L-谷氨酸产生菌的选育及其代谢调控的研究
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摘要
本文根据代谢工程的基本原理,对谷氨酸棒杆菌(Corynebacterium glutamicum)TWQ080进行逐级诱变处理,最终选育出一株谷氨酸产生菌GW46-4。然后对其摇瓶发酵条件及7L罐发酵条件进行了优化,最后又对出发菌株和突变菌株的代谢流量进行了分析。主要研究内容和结论如下：
(1)以本实验室保藏菌株谷氨酸棒杆菌TWQ080为出发菌株,经过紫外线(UV)和硫酸二乙酯(DES)逐级诱变处理,以高浓度糖、高浓度味精和琥珀酸为唯一碳源的平板进行定向筛选,最终选育出一株谷氨酸产生菌GW46。然后对该菌株进行了耐高温驯化,驯化过程中不断提高培养温度,经过多次驯化,育得一耐高温菌株GW46-4,在未经优化的条件下产酸92.5g/L,比出发菌株提高了31.2%。
(2)对菌株GW46-4的种子培养基及培养条件进行了优化,发现最佳种子培养基配方：葡萄糖25g/L、玉米浆30g/L、磷酸氢二钾1.5g/L、尿素6g/L、硫酸镁0.4g/L；最佳培养条件：初始pH 7.0,装液量30mL/250mL。然后对其发酵培养基及培养条件进行了优化,发现最佳发酵培养基配方：葡萄糖158.3g/L、玉米浆3.2g/L、磷酸氢二钾1.5g/L、硫酸镁0.8g/L、硫酸锰20mg/L、硫酸亚铁20mg/L、VB1200μg/L；最佳发酵条件：装液量20mL/500mL,接种量8%,种龄7h。在此条件下发酵产酸达102.6g/L,比未优化前提高了10.9%。
(3)对菌株GW46-4补料分批发酵条件进行了初步研究,结果发现：最佳初糖浓度100g/L,最佳残糖维持浓度10-20g/L。对菌株GW46-4进行了7L罐发酵条件研究,考察了不同溶氧控制策略和温度控制策略对发酵的影响。结果表明,当溶氧控制在30-40%时效果较好；当温度0-8h控制在32℃,8-16h控制在34℃,16-30h控制在36℃时效果最佳。在优化条件下,经7L发酵罐发酵,产酸达114.2g/L,比未优化前提高了23.5%,转化率达59.4%。
(4)建立了L-谷氨酸生物合成的代谢网络模型,对出发菌株和突变菌株的代谢流量分布情况进行了研究,并对其主要节点进行了分析。经丙酮酸节点分析发现,突变菌株较出发菌株CO2固定反应得到加强,而乙醛酸循环相对减弱。经异柠檬酸节点分析发现,突变株合成谷氨酸的流量增加。与出发菌株相比,谷氨酸作为前体物质合成其它氨基酸的流量也相应的减少。
The original strain Corynebacterium glutamicum TWQ080 were treated gradually by mutagen, and a L-glutamic acid producer GW46-4 was obtained based on metabolic engineering principle. Then, fermentation conditions in flask and fermentor were studied, and the metabolic flux distribution of original strain and mutant were also studied. The research and its main results are as follows:
(1) The original strain Corynebacterium glutamicum TWQ080 was treated gradually by UV and DES and then was screened by high concentrations of glucose and glutamate.As a result, a L-glutamic acid producing strain GW46 was obtained. The thermotolerant property of Corynebacterium glutamicum was improved gradually via a passage transfer approach. After several times of domestication, a thermotolerant strain GW46-4 was obtained. An accumulation of 92.5g/L glutamic acid was obtained in the original culture condition, which was 31.2% higher than the original strain.
(2) The seed medium and culture conditions were studied. It was found that the optimal medium components were glucose 25g/L,corn steep liquor 30g/L, K2HPO4·3H2O 1.5g/L, urea 6g/L, MgSO4·7H2O 0.4g/L and the optimal culturing conditions were initial pH7.0, medium volume 30mL/250mL. The fermentation medium and conditions in shaker flask were studied. The optimal medium components was glucose 158.3g/L, corn steep liquor 3.2g/L, K2HPO4·3H2O 1.5g/L, MgSO4·7H2O 0.8g/L, MnSO4·4H2O 20mg/L, FeSO4·7H2O 20mg/L, VB1200μg/L. The optimal fermentation conditions were medium volume 20mL/500mL, inoculated volume 8%, seed age 7h. L-glutamic acid concentration reached 102.6g/L in flask, which was 10.9% higher than before optimization.
