甘油生物转化联产乳酸及其盐析萃取
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摘要
1,3-内二醇(1,3-Propanediol,1,3-PD)和乳酸都是应用广泛并具高附加值的大宗生物基化学品,二者均可用于化妆品、药品等领域,且在新型材料领域应用尤为突出。1,3-丙二醇和乳酸分别是聚对苯二甲酸丙二酯(PTT)和聚乳酸的合成单体,市场应用前景广阔。目前,利用微生物发酵生产大宗化学品,大多追求的是单一产品,存在原料利用率低,副产物浪费等问题。本课题组前期研究发现,克雷伯氏杆菌(Klebsiella pneumoniae)代谢甘油不仅能主产1,3-丙二醇,还能副产2,3-丁二醇、乳酸、琥珀酸、乙酸、乙醇等,但甘油转化率不足50%。为提高原料利用率,降低生产成本,本论文通过采用碳酸钠控制发酵过程pH、增加发酵罐二氧化碳的分压(通过减少尾气排放二氧化碳方式)、流加琥珀酸以及以粗甘油为原料来调控克雷伯氏杆菌代谢甘油的过程,提高1,3-丙二醇和乳酸的总产率,实现1,3-丙二醇和乳酸联产。
首先,采用批式流加发酵方式,以氢氧化钠控制发酵过程pH作为对照,探索了碳酸钠控制发酵过程pH对克雷伯氏杆菌代谢甘油联产1,3-丙二醇和乳酸的克行性。实验结果表明,利用碳酸钠控制发酵过程pH,一方面促进菌体自身生长,另一方面利于1,3-丙二醇和乳酸的联产。其中,发酵过程中菌体OD值最高可达13.6,比对照组提高了25%；发酵33h后,1,3-丙二醇和乳酸浓度分别为79.48g/L,和41.97g/L,分别比对照组提高38%和28%;1,3-丙二醇和乳酸的质量转化率分别为41.46%和21.89%,二者总转化率为63.35%,比对照组提高4%。
其次,在碳酸钠控制发酵过程pH基础L,分别考察了采用增加发酵罐二氧化碳分压、流加琥珀酸以及以粗甘油为原料的策略来调控克雷伯氏杆菌代谢甘油的过程。实验结果表明,与仅利用碳酸钠控制发酵过程pH相比,增大罐内压力至4kPa(表压)时,菌体的OD值最高为9.6,1,3-丙二醇浓度和乳酸浓度分别为52.81g/L和56.02g/L,质量转化率分别为34.70%和36.85%,总转化率为71.55%；流加琥珀酸时,菌体OD值最高为10.54,1,3-丙二醇浓度和乳酸浓度分别为52.11g/L和54.85g/L,质量转化率分别为33.83%和35.62%,总转化率为69.45%；以粗甘油为发酵原料时,菌体OD值最高为9.61,1,3-丙二醇浓度和乳酸浓度分别为46.67g/L和56.13g/L,质量转化率分别为29.69%和35.71%,总转化率为65.40%。增加发酵罐二氧化碳分压、流加琥珀酸以及以粗甘油为原料不利于碳源流向菌体生长和1,3-丙二醇的生成通路,但有利于其流向乳酸的生成通路,提高了1,3-丙二醇和乳酸的总质量转化率。
最后,在课题组前期研究发现盐析萃取1,3-丙二醇效果显著的基础上,以乳酸发酵液为对象,探索了盐析萃取乳酸的可行性,以期为利用盐析萃取技术同时分离提取1,3-丙二醇和乳酸奠定基础。通过系统考察乳酸在不同盐析萃取体系中的分配规律,发现K2HPO4/甲醇和K2HPO4/乙醇体系适合分离发酵液中的乳酸。发酵液中乳酸浓度为167g/L时,采用25%(w/w)K2HPO4/26%(w/w)甲醇盐析萃取体系,乳酸的分配系数和回收率分别为4.01和86.0%；采用14%(w/w)K2HPO4/30%(w/w)乙醇盐析萃取体系,乳酸的分配系数和回收率分别为3.23和90.6%.此时上相中残余葡萄糖、菌体和可溶性蛋白的去除率分别达到67.3%、100%和85.9%。实验结果表明,盐析萃取同样适用于分离乳峻,为利用多步盐析萃取从Klebsiella pneumoniae代谢甘油联产1,3-丙二醇和乳酸的发酵液中有效分离目标产物提供了操作上的可能性。
Much attention has recently been paid to the bulk bio-based chemicals, including1,3-propanediol(1,3-PD) and lactic acid, which have tremendously applied in cosmetics, foods, medicines and especially in the production of novel polymers.1,3-Propanediol and lactic acid can be used as the monomer to synthesize polytrimethylene terephthalate (PTT) and polylactic acid (PLA), respectively, which will lead to prospective market. At present, pursuiting of the single product via microbial production is limited by low utilization efficiency of raw materials and the waste of by-products. In the previous research, the by-products in bioconversion of glycerol to1,3-PD by Klebsiellia pneumonia include2,3-butanediol, lactic acid, succinic acid, acetic acid and ethanol. The yield of1,3-PD on glycerol is less than50%. In order to increase the utilization