物生产中仅实现了有限的改进。然而，在有氧补料分批程序中可以达到高达27 g/L，伴随着严重的泡沫形成，这导致消泡剂的过量添加以及不足以令人信服的比产物产率（YP/X）。与此相反，我们以前建立了一个有前途的替代厌氧培养策略，导致高YP/X值，但受到其他限制。在本申请中，将开发具有集成产物分离的新型好氧至微好氧培养转换工艺。将通过泡沫柱对进料和流出速率进行基于模型的生物工艺控制，以实现延长的分批进料工艺。目的是联合收割机结合好氧和厌氧培养的优点，同时避免各自的缺点。这将使比表面活性素产量倍增。此外，泡沫分离和细胞再循环将在一个组合的动态模型中进行描述。所使用的设计允许用于泡沫分离的气流独立于生物反应器通气。初步工作已经表明，亚硝酸盐还原酶PnasD的启动子在微需氧和厌氧条件下高度表达，并可用于表面活性素生物合成的微需氧诱导。因此，在生物学上和技术上要澄清的是，一方面如何通过微需氧条件使生物反应器内的泡沫形成最小化，另一方面如何实现最大诱导，使得用PnasD替换天然启动子PsrfA导致微需氧的、自诱导的表面活性素生产方法，其具有以前从未实现的生产率。总之，该项目研究了一种非常规的新方法，以实现高效的表面活性素生产工艺，该工艺结合了高效的好氧生物质生长阶段和微好氧产物形成阶段，具有最高的比产物产率（YP/X）。为了探索微需氧条件下细胞的生产潜力，将结合集成泡沫分离使用基于模型的工艺。知识的根本收获在于一方面在探索的潜力，氧气有限的细胞，另一方面在了解这种新的耦合过程的概念的动态行为。

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.