发酵液中1，3-丙二醇和2，3-丁二醇的双水相萃取研究
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摘要
近三十年来,二元醇的生物转化在1,3-丙二醇(1,3-propanediol)和2,3-丁二醇(2,3-Butanediol)这两种高附加值的大宗化学品上取得了重大突破,并受到了广泛关注。但由于这两种二元醇的高沸点和强亲水性以及发酵液成分过于复杂,使该产品下游分离非常困难。尽管国内外进行了大量的研究,然而以高能耗的蒸馏为主体的传统分离方法依然无法被替代,致使1,3-丙二醇和2,3-丁二醇的分离成本超过了其总生产成本的30%,成为制约其工业化发展的主要瓶颈。针对上述问题,本论文研究了一种新型双水相体系用于萃取发酵液中的1,3-丙二醇和2,3-丁二醇,并考察了双水相中盐的回收工艺,最后考察了将双水相下相代替现有的氢氧化钠碱液来调节1,3-丙二醇和2,3-丁二醇发酵过程的pH值,从而回用于发酵的可行性。
首先,考察了加盐有机溶剂萃取、盐析分离和新型双水相萃取对发酵液中1,3-丙二醇和2,3-丁二醇的分离效果。发现由强极性萃取剂和高价阴离子盐组成的双水相体系效果最佳,其1,3-丙二醇和2,3-丁二醇的最高分配系数分别达到了11.4和31.9。综合考虑萃取效果和原料成本后,选定乙醇/硫酸铵、乙醇/碳酸钠和甲醇/磷酸氢二钾三种体系做进一步研究。
其次,考察了成相物质的浓度、pH值以及产物浓度等条件对双水相萃取发酵液中1,3-丙二醇和2,3-丁二醇的影响。结果表明,有机溶剂和盐的浓度对萃取的影响尤为关键。经过优化后,三个体系的产物分配系数均高于4.5,单次萃取回收率均高于90%。研究中还发现新型双水相在萃取产物的同时还可以将菌体和蛋白通过富集在两相之间的方式加以去除,在最佳萃取条件下,各体系对蛋白和菌体的去除率均高于79%和98%。
再次,研究了三种体系下相中硫酸铵、磷酸氢二钾和碳酸钠的回收工艺。通过采用甲醇溶析结晶、磷酸调节pH与甲醇溶析结晶相结合以及与二氧化碳反应沉淀的方法分别成功地回收了三种体系中的盐,回收率均高于92%。在通过ProⅡ软件的模拟与优化后,乙醇/碳酸钠因其较低的生产成本而被确定为最适合工业化的分离体系。
在此基础上,进一步研究了1,3-丙二醇发酵液的多级萃取,使产物的回收率和浓缩倍数分别提高到98%和3,并成功地将75%以上的有机酸富集在下相,极大地简化了分离工艺。
随后,研究了将双水相下相代替氢氧化钠溶液回用于发酵以调节发酵过程pH值的可行性,结果发现此工艺可将2,3-丁二醇发酵的产物浓度和转化率分别提高14.5%和2.5%；而对于1,3-丙二醇发酵,此工艺不但能使1,3-丙二醇的浓度增加16%,而且可将副产物乳酸的浓度和转化率分别提高126%和60%,而1,3-丙二醇、2,3-丁二醇、乙醇和乳酸等几种主要产物对甘油的转化率达到了94.1%。通过对代谢途径进行分析,发现碳酸盐在代谢中起到了较为重要的作用：可抑制甲酸和乙酸的代谢,导致丙酮酸积累,促进乳酸的形成。
最后通过ProⅡ和Aspen软件对双水相工艺与超滤-醇沉和汽提工艺的过程进行了模拟分析,结果表明双水相工艺可以节省大量的能耗,其最终生产成本比超滤-醇沉和汽提工艺降低了16.7%和17.0%。此外,双水相工艺可以通过降低能耗大幅减少二氧化碳的产生,并在除盐过程中吸收大量的二氧化碳,最终使二氧化碳排放量比超滤-醇沉和汽提工艺分别降低了75.6%和77.4%。综上所述,双水相工艺具有分离工艺简单、成本低、底物利用率高和环境友好等优点,具有较好的工业化应用前景。
In the past thirty years, microbial route for manufacturing diols has attracted great attention, with significant progress achieved in the fermentations of 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD), two important bulk chemicals. However, high boiling point and hydrophilicity as well as the complexity in the composition of the fermentation broths have restricted the development of efficient processes for their separation, and energy-intensive steam stripping and vacuum distillation are still dominated their industrial production. Currently, the cost associated with the downstream separation is as high as 30% of the total production cost. In this thesis, a novel aqueous two-phase system (ATPS) was developed for separating 1,3-PD and 2,3-BD from the fermentation broths. In addition, salt recovery was also studied for the ATPS system.
