【作者】 李国华；

【导师】 陈宁；

【作者基本信息】 天津科技大学 ， 生物工程， 2021， 硕士

【摘要】 L-酪氨酸是一种在医药、化工、食品等领域广泛应用的芳香族条件必需氨基酸。利用微生物发酵法生产L-酪氨酸是未来工业生产的主要研究方向,但是目前受限于生产成本和生产效率等因素制约了微生物发酵法的应用。为了提高生产效率,降低生产成本,本文利用在线活细胞分析仪监控种子培养过程和分批补料发酵过程,解析了电容电导与其他发酵过程参数的关系,开发并验证了适合L-酪氨酸的基于电容法控制葡萄糖流加速率的算法;为进一步提高发酵强度和底物转化率,开发了适合L-酪氨酸的重复分批补料发酵工艺;为了能够从发酵液中提取到高品质的产品,开发了从大肠杆菌发酵液中提取L-酪氨酸的工艺技术。（1）利用在线活细胞分析仪的在线电容测量生物量和传统OD₆₀₀、DCW和CFU离线测量生物量的方法进行比较。结果表明,在线电容与CFU表现出良好的线性关系（R²=0.9974）,比DCW（R²=0.9807）和OD₆₀₀（R²=0.9613）的线性关系要好,显示出电容法在准确性上高于其他两种方式。（2）利用在线活细胞分析仪在线监控L-酪氨酸种子培养和分批补料发酵过程,解析过程中电容、电导、葡萄糖、氨氮、溶磷和L-酪氨酸的之间参数变化规律。当接种比例为15%,种子培养到电容值为2.0 p F/cm时,L-酪氨酸产量最高。发现L-酪氨酸发酵过程属于半生长偶联型,并且葡萄糖补料速率和电容存在一定的线性关系。比较了不同的葡萄糖补料策略对L-酪氨酸分批补料发酵的影响,采用电容法与现场控制法补糖发酵水平最高,L-酪氨酸产量42.2 g/L,发酵强度1.76 g/L/h,糖酸转化率17.3%。（3）为了评价该生产菌及工艺的发酵水平,对目前国内外已经报道的几株L-酪氨酸生产菌进行了过程评价和经济分析。结果表明,目前本生产菌及工艺与国内其他菌种基本持平,与国外先进水平仍有较大的差距。并且,对发酵成本影响最大的因素是糖酸转化率。（4）为了进一步提高L-酪氨酸发酵水平和生产效率,开发了利用离心机偶联生物反应器的重复分批补料发酵工艺。确定了L-酪氨酸结晶、大肠杆菌细胞和废液的分离条件。重复分批发酵在98 h内进行了4批发酵,其第三批的发酵强度和转化率分别为1.70 g/L/h和28.6%,比分批补料发酵提高44.0%和69.0%。（5）研究了从发酵液中分离提取L-酪氨酸的生产工艺。发酵液酸化、脱色、过滤、结晶和干燥等工序制成产品。L-酪氨酸提取过程中回收率92.8%,纯度98.7%。更多还原

【Abstract】 L-tyrosine is a kind of essential amino acid of aromatic group widely used in medicine,chemical industry and food industry.The production of L-tyrosine by microbial fermentation is the main research direction of industrial production in the future,but the application of microbial fermentation is restricted by factors such as production cost and production efficiency.In order to improve production efficiency and reduce production cost,this paper used live cell biomass sensor to monitor the seed culture process and batch feeding fermentation process,analyzed the relationship between capacitance conductance and other state parameters,and developed and verified an algorithm based on capacitance to control the glucose flow acceleration rate suitable for L-tyrosine.In order to further improve the fermentation strength and substrate conversion,a repeated batch fermentation process suitable for L-tyrosine was developed.In order to extract high quality products from the fermentation broth of E.coli,a technology for extracting L-tyrosine from the fermentation broth of E.coli was developed.（1）The online measurement of biomass by the capacitance of the live cell biomass sensor was compared with the offline measurement of biomass by the methods of OD600,DCW and CFU.It was found that the linear relationship between the online capacitance and CFU（R²=0.9974）was better than that between DCW（R²=0.9807）and OD₆₀₀（R²=0.9613）,indicating that the accuracy of the capacitance method was higher than the other two methods.Moreover,the measurement of biomass by capacitance method has the advantages of real-time on-line,accurate and reliable,simple and convenient.（2）Online biomass sensor was used to monitor seed culture and fed-batch fermentation process,and the parameters of capacitance,conductance,glucose,ammonia nitrogen,dissolved phosphorus and L-tyrosine during the process were analyzed.The L-tyrosine yield was the highest when the seed was cultured to 2.0 p F/cm with 15%inoculation ratio.It was found that L-tyrosine fermentation process was of semi-growth coupling type,and there was a linear relationship between glucose feeding rate and capacitance.The effects of different glucose feeding strategies on the batch feeding fermentation of L-tyrosine were compared.The highest level of sugar feeding fermentation was achieved by the two methods,the yield of L-tyrosine was 42.2 g/L,the fermentation intensity was 1.76 g/L/h,and the conversion rate of sugar and acid was 17.3%.（3）In order to further improve the fermentation level and production efficiency of L-tyrosine,a two-stage centrifugal fermentation process with repeated batch feeding was developed.A set of experimental apparatus was developed to determine the separation conditions of L-tyrosine crystals,E.coli cells and waste liquid.Four batches of repeated batch fermentation were carried out within 98 h.The fermentation intensity and conversion rate of the third batch were 1.7 g/L/h and 28.6%,respectively,which were 44.0%and69.0%higher than those of the batch feeding fermentation（4）In order to further improve the level and production efficiency of L-tyrosine fermentation,a centrifuge coupled bioreactor with repeated batch feeding fermentation process was developed.The separation conditions of L-tyrosine crystallization,E.coli cells and waste liquid were determined.Four batches of repeated batch fermentation were carried out within 98 h.The fermentation intensity and conversion rate of the third batch were 1.7g/L/h and 28.6%,respectively,which were 44.0%and 69.0%higher than those of the batch feeding fermentation.（5）The separation and extraction of L-tyrosine from fermentation broth was studied.The fermentation liquid was acidified,decolorized,filtered,crystallized and dried to produce the final product..The recovery of L-tyrosine was 92.8%and the purity was98.7%.更多还原