(3) The fed-batch fermentation was studied. It was found that the optimal initial glucose was 100g/L and the residual glucose concentration was 10-20g/L. The fermentation in fermentor was also studied and found the optimal DO control strategy was controlled at 30-40%. The optimal temperature control mode was 0-8h at 32℃,8-16h at 34℃,16-30h at 36℃. In 7L fermentor, L-glutamic acid concentration was 114.2g/L, which was 23.5% higher than before optimization. In addition, the conversion rate was 59.4%.
(4) The metabolic network of Corynebacterium glutamicum was established and the metabolic flux distribution of the original strain and mutant were studied. It was found that the CO2 fixed equation was strengthened and the glyoxy cycle was decreased. The flux to glutamic acid from the node of isocitrate was increased. Compared with the original strain, the flow to synthesis of other amino acids by using glatamic acid as precursor was decreased.
(1)以本实验室保藏菌株谷氨酸棒杆菌TWQ080为出发菌株,经过紫外线(UV)和硫酸二乙酯(DES)逐级诱变处理,以高浓度糖、高浓度味精和琥珀酸为唯一碳源的平板进行定向筛选,最终选育出一株谷氨酸产生菌GW46。然后对该菌株进行了耐高温驯化,驯化过程中不断提高培养温度,经过多次驯化,育得一耐高温菌株GW46-4,在未经优化的条件下产酸92.5g/L,比出发菌株提高了31.2%。
(2)对菌株GW46-4的种子培养基及培养条件进行了优化,发现最佳种子培养基配方：葡萄糖25g/L、玉米浆30g/L、磷酸氢二钾1.5g/L、尿素6g/L、硫酸镁0.4g/L；最佳培养条件：初始pH 7.0,装液量30mL/250mL。然后对其发酵培养基及培养条件进行了优化,发现最佳发酵培养基配方：葡萄糖158.3g/L、玉米浆3.2g/L、磷酸氢二钾1.5g/L、硫酸镁0.8g/L、硫酸锰20mg/L、硫酸亚铁20mg/L、VB1200μg/L；最佳发酵条件：装液量20mL/500mL,接种量8%,种龄7h。在此条件下发酵产酸达102.6g/L,比未优化前提高了10.9%。
(3)对菌株GW46-4补料分批发酵条件进行了初步研究,结果发现：最佳初糖浓度100g/L,最佳残糖维持浓度10-20g/L。对菌株GW46-4进行了7L罐发酵条件研究,考察了不同溶氧控制策略和温度控制策略对发酵的影响。结果表明,当溶氧控制在30-40%时效果较好；当温度0-8h控制在32℃,8-16h控制在34℃,16-30h控制在36℃时效果最佳。在优化条件下,经7L发酵罐发酵,产酸达114.2g/L,比未优化前提高了23.5%,转化率达59.4%。
(4)建立了L-谷氨酸生物合成的代谢网络模型,对出发菌株和突变菌株的代谢流量分布情况进行了研究,并对其主要节点进行了分析。经丙酮酸节点分析发现,突变菌株较出发菌株CO2固定反应得到加强,而乙醛酸循环相对减弱。经异柠檬酸节点分析发现,突变株合成谷氨酸的流量增加。与出发菌株相比,谷氨酸作为前体物质合成其它氨基酸的流量也相应的减少。
The original strain Corynebacterium glutamicum TWQ080 were treated gradually by mutagen, and a L-glutamic acid producer GW46-4 was obtained based on metabolic engineering principle. Then, fermentation conditions in flask and fermentor were studied, and the metabolic flux distribution of original strain and mutant were also studied. The research and its main results are as follows:
(1) The original strain Corynebacterium glutamicum TWQ080 was treated gradually by UV and DES and then was screened by high concentrations of glucose and glutamate.As a result, a L-glutamic acid producing strain GW46 was obtained. The thermotolerant property of Corynebacterium glutamicum was improved gradually via a passage transfer approach. After several times of domestication, a thermotolerant strain GW46-4 was obtained. An accumulation of 92.5g/L glutamic acid was obtained in the original culture condition, which was 31.2% higher than the original strain.
