| Acrylamide is an important chemical widely used in many industrial fields. The consumptive market of acrylamide is growing quickly, accompanying with the development of its potential applications. However, many shortages are still in existence in the traditional bio-process of acrylamide, in which the immobilized cells are used as biocatalysts. To further promote the industrial productivity of acrylamide, the fermentative techniques of Norcardia strain were improved, the kinetic models of nitrile hydratase (NHase) in free cells were studied, the activity stable methods of NHase was explored, and a new technology of bioconversion coupled with the membrane separation, in which a hollow-fiber membrane bioreactor was designed and the free cells were used as biocatalysts in the microbial bioconversion of acrylonitrile to acrylamide, was presented to replace the traditional technology.Firstly, the cultural conditions for the growth of Norcardia cells were focused. The fermentative procedures were carefully investigated, including the controlling of pH value and the designing of supplemental scheme of glucose and inducer. By the above improvement the activity of NHase reached over 6000 U/ml, which was much higher than that in today's industry. A better inducer was also developed to replace the commercial one whose components remained unknown till now. When using the new inducer, the final total enzyme activity and the special enzyme activity of NHase were enhanced to 7800 U/mL and 390 U/mg, respectively.Then, the kinetic models of NHase in the free cells were studied. It is the first time to introduce the assumption of "two-steady state" in the enzyme catalytic reaction to describe the reaction mechanism of the free NHase. The new concept of micro-encapsulated-immobilized enzyme was adopted to simulate the situation of NHase inside individual free cell. Based on these assumptions, the kinetic models for fee cells were successfully constructed, by which the behaviors of the catalytic kinetics and enzyme activity-lose kinetics of free cells were accurately simulated.The new way to improve the stability of NHase in Norcardia was further explored. It was found that some additives, such as glucose, trehalose and butyric acid, could strengthen the stability of NHase. Through domestication and acclimation of Nocardia sp. by feeding acrylonitrile into the culture medium, a superior acclimated Nocardia strain was obtained, in which the stability of NHase was greatly improved and the bearable concentration of acrylamide was reached on the 600g/L level which was increased about 85% comparing to the original control.Based on the fundamental researches above, a novel technology was presented, in which the free cells was used as catalysts in the bioconversion and the membrane separation was coupled to remove the products. Then a new membrane bioreactor device was designed. The polysulfone (PS) and polyvinylidene fluoride (PVDF) hollow-fiber membrane were respectively tested and chosen, and serially connected to the bioreactor. It was proved that the new process has many advantages such as simple composition, easy operation and stable filtration. Finally it was put into the practice.For the new bioconversion system coupling with the membrane separation three different operation modes were simultaneously developed, i.e. single-stage un-steady operation, single-stage false-steady operation, multi-stage steady and continuous operation. The feasibility and superiority of these operation modes in membrane bioreactor were confirmed in lab-scale, pilot-scale and industrial-scale step by step.The operation modes were simulated and optimized by computer program, following the analysis of the characteristics of operation modes and construction of the mathematic models. The control strategy of the multi-stage membrane bioconversion was also optimized by Dynamic Programming Algorithm, and the superiorities of the new process were strengthened further more.Finally, the new process of membrane bioconversion using free...
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