海洋克雷伯氏菌Klebsiella pneumoniae HSL4是一株分离于海洋沉积物环境的用于发酵生产1,3-丙二醇(1,3-propanediol, 1,3-PDO)的优良菌株。K. pneumoniae HSL4发酵生产1,3-PDO的培养基包含多种营养因子, 其中磷酸根对发酵过程影响较大。利用5L发酵罐进行补料发酵实验, 分别考察培养基中低磷(1.6×10-4mol·L-1 PO43-)和高磷(5.0×10-3mol·L-1 PO43-)浓度条件下K. pneumoniae HSL4发酵生产1,3-PDO过程中细胞生长、1,3-PDO及多种副产物生成的规律, 分析初始磷酸根浓度差异对K. pneumoniae HSL4不同发酵阶段代谢流分布的影响。结果表明, 培养基中高、低初始磷酸根浓度条件下, 1,3-PDO的终浓度均在75g·L-1左右。在低磷条件下, 底物甘油消耗量较少, 具有较高的1,3-PDO转化率; 除2,3-丁二醇外, 其他副产物在发酵液中的积累均小于高磷条件下副产物的积累。高磷条件促进乙酸、乙醇、乳酸等副产物生成, 改变中、后期发酵环境, 增加下游分离提取1,3-PDO的压力。根据高、低初始磷酸根浓度条件下菌体生物量、产物、副产物合成的差异, 对细菌4个发酵时期(延滞期、对数生长期、平台期和衰亡期)的代谢流分布及其变化进行了分析。研究结果可以为合理配比培养基中磷酸盐浓度、进行发酵生产1,3-PDO过程控制与代谢调控提供理论依据。
李莉莉
,
周胜
,
高品
,
郭传宇
,
俞也频
,
尼松伟
,
李鹏飞
,
秦启伟
. 磷酸根对海洋克雷伯氏菌合成1,3-丙二醇代谢的影响[J]. 热带海洋学报, 2015
, 34(5)
: 27
-35
.
DOI: 10.11978/2014136
Klebsiella pneumoniae HSL4 is an excellent strain for the fermentation of 1,3-propanediol (1,3-PDO), which was isolated from coastal mangrove sediment environment. Many nutrient factors are involved in the culture medium of K. pneumoniae HSL4. Among them, phosphate is one of the main factors, which have a significant impact on the fermentation. The effects of the low concentration of phosphate (LP: 1.6×10-4mol·L-1 PO43-) and the high concentration phosphate (HP: 5.0×10-3mol·L-1 PO43-) on cell growth, 1,3-PDO and by-product (acetic succinic acid, lactic acid, acid, 2,3-butanediol, and ethanol) production were studied. The impacts of phosphate on the metabolism of K. pneumoniae HSL4 were characterized by metabolic flux analysis. The results showed that the final concentration of 1,3-PDO was 75g·L-1 under both LP and HP in the culture medium. Significantly, the 1,3-PDO conversion was higher, but the substrate glycerol consumption and by-products (except for 2,3-butanediol) accumulation were less under LP condition compared to HP condition. The productions of acetic acid and ethanol were higher under HP condition. The accumulations of acetic acid and ethanol will change the environment of fermentation and increase the difficulty of product separation in the downstream. Metabolic flux distributions in four fermentation periods (adjustmental phase, log phase, stationary phase, and decline phase) were analyzed. This study will help researchers to choose reasonable phosphate concentration in the culture broth and provide information for process control and metabolic regulation in 1,3-PDO fermentation by K. pneumoniae HSL4.
[1] 丛春水, 苏志国,登继先. 1998. 重组大肠杆菌的分批补料培养方法[J]. 微生物学杂志,18 (4): 44-47.
[2] 管桂萍, 王红兵, 田杰生. 2008. 琥珀酸对产酸克雷伯氏菌好氧发酵甘油产1,3-丙二醇的影响[J]. 食品与发酵工程, 34(5): 14-17.
[3] 金平, 张建刚, 佟明友, 等. 2004. 甘油发酵制取1,3-丙二醇菌株筛选[J]. 精细与专用化学品, 12 (16): 13-15.
[4] 闵恩泽．2006. 利用可再生农林生物质资源的炼油厂[J]．化学进展, 18(2/3): 132-141.
[5] 孙家凯, 吴晓娇, 王晶等. 2012. 磷酸盐对大肠杆菌发酵异亮氨酸的影响[J]. 食品与发酵工业, 38(1): 20-24.
[6] 谢家明, 徐泽辉, 夏蓉晖, 等. 2005. 1,3-丙二醇制备工艺的研究进展[J]．合成纤维, 34(2): 13-16.