(2) The seed medium and culture conditions were studied. It was found that the optimal medium components were glucose 25g/L,corn steep liquor 30g/L, K2HPO4·3H2O 1.5g/L, urea 6g/L, MgSO4·7H2O 0.4g/L and the optimal culturing conditions were initial pH7.0, medium volume 30mL/250mL. The fermentation medium and conditions in shaker flask were studied. The optimal medium components was glucose 158.3g/L, corn steep liquor 3.2g/L, K2HPO4·3H2O 1.5g/L, MgSO4·7H2O 0.8g/L, MnSO4·4H2O 20mg/L, FeSO4·7H2O 20mg/L, VB1200μg/L. The optimal fermentation conditions were medium volume 20mL/500mL, inoculated volume 8%, seed age 7h. L-glutamic acid concentration reached 102.6g/L in flask, which was 10.9% higher than before optimization.
(3) The fed-batch fermentation was studied. It was found that the optimal initial glucose was 100g/L and the residual glucose concentration was 10-20g/L. The fermentation in fermentor was also studied and found the optimal DO control strategy was controlled at 30-40%. The optimal temperature control mode was 0-8h at 32℃,8-16h at 34℃,16-30h at 36℃. In 7L fermentor, L-glutamic acid concentration was 114.2g/L, which was 23.5% higher than before optimization. In addition, the conversion rate was 59.4%.
(4) The metabolic network of Corynebacterium glutamicum was established and the metabolic flux distribution of the original strain and mutant were studied. It was found that the CO2 fixed equation was strengthened and the glyoxy cycle was decreased. The flux to glutamic acid from the node of isocitrate was increased. Compared with the original strain, the flow to synthesis of other amino acids by using glatamic acid as precursor was decreased.
引文
1.Lothar Eggeling, Michael Bott. Handbook of Corynebacterium glutamicum[M], USA, CRC Press,2005.3-5.
2.张克旭.氨基酸发酵工艺学[M],北京,中国轻工业出版社,1995.2-3.
3.王宏龄,富春江.国内外主要发酵类氨基酸产品发展现状[J].精细与专用化学品,2007,15(24)：1-5.
4.王家勤.发酵法谷氨酸生产味精的发展综述.全国氨基酸生产技术交流会,2005.5-10.
5.于信令.味精工业手册[M],北京,中国轻工业出版社,1995.282-287.
6.王天贵,王诗瑜,张引沁.葡萄糖发酵制取谷氨酸的研究进展[J].平原大学学报,2000,17(3)：89-90.
7.胡学智,沈天益.617短杆菌发酵生产谷氨酸的研究历程[J].江苏调味副食品,2006,23(3)：4-6.
8.李华玮,苏庆辉,李志江等.谷氨酸生产行业现状综合分析[J].农产品加工学刊,2005,(8)：65-67.
9. Eiichiro Kimura. Metabolic Engineering of glutamate production. Advances in Biochemical Engineering/Biotechnology,2003,79(1):37-57.
10.余秉琦,沈微,王正祥等.谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成[J].生物工程学报,2005,21(2)：270-274.
11. Eikmanns BJ, Thum-Schmitz N, Eggeling L, et al. Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology,1994,140(8):1817-1828.
12. Christoph Wittmann. Analysis and engineering of metabolic pathway fluxes in Coryne-bacterium glutamicum. Adv Biochem Engin/Biotechnol,2010,120(2):21-49.
13.杜军,徐庆阳,谢希贤等.谷氨酸高产菌GDK-9的定向选育及其发酵过程研究[J].食品与发酵工业,2008,34(7)：17-19.
14.甄卫军,张石峰,曾宪贤.高产谷氨酸菌选育研究进展[J].新疆大学学报(自然科学版),2002,19(2)：209-211.
15.张克旭,陈宁,张蓓.代谢控制发酵[M],北京,中国轻工业出版社,1998.54-55.
16.张克旭.关于谷氨酸高产菌WTH-1的选育[J].食品与发酵工业,1985,(5)：36-39.
17.郑善良.L-谷氨酸一次性高糖发酵产生菌FM84-415的选育及其特性的研究[J].氨基酸和生物资源,1986,4(2)：15-19.
18.云逢霖.高产谷氨酸菌S9114的选育及生产[J].发酵科技通讯,1993,22(1)：3-7.
