During the production of cytosolic choline by the fermentation method using choline chloride, potassium dihydrogen phosphate, and sodium dihydrogen phosphate as substrates, the high concentration of salt ions in the later stage leads to the decrease of bacterial viability or even the death of the bacteria, which is the main problem limiting the industrialization of cytosolic choline.To solve this problem, the effects of different substrates on cytosolic choline fermentation were first determined.Secondly, to eliminate the effects of Cl-, K+, and Na+ on fermentation, electrodialysis and ion-exchange resin were used to pre-treat the substrates and to investigate the separation efficiency.Results showed that ion-exchange resin was superior to electrodialysis in terms of efficiency and cost.Therefore, to realize the large-scale production of cytosolic choline, different strengths of ion exchange resins were investigated on this basis, and the optimum resin type, sample volume, sample concentration, eluent concentration, regeneration solution type, and stability of the resin were determined through experiments, which solved the high osmolality problem caused by high salt ions in the later stage, and made it possible to efficiently produce cytosolic choline with inexpensive raw materials.Finally, verified by a scale-up of a 5 L fermenter, the final cytosolic choline yield reached 32.1 g/L, and the sugar-acid conversion rate was 25.4%, which was the highest level of cytosolic choline production by fermentation method in China at present, taking the cytosolic choline yield, the biomass and the sugar-acid conversion rate as indicators.Compared with the direct use of choline phosphate as the substrate for the production of cytosine, the cost of cytosine was reduced by 85%, which also proved the feasibility of this experiment, providing some guiding significance for the large-scale industrialized production of cytosine.
[1] GIBELLINI F, SMITH T K.The Kennedy pathway: De novo synthesis of phosphatidylethanolamine and phosphatidylcholine[J].IUBMB Life, 2010, 62(6):414-428.
[2] SALVADORINI F, GALEONE F, NICOTERA M, et al.Clinical evaluation of CDP-choline (Nicholin):efficacy as antidepressant treatment[J].Current Therapeutic Research, Clinical and Experimental,1975, 18(3):513-520.
[3] TSUBAKI T, KASE M, ANDO K, et al.Therapeutic effects of Nicholin (CDP-choline):Parkinson's syndrome: A double-blind study[J].Nihon Rinsho.Japanese Journal of Clinical Medicine, 1974, 32(11):3435-3450.
[4] MIYAKE H, HAYAKAWA I, TAKAKURA K.Treatment of head injuries with intermediate substances of the metabolic cycle of the brain.1.the use of Cdp-choline[J].Roczniki Akademii Medycznej im.Juliana Marchlewskiego w Białymstoku, 1964, 16:873-878.
[5] WATANABE S, SHIROTA S, HANEDA K, et al.Effects of cultural conditions on CDP-choline production by hydrocarbon assimilating yeasts:Enzymatic studies on nucleic acid related compounds of microorganisms (IV)[J].Journal of Fermentation Technology, 1981, 59:191-195.
[6] 胡晨龙. 代谢工程改造大肠杆菌制备胞二磷胆碱[D].天津:天津科技大学, 2021.
HU C L.Metabolic engineering of Escherichia coli for CDP-choline production[D].Tianjin:Tianjin University of Science and Technology, 2021.
[7] 陈燕飞. 渗透压对细菌的影响[J].太原师范学院学报(自然科学版), 2012, 11(1):136-139.
CHEN Y F.Osmosis for the influence of bacteria[J].Journal of Taiyuan Normal University (Natural Science Edition), 2012, 11(1):136-139.
[8] 郭勤, 韩文艳, 李江, 等.氯离子对氧化亚铁硫杆菌和氧化亚铁钩端微螺菌活性的影响[J].有色金属(冶炼部分), 2015(1):42-45;53.
GUO Q, HAN W Y, LI J, et al.Effect of chloride ions on activity of Acidimicrobium ferrooxidans and Leptospirillum ferriphilum ferrous[J].Nonferrous Metals(Extractive Metallurgy), 2015(1):42-45;53.
[9] 张亚南, 马来波, 高春娟, 等.不同电流和电压对电渗析分离深层海水的影响[J].盐科学与化工, 2023, 52(1):26-29.
ZHANG Y N, MA L B, GAO C J, et al.Effect of different current and voltage on electrodialysis separation of deep sea water[J].Journal of Salt Science and Chemical Industry, 2023, 52(1):26-29.
[10] 熊海波. L-异亮氨酸发酵过程控制优化及分离提取工艺研究[D].天津:天津科技大学, 2021.
XIONG H B.Optimization of L-isoleucine fermentation process and study on separation and extraction technology[D].Tianjin:Tianjin University of Science and Technology, 2021.
[11] 易凤炎, 陈燕妮, 邱蔚然, 等.酿酒酵母生物合成胞磷胆碱的条件优化[J].工业微生物, 2016, 46(3):36-41.
YI F Y, CHEN Y N, QIU W R, et al.Optimization of fermentation conditions for citicoline biosynthesis by Saccharomyces cerevisiae[J].Industrial Microbiology, 2016, 46(3):36-41.
