Optimization and scale-up production process of 2,3-butadeniol from maltodextrin by metabolically engineered klebsiella oxytoca KMS005

摘要

An optimization process with a cheap and abundant substrate is considered one of the factors affecting the price of commercial 2,3-Butanediol (2,3-BD) production. The optimized levels of pH, aeration rate, agitation speed, and substrate concentration (maltodextrin)were optimized by a conventional method and Response Surface Methodology (RSM) with Box-Behnken design in which metabolically engineered Klebsiell oxytoca KMS005 utilized maltodextrin to produce 2,3-BD. Results revealed that pH, aeration rate, agitation speed,and maltodextrin concentration at levels of 6.0, 0.8 vvm, 400 rpm, and 150 g/L, respectively, were the optimal conditions. RSM indicated that the agitation speed was the most influential parameter when either agitation and aeration interaction or agitation and substrate concentrationinteraction played important roles for 2,3-BD production. Under interim fedbatch fermentation, 2,3-BD concentration, yield, and productivitywere obtained at 88.1±0.2 g/L, 0.412±0.001 g/g sugar supplied, and 1.13±0.01 g/L/h, respectively, within 78 h. The influence of micro-aerobicconditions on microbial growth and 2,3-BD production was also studied. In batch bioreactors, air flow rate and agitation rate characterized through kLa measurement were tested. The optimal amount of oxygen supply was evaluated at 9.5 g corresponding to a kLa of 25.2 h-1 for cell growth and 2,3-BD production. Then, a fed-batch process was investigated by different glucose feeding rate strategies. Fedbatch with a glucose feeding rate of 2 g/h starting at the end of the growth phase during 48 h, followed by a final batch phase of 40 h was found satisfactory. It resulted in a final 2,3- BD concentration of 74.7 g/L with a productivity of 0.64 g/L/h but few byproducts formed (about 3 g/L including succinate, acetate and ethanol). Validated information in the 2 L bioreactor was further applied in a larger scale production of 2,3-BD with series of bioreactors from 10, 90 and 300 L vessels. Batch experiments were conducted based on various agitation speeds with the fixedaeration rate at 0.8 vvm. As a result, 2,3-BD concentration, and yield were achieved at 53.8 g/L, and 0.40 g/g sugar supplied within 48 h, respectively, under the constant tip speed at 295 rpm using a 10 L vessel. Its concentration of 52.53 g/L and yield of 0.43 g/g sugar consumedwithin 72 h were attained under the condition of the constant tip speed at 130 rpm using a 90 L fermenter. An appropriate seed inoculum condition was found with an optical cell density (OD550) around 4 at the log phase (12 h incubation) prior to transferring of the inoculum into the 90 L fermenter. Under the constant tip speed at 70 rpm, 2,3-BD concentration and yield were obtained at 45.02 g/L and 0.43 g/g sugar consumed in the pilot scale of 300 L bioreactor after 72 h incubation.