19.毛富根.耐高糖谷氨酸产生菌Ls168的激光育种[J].发酵科技通讯,1995,24(1)：5-9.
20.王岁楼,张一震.谷氨酸高产菌TZ2310的诱变选育及其发酵的研究[J].中国调味品,1997,(9)：16-18.
21.袁品坦.谷氨酸菌种选育的进展及其回顾[J].发酵科技通讯,2000,39(1):15-20.
22.张克旭,陈宁,张永志等.用原生质体融合技术选育谷氨酸高产菌[J].微生物学报,1991,(2)：108-114.
23.张蓓,张克旭,陈宁.谷氨酸高产菌FTN9108的细胞融合育种及发酵条件的研究[J].食品与发酵工业,1992,(5)：21-25.
24.陈宁,张克旭.谷氨酸温度敏感型菌株CN1021的原生质体融合育种[J].发酵科技通讯,2003,32(3)：11-14.
25.于信令,于军.对中国味精工业今后发展趋势的研讨[J].发酵科技通讯,2005,34(1)：12-14.
26.秦海斌.L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D]：[硕士学位论文].江南大学生物工程学院,2009.
27.诸葛健,李华忠.微生物学[M].北京：科学出版社.2005,316-321.
28.诸葛健,王正祥.工业微生物实验技术手册[M].北京：中国轻工业出版社,1994,391-392.
29.张克旭,陈宁,张蓓等.代谢控制发酵[M],北京,中国轻工业出版社,1998.57-61.
30. KiBeom Lee. Enhanced production of lactid acid by an adapted strain of Lactobaclllusdel-brueckii subsp. Lactis[J]. World J Microbiol Biotechnol,2007,23 (2):1317-1320.
31.杨森莲,张克旭,陈宁.谷氨酸温度敏感突变株的驯化及发酵条件研究[J].中国酿造,2006,(7)：18-20.
32.张克旭.谷氨酸的代谢控制发酵育种[J].发酵科技通讯,2004,33(2)：11-13.
33.马立安,贺飞英.耐高温柠檬酸菌种驯化及筛选[J].中国酿造,2007,(11)：57-58.
34.赵宇星,周惠明,钱海峰.酸豆乳生产菌种的驯化研究[J].食品科技,2004,(11)：28-30.
35.张克旭.氨基酸发酵工艺学[M],北京,中国轻工业出版社,1995.126.
36.邓毛程,王瑶,阳元娥.高初糖谷氨酸发酵的研究[J].广东轻工职业技术学院学报,2004,3(2)：12-14.
37.张伟国,钱和.氨基酸生产技术及其应用[M],北京,中国轻工业出版社,1997.27.
38.陈碧娥.谷氨酸生产菌培养基最优配比设计[J].华侨大学学报(自然科学版),2001,22(2)：199-201.
39. Mohamed Hajji, Ahmed Rebai, Neji Gharsallah, et al. Optimization of alkaline protease production by Aspergillus clavatus ESI in Mirabilis jalapa tuber powder using statistical experimental design [J].Appl Microbiol Biotechnol,2008,79 (6):915-923.
40.卞芙蓉,劳兴珍,郑绗等.酵母发酵生产谷胱甘肽的培养基优化[J].中国生化药物杂志,2009,30(3)：184-186.
41.毕静莹,卢丽娟,秦义等.Plackett-Burman设计法筛选影响灵芝富锌能力的主要培养基组分[J].安徽农业科学,2010,38(10)：5335-5337.
42. Lei Yu, Ting Lei, Xiao-lin Pei. Rapid screen of medium components for L(+)lactic acid production by Lactobacillus rhamnosus[J]. Food Science,2007,28(11):382-385.
43.刘春辉.L-谷氨酰胺产生菌的选育及其代谢调控的初步研究[D]：[硕士学位论文].江南大学生物工程学院,2009.
44.陈银芳.L-赖氨酸产生菌的选育及其发酵条件的调控[D]：[硕士学位论文].江南大学生物工程学院,2009.
45.冯容保.发酵谷氨酸生产新工艺对比[J].发酵科技通讯,2003,32(1)：15-16.
46.于信令,于军.氨基酸发酵生产的调控优化[J].发酵科技通讯,2006,35(1)：28-30.
47.李艳.发酵工程原理与技术[M],北京,高等教育出版社,2006.221-222.
48.马占龙.谷氨酸发酵过程的控制[J].发酵科技通讯,2006,35(3)：1-2.