ratio of raw material and reduce the production cost, this paper is focused on improving the yield of1,3-PD and lactic acid. The strategies, such as adjusting the pH of the fermentation process with sodium carbonate, increasing partial pressure of carbon dioxide in the fermentor, adding succinic acid and changing the raw materials, were used to achieve the combined production of1,3-propanediol and lactic acid with improving the total yield.
Firstly, fed-batch fermentations for the coproduction of1,3-PD and lactic acid by K. pneumoniea were carried out by adjusting the pH with sodium carbonate during fermentation, using sodium hydroxide as control. The results demonstrated that adjusting the pH with sodium carbonate was not only beneficial to promote cell growth, but also increased1,3-PD and lactic acid production. The highest optical density (OD) raised to13.6, which was25%higher than the control. The concentractions of1,3-propanediol and lactic acid were79.48g/L and41.97g/L at33h, increasing by38%and28%respectively, compared to the control. The yields of1,3-PD and lactic acid were up to41.46%and21.89%respectively. The total yield of1,3-PD and lactic acid reached63.35%increasing by4%compared to the control.
Secondly, based on adjusting the pH with sodium carbonate, the metabolic regulation of the conversion of glycerol to1,3-PD and lactic acid was investigated by increasing partial pressure of carbon dioxide in the fermentor, adding succinic acid and changing the raw materials. When the partial pressure of carbon dioxide in the fermentor was adjusted to4kPa (pressure in meter), the highest OD was9.6, the concentrations of1,3-PD and lactic acid were52.81g/L and56.02g/L respectively, the yields of1,3-PD and lactic acid were34.70%and 36.85%respectively and the total yield was71.55%. When succinic acid was added, the highest OD was10.54, the concentrations of1,3-propanediol and lactic acid were52.11g/L and54.85g/L respectively, the yields of1,3-PD and lactic acid were33.83%and35.62%respectively and the total yield was69.45%. When crude glycerin was used as raw material for fermentation, the highest OD was9.61, the concentrations of1,3-PD and lactic acid were46.67g/L and56.13g/L. The yields of1,3-PD and lactic acid were29.69%and35.71%respectively and the total yield was65.40%. The results revealed that designed strategies were beneficial to improve the total yield of1,3-PD and lactic acid, although against cell growth and1,3-PD accumulation.