[12] 陈志超, 王金多, 徐庆阳.微量元素与生长因子对L-苯丙氨酸发酵的影响[J].食品与发酵工业, 2022, 48(8):82-89.
CHEN Z C, WANG J D, XU Q Y.Effects of trace elements and growth factors on L-phenylalanine fermentation[J].Food and Fermentation Industries, 2022, 48(8):82-89.
[13] 李澜潇, 王硕, 徐庆阳.金属离子及生长因子对核黄素发酵影响的研究[J].食品与发酵工业, 2023, 49(10):177-184.
LI L X, WANG S, XU Q Y.Effects of metal ions and growth factors on riboflavin fermentation[J].Food and Fermentation Industries, 2023, 49(10):177-184.
[14] 陈志超, 李澜潇, 徐庆阳.大肠杆菌发酵生产L-苯丙氨酸新工艺研究[J].中国调味品, 2022, 47(12):84-89.
CHEN Z C, LI L X, XU Q Y.Study on new technology of producing L-phenylalanine by fermentation of Escherichia coli[J].China Condiment, 2022, 47(12):84-89.
[15] 王娇, 代新英, 赵博雅, 等.离子交换树脂脱除氢氧化胆碱中Sb2+、Fe3+的研究[J].应用化工, 2023,52(5):1410-1413;1419.
WANG J, DAI X Y, ZHAO B Y, et al.Study on the removal of Sb2+ and Fe3+ in choline hydroxide by ion exchange resin[J].Applied Chemical Industry, 2023, 52(5):1410-1413;1419.
[16] 郭继鸿. 离子泵(210)[J].临床心电学杂志, 2022, 31(1):75.
GUO J H.Ion pump(210)[J].Journal of Clinical Electrocardiology, 2022, 31(1):75.
[17] 李世霖, 胡景泽, 王毅霖, 等.电渗析分离提取高值组分的研究进展[J].化工进展, 2023, 42(S1):420-429.
LI S L, HU J Z, WANG Y L, et al.Research progress in separation and extraction of high value components by electrodialysis[J].Chemical Industry and Engineering Progress, 2023, 42(S1):420-429.
[18] 白亚磊, 冯宁, 孙家凯, 等.发酵液中L-异亮氨酸提取工艺研究[J].发酵科技通讯, 2010, 39(4):13-15.
BAI Y L, FENG N, SUN J K, et al.Study on the extraction technology of L-isoleucine from fermentation broth[J].Bulletin of Fermentation Science and Technology, 2010, 39(4):13-15.
[19] 高健. 探讨离子交换树脂在水处理中的应用[J].皮革制作与环保科技, 2022, 3(2):10-12.
GAO J.Discuss the application of ion exchange resin in water treatment[J].Leather Manufacture and Environmental Technology, 2022, 3(2):10-12.
[20] 吴欣森, 杨晔, 张杨, 等.离子交换树脂脱除高盐废水中Ca2+的特性研究[J].中国胶粘剂, 2023, 32(1):39-43;54.
WU X S, YANG Y, ZHANG Y, et al.Study on characteristics of removing Ca2+from high-salt wastewater by ion exchange resin[J].China Adhesives, 2023, 32(1):39-43;54.
[21] 李雅, 张盈, 王美娇, 等.离子交换树脂除废水中Ni技术研究[J].有色金属(冶炼部分), 2021(10):121-125.
LI Y, ZHANG Y, WANG M J, et al.Study on removal of Ni in wastewater by ion exchange resin[J].Nonferrous Metals(Extractive Metallurgy), 2021(10):121-125.
[22] 许航, 彭人民.奥氏体不锈钢发酵罐应力腐蚀裂纹探讨[J].机电技术, 2003, 26(2):36-39.
XU H, PENG R M.Discussion on stress corrosion cracks in austenitic stainless steel fermentation tank[J].Mechanical & Electrical Technology, 2003, 26(2):36-39.
[23] 王卉. 氯离子对肝细胞癌细胞生物学行为的影响及机制的研究[D].遵义:遵义医科大学, 2022.
WANG H.Study on the effect of chloride ion on biological behavior of hepatocellular carcinoma cells and its mechanisms[D].Zunyi:Zunyi Medical University, 2022.
[24] 韩彦飞. 三氯化铝对体外培养大鼠成骨细胞的毒性作用[D].哈尔滨:东北农业大学,2013.
HAN Y F.Toxicity of AlCl3 on osteoblast of rats cultured in vitro[D].Harbin:Northeast Agricultural University, 2013.
[25] 李晓丹, 夏冰, 汪仁.酿酒酵母磷酸胆碱胞苷转移酶基因(cct)的克隆表达及活性测定[J].药物生物技术, 2014, 21(2):99-102.
LI X D, XIA B, WANG R.Cloning, expression of cholinephosphate cytidylyltransferase of Saccharomyces cerevisiae in Escherichia coli and evaluation of the expressed product[J].Chinese Journal of Pharmaceutical Biotechnology, 2014, 21(2):99-102.