49.邓毛程,吴亚丽,梁世中等.高生物素谷氨酸发酵中溶氧水平与生物素用量的研究[J].食品与机械,2008,24(3)：10-13.
50.王营,董亮,张雁铃等.谷氨酸发酵标准溶解氧水平的确定[J].食品与发酵工业,2005,31(9)：42-44.
51.赵克勤,刘毅,邹祖然.谷氨酸发酵流加糖工艺的生产研究[J].食品工业科技,2005,26(3)：124-125.
52. Shintaro Iwatani,Yohei Yamada,Yoshihiro Usuda.Metabolic flux analysis in biotechnology processes. Biotechnol Lett,2008,30(5):791-799.
53.杜军,陈宁,刘辉等.基于代谢流量分析的L-谷氨酸发酵过程优化[J].中国食品学报,2008,8(2)：30-34.
2.张克旭.氨基酸发酵工艺学[M],北京,中国轻工业出版社,1995.2-3.
3.王宏龄,富春江.国内外主要发酵类氨基酸产品发展现状[J].精细与专用化学品,2007,15(24)：1-5.
4.王家勤.发酵法谷氨酸生产味精的发展综述.全国氨基酸生产技术交流会,2005.5-10.
5.于信令.味精工业手册[M],北京,中国轻工业出版社,1995.282-287.
6.王天贵,王诗瑜,张引沁.葡萄糖发酵制取谷氨酸的研究进展[J].平原大学学报,2000,17(3)：89-90.
7.胡学智,沈天益.617短杆菌发酵生产谷氨酸的研究历程[J].江苏调味副食品,2006,23(3)：4-6.
8.李华玮,苏庆辉,李志江等.谷氨酸生产行业现状综合分析[J].农产品加工学刊,2005,(8)：65-67.
9. Eiichiro Kimura. Metabolic Engineering of glutamate production. Advances in Biochemical Engineering/Biotechnology,2003,79(1):37-57.
10.余秉琦,沈微,王正祥等.谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成[J].生物工程学报,2005,21(2)：270-274.
11. Eikmanns BJ, Thum-Schmitz N, Eggeling L, et al. Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology,1994,140(8):1817-1828.
12. Christoph Wittmann. Analysis and engineering of metabolic pathway fluxes in Coryne-bacterium glutamicum. Adv Biochem Engin/Biotechnol,2010,120(2):21-49.
13.杜军,徐庆阳,谢希贤等.谷氨酸高产菌GDK-9的定向选育及其发酵过程研究[J].食品与发酵工业,2008,34(7)：17-19.
14.甄卫军,张石峰,曾宪贤.高产谷氨酸菌选育研究进展[J].新疆大学学报(自然科学版),2002,19(2)：209-211.
15.张克旭,陈宁,张蓓.代谢控制发酵[M],北京,中国轻工业出版社,1998.54-55.
16.张克旭.关于谷氨酸高产菌WTH-1的选育[J].食品与发酵工业,1985,(5)：36-39.
17.郑善良.L-谷氨酸一次性高糖发酵产生菌FM84-415的选育及其特性的研究[J].氨基酸和生物资源,1986,4(2)：15-19.
18.云逢霖.高产谷氨酸菌S9114的选育及生产[J].发酵科技通讯,1993,22(1)：3-7.
19.毛富根.耐高糖谷氨酸产生菌Ls168的激光育种[J].发酵科技通讯,1995,24(1)：5-9.
20.王岁楼,张一震.谷氨酸高产菌TZ2310的诱变选育及其发酵的研究[J].中国调味品,1997,(9)：16-18.
21.袁品坦.谷氨酸菌种选育的进展及其回顾[J].发酵科技通讯,2000,39(1):15-20.
22.张克旭,陈宁,张永志等.用原生质体融合技术选育谷氨酸高产菌[J].微生物学报,1991,(2)：108-114.
23.张蓓,张克旭,陈宁.谷氨酸高产菌FTN9108的细胞融合育种及发酵条件的研究[J].食品与发酵工业,1992,(5)：21-25.
24.陈宁,张克旭.谷氨酸温度敏感型菌株CN1021的原生质体融合育种[J].发酵科技通讯,2003,32(3)：11-14.
25.于信令,于军.对中国味精工业今后发展趋势的研讨[J].发酵科技通讯,2005,34(1)：12-14.
