【作者】 修志龙；

【导师】 安利佳； 曾安平；

【作者基本信息】 大连理工大学 ， 生物化工， 2000， 博士

【摘要】 微生物细胞连续培养过程中多态和振荡行为的研究是本文的核心内容。全文共分两部分：实验部分和理论分析部分。在第一部分中，以甘油微生物连续转化为1，3-丙二醇为研究对象，考察了培养基组成和pH调控用碱中铵、钠、钾三种一价阳离子对肺炎杆菌细胞代谢的影响，同时考察了代谢中间产物（如二羟基丙酮）的加入对代谢流量的影响：接着检验了系统对底物初始浓度不同变化方式的稳态响应以及对稀释速率变化的动态响应；最后用过量动力学方程对文献和自己的实验数据进行了描述。 实验结果表明，无铵培养基配合氨水调节pH值的培养策略是可行的，尤其对细胞生长有利。过量钠离子对甘油脱氢酶的抑制作用只有在底物过量的条件才能表现出来。向底物过剩的培养液中添加二羟基丙酮，有利于生物能的产生和细胞的生长，同时也有利于1，3-丙二醇的生产，说明在底物过量条件下甘油脱氢酶的活性受到严重抑制。当稀释速率为0.35h^-1时，系统在初始甘油浓度为400－1400mM的范围内至少存在两个稳定的状态。当发酵系统在40g/L的甘油初始浓度下从稀释速率为0.1h^-1的稳态过渡到0.22h^-1稳态时，反应器中甘油的残余浓度先增后降，生物量、1，3-丙二醇和乙酸的浓度增大，而乙醇浓度则下降，这与菌体代谢特性以及稳态变化前后的操作条件有关。 过量动力学模型较好地描述了甘油生物转化过程中甘油的比消耗速率、1，3-丙二醇和乙酸的比生成速率，但不适用于乙醇的形成，乙醇的产率与反应器中残余的甘油浓度变化速率有关。用上述动力学模型描述连续培养过程发现，在一定条件下系统存在两个稳定状态，但出现多稳态的范围要比实验的范围小得多。过量动力学模型还适用于间歇培养过程的适应期与对数生长期的描述。稳态模型不能很好地描述甘油歧化过程的过渡行为，用甘油浓度变化速率对生长模型进行简单修正后，新模型能较好地模拟大部分实验结果。 人连理工人学博1：论文 在论文的第二部分内容中，分析了过量动力学模型产生多态、滞后和振荡的原囚，根据系统的稳定性对操作区域进行了划分。另外在基因调控的水平上考察了生长速率和胞内色氨酸浓度对色氨酸操纵子稳定性和动态行为的影响。 具有代谢过量以及底物和产物抑制特征的连续培养系统的模型分析表明，在一定操作条件范围内可以得到三个非洗出稳态解。局部稳定性分析说明其中的两个解是稳定的，导致多态性、滞后和振荡现象。对于所研究的培养系统和模型参数，产物抑制和在底物过剩条件下增加的产物形成速率的综合作用是引起多态和滞后的主要原因。底物引起的生长抑制和增加的底物消耗速率似乎不是必要条件。产物抑制和增加的产物形成速率的综合作用也能导致非正常的动态行为，如延长达到新稳态的时间，从一个稳态到另一个稳态的振荡过渡以及持续振荡。用多态和振荡出现作为判据可以将连续培养的操作范围划分为四个区域：一个是多态和振荡区，一个是具有唯一稳态但可能有振荡行为的区域，另外两个区域具有唯一且稳定的稳态。模型预测与最近发表的实验结果是吻合的。HOpf分叉出现在多态区域，并且位于第二个稳定状态，这与甘油发酵过程中出现的振荡现象是吻合的。理论分析与实验结果的差异在于，前者是系统自身的特性，而后者则与操作条件密不可分。 对色氨酸操纵于的理论分析表明，生长速率是影响色氨酸生产的重要因素之一。胞内色氨酸浓度随生长速率增大而明显降低，尤其在低生长速率下。当生长速率增大到一定值后，色氨酸浓度的衰减幅度大大降低。胞内色氨酸的水平是影响色氨酸操纵于稳定性的决定因素，在相对较低的胞内色氨酸浓度范围内操纵子是不稳定的，出现振荡现象，而在很高或很低的色氨酸浓度区域则是稳定的。操纵子的稳定性与生长速率和菌种水平无X。更多还原

【Abstract】 Multiplicity and oscillation in continuous microbial cultures were studied in this work. The work was divided into two parts: experimental results and theoretical analysis. In the first part, the microbial conversion of glycerol to 1,3- propanediol by Klebsie//a pneumoniae was used to investigate the effects of three monovalent cations in media and bases being used to control pH on metabolic flow, to verify multiple steady states and transient response from one steady state to another. In addition, the excessive kinetics was used to simulate the experimental results. The influences of three monovalent cations (Nat, K, and NH4) on bioconversion of glycerol to 1 ,3-propanediol by K/ebsiella pneumoniae were investigated by designing three different media and pH control strategies and discussed in terms of metabolic control. Experimental results showed that glycerol dehydrogenase, one of the key enzymes of glycerol conversion, was inhibited by high concentration of Nat, leading to a decline of yields of biomass and l,3-propanediol on glycerol. It is feasible to adjust pH by using KOH in a medium consisting of enough NH4 or using ammonia in a medium without NH4~ and Na~ in continuous culture. Under substrate sufficient conditions, the generation of bioenergy ATP, cell growth and the production of I ,3-propanediol were increased by adding dihydroxyacetone into glycerol fermentation. This showed that the activity of glycerol dehydrogenase was strongly inhibited under substrate excess. At a dilution rate of 0.35 h1, two steady states occurred under the same conditions when the initial glycerol concentration changed from low to high or from high to low in the range of 400 to 1400mM. If the dilution rate shifted from 0.1 hup to 0.22 fr1 at an initial glycerol concentration of 40 gIL, the residual glycerol concentration in reactor initially increased and thendecreased, and the concentrations of l,3-propanediol and acotate alwaysincreased, but the ethanol concentfation declined.The excess kinetic models fOr substrate consumPtion and product formationwere reexamined for the bioconversion of glycerol to 1,3-propanediol byKlebsiella pneumoniae. Whereas these kinetic models described the substrateconsumption and the formaton of l,3Tropanediol and acetic acid reasonablywell, relative large deviations were observed for the formation of ethanol,especially at low substrate concentrations. A new term, i.e. the substrateconcentration change ratio (Cs. D) was found in this work to be useful fOrdescribing ethanol fOrmation in continuous culture. The resulting kinetic modelsand parameters satisfactorily described both continuous and batch cultures ofglycerol fermentation by K pneumoniae under various conditions. The modelalso predicted the occurence of multiplicity in the bioprocess. Alteration ofmetabolic pathways associated with ethanol fOrmation aPpeared to be themain reason causing multiplicity in a fermentation When transferring fromsubstrate-limiting to substrate-sufficient conditions.The transient responses of Klebsiella pneumoniae to dilUtion rate and glycerolconcentration step changes in continuous bioconversion of glycerol to l,3-propanediol showed that distinguished differences lay in cell growth andintracellular metabolism under differeni conditions. The tfansient behaviordepended on the both states before and after a step change of operationconditions. When substrate concentration transferred from a limiting conditionto a excessive condition, the residual concentration of glycerol in reactorconstamly increased, bat ethanol conceotration decreased gradually asfermelltation time. Simultaneously biomass, l,3-propanediol and acetateconcentration increased firstIy and then decreased.更多还原