Compared with hydrophobic organic solvent/salt extraction and salting-out, ATPS composed of multivalent anion salts and strong polar solvents was validated to be more efficient for the separation of 1,3-PD and 2,3-BD from the fermentation broths, and their partition coefficients of 11.4 and 31.9, respectively for the ethanol/potassium phosphate system. Furthermore, the ethanol/ammonium sulfate, methanol/potassium dihydrogen phosphate and ethanol/sodium carbonate systems were investigated, due to low costs of solvents and salts for commercial applications.
The effects of salt, solvent and product concentrations and pH of the systems on the extraction of 1,3-PD and 2,3-BD were studied. It was found that the salt and solvent concentrations affected the extraction efficiency more significantly. Under optimum conditions, the partition coefficients and yields of the product recovery were over 4.5 and 90% for all three ATPSs. Meanwhile, most cells (79%) and proteins (99.5%) were concentrated at the interphase, which could be removed conveniently.
Salt recovery is the major concern for the ATPSs. Experimental results indicated that the salts in these three ATPSs could be successfully recovered through methanol dilution crystallization for the ethanol/ammonium sulfate system, methanol dilution crystallization after adjusting pH for the methanol/potassium dihydrogen phosphate system, and crystallization by reacting with CO2 for the ethanol/sodium carbonate system, and more than 92% salt was recovered. The economic analysis performed by the program ProⅡshowed that the ethanol/sodium carbonate system is more competitive, and thus it was recommended for commercial application.
The multi-stage extraction of 1,3-PD from the fermentation broth was also performed, and the yield and enriched factor was increased to 98% and 3, respectively. In addition, more than 75% organic acids were enriched in the bottom phase, which greatly simplified the separation process.
The bottom phase of the ethanol/sodium carbonate system was explored for adjusting the pH of the fermentation culture instead of sodium hydroxide, and experimental results indicated that the concentration and yield of 2,3-BD increased 24.8% and 6.5%, respectively, when the pH of the 2,3-BD fermentation was adjusted with the bottom phase. At the same time, the method not only increased 1,3-PD concentration by 16% when it was used in the 1, 3-PD fermentation, but also increased the concentration and yield of lactic acid (a by-product of great value) by 126% and 60%, respectively. The reason of this phenomenon was explored by the metabolic flux analysis, and it was found that carbonate was the main factor responsible for this change, since it controlled the metabolic fluxes of formic acid and acetic acid, and this in turn induced the accumulation of pyruvic acid, which promoted the generation of lactic acid.
At the end, the process simulations in the ATPS, the steam stripping and the combination of ultra filtration and alcohol precipitation were performed by Pro II and Aspen. The results showed that the production costs of 1,3-PD and 2,3-BD in the ATPS were increased from 16.7% and 17.0%, respectively, due to the drastic decrease in the energy expenditure. Furthermore, the carbon dioxide emission of 1,3-PD and 2,3-BD from ATPS can be decreased by 75.6% and 77.4%, due to its absorption during the process of salt recovery and lower generation as a result of lesser energy consumption. Thus, ATPS would be a potential forefront technology for industrial application as it offers high substrate utilization ratio, lower cost and benefit for environment protection.
首先,考察了加盐有机溶剂萃取、盐析分离和新型双水相萃取对发酵液中1,3-丙二醇和2,3-丁二醇的分离效果。发现由强极性萃取剂和高价阴离子盐组成的双水相体系效果最佳,其1,3-丙二醇和2,3-丁二醇的最高分配系数分别达到了11.4和31.9。综合考虑萃取效果和原料成本后,选定乙醇/硫酸铵、乙醇/碳酸钠和甲醇/磷酸氢二钾三种体系做进一步研究。
其次,考察了成相物质的浓度、pH值以及产物浓度等条件对双水相萃取发酵液中1,3-丙二醇和2,3-丁二醇的影响。结果表明,有机溶剂和盐的浓度对萃取的影响尤为关键。经过优化后,三个体系的产物分配系数均高于4.5,单次萃取回收率均高于90%。研究中还发现新型双水相在萃取产物的同时还可以将菌体和蛋白通过富集在两相之间的方式加以去除,在最佳萃取条件下,各体系对蛋白和菌体的去除率均高于79%和98%。
再次,研究了三种体系下相中硫酸铵、磷酸氢二钾和碳酸钠的回收工艺。通过采用甲醇溶析结晶、磷酸调节pH与甲醇溶析结晶相结合以及与二氧化碳反应沉淀的方法分别成功地回收了三种体系中的盐,回收率均高于92%。在通过ProⅡ软件的模拟与优化后,乙醇/碳酸钠因其较低的生产成本而被确定为最适合工业化的分离体系。
在此基础上,进一步研究了1,3-丙二醇发酵液的多级萃取,使产物的回收率和浓缩倍数分别提高到98%和3,并成功地将75%以上的有机酸富集在下相,极大地简化了分离工艺。
随后,研究了将双水相下相代替氢氧化钠溶液回用于发酵以调节发酵过程pH值的可行性,结果发现此工艺可将2,3-丁二醇发酵的产物浓度和转化率分别提高14.5%和2.5%；而对于1,3-丙二醇发酵,此工艺不但能使1,3-丙二醇的浓度增加16%,而且可将副产物乳酸的浓度和转化率分别提高126%和60%,而1,3-丙二醇、2,3-丁二醇、乙醇和乳酸等几种主要产物对甘油的转化率达到了94.1%。通过对代谢途径进行分析,发现碳酸盐在代谢中起到了较为重要的作用：可抑制甲酸和乙酸的代谢,导致丙酮酸积累,促进乳酸的形成。
最后通过ProⅡ和Aspen软件对双水相工艺与超滤-醇沉和汽提工艺的过程进行了模拟分析,结果表明双水相工艺可以节省大量的能耗,其最终生产成本比超滤-醇沉和汽提工艺降低了16.7%和17.0%。此外,双水相工艺可以通过降低能耗大幅减少二氧化碳的产生,并在除盐过程中吸收大量的二氧化碳,最终使二氧化碳排放量比超滤-醇沉和汽提工艺分别降低了75.6%和77.4%。综上所述,双水相工艺具有分离工艺简单、成本低、底物利用率高和环境友好等优点,具有较好的工业化应用前景。
