Efficient Production Of ε-poly-l-lysine Through Bioprocess Optimization And Regulation At Pilot Scale

ε-Poly-L-lysine(ε-PL) is a natural food preservative produced by microbial fermentation,which has been widely used in the food industries of Japan, Korea, Europe and US. The global ε-PL production is mainly monopolized by the Japanese company of JNC, whereas,ε-PL production in domestic is only under small-scale in a few companies. On the whole,ε-PL research in our country is still on laboratory and pilot scales. However, the recent approval of ε-PL as a food preservative in starch, meat, fruit and other foods by China has expanded the demand for industrial ε-PL production to a high level.The strain Streptomyces albulus M-Z18 was used as the study object throughout this study, which was a potential industrial production strain. We first optimized the expansion of seed cultivation, and then gradually scaled up ε-PL production from 5 L to 50 L and 1 m3 fermenter by the optimization of fermentation conditions in lab(50 L) and pilot(1 m3) scales.Finally, two strategies(p H shift and yeast extract feeding) were successfully employed to solve the decline of ε-PL productivity in the middle or late phase of the fed-batch fermentation and improved ε-PL productivity. The specific results were depicted as follows:(1) The optimal conditions for spore pretreatment and seed expansion cultivation were sequentially established. After studying the effect of spore pretreatment on seed cultivation and batch fermentation, ultrasonic for 1 min was considered as the best condition. On this base, we further optimized the inoculation age and amount, and the optimal condition for the first seed cultivation was established: late-log inoculation age, 12%(v/v) inoculation amount.Under the best seed cultivation condition, ε-PL production reached 48.9 g·L-1 after 192 h of fed-batch fermentation in 5 L fermenter, which was 32.3% higher than that of the control(shake-flask culture seed). Based on the first seed cultivation, the optimal condition for the inoculation age of the secondary seed cultivation was determined to be the mid-log phase.The ε-PL production was 12.47 g·L-1 in batch fermentation, which was enhanced by 21.1%,compared with the inoculation of first seed cultivation.(2) ε-PL production was successfully scaled up from 5 L to 50 L and 1 m3 fermenters.We investigated the effect of culture temperature, impeller number and liquid volume on ε-PL fermentation in 50 L fermenter, and determined the optimal condition: culture temperature30 oC, two impellers and 70% of liquid volume. In addition, tap water showed no significant effect on ε-PL fermentation. The optimal ε-PL production and productivity reached 36.22g·L-1 and 4.53 g·L-1·d-1 at 192 h in 50 L fermenter, respectively. According to the laboratory amplification technology and the optimization of spore inoculation quantity in 100 L, we realized the ε-PL production more than 30 g·L-1by three-time repeated fed-batch fermentation in 1 m3 fermenter.(3) The research enhanced ε-PL productivity in the late phase of fed-batch fermentation by p H shift and yeast extract feed strategies. The final ε-PL production reached 35.87 g·L-1and 41.32 g·L-1 at 192 h with both strategies, which were 23.7% and 42.5% higher than that of the typical ε-PL fed-batch fermentation, respectively. The results of CTC and key enzyme activity analyses demonstrated that the rise of dissolved oxygen and the decline of ε-PLproductivity were caused by the decline of cell viability and metabolic activities of the central metabolic pathways. Two strategies enhanced the ε-PL production by improving cell viability and key enzyme activities.