Recombinant E. Coli For Production Of Human Collagen Fermentation Control Strategies And 500l In The Pilot Scale Up Optimization

The aim of this investigation was to develop a process for production of human-like collagen (HLC) by recombinant Escherichia coli on pilot-scale. In addition, the study would lay a sound basis for scaling-up to production scale further, through scale-up investigations for unit operations of the process.High cell density culture of recombinant E. coli is the key unit for HLC production and its scale-up performance would determine the whole process efficiency. To find the key factors influencing the fermentation process and thus chose the scale-up criteria and method properly, the effects of carbon dioxide and acetate on HLC production was studied firstly.Carbon dioxide plays a major role in both aerobic and anaerobic fermentations. However, few literatures focused on the effects of carbon dioxide on recombinant E. coli culture and target protein production. In this paper, a systematic study was initially carried out with the carbon dioxide pulse injection method to investigate the influence of carbon dioxide on the cultivation system of recombinant E. coli. The CO₂ pulse injection experiments showed that: 1) a 20% CO₂ pulse introduced in the batch cultivation phases inhibited cell growth obviously while that introduced in the fed-batch cultivation phases stimulated cell growth slightly; and 2) inhibitory effect of CO₂ on HLC expression occurred only in the expression phase, where the final HLC concentration decreased by 33.9% under a 3-h 20% CO₂ pulse. Meanwhile, the higher the CO₂ concentration and/or the longer the duration of the CO₂ pulses, the stronger the stimulatory or inhibitory effects. However, the CO₂ concentration below or equal to 10% had little influence on the HLC production.Then, a systematic study was carried out to determine the effects of acetate on the high cell density culture of recombinant E. coli containing HLC mRNA. In this study, the effects of acetate assimilation through a glucose starvation period at different cell growth phases were initially investigated in a fed-batch culture of recombinant E, coli. The results showed that acetate assimilation was observed without negative effects on the process during a glucose starvation period introduced at all defined cell growth phases except for the post-induction phase. In addition, a new method for evaluating the effects of acetate was development based on the above findings: the acetate concentration was controlled by adding acetate into culture media and/or employed a glucose starvation and thus the effects of acetate at different cell growth phase were determined. Experimental results show that obvious acetate inhibition on cell growth occurred in the phases of batch culture while its inhibitory effect on HLC expression occurred only in the expression phase.The development of a suitable inoculum is important to the fermentation process. To optimize the inoculum preparation process, the third inoculum transfer stage and medium were determined. The results showed that HLC productivity was highest when the third inoculum was transferred at lately exponential stage; in addition, when the glucose concentration was 20 g/L, the following fermentation time was shortest and the final HLC concentration was highest, and thus the HLC average productivity was highest (achieved 0.518 g/L/h).Acetate had a strong negative influence on the HLC production according to this work, so a feed-back controlled method that the probing feeding strategy was developed to control the acetate production. The method was employed to maintain dissolved oxygen concentration at the same level on both laboratory scale and pilot scale fermentations. The final DCW of 69.1 g/L and HLC concentration of 13.1 g/L were obtained on the laboratory scale and the results are similar to that optimized in the previous study. The final DCW of 51.7 g/L and HLC concentration of 9.6 g/L were obtained on the pilot-scale upon optimization of induction timing.The reason for the decrease in the HLC production on the pilot scale may be the lower oxygen transfer rate for the pilot-scale fermentor. Therefore, the volumetric mass transfer coefficient k_La on different scale fermentors was determined and the correlations between the k_La and operation parameters (aeration, stirrer rate and initial medium volume) were developed. And thus the oxygen transfer capacities for different fermentors could be compared. The correlations for k_La on the lab-scale fermentor is,And the correlations for k_La on the pilot-scale fermentor is, Finally, the scale-up fermentations based on k_La and p*k_La were carried out respectively, according to the correlations for k_La calculation. Because the recombinant system was less sensitivity to the CO₂, pressurized culture was successful for the HLC production. That is, for the non-induced cultivation on the pilot-scale based on the constant p*k_La scale-up criteria, the final DCW was 77.9 g/L, which was similar to that (80.3 g/L) obtained on the lab-scale cultivation and lower than the value (90.0 g/L) obtained in the pilot-scale based on the constant k_La scale-up criteria. However, the initial medium volume for the former (285 L) was much higher than the latter (105 L). The final DCW and HLC concentration were 68.4 g/L and 13.0 g/L repectively, which were similar to those obtained on the lab-scale, for the induced cultivation on the pilot-scale.The process for HLC purification on the pilot-scale was also developed in this study. The major operation units influencing HLC recovery and purity were homogenization, precipitation, ultrafiltration and CM52 chromatography. The scale-up investigation showed that, 1) the operation parameters could be directly transferred to the larger scale for the homogenization; 2) the operation parameters related to the membrane area were increased proportionally for scaling-up the ultrafiltration process; 3) the precipitation time was optimized on the pilot-scale and the optimized precipitation was 70 min, which is little higher than that on the lab-scale; 4) CM52 chromatography scaling-up based on constant linear velocity was successful. The purity and recovery of HLC reached 95.4%, 65.4%, repectively, which were decreased slightly compared to those (the putity and recovery were 96.4%,71.7%, respectively) obtained on the lab-scale.