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

表面活性物质是由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，这种脂肽的生产不是微不足道的，尽管进行了广泛的努力，但迄今为止在其生物生产方面只取得了有限的改进。但是，在好氧流加分批工艺中，消泡剂的投加量高达27g/L，但伴随着严重的泡沫形成，导致消泡剂的投加量过大，比产物产率(Yp/X)不够令人信服。与此形成对比的是，我们之前已经建立了一种有希望的替代厌氧培养策略，该策略导致了高Yp/X值，但受到其他限制。在目前的应用中，将开发一种新的好氧到微好氧培养切换工艺，并集成产品分离。将通过泡沫柱对进料和流出物速率进行基于模型的生物过程控制，以实现扩展的进料间歇过程。其目的是结合好氧培养和厌氧培养的优点，同时避免各自的缺点。这应该会使特定的表面素产率成倍增加。此外，泡沫分离和细胞回收将在一个组合的动态模型中描述。所采用的设计允许泡沫分级的气流独立于生物反应器的曝气。初步研究表明，亚硝酸还原酶PnasD启动子在微好氧和厌氧条件下均能高效表达，可用于表面素微好氧合成的诱导。因此，一方面，如何在微好氧条件下最大限度地减少生物反应器内泡沫的形成，另一方面如何获得最大的诱导作用，从而使PnasD取代天然启动子PsrfA，从而产生一种前所未有的微好氧、自诱导表面蛋白生产过程，这是一个需要从生物学和技术上阐明的问题。综上所述，该项目研究了一种非传统的新方法，以实现高效的表面活性物质生产过程，该过程结合了高效的好氧生物质生长阶段和具有最高比产品产率(yP/X)的微好氧产物形成阶段。为了探索细胞在微好氧条件下的生产力潜力，将使用基于模型的过程和综合泡沫分级。知识的基本收获一方面在于探索氧气限制电池的潜力，另一方面在于理解这一新的耦合过程概念的动态行为。

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.