Production of ethyl-butyrate in a self-sufficient reaction cascade (Acronym PEBcascade)

通过该提议，申请人希望研究自给自足的生物技术反应级联以生产燃料丁酸乙酯。一般来说，酯的生产是一个复杂的过程，涉及多个步骤，如前体（醇和羧酸）的生产，化学或酶促酯化，以及最终产物的分离。在我们的方法中，梭菌和酵母的人工厌氧混合培养物，或细菌与表面展示脂肪酶的组合将用于在一个反应系统中生产酯。虽然梭菌负责丁酸的生产，但酵母或细菌生产乙醇。脂肪酶将在产乙醇菌株的细胞表面表达。这种方法有望实现优化的生产率，因为每种生物体都用于生产一种组分，因此可以进入最佳工艺条件。为了在PEBcascade项目中实现生物燃料工艺开发的新生产路线，生物研究（微生物学、分子生物学）将与生物化学工程（工艺开发、建模）相结合。该项目包括工艺开发和建模，放大（至50升），以及燃料在单缸汽油发动机中的应用。在第一步中，将分别详细表征生物催化剂、梭菌丁酸盐生产、酵母和细菌乙醇生产以及不同脂肪酶的酯形成（WP 1）。动力学数据将使得能够选择“最佳”催化剂。这些数据还将用于第一个数学模型，以描述建立级联反应的需求。WP 2侧重于选择用于丁酸乙酯的原位产物去除的有机溶剂。特别感兴趣的是生物催化剂的稳定性和丁酸、乙醇、丁酸乙酯和副产物的分配系数。为了满足所有生物催化剂的要求，将进行培养基优化（WP 3）。为了鉴定脂肪酶的合适表面表达，将在WP 4中比较几种菌株和转运蛋白系统。最后，应将已鉴定的菌株整合到一个反应器系统中。在这里，将研究混合培养物的组成和自给自足的反应级联的性能。获得的数据将用于数学模型（WP 5）的完善。在WP+中，将研究丁酸乙酯的基本燃烧特性。这将与车辆推进系统研究所（TUK）合作完成。在项目开始时，计划测试商业丁酸乙酯，以确定发动机设置，并在项目结束时，与生物技术生产的产品进行比较测试。

With this proposal, the applicants want to investigate a self-sufficient biotechnological reaction cascade to produce the fuel ethyl-butyrate. In general, ester production is a complex process involving multiple steps, such as the production of the precursors (an alcohol and a carboxylic acid), the chemical or enzymatic esterification, and the final product separation. In our approach, an artificial anaerobic mixed culture of Clostridia and yeast, or bacteria in combination with a surface displayed lipase will be used for the production of the ester in one reaction system. While the Clostridia are responsible for the production of butyrate, the yeast or bacteria produce ethanol. The lipase will be expressed at the cell-surface of the ethanol-producing strain. This approach is expected to achieve optimized productivities, as each organism is used to produce a component, and can thus be brought into an optimal process condition. In order to realize a novel production route of a biofuel process development in the project PEBcascade, biological research (microbiology, molecular biology) will be combined with biochemical engineering (process development, modelling). The project includes process development and modeling, a scale-up (to 50 liters), and an application of the fuel in a single-cylinder gasoline engine. In a first step, the biocatalyst, the butyrate production with clostridia, the ethanol production with yeast and bacteria, and the ester formation by different lipases, will be characterised separately in detail (WP1). The kinetic data will enable a selection of the "best" catalysts. The data will also be used for a first mathematical model to describe the demands for the establishment of a cascade reaction. WP2 focuses on the selection of an organic solvent for an in-situ product removal of the ethyl-butyrate. Of particular interest are the stability of the biocatalysts and the partitioning coefficients of the butyrate, ethanol, ethyl-butyrate and side-products. In order to fulfil the requirements of all biocatalysts, a media optimisation will be performed (WP3). In order to identify a suitable surface expression of the lipase, several strains and transporter systems will be compared in WP4. Lastly, identified strains shall be integrated in one reactor system. Here, the composition of the mixed culture and the performance of the self-sufficient reaction cascade will be investigated. The obtained data will be used for the refinement of the mathematical model (WP5). In WP+, the basic combustion properties of the ethyl butyrate will be investigated. This will be done in collaboration with the Institute of Vehicle Propulsion Systems (TUK). At the beginning of the project, it is planned to test commercial ethyl butyrate to determine the engine settings and, at the end of the project, to carry out comparative tests with the biotechnologically produced product.