| 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.
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