Finally, based on the previous research obtained by salting-out extraction of1,3-PD from fermentation broths efficiently, the feasibility of salting-out extraction of lactic acid was studied to lay the foundation for the separation of the combined production of1,3-propanediol and lactic acid. Different systems composed of hydrophilic organic solvents/inorganic salts were investigated for the extraction ability of lactic acid. The results showed that the systems composed of dipotassium hydrogen phosphate/methanol and dipotassium hydrogen phosphate/ethanol appeared to be favorable. The partition coefficient and recovery of lactic acid reached up to4.01and86.0%for the system composed of25%(w/w) dipotassium hydrogen phosphate and26%(w/w) methanol, when the concentration of lactic acid is167g/L in the fermentation broth. For the system of14%(w/w) dipotassium hydrogen phosphate and30%(w/w) ethanol, the partition coefficient and recovery of lactic acid reached up to3.23and90.6%. The removal ratio of glucose, cells and proteins reached67.3%,100%and85.9%, respectively. The experimental results indicated that the salting-out extraction was also available for the separation of lactic acid, which can provided a possibility for multistep salting-out of1,3-PD and lactic acid from glycerol-based fermentation broths
首先,采用批式流加发酵方式,以氢氧化钠控制发酵过程pH作为对照,探索了碳酸钠控制发酵过程pH对克雷伯氏杆菌代谢甘油联产1,3-丙二醇和乳酸的克行性。实验结果表明,利用碳酸钠控制发酵过程pH,一方面促进菌体自身生长,另一方面利于1,3-丙二醇和乳酸的联产。其中,发酵过程中菌体OD值最高可达13.6,比对照组提高了25%；发酵33h后,1,3-丙二醇和乳酸浓度分别为79.48g/L,和41.97g/L,分别比对照组提高38%和28%;1,3-丙二醇和乳酸的质量转化率分别为41.46%和21.89%,二者总转化率为63.35%,比对照组提高4%。
其次,在碳酸钠控制发酵过程pH基础L,分别考察了采用增加发酵罐二氧化碳分压、流加琥珀酸以及以粗甘油为原料的策略来调控克雷伯氏杆菌代谢甘油的过程。实验结果表明,与仅利用碳酸钠控制发酵过程pH相比,增大罐内压力至4kPa(表压)时,菌体的OD值最高为9.6,1,3-丙二醇浓度和乳酸浓度分别为52.81g/L和56.02g/L,质量转化率分别为34.70%和36.85%,总转化率为71.55%；流加琥珀酸时,菌体OD值最高为10.54,1,3-丙二醇浓度和乳酸浓度分别为52.11g/L和54.85g/L,质量转化率分别为33.83%和35.62%,总转化率为69.45%；以粗甘油为发酵原料时,菌体OD值最高为9.61,1,3-丙二醇浓度和乳酸浓度分别为46.67g/L和56.13g/L,质量转化率分别为29.69%和35.71%,总转化率为65.40%。增加发酵罐二氧化碳分压、流加琥珀酸以及以粗甘油为原料不利于碳源流向菌体生长和1,3-丙二醇的生成通路,但有利于其流向乳酸的生成通路,提高了1,3-丙二醇和乳酸的总质量转化率。
最后,在课题组前期研究发现盐析萃取1,3-丙二醇效果显著的基础上,以乳酸发酵液为对象,探索了盐析萃取乳酸的可行性,以期为利用盐析萃取技术同时分离提取1,3-丙二醇和乳酸奠定基础。通过系统考察乳酸在不同盐析萃取体系中的分配规律,发现K2HPO4/甲醇和K2HPO4/乙醇体系适合分离发酵液中的乳酸。发酵液中乳酸浓度为167g/L时,采用25%(w/w)K2HPO4/26%(w/w)甲醇盐析萃取体系,乳酸的分配系数和回收率分别为4.01和86.0%；采用14%(w/w)K2HPO4/30%(w/w)乙醇盐析萃取体系,乳酸的分配系数和回收率分别为3.23和90.6%.此时上相中残余葡萄糖、菌体和可溶性蛋白的去除率分别达到67.3%、100%和85.9%。实验结果表明,盐析萃取同样适用于分离乳峻,为利用多步盐析萃取从Klebsiella pneumoniae代谢甘油联产1,3-丙二醇和乳酸的发酵液中有效分离目标产物提供了操作上的可能性。
Much attention has recently been paid to the bulk bio-based chemicals, including1,3-propanediol(1,3-PD) and lactic acid, which have tremendously applied in cosmetics, foods, medicines and especially in the production of novel polymers.1,3-Propanediol and lactic acid can be used as the monomer to synthesize polytrimethylene terephthalate (PTT) and polylactic acid (PLA), respectively, which will lead to prospective market. At present, pursuiting of the single product via microbial production is limited by low utilization efficiency of raw materials and the waste of by-products. In the previous research, the by-products in bioconversion of glycerol to1,3-PD by Klebsiellia pneumonia include2,3-butanediol, lactic acid, succinic acid, acetic acid and ethanol. The yield of1,3-PD on glycerol is less than50%. In order to increase the utilization ratio of raw material and reduce the production cost, this paper is focused on improving the yield of1,3-PD and lactic acid. The strategies, such as adjusting the pH of the fermentation process with sodium carbonate, increasing partial pressure of carbon dioxide in the fermentor, adding succinic acid and changing the raw materials, were used to achieve the combined production of1,3-propanediol and lactic acid with improving the total yield.
