疏水基质晶胶的成形机理及对固液分配反应器内微生物合成高值有机酸的调控与分离特性

Microbial synthesis of high value-added organic acids in industrial scale is still a challenge because the biosynthesis process always suffers either the inhibition of substrates, products and by-products, or the lack of effective separation techniques and thus a low productivity and low concentration of organic acids are obtained in the fermentation broth..In this project, a novel method by employing supermacroporous hydrophobic cryogel beads as solid carriers for the controlled delivery of the substrate and the partition of by-products in solid–liquid partitioning bioreactors, using the packed-bed of hydrophilic cryogel monolith disks as the filter media for the prefiltration of cells and the monolithic cryogel as the adsorbent for the simultaneous chromatographic separation of the target products of organic acids from the fermentation broth, as well as constructing the organic acid-producing strains by metabolic engineering methods, has been proposed to settle the inhibition and separation issues regarding the microbial synthesis of high value-added organic acids..The monolithic cryogels will be prepared by cryo-polymerization of microemulsions containing gel-forming hydrophobic reagents under rapid freezing conditions, while the hydrophobic cryogel beads will be produced by a new method, i.e., the combination of the cryo-polymerization and the liquid-flow focusing in a novel multiple microtube reactor make up of dual-microtubes. The microbial synthesis of two typical high-value organic acids so called 2-ketoisocaproate by Corynebacterium glutamicum strains and phenyllactic acid by Lactobacillus casei, will be employed as examples to demonstrate this new method, i.e., by simultaneous combination of the delivery, the partition, the prefiltration, the separation using cryogels as well as the metabolic engineering construction aimed to reduce the inhibition of substrates, products and by-products during the processes of the organism growth and metabolism and, thus to improve the bio-productivities of these two important high-value organic acids. The formation mechanisms of supermacropores and gel skeleton of the hydrophobic cryogels, the simultaneous combination characteristics, the dynamic behaviors, the mass transfer and the separation performance with the biosynthesis process as well as the mathematical model for the describing the mass transfer behaviors during the microbial biosynthesis process combing with the substrate delivery and the product separation using cryogels in solid–liquid two-phase partitioning bioreactors will also be addressed..By this project, it is expected to reveal the related mechanisms and mass transfer performance as well, which are then considered as being fundamental for this method to be applied in industrial microbial synthesis areas.

微生物法合成高值有机酸时，常因底物和产物抑制及缺乏高效分离方法，使发酵液中产物浓度小，产率低，成为限制工业化的关键瓶颈。.针对此现状，本申请探索一种利用固−液分配生物反应器、通过晶胶载体对底物控释补给及代谢副产物分配富集、菌体晶胶粗滤、产物晶胶同步层析分离及结合代谢工程改造实现微生物合成高值有机酸的新方法。以超声乳化疏水单体微乳为反应液，通过剧冷相变结晶致孔与聚合反应和微流成滴方法，制备疏水基质的晶胶微球载体和整体分离介质；以棒杆菌合成2-酮异己酸及干酪乳杆菌合成苯乳酸等两种典型高值有机酸为例，以晶胶微球为分配反应器固相载体，通过代谢工程改造、晶胶控释补给、分配、分离及代谢合成过程的协同进行代谢流调控，解除抑制，提高有机酸产率。重点研究疏水晶胶成形机理及控释、富集、分离和代谢合成过程协同作用的机制、动力学特性、分离规律和相关模型。.通过本工作可望揭示相关机理和规律，为工业应用打下基础。

微生物法合成高值有机酸时，常因底物和产物抑制及缺乏高效的分离方法，使发酵液中产物浓度小，产率低，成为限制工业化的关键瓶颈。.针对此现状，项目利用固−液分配生物反应器，通过晶胶载体对底物控释及产物晶胶层析分离，结合代谢工程改造实现微生物合成高值有机酸的新方法。主要内容包括：疏水基质晶胶基质和晶胶微球的制备方法与成形机理；双模式晶胶分离介质的制备及功能化修饰；晶胶微球载体对固−液分配反应器内微生物合成高值有机酸过程的调控和传质规律；固−液分配反应器内高值有机酸微生物合成过程、晶胶层析分离特性与数学模型。项目提出了制备疏水基质晶胶的创新方法：即超声乳化−结晶致孔−聚合反应法、超声乳化−微流成滴−微乳滴剧冷结晶致孔−聚合反应法，并通过疏水单体微乳液结晶致孔和聚合反应，结合微流控成形，制备了具有微米量级超大孔隙的半疏水晶胶微球，揭示了疏水晶胶成形的相关机理；针对酮异己酸和苯乳酸的分子结构特点，设计制备具有疏水和离子交换双模式功能基团的晶胶分离介质；以晶胶微球为分配反应器固相载体，利用筛选获得的干酪乳杆菌、副干酪乳杆菌、布氏乳杆菌等新菌株，结合代谢工程改造、晶胶控释、分离及代谢流调控，解除抑制，提高了有机酸产率。项目还研究了疏水晶胶控释、代谢途径、合成过程动力学特性、分离规律、数学模型和菌体生长代谢过程关键酶系的变化规律。以晶胶为载体，在固-液分配反应器内实现了微生物的高密度培养，生物量为常规培养条件下的6倍；建立了以廉价苯丙氨酸为底物，通过副干酪乳杆菌转化合成苯乳酸的新途径。其中，全细胞转化合成过程中，苯丙氨酸底物的转化率达到83%，产物浓度达3.4g/L；同时，用双模式晶胶层析分离苯乳酸，获得纯度96.7%的苯乳酸，可重复使用20次以上。研究结果对于发展高值有机酸生物合成、分离和工业应用方法，都具有重要意义。