微生物发酵制备2，3-丁二醇及其双水相萃取
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摘要
2,3-丁二醇(2,3-Butanediol)是一种重要的化工原料和液体燃料,被广泛应用于化工、食品、医药、燃料及航空航天等多个领域,其生产方法主要为生物转化法。目前生物转化法生产2,3-丁二醇大多用葡萄糖作为碳源,而葡萄糖的价格较高,且存在与人争粮、与粮争地的问题。此外,由于2,3-丁二醇具有较强的亲水性和较高的沸点,且发酵液成分复杂,因而该产品下游分离比较困难。原料成本高、产品分离困难已经成为2,3-丁二醇大规模工业化生产的瓶颈。针对这些问题,本论文研究了生物转化法生产2,3-丁二醇的工艺,并采用新型双水相体系对发酵液中的2,3-丁二醇进行分离萃取,最后考察了利用廉价的非粮原料菊芋作为底物发酵2,3-丁二醇的可行性。
首先,以Klebsiella pneumoniae DSM 2026为菌种,通过批式发酵实验,确定了生物转化法生产2,3-丁二醇的最适条件为:初始底物浓度8%,微氧发酵,通气量为0.04vvm,pH值为5.5。在该条件下进行批式流加发酵,可以获得目标产物浓度为42.60 g/L,生产强度为0.76g/(L·h),葡萄糖的质量转化率为23%。在此基础上,考察了微氧条件下K.pneumoniae DSM 2026在连续发酵中的动态行为,结果表明,较大程度地扰动底物浓度并不能诱发长周期振荡现象的产生,但却在底物浓度没有扰动情况下出现了CO_2短周期振荡现象,且仅当稀释速率在0.20～0.25 h~(-1)范围内才能出现这种振荡,振荡周期为9.3～15.0 min。
其次,以K.pneumoniae CICC 10011为菌种,通过批式发酵实验,确定了生物转化法生产2,3-丁二醇的最适通气量为0.10 vvm,最适pH为5.8。在该条件下进行批式流加发酵,可以获得目标产物浓度为85.61 g/L,生产强度为1.53 g/(L·h),葡萄糖的质量转化率为45%。
通过对批式流加发酵过程中两株克雷伯氏菌代谢流量的分析表明,K.pneumoniaeCICC 10011代谢产生的各种有机酸和乙醇浓度均明显低于K.pneumoniae DSM 2026,而目标产物浓度以及产物转化率却相对较高,是发酵2,3-丁二醇的优良菌株。
再次,对新型双水相分离体系—异丙醇/硫酸铵体系从发酵液中萃取分离2,3-丁二醇进行了研究。结果表明,目标产物2,3-丁二醇和乙偶姻主要富集在上相中,而葡萄糖主要富集在下相中;当异丙醇和硫酸铵的质量浓度分别是34%和20%时,2,3-丁二醇和乙偶姻的分配系数分别为8.29和8.98,回收率分别达到91%和92%;采用该条件对发酵液直接进行萃取,细胞的去除率在99%以上,可溶性蛋白去除率可达85%。双水相萃取2,3-丁二醇除去了部分水和一些大分子物质,将固液分离和浓缩两步集成化,降低了后续蒸馏时的粘度,极大地简化了分离工艺。而且经过双水相萃取后所得的菌体,可以直接作为下一批发酵的种子继续使用。细胞循环发酵可以减少种子培养步骤,节约发酵时间。
最后,探索了利用非粮廉价的菊芋为原料生物转化法生产2,3-丁二醇的可行性。通过摇瓶发酵实验考察了不同底物、不同水解方式以及微量元素对发酵的影响,结果表明,菊芋是良好的发酵生产2,3-丁二醇的底物,采用酸水解或酶水解两种方式制得的糖液发酵效果基本一致,菊芋中含有的微量元素完全能够满足发酵的需要,发酵过程中无需添加微量元素。在此基础上,在5L发酵罐中分别采用分步糖化发酵和同步糖化发酵两种工艺发酵生产2,3-丁二醇,结果表明,同步糖化发酵不但省去了单独糖化步骤,而且还提高了产物浓度和生产效率,采用批式流加发酵方式,发酵40 h,目标产物浓度和生产强度分别达到91.63 g/L和2.29g/(L·h)。与分步糖化发酵相比,发酵时间从56h缩短到40h,产物浓度提高了30%,生产强度提高了83%。实验结果表明以菊芋为原料发酵生产2,3-丁二醇具有一定的产业化前景。
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 CO_2 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/(NH_4)_2SO_4 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)(NH_4)_2SO_4,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.