26.秦海斌.L-谷氨酸产生菌选育、发酵条件研究及其代谢流分析[D]：[硕士学位论文].江南大学生物工程学院,2009.
27.诸葛健,李华忠.微生物学[M].北京：科学出版社.2005,316-321.
28.诸葛健,王正祥.工业微生物实验技术手册[M].北京：中国轻工业出版社,1994,391-392.
29.张克旭,陈宁,张蓓等.代谢控制发酵[M],北京,中国轻工业出版社,1998.57-61.
30. KiBeom Lee. Enhanced production of lactid acid by an adapted strain of Lactobaclllusdel-brueckii subsp. Lactis[J]. World J Microbiol Biotechnol,2007,23 (2):1317-1320.
31.杨森莲,张克旭,陈宁.谷氨酸温度敏感突变株的驯化及发酵条件研究[J].中国酿造,2006,(7)：18-20.
32.张克旭.谷氨酸的代谢控制发酵育种[J].发酵科技通讯,2004,33(2)：11-13.
33.马立安,贺飞英.耐高温柠檬酸菌种驯化及筛选[J].中国酿造,2007,(11)：57-58.
34.赵宇星,周惠明,钱海峰.酸豆乳生产菌种的驯化研究[J].食品科技,2004,(11)：28-30.
35.张克旭.氨基酸发酵工艺学[M],北京,中国轻工业出版社,1995.126.
36.邓毛程,王瑶,阳元娥.高初糖谷氨酸发酵的研究[J].广东轻工职业技术学院学报,2004,3(2)：12-14.
37.张伟国,钱和.氨基酸生产技术及其应用[M],北京,中国轻工业出版社,1997.27.
38.陈碧娥.谷氨酸生产菌培养基最优配比设计[J].华侨大学学报(自然科学版),2001,22(2)：199-201.
39. Mohamed Hajji, Ahmed Rebai, Neji Gharsallah, et al. Optimization of alkaline protease production by Aspergillus clavatus ESI in Mirabilis jalapa tuber powder using statistical experimental design [J].Appl Microbiol Biotechnol,2008,79 (6):915-923.
40.卞芙蓉,劳兴珍,郑绗等.酵母发酵生产谷胱甘肽的培养基优化[J].中国生化药物杂志,2009,30(3)：184-186.
41.毕静莹,卢丽娟,秦义等.Plackett-Burman设计法筛选影响灵芝富锌能力的主要培养基组分[J].安徽农业科学,2010,38(10)：5335-5337.
42. Lei Yu, Ting Lei, Xiao-lin Pei. Rapid screen of medium components for L(+)lactic acid production by Lactobacillus rhamnosus[J]. Food Science,2007,28(11):382-385.
43.刘春辉.L-谷氨酰胺产生菌的选育及其代谢调控的初步研究[D]：[硕士学位论文].江南大学生物工程学院,2009.
44.陈银芳.L-赖氨酸产生菌的选育及其发酵条件的调控[D]：[硕士学位论文].江南大学生物工程学院,2009.
45.冯容保.发酵谷氨酸生产新工艺对比[J].发酵科技通讯,2003,32(1)：15-16.
46.于信令,于军.氨基酸发酵生产的调控优化[J].发酵科技通讯,2006,35(1)：28-30.
47.李艳.发酵工程原理与技术[M],北京,高等教育出版社,2006.221-222.
48.马占龙.谷氨酸发酵过程的控制[J].发酵科技通讯,2006,35(3)：1-2.
49.邓毛程,吴亚丽,梁世中等.高生物素谷氨酸发酵中溶氧水平与生物素用量的研究[J].食品与机械,2008,24(3)：10-13.
50.王营,董亮,张雁铃等.谷氨酸发酵标准溶解氧水平的确定[J].食品与发酵工业,2005,31(9)：42-44.
51.赵克勤,刘毅,邹祖然.谷氨酸发酵流加糖工艺的生产研究[J].食品工业科技,2005,26(3)：124-125.
52. Shintaro Iwatani,Yohei Yamada,Yoshihiro Usuda.Metabolic flux analysis in biotechnology processes. Biotechnol Lett,2008,30(5):791-799.
53.杜军,陈宁,刘辉等.基于代谢流量分析的L-谷氨酸发酵过程优化[J].中国食品学报,2008,8(2)：30-34.