In the past thirty years, microbial route for manufacturing diols has attracted great attention, with significant progress achieved in the fermentations of 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD), two important bulk chemicals. However, high boiling point and hydrophilicity as well as the complexity in the composition of the fermentation broths have restricted the development of efficient processes for their separation, and energy-intensive steam stripping and vacuum distillation are still dominated their industrial production. Currently, the cost associated with the downstream separation is as high as 30% of the total production cost. In this thesis, a novel aqueous two-phase system (ATPS) was developed for separating 1,3-PD and 2,3-BD from the fermentation broths. In addition, salt recovery was also studied for the ATPS system.
Compared with hydrophobic organic solvent/salt extraction and salting-out, ATPS composed of multivalent anion salts and strong polar solvents was validated to be more efficient for the separation of 1,3-PD and 2,3-BD from the fermentation broths, and their partition coefficients of 11.4 and 31.9, respectively for the ethanol/potassium phosphate system. Furthermore, the ethanol/ammonium sulfate, methanol/potassium dihydrogen phosphate and ethanol/sodium carbonate systems were investigated, due to low costs of solvents and salts for commercial applications.
The effects of salt, solvent and product concentrations and pH of the systems on the extraction of 1,3-PD and 2,3-BD were studied. It was found that the salt and solvent concentrations affected the extraction efficiency more significantly. Under optimum conditions, the partition coefficients and yields of the product recovery were over 4.5 and 90% for all three ATPSs. Meanwhile, most cells (79%) and proteins (99.5%) were concentrated at the interphase, which could be removed conveniently.
Salt recovery is the major concern for the ATPSs. Experimental results indicated that the salts in these three ATPSs could be successfully recovered through methanol dilution crystallization for the ethanol/ammonium sulfate system, methanol dilution crystallization after adjusting pH for the methanol/potassium dihydrogen phosphate system, and crystallization by reacting with CO2 for the ethanol/sodium carbonate system, and more than 92% salt was recovered. The economic analysis performed by the program ProⅡshowed that the ethanol/sodium carbonate system is more competitive, and thus it was recommended for commercial application.
The multi-stage extraction of 1,3-PD from the fermentation broth was also performed, and the yield and enriched factor was increased to 98% and 3, respectively. In addition, more than 75% organic acids were enriched in the bottom phase, which greatly simplified the separation process.
The bottom phase of the ethanol/sodium carbonate system was explored for adjusting the pH of the fermentation culture instead of sodium hydroxide, and experimental results indicated that the concentration and yield of 2,3-BD increased 24.8% and 6.5%, respectively, when the pH of the 2,3-BD fermentation was adjusted with the bottom phase. At the same time, the method not only increased 1,3-PD concentration by 16% when it was used in the 1, 3-PD fermentation, but also increased the concentration and yield of lactic acid (a by-product of great value) by 126% and 60%, respectively. The reason of this phenomenon was explored by the metabolic flux analysis, and it was found that carbonate was the main factor responsible for this change, since it controlled the metabolic fluxes of formic acid and acetic acid, and this in turn induced the accumulation of pyruvic acid, which promoted the generation of lactic acid.
At the end, the process simulations in the ATPS, the steam stripping and the combination of ultra filtration and alcohol precipitation were performed by Pro II and Aspen. The results showed that the production costs of 1,3-PD and 2,3-BD in the ATPS were increased from 16.7% and 17.0%, respectively, due to the drastic decrease in the energy expenditure. Furthermore, the carbon dioxide emission of 1,3-PD and 2,3-BD from ATPS can be decreased by 75.6% and 77.4%, due to its absorption during the process of salt recovery and lower generation as a result of lesser energy consumption. Thus, ATPS would be a potential forefront technology for industrial application as it offers high substrate utilization ratio, lower cost and benefit for environment protection.
引文
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