Firstly, fed-batch fermentations for the coproduction of1,3-PD and lactic acid by K. pneumoniea were carried out by adjusting the pH with sodium carbonate during fermentation, using sodium hydroxide as control. The results demonstrated that adjusting the pH with sodium carbonate was not only beneficial to promote cell growth, but also increased1,3-PD and lactic acid production. The highest optical density (OD) raised to13.6, which was25%higher than the control. The concentractions of1,3-propanediol and lactic acid were79.48g/L and41.97g/L at33h, increasing by38%and28%respectively, compared to the control. The yields of1,3-PD and lactic acid were up to41.46%and21.89%respectively. The total yield of1,3-PD and lactic acid reached63.35%increasing by4%compared to the control.
Secondly, based on adjusting the pH with sodium carbonate, the metabolic regulation of the conversion of glycerol to1,3-PD and lactic acid was investigated by increasing partial pressure of carbon dioxide in the fermentor, adding succinic acid and changing the raw materials. When the partial pressure of carbon dioxide in the fermentor was adjusted to4kPa (pressure in meter), the highest OD was9.6, the concentrations of1,3-PD and lactic acid were52.81g/L and56.02g/L respectively, the yields of1,3-PD and lactic acid were34.70%and 36.85%respectively and the total yield was71.55%. When succinic acid was added, the highest OD was10.54, the concentrations of1,3-propanediol and lactic acid were52.11g/L and54.85g/L respectively, the yields of1,3-PD and lactic acid were33.83%and35.62%respectively and the total yield was69.45%. When crude glycerin was used as raw material for fermentation, the highest OD was9.61, the concentrations of1,3-PD and lactic acid were46.67g/L and56.13g/L. The yields of1,3-PD and lactic acid were29.69%and35.71%respectively and the total yield was65.40%. The results revealed that designed strategies were beneficial to improve the total yield of1,3-PD and lactic acid, although against cell growth and1,3-PD accumulation.
Finally, based on the previous research obtained by salting-out extraction of1,3-PD from fermentation broths efficiently, the feasibility of salting-out extraction of lactic acid was studied to lay the foundation for the separation of the combined production of1,3-propanediol and lactic acid. Different systems composed of hydrophilic organic solvents/inorganic salts were investigated for the extraction ability of lactic acid. The results showed that the systems composed of dipotassium hydrogen phosphate/methanol and dipotassium hydrogen phosphate/ethanol appeared to be favorable. The partition coefficient and recovery of lactic acid reached up to4.01and86.0%for the system composed of25%(w/w) dipotassium hydrogen phosphate and26%(w/w) methanol, when the concentration of lactic acid is167g/L in the fermentation broth. For the system of14%(w/w) dipotassium hydrogen phosphate and30%(w/w) ethanol, the partition coefficient and recovery of lactic acid reached up to3.23and90.6%. The removal ratio of glucose, cells and proteins reached67.3%,100%and85.9%, respectively. The experimental results indicated that the salting-out extraction was also available for the separation of lactic acid, which can provided a possibility for multistep salting-out of1,3-PD and lactic acid from glycerol-based fermentation broths
引文
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