Microbial Fermentation And Aqueous Two-phase Extraction Of 2,3-Butanediol

2,3-Butanediol(2,3-BD) is one of important bulk chemicals and liquid fuels,which exhibits a wide range of potential utilizations in cosmetics,foods,medicines,as well as polymers.2,3-BD is mainly manufactured with microbial method by using glucose currently as the most common feedstock.While the fermentation of 2,3-butanediol from glucose takes high costs,moreover,it competes with grain supplies and takes up arable land.In addition,a major difficulty still exists in downstream processing because 2,3-BD has a high-boiling point and a high affinity with water.It is clear that a large-scale production of 2,3-BD will require low-cost raw materials and effective downstream process.In this paper,microbial production of 2,3-BD was performed by Klebsiella pneumoniae using glucose or Jerusalem artichoke tubers as carbon source.Additionally,a novel aqueous two-phase system(ATPS) for the recovery of 2,3-BD from fermentation broth was developed employing short chain alcohol/inorganic salt.Firstly,batch fermentation for 2,3-BD by K.pneumoniae DSM 2026 was investigated to obtain the optimized fermentative conditions.The results showed that the optimum conditions were 8%of initial glucose concentration,0.04 vvm of air flow,and pH 5.5.The final concentration of target products(2,3-BD and acetoin) in a fed-batch fermentation carried out under the optimized conditions was 42.60 g/L with a productivity of 0.76 g/L·h in 56 h, corresponding to 23%of yield from glucose.Continuous fermentations were carried out under micro-aerobic conditions.The dynamic behavior of fermentation was studied by a large perturbation of substrate concentration.The results showed that no long period oscillation was observed,but a short oscillatory behavior in CO₂ was occurred when dilution rate was between 0.20 and 0.25 h^-1,and the period was about 9.3～15.0 minutes.Secondly,the optimum conditions for 2,3-BD production by K.pneumoniae CICC 10011 were investigated.The results showed that the optimum conditions for fermentation were 0.10 vvm of air flow,5.8 of pH value.The final concentration of target products (2,3-BD and acetoin) in a fed-batch fermentation carried out under the optimized conditions was 85.61 g/L with a productivity of 1.53 g/L.h in 56 h,corresponding to 45%of yield from glucose.Comparison of the metabolic flux distributions between K.pneumoniae CICC 10011 and DSM 2026 showed that the strain CICC 10011 produced less organic acids and ethanol,but more target products and more yield could be obtained than those of DSM 2026.Therefore,K. pneumoniae CICC 10011 is a potential strain for 2,3-BD production.Thirdly,a novel aqueous two-phase system for the recovery of 2,3-BD from fermentation broth was developed employing isopropanol/(NH₄)₂SO₄ system.The results showed that most of target products(2,3-BD and acetoin) can be partitioned efficiently into the top phase,while most of glucose can be partitioned into the bottom phase.The optimized system was composed of 34%(w/w) isopropanol and 20%(w/w)(NH₄)₂SO₄,yielding the partition coefficients of 8.29 and 8.98 for 2,3-BD and acetoin,respectively.The recoveries for 2,3-BD and acetoin were 91%and 92%,respectively.Aqueous two-phase extraction of 2,3-BD from real fermentation broth,the removal ratio of cells was above 99%,and the soluble proteins were about 85%in the top phase,which resulted in easy recovery of 2,3-BD from the top phase by vaporization because a viscous slurry was not formed by glucose,cells and soluble proteins at high temperature as in traditional separation methods.In addition,the cells recovered from the fermentation broth could be used as inoculum for further fermentation.Subsequent fed-batch fermentation eliminated the need for separately prepared inoculums,saving the cost and time of fermentation.Lastly,2,3-BD production from Jerusalem artichoke tubers was investigated by K. pneumoniae CICC 10011.Shake flask cultures were performed in order to study the effects of different carbon sources,different hydrolysis modes and trace elements on the fermentation of 2,3-BD.The results showed that Jerusalem artichoke was an excellent carbon source for the production of 2,3-BD.There was no distinct difference between enzyme hydrolysis and acid hydrolysis for the 2,3-BD fermentation.Moreover,no difference was observed between trace elements added and not added into the medium,which proved that the trace elements contained in Jerusalem artichoke tubers are sufficient for 2,3-BD production.On the basis of the results,separate hydrolysis and fermentation(SHF) and simultaneous saccharification and fermentation(SSF) were performed,respectively.The results showed that SSF process was preferable for high concentration and high efficiency of 2,3-BD production.Without considering hydrolysis(12 h in SHF) outside the bioreactor,fermentation time decreased from 56 h for SHF to 40 h for SSF.Regardless of the time for enzymatic hydrolysis,a remarkable increase in productivity for SSF was observed.For example,fed-batch SSF provides 30%higher concentration of target products and 83%higher productivity than fed-batch SHF.The results show that Jerusalem artichoke tuber is a potential material for industrial production of 2,3-BD.