首先,以Klebsiella pneumoniae DSM 2026为菌种,通过批式发酵实验,确定了生物转化法生产2,3-丁二醇的最适条件为:初始底物浓度8%,微氧发酵,通气量为0.04vvm,pH值为5.5。在该条件下进行批式流加发酵,可以获得目标产物浓度为42.60 g/L,生产强度为0.76g/(L·h),葡萄糖的质量转化率为23%。在此基础上,考察了微氧条件下K.pneumoniae DSM 2026在连续发酵中的动态行为,结果表明,较大程度地扰动底物浓度并不能诱发长周期振荡现象的产生,但却在底物浓度没有扰动情况下出现了CO_2短周期振荡现象,且仅当稀释速率在0.20～0.25 h~(-1)范围内才能出现这种振荡,振荡周期为9.3～15.0 min。
其次,以K.pneumoniae CICC 10011为菌种,通过批式发酵实验,确定了生物转化法生产2,3-丁二醇的最适通气量为0.10 vvm,最适pH为5.8。在该条件下进行批式流加发酵,可以获得目标产物浓度为85.61 g/L,生产强度为1.53 g/(L·h),葡萄糖的质量转化率为45%。
通过对批式流加发酵过程中两株克雷伯氏菌代谢流量的分析表明,K.pneumoniaeCICC 10011代谢产生的各种有机酸和乙醇浓度均明显低于K.pneumoniae DSM 2026,而目标产物浓度以及产物转化率却相对较高,是发酵2,3-丁二醇的优良菌株。
再次,对新型双水相分离体系—异丙醇/硫酸铵体系从发酵液中萃取分离2,3-丁二醇进行了研究。结果表明,目标产物2,3-丁二醇和乙偶姻主要富集在上相中,而葡萄糖主要富集在下相中;当异丙醇和硫酸铵的质量浓度分别是34%和20%时,2,3-丁二醇和乙偶姻的分配系数分别为8.29和8.98,回收率分别达到91%和92%;采用该条件对发酵液直接进行萃取,细胞的去除率在99%以上,可溶性蛋白去除率可达85%。双水相萃取2,3-丁二醇除去了部分水和一些大分子物质,将固液分离和浓缩两步集成化,降低了后续蒸馏时的粘度,极大地简化了分离工艺。而且经过双水相萃取后所得的菌体,可以直接作为下一批发酵的种子继续使用。细胞循环发酵可以减少种子培养步骤,节约发酵时间。
最后,探索了利用非粮廉价的菊芋为原料生物转化法生产2,3-丁二醇的可行性。通过摇瓶发酵实验考察了不同底物、不同水解方式以及微量元素对发酵的影响,结果表明,菊芋是良好的发酵生产2,3-丁二醇的底物,采用酸水解或酶水解两种方式制得的糖液发酵效果基本一致,菊芋中含有的微量元素完全能够满足发酵的需要,发酵过程中无需添加微量元素。在此基础上,在5L发酵罐中分别采用分步糖化发酵和同步糖化发酵两种工艺发酵生产2,3-丁二醇,结果表明,同步糖化发酵不但省去了单独糖化步骤,而且还提高了产物浓度和生产效率,采用批式流加发酵方式,发酵40 h,目标产物浓度和生产强度分别达到91.63 g/L和2.29g/(L·h)。与分步糖化发酵相比,发酵时间从56h缩短到40h,产物浓度提高了30%,生产强度提高了83%。实验结果表明以菊芋为原料发酵生产2,3-丁二醇具有一定的产业化前景。
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 CO_2 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/(NH_4)_2SO_4 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)(NH_4)_2SO_4,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.
引文
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