Process Scaled-up And Metabolic Analysis Of Efficient γ-PGA Production In Bacillus Licheniformis MEL-02

Poly y-glutamic acid (y-PGA), a kind of glutamic acid homopolymer, is generally synthesized by microbes. Because of its high added-value, y-PGA has presented great potential in industrial applications like pharmaceutical manufacturing, environment management, light chemical industries, agriculture and so on.To improve the yield of y-PGA and decrease the cost of raw material, the essential substance concentrations of y-PGA production of Bacillus licheniformis MEL-02 were optimized while several parameters were considered simultaneously. The optimization was performed based on the medium result from orthogonal design with single target. An efficient and cost-effective medium was obtained below (g/L):sodium glutamate 30, glucose 90, sodium citrate 10.0, NaNO3 10.0, K2HPO4 0.5, MgSO4·7H2O 0.5, FeCl3·6H2O 0.04, CaCl2·2H2O 0.15, MnSO4·H2O 0.104. The main raw material cost decreased 63% after optimization, while the production rate increased from 0.95 g·L-1·h-1 to 1.27 g·L-1·h-1. The scaled-up was done in the 180 L fermentor next, resulted in a further reduce of glutamate to 20 g/L.A network of B. licheniformis MEL-02 was structured for the flux analysis of glucose metabolic during γ-PGA fermentation. Comparing the flux simulated with the data determined, the main byproduct —2,3-butanediol was discovered, with a final concentration of 26.44 g/L. Furthermore, the physiological function of 2,3-butanediol was revealed to be preventing intracellular acidification and the regeneration of the excess reducing power.Fed-batch culture strategy was optimized and chosen to be maintaining 50 g glucose/L in medium, which increased the titer to 40.8 g/L,15% higher than that of batch fermentation. Metabolic flux analysis was carried out both in batch and fed-batch fermentation, which indicated that:both the glucose dosage increasing in optimized medium and the fed-batch culture were belong to the flux change of glucose consumption, which failed to lead a perfect flux distribution both at the nod of pyruvate and the nod of 2-Oxoglutarate.At last, a medium for co-producing γ-PGA and 2,3-butanediol was developed as below (g/L):sodium glutamate 40, glucose 150, sodium citrate 10.0, NaNO3 10.0, K2HPO4 0.5, MgSO4·7H2O 0.5, FeCl3·6H2O 0.04, CaCl2·2H2O 0.15, MnSO4·H2O 0.104. The volume of production ofy-PGA and 2,3-butanediol reached 52.20 g/L and 73.68 g/L, respectively, making a significant increase of 47% and 179%. The total yield increased from 0.68 g/g glucose to 0.89 g/g.