Pulse Filling Material For High Density Fermentation Type Collagen Engineering Bacterium

In high-density fed-batch cultivation of Recombinant Escherichia coli BL21,12different pulse feeding models were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth and human-like collagen (HLC) synthesis; mass spectrum was applied to research the fermentation gas and its related parameters such as oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and respiratory quotient (RQ); the plasmid stability of Esherichia.coli BL21was primarily discussed by the method of plasmid replicating. Compared to the pulse feeding strategy, the effect of DO-pulse feeding and the stable substrate concentration feeding were observed on cell growth and HLC production. The results are as the follows:1. The whole fermentation process can be identified as three phases:the batch culture phase, and before induction phase and after induction phase of fed-batch culture, which was in accordance with cell growth and metabolic illustration. The DCW of six pulse modes at14th were51.67g/L,49.88g/L,46.45g/L,66.85g/L.61.28g/L, and57.22g/L, respectively (R1-R6), which increased1.61-fold.1.81-fold.1.59-fold,2.12-fold.1.54-fold and1.50-fold greater than the biomass attained at the beginning of feeding, meanwhile, no significant increased of cell growth in the after induction because of the cell growth attached to stationary phase. The accumulation of acetate increased to the maximum value near to the end of batch culture gradually, and then, it was consumed when a starvation period after the batch phase was introduced. Furthermore, the acetate concentration was kept at lower level (below0.5g/L) in all cases when pulse feeding was used before induction. It was suggestion that acetate assimilation and cell growth after glucose exhaustion. The final biomass and HLC could reach to75.46g/L and7.26g/L respectively in the model of feeding5.6ml per188s s of before induction phase and feeding2.1ml per27s of induction phase.2. The presence of sufficient oxygen throughout the fermentation could improve cell growth and metabolism in DO-pulse feeding strategies, in which the toxic of excess oxygen on cell was also reduced. Thus, the glucose concentration can be maintained in limiting level to avoid acetate extraction successfully when the critical specific uptake oxygen rate to cause the critical specific uptake oxygen rate decreased after induction of human-like collagen production. Consequently, the relatively lower acetic concentration was obtained, and the acetate concentration, biomass and protein concentration were1.72g/L,73.45g/L and7.89g/L, respectively. Accordingly,2.41g/L of acetate concentration was attained in stable substrate concentration fermentation strategy; however, this feeding strategy couldn’t be implicated to large-scale fermentation because the substrate control was not easy to be achieved. Using stable feeding rate, the nutrient was sufficient at begining of fermentation. But the inhibition of lack substrate was observed around the fermentation, in which the highest acetate accumulated was about4.23g/L in comparison with acetate concentration in DO-pulse feeding fermentation and stable substrate concentration. Meanwhile, the final concentration of biomass and HLC concentration were48.62g/Land2.89g/L, respectively.3. In model R4, the overall yield coefficient Yx/s increased to around0.9during the first7.5h of culture, and then decrease to0.42g/g at the end of the culture. It was suggested that the effect of substrate to cell growth decline with the feeding start. Base on these finding, the major work of substrate was to supply enough energy for cell, adjusting to production of target protein. By summing up the carbon in biomass, CO2, and acetate it became clear that only90%of substrate carbon was recovered in these products at the end of culture. Obviously, other products than biomass and CO2must have been formed. The missing carbon remained as soluble organic compounds in the medium. Indeed, acetate, lactate, formate, and pyromaniac acid were detected in the culture medium, but made up only a few percent of the extracellular carbon mass.