【作者】 刘龙；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 透明质酸（HA）是由葡萄糖醛酸和乙酰氨基葡萄糖通过β-（1→3）和β-（1→4）糖苷键连接而成的粘多糖,具有高保湿性、粘弹性和生物相容性等许多优良性质,在生物医药、化妆品和保健食品等领域具有广泛应用。目前微生物发酵法已经取代动物组织提取法成为生产HA的主要方法,随着HA市场需求的不断扩大,有效提高发酵法生产HA的产率和生产强度至关重要。本论文依据HA发酵系统的混合与传质特性以及生产菌株兽疫链球菌Streptococcus zooepidemicus自身的生理代谢特性,提出了一系列发酵优化与控制策略,实现了微生物发酵法生产HA的高产量、高底物转化率与高生产强度的相对统一,其主要研究内容和结果如下:（1） HA发酵属于高粘度发酵,混合性能差和传质效率低是HA发酵的一个重要瓶颈,因此优化HA发酵体系的混合与传质特性至关重要。首先研究了HA发酵过程的混合与传质特性,然后运用径向基函数神经网络-具有量子行为的粒子群优化算法（RBF-QPSO）对影响混合与传质特性的三个主要因素（搅拌转速、通气速率与搅拌器个数）进行优化。得到的优化条件为:搅拌转速294 rpm,通气速率1.5 vvm,搅拌器个数3,在此条件下HA产量为5.58 g/L,比优化前提高了12%（以5.0 g/L为对照）;（2）进一步研究发现在上述优化条件下HA发酵体系仍没有处于全混合状态,于是进一步研究了全混合状态对HA发酵的影响。结果表明在全混合状态下虽然混合与传质效率得到明显提高,但是HA产量反而下降,这可能是因为在全混合状态下高搅拌转速（高剪切力）对细胞的剪切作用较大,降低了细胞的生理代谢活性,表明低剪切、高传质效率和高溶氧水平是HA发酵的较理想方式;（3）基于上述研究结果,进一步研究了添加氧载体正十二烷对HA发酵过程的影响,结果表明,当添加的正十二烷浓度为5%（v/v）时,8-16 h期间的平均体积溶氧传递系数（KLa）为37 h^-1,是添加前的3.7倍;HA产量达到6.3 g/L,提高了26%;（4）在上述研究基础上进一步探讨了在发酵过程中通过降解HA提高发酵体系混合与传质效率的可能性。考察了在发酵过程中添加透明质酸酶对HA发酵过程的影响,研究结果表明在发酵过程中添加透明质酸酶降解透明质酸能够显著改善发酵体系的混合与传质特性,当添加的透明质酸酶浓度为0.25 g/L时,HA分子量由1300 kDa降为21kDa,发酵体系处于完全混合状态,KLa为100 h-1,HA产量为6.0 g/L,提高了20%;（5）出于对生产成本的考虑,进一步考察了在发酵过程中同时添加过氧化氢和抗坏血酸对HA发酵过程的影响,在发酵8 h和12 h两次添加过氧化氢和抗坏血酸以氧化还原降解HA也能够显著改善发酵体系的混合与传质特性,当添加的过氧化氢浓度为1.0mmol/g HA、抗坏血酸浓度为0.5 mmol/g HA时,KLa从3.7 h^-1提高到49 h^-1,HA产量为6.5 g/L,提高了26%;（6）基于HA在兽疫链球菌中的生理功能以及细胞在胁迫环境下的物质及能量代谢的应答机理,考察了流加H₂O₂进行氧化胁迫对HA发酵过程的影响,研究发现在分批培养过程中流加H₂O₂进行氧化胁迫能够转变细胞产能途径、提高细胞产能效率和提高HA合成速率,当H₂O₂流加速率为0.06 mmol L-1 h^-1时,HA的产量达到6.0 g/L,比对照提高了20%;（7）提出了一种间歇性pH胁迫策略:前0-6 h期间控制pH为7.0,在6-7 h期间控制pH为8.5,在7-8 h期间控制pH为7.0,在8-9 h期间再将pH调到8.5,如此循环下去,直至发酵结束。在此胁迫策略控制下,HA产量提高到6.5 g/L,提高了30%;（8）考察了HA发酵过程中常见氨基酸的代谢动力学,确定精氨酸、半胱氨酸和赖氨酸是影响细胞生长和HA合成的关键氨基酸;考察了添加核苷酸碱基（腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶、尿嘧啶）对HA发酵过程的影响,发现尿嘧啶对HA发酵具有重要影响。运用RBF-QPSO算法对关键营养因子的添加浓度进行了优化,最佳添加浓度为:精氨酸0.062 g/L,半胱氨酸0.036 g/L,赖氨酸0.043 g/L,尿嘧啶0.06 g/L时,在此条件下,HA产量提高到6.3 g/L,提高了26%;（9）研究了不同培养模式对HA发酵过程的影响,发现采用分批培养可获得较高的碳源对HA的转化率,而补料分批培养可获得较高的细胞比生长速率,于是提出了一种两阶段培养模式:在第一阶段内（0-8 h）进行指数流加培养,在8 h提高碳源浓度至25 g/L,进行第二阶段的分批培养。在此两阶段培养模式中,HA产量提高至5.98 g/L,比单一分批培养模式提高了20%。为进一步提高HA产量,在第二阶段培养期间应用间歇性pH胁迫策略转变细胞产能途径、提高细胞产能效率,HA产量进一步提高至6.6 g/L,提高了32%,碳源对HA的转化率（YHA）提高了19%,而生产强度提高了32%,实现了HA高产量、高底物转化率与高生产强度的相对统一。更多还原

【Abstract】 Hyaluronic acid （HA） is a natural biopolymer composed of glucuronic acid and N-acetylglucosamine joined alternatively byβ-（1→3） andβ-（1→4） glycosidic bonds. With its unique physico-chemical and biological properties including high water-holding capacity, viscoelasticity, and biocompatibility, HA is widely applied in biomedical, cosmetic and healthcare fields. Currently the microbial fermentation is replacing the animal tissue extraction as the predominant technique to produce HA. With the increasing HA markrt demand, it is very crucial to improve HA productivity. In this thesis, based on the mixing performance and oxygen mass transfer characteristics of the culture system and the physiological and metabolic characteristics of Streptococcus zooepidemicus, a series of process control and optimization strategies were developed as listed in the following:（1） Microbial HA production was a kind of high viscosity fermentation, and the poor mixing performance and low mass transfer efficiency were the bottlenecks of HA production. The mixing performance and oxygen mass transfer characteristics were studied and the main factors influencing mixing performance and oxygen mass transfer （agitation speed, aeration rate and stirrer number） were optimized with the radial basis function neural network coupling quantum-behaved particle swarm optimization （RBF-QPSO） algorithm. Under the optimal conditions （agitation speed: 294 rpm, aeration rate: 1.5 vvm, stirrer number: 3）, HA production reached 5.58 g/L, and increased by 12%;（2） The further study indicated that the culture system was not in a status of complete mixing even under the above optimized culture conditions, and then the effect of complete mixing on the microbial HA production was investigated. Though the mixing performance and oxygen mass transfer efficiency were improved significantly when the culture system was in a status of complete mixing, HA production decreased, possibly due to the harmful effect of strong shear stress on the cell growth and metabolism. It was indicated that low shear stress, high oxygen mass transfer efficiency and high dissolved oxygen level were the ideal culture conditions for microbial HA production.