[7] 张延平, 刘铭, 杜晨宇, 等. 2006. 代谢副产物对 Klebsiella pneumoniae 生长及合成1,3-丙二醇的影响[J]. 过程工程学报, 6(5): 804-808.
[8] 赵春光, 谢希贤, 徐庆阳, 等. 2009. 磷酸盐对 Escherichia coli TRJH L-色氨酸发酵代谢流分布的影响[J]. 天津科技大学学报, 24(5): 10-13.
[9] 中华人民共和国国家质量监督检验检疫总局. 2008. GB/T 9727-2007. 中华人民共和国国家标准—化学试剂 磷酸盐测定通用方法 [S]. 北京: 中国标准出版社: 1-2.
[10] 周胜, 秦启伟, 魏京广, 等. 2012-07-25 [2013-03-27]. 一种红树林克雷伯氏菌及其在发酵法生产1,3-丙二醇中的应用: 中国: ZL201210053231.X[P].
[11] BARBIRATO F, CLARET C C, GRIVET J P, et al. 1995. Glycerol fermentation by a new 1,3-propanediol-producing microorganism: Enterobacter agglomerans [J]. Appl Microbiol Biotechnol, 43: 786-793.
[12] BIEBL H. 2001. Fermentation of glycerol by Clostridium pasteurianum : Batch and continue culture studies [J]. J Ind Microbiol Biot, 27(1): 18-26.
[13] BOENIGK R, BOWIEN S, GOTTSEHALK G. 1993. Fermentation of glycerol to l,3-propanediol in continuous cultures of Citrobacter freundi [J]. Appl Microbiol Biotechnol, 38: 453-457.
[14] CELINSKA E, GRAJEK W. 2009. Biotechnological production of 2,3-butanediol-Current state and prospects[J]. Biotechnol Adv, 27(6): 715-25.
[15] GEN L, MA B B, XU X L, et al. 2007. Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae [J]. Biochem Eng J, 37(3): 256-260.
[16] HAN K. 1991. Acetate acid formation in Escherichia coli fermantation[J]. Biotechnol Bioeng, 39: 663-671.
[17] LI L L, ZHOU S, JI H S, et al. 2014. Optimization of fermentation conditions for 1,3-propanediol production by marine Klebsiella pneumonia HSL4 using response surface methodology[J]. Chin J Oceanol Limnol, 32(5): 1036-1045.
[18] MAR A G P, ISABELLE M S, FILIPA M, et al. 2006. Microbial conversion of glycerol to 1,3-propanediol: Physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1 (pSPD5)[J]. Appl Microbiol Biotechnol, 72(1): 96-101.
[19] PFLEGER D, GOMES S, GILBERT N, et al. 1999. Hydrodynamic simulations of laboratory scale bubble columns fundamental studies of the Eulerian-Eulerian modelling approach[J]. Chem Eng Sci, 54(21): 5091-5099.
[20] STREEKSTRA H, TEIXEIRA DE MATTOS M J, NEIJSSEL O M, et al. 1987. Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostat culture[J]. Arch Microbiol, 147: 268-275.
[21] WITT U, MUELLER R J, AUGUSTA J, et al. 1994. Synthesis, properties and biodegradability of polyesters based on 1,3-propanediol[J]. Macromolecular Chemistry and Physics, 195(2): 793-802.
[22] XU YZ, GUO N N, ZHENG Z M, et al. 2009. Metabolism in 1,3-propanediol fed-batch fermentation by a D-lactate deficient mutant of Klebsiella pneumoniae [J]. Biotechnol Bioeng, 104: 965-972.
[23] ZENG A P, ROSS A, BIEBL H, et al. 1994. Multiple product inhibition and growth modeling of Clostridium butyricum and Klebsiella pneumoniae in glycerol fermentation[J]. Biotechnol Bioeng 44: 902-911.
[24] ZENG A P, BIEBL H. 2002. Bulk chemicals from biotechnology: The ease of l,3-propanediol production and the new trends[J]. Adv Biochem Eng Biotechnol, 74: 239-259.
[25] ZENG A P, SABRA W. 2011. Microbial production of diols as platform chemicals: Recent progresses[J]. Curr Opin Biotechnol, 22: 749-757.
[26] ZHENG Z M, XU Y Z, LIU H J, et al. 2008. Physiologic mechanisms of sequential products synthesis in 1,3- propanediol fed-batch fermentation by Klebsiella pneumoniae [J]. Biotechnol Bioeng, 100(5): 923-932.
[27] ZHOU S, LI L L, WEI J G, et al. 2013. Genome sequence of Klebsiella pneumoniae HSL4 a new strain isolated from mangrove sediment for biosynthesis of 1,3-propanediol[J]. Genome Announ, 1(3): e00177-13. doi: 10.1128/genomeA. 00177-13.
