L-缬氨酸5L罐发酵条件研究
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摘要
本论文根据代谢控制发酵和代谢流分析理论，研究了L-缬氨酸高产菌5L罐的发酵条件。主要研究内容和结果如下：
(1)以已有的摇瓶分批发酵最优条件为基础，对菌株TV2564进行了5L发酵罐分批发酵试验。首先研究了不同碳氮比对缬氨酸发酵的影响，结果表明高碳氮比或高浓度初糖不利于缬氨酸发酵。
(2)根据5L发酵罐中溶氧变化情况及不同K_La对缬氨酸发酵的影响，结合缬氨酸发酵的生长与生产耦合性研究及发酵过程中代谢流分配的特点，得到了两阶段供氧的控制方式。以5L罐分批发酵试验数据为依据，利用MATLAB工具软件建立了菌体生长、产物形成和底物消耗的动力学模型，实验值与拟合值接近，拟合情况较好。
(3)构建L-缬氨酸生产菌TV2564的代谢网络，依据代谢流分析原理，应用MATLAB线性规划计算得到了L-缬氨酸生产菌不同发酵时期的代谢流分配。通过分析葡萄糖经EMP、HMP与TCA循环以及关键性节点GLC6P、PEP和PYR的代谢流分配，为发酵过程控制提供理论指导。
(4)以分批发酵最优条件为依据，对菌株TV2564进行了5L罐补料分批发酵研究。进一步研究了溶氧在补料发酵方式下的变化情况，寻找到适宜的供氧方式，即根据菌体生长的四个不同阶段：延滞期、对数生长期、稳定期和衰亡期分别调节不同转速，以满足菌体生长和产物合成的溶氧需求。
(5)确定了最佳发酵工艺，即初始低糖低铵，产酸期控制糖浓度在2.0g／L以下，发酵前期及后期补加少量玉米浆，自动流加氨水控制pH值，发酵过程根据菌体生长的四个阶段分别调节不同转速，以满足生长和产酸的溶氧需求，在此工艺下，菌体生长良好，发酵后劲足，产酸期延长，到后期流加80％的浓糖，发酵72h产L-缬氨酸52.68g／L。
(6)利用不锈钢膜过滤技术对缬氨酸发酵液进行前处理，去除菌体和蛋白质，效果良好，去除蛋白率可达79.89％。并初步研究了缬氨酸的提取工艺。
According to the theory of metabolic control fermentation and metabolic flux analysis, the dissertation focuses on the fermentation conditions of L-valine high-yielding strain .The main research contents and results are as follows:
(1) Based on the optimized conditions of flask, Fermentation in 5L fermenter of L-valine by TV2564 has proceeded. The effect of different C/N on fermentation was studied, and the results show high C/N or high concentration of initial glucose is adverse to fermentation of valine.
(2) According to the change of DO in 5L fermenter, with dynamics analysis during fermentation process, two stages oxygen supply was provided .The L-valine batch fermentation kinetics was studied by MATLAB tool based on the experimental data from 5-liter fermenter's batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.
(3) The metabolic network was proposed for L-valine producing strain. The metabolic flux distributions of Brevibacterium flavum TV2564 during the different fermentation period were calculated by linear program of MATLAB software according to metabolic flux analysis theory. The metabolic flux distributions of EMP, HMP, TCA cycle and the key principle nodes of GLC6P, PEP and PYR were analyzed. At the same time it can be indicated to provide theory guide for the fermentation process.
(4) Fed batch fermentation was studied with TV2564 according to the optimum condition of batch fermentation. According to the change of DO in fed-batch fermentation, the optimized oxygen supply was provided. The optimum conditions was obtained, which were the technology of low glucose and amino, feeding corn syrup liquid in the former and later phase of fermentation, controling pH by feeding amino liquid, feeding 80% glucose in later phase, production of L-valine reached 52.68g/L after fermentation for 72 hours under the optimized conditions.
(5) By membrane filtration, the cells and protein were leached. The results are perfect, and the removal rate of protein is 79.89%.
(1)以已有的摇瓶分批发酵最优条件为基础，对菌株TV2564进行了5L发酵罐分批发酵试验。首先研究了不同碳氮比对缬氨酸发酵的影响，结果表明高碳氮比或高浓度初糖不利于缬氨酸发酵。
(2)根据5L发酵罐中溶氧变化情况及不同K_La对缬氨酸发酵的影响，结合缬氨酸发酵的生长与生产耦合性研究及发酵过程中代谢流分配的特点，得到了两阶段供氧的控制方式。以5L罐分批发酵试验数据为依据，利用MATLAB工具软件建立了菌体生长、产物形成和底物消耗的动力学模型，实验值与拟合值接近，拟合情况较好。
(3)构建L-缬氨酸生产菌TV2564的代谢网络，依据代谢流分析原理，应用MATLAB线性规划计算得到了L-缬氨酸生产菌不同发酵时期的代谢流分配。通过分析葡萄糖经EMP、HMP与TCA循环以及关键性节点GLC6P、PEP和PYR的代谢流分配，为发酵过程控制提供理论指导。
(4)以分批发酵最优条件为依据，对菌株TV2564进行了5L罐补料分批发酵研究。进一步研究了溶氧在补料发酵方式下的变化情况，寻找到适宜的供氧方式，即根据菌体生长的四个不同阶段：延滞期、对数生长期、稳定期和衰亡期分别调节不同转速，以满足菌体生长和产物合成的溶氧需求。
(5)确定了最佳发酵工艺，即初始低糖低铵，产酸期控制糖浓度在2.0g／L以下，发酵前期及后期补加少量玉米浆，自动流加氨水控制pH值，发酵过程根据菌体生长的四个阶段分别调节不同转速，以满足生长和产酸的溶氧需求，在此工艺下，菌体生长良好，发酵后劲足，产酸期延长，到后期流加80％的浓糖，发酵72h产L-缬氨酸52.68g／L。
(6)利用不锈钢膜过滤技术对缬氨酸发酵液进行前处理，去除菌体和蛋白质，效果良好，去除蛋白率可达79.89％。并初步研究了缬氨酸的提取工艺。
According to the theory of metabolic control fermentation and metabolic flux analysis, the dissertation focuses on the fermentation conditions of L-valine high-yielding strain .The main research contents and results are as follows:
(1) Based on the optimized conditions of flask, Fermentation in 5L fermenter of L-valine by TV2564 has proceeded. The effect of different C/N on fermentation was studied, and the results show high C/N or high concentration of initial glucose is adverse to fermentation of valine.