（3） Based on the above results, the influence of oxygen vector n-dodecane addition on microbial HA production was studied and the results showed that, with an n-dodecane concentration of 5% （v/v）, the volumetric oxygen mass transfer coefficient KLa during 8-16 h reached 37 h^-1, increased by 3.7 folds and HA production achieved 6.3 g/L, increased by 26%. Though HA production was improved significantly via the addition of oxygen vector n-dodecane, the purification process was more complicated and the industrial HA production was not applicable. Therefore, the possibility of increasing mixing performance and oxygen mass transfer efficiency via the HA degradation was further explored.（4） Effects of hyaluronidase addition on the microbial HA production were investigated and it was indicated that the hyaluronidase addition could improve the mixing performance and oxygen mass transfer significantly. When hyaluronidase concentration was 0.25 g/L, HA molecular weight decreased from 1300 kDa to 21 kDa, making the culture system in a status of complete mixing, and KLa reached 100 h^-1. HA production reached 6.3 g/L, enhanced by 26%. The production cost was high and the industrial HA production was not feasible;（5） Effect of hydrogen peroxide and ascorbate addition on microbial HA production were studied and the results indicated that, the twice addition of hydrogen peroxide and ascorbate at 8 h and 12 h could significantly improve the mixing performance and oxygen mass transfer. When the hydrogen peroxide concentration was 1.0 mmol/g HA and ascorbate concentration was 0.5 mmol/ g HA, KLa reached 49 h^-1 and HA production was 6.0 g/L, enhanced by 20%;（6） Based on the physiological function of HA capsule in Streptococcus zooepidemicus and the subatrate and energy response mechanism of the cells subject to stress environments, Impact of oxidative stress via feeding hydrogen peroxide on microbial HA production was studied, and it was found that the oxidative stress could transform the energy-yielding pathway and improve the energy-yielding efficiency, contributed to the enhancement of HA synthesis rate. At a hydrogen peroxide feeding rate of 0.06 mmol L-1 h^-1, HA production reached 6.0 g/L and was enhanced by 20%;（7） An intermittent alkaline stress strategy was proposed: pH value was kept at 7.0 for the first 6 h, and then intermittently switched to 8.5 for 1 h and back to 7.0 for 1 h, the cycle was repeated until the end of the fermentation. HA production reached 6.5 g/L and was enhanced by 30% with the improved energy status by the intermittent alkaline stress strategy;（8） The metabolic kinetics of amino acids during the microbial HA production process was investigated, and the arginine, cysteine and lysine were found to be the key amino acids for cell growth and HA synthesis. The influence of nucleotides bases addition on the microbial HA production was also studied and the uracil had a positive effect on the microbial HA production. Under the optimal conditions （by adding arginine 0.062 g/L, cysteine 0.036 g/L, lysine 0.043 g/L and uracil 0.06 g/L） obtained by RBF-QPSO approach, HA production reached 6.3 g/L, enhanced by 26%;（9） Effect of different culture modes on the microbial HA production were studied and the results showed that batch culture had a higher HA synthesis rate while fed-batch culture had a higher specific cell growth rate. A two-step fed-batch and batch culture approach was proposed: the exponential feeding was carried out during 0-8 h and then the batch culture was started with an initial sucrose concentration of 25 g/L. HA production increased to 5.98 g/L, enhanced by 20 %. To further decrease the inhibition of lactic acid on the cell growth and HA synthesis, the intermittent alkaline stress strategy was applied in the second stage and HA production increased to 6.6 g/L, enhanced by 32%. The transformation yield of carbon source to HA and HA productivity were increased by 19% and 32%, respectively, achieving the unification of high HA production, high carbon transformation rate and high productivity.更多还原