Process Optimization And Control For L-phenylalanine Production

L-Phenylalanine(L-Phe) is a valuable chemical product that is widely used in food, animal feed-stock and pharmaceutical industries and is one of the eight essential amino acids. At present it is mainly produced by microbial fermentation process, which is considered more economical and eco-friendly than other industrial processes. The existing problems of fermentation process are: The production strain E. coli is often infected by bacterial phages; The feeding strategy of L-Tyrosine(L-Tyr), which is an important limiting substrate for an L-Tyr auxotrophic strain, is not well studied; Glucose, as the most frequently used carbon source, has its shortcomings while there is no available report on possible alternative substance that could be used in fed-batch fermentation. Based on the problems mentioned above, the main contents of this dissertation are as follows:1. Two different bacterial phages BP-1 and BP-2 were isolated from the abnormal fermentation broth of a sensitive strain E.coli WSH-Z06 (pAP-B03). After the exposure to ultraviolet rays, E.coli WSH-Z06 (pAP-B03) was mutated so as to screen a bacterial phage resistant mutant. The isolated bacterial phages were added directly into the culture after mutation to exert selective pressure for those non-resistant mutants. Finally an E.coli strain named as WSH-BR165 (pAP-B03) was successfully screened and its performance as an L-Phe production strain was comparable to the parent strain.2. The growth of E.coli WSH-BR165 (pAP-B03) can be maintained by feeding L-Tyr at a constant rate after induction point, and as a result, cell density and L-Phe productivity was increased. The constant feeding rate led to a weakening cell growth where specific growth rate was decreasing. Dual exponential feedings of L-Tyr and glucose were found to increase cell density at a constant specific growth rate. Via dual exponential feedings after/before induction point, L-Phe production process and cell growth were integrated/separated. The results of the comparative study between the two different feeding modes indicated that when L-Phe production was accompanied by continuous cell growth, L-Phe fermentation process could be enhanced without extra substrate consumption. Not only was the increased cell density the reason for enhanced productivity, also the increased specific production rate could be counted as an important factor. At a specific growth rate set point of 0.09 h-1 after induction point, maximum dry cell weight reached 43.16 g/L (3.04 times that of the control) with a final L-Phe titer of 44.53 g/L (1.06 times that of the control) and an L-Phe productivity of 1.484 g/L/h (1.69 times that of the control). High cell density cultivation via feeding L-Tyr and glucose exponentially after the induction point proved to be an efficient approach to enhance L-Phe production.3. The feasibility of glycerol as an alternative carbon source for L-Phe fed-batch fermentation process was evaluated. The initial glycerol concentration was optimized to be at 30 g/L during batch cultures. Under fed-batch cultivation conditions, three different glycerol feeding schemes (i.e. pH-stat, feedings at constant feeding rates, feedings that kept residue glycerol concentration at constant) were investigated. The results indicated that under the feeding strategy where residue glycerol concentration was kept at 5 g/L, it was most favorable for L-Phe production. Its maximum L-Phe titer reached 48.61 g/L (1.32 times that of the control) with a productivity at 0.972 g/L/h (1.42 times that of the control) when pH-stat was chosen as the control for glycerol feeding runs. On the other hand, while compared to the fermentation process under the same substrate control strategy using glucose as the carbon source (i.e. residue substrate level was kept constant at 5 g/L), L-Phe titer was increased by 15.6%, while L-Phe mass yield on substrate was increased by 18.9%, which indicated that glycerol was better than glucose as a carbon source during L-Phe production. According to the study of the correlation between the glycerol consumption and ammonium water consumption, a linear-fitted equation y=6.686x+89.329 was deduced. In application, it implied that via setting glycerol feeding rate positively correlated with the assumption rate of ammonium water, substrate feeding could be adjusted automatically to maintain residue glycerol concentration at a relatively low concentration.