(2) According to the change of DO in 5L fermenter, with dynamics analysis during fermentation process, two stages oxygen supply was provided .The L-valine batch fermentation kinetics was studied by MATLAB tool based on the experimental data from 5-liter fermenter's batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.
(3) The metabolic network was proposed for L-valine producing strain. The metabolic flux distributions of Brevibacterium flavum TV2564 during the different fermentation period were calculated by linear program of MATLAB software according to metabolic flux analysis theory. The metabolic flux distributions of EMP, HMP, TCA cycle and the key principle nodes of GLC6P, PEP and PYR were analyzed. At the same time it can be indicated to provide theory guide for the fermentation process.
(4) Fed batch fermentation was studied with TV2564 according to the optimum condition of batch fermentation. According to the change of DO in fed-batch fermentation, the optimized oxygen supply was provided. The optimum conditions was obtained, which were the technology of low glucose and amino, feeding corn syrup liquid in the former and later phase of fermentation, controling pH by feeding amino liquid, feeding 80% glucose in later phase, production of L-valine reached 52.68g/L after fermentation for 72 hours under the optimized conditions.
(5) By membrane filtration, the cells and protein were leached. The results are perfect, and the removal rate of protein is 79.89%.
引文
[1] 张炳荣．氨基酸工业大全(技术与市场)．北京：轻工业出版社，1991．
[2] 欧阳平凯．生物化工产品．北京：化学工业出版社，1999．
[3] 李良铸，李明华．最新生化药物制备技术．中国药科技术出版社，2001．
[4] Iborra J L et al. Analysis of a laminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase. Biotechnol. Appl. Biochem., 1992, 15 (1): 22-30.
[5] 王平，何英等．L-缬氨酸菌种的选育及发酵条件．大连轻工业学院学报，2002，21(2)：116-119．
[6] 氨基酸发酵生产的研究进展．氨基酸和生物资源，1999，21(4)：19～23．
[7] Polanuer B M, Ivanov S V. High-performance liquid chromatography of amino acids in copper complex form: application to valine fermentation samples-valine analysis during fermentation by HPLC. J. Chromatogr. 1996,722:311-315.
[8] 朱曙东，赵昇皓．氨基酸的高效液相色谱分析．色谱，1994,12(1)：20-23．
[9] 蒋新宇，周春山．氨基酸的柱前衍生高效液相色谱分析述评．湖南化工，1999，29(4)：9-11．
[10] 张克旭，陈宁，张蓓等．代谢控制发酵．北京：中国轻工业出版社，1991．
[11] 屈明波，龚建华，黄和容．L-缬氨酸发酵供氧与补料过程控制的研究．生物工程学报，1992,8(2)：184-191．
[12] Ajinomoto. Enhanced L-valine production-by mutagenesis of Brevibacterium lactofermentum and corynebacterium glutamicum. EP287123,1988.
[13] Iborra J L et al. Analysis of a laminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase. Biotechnol. Appl. Biochem., 1992, 15 (1): 22-30.
[14] Tomota F et al. Methods for producing L-valine by fermentation. UP 5888783,1999.
[15] Mitsubishi-Petrochem et al. Production of L-valine using glucose-containing aqueous solution with biotin auxotrophic coryneform bacteria,e.g. Corynebacterium, Brevibacterium, etc.-Brevibacterium flavum fermentation in glucose culture medium. JP 094373, 1989.
[16] A. G. Khachatryan et al. Dependence of valine production by serratia marcescens on the ion composition of the fermentation. Microbiology, 1986,
22(4):554-556.
[17] Chattopadhyay et al. Production of valine by a Bacillus sp. Z.Allg. Microbiology, 1978,18(4):243-254.
[18] Katsurada et al. Process for producing L-valine by fermentation. UP 51889413, 1993.
[19] 徐旭东，肖灿鹏等．L-缬氨酸生产菌的选育．药物生物技术，1999，6(1)：24～27．
[20] 富田房男，横田笃等．L-缬氨酸和L-亮氨酸的生产方法．CN1162976，1997．
[21] Hoechst et al. New extrachromosomal element-containing aliphatic-transaminase gene-from Escherichia coli; alanine, valine and isoleucine over production. EP265852, 1986.
[22] Marquardt R,Then J.Cloning and application of the ilve transaminase gene. EP0265852,1986.
[23] Nakayama K, Kitada. L-valine production using microbial auxotroph. J. Gen.Appl.Microbiol. Tokyo, 1961,7:52-69.
[24] Kisumi M, S Komatsubara, and H Sahm. Vline accumulation by α-aminobutyric acid-resistant mutants of Serratia marcescens. J.Bacteriol, 1971,106:493-499.
[25] 中国科学院微生物研究所氨基酸组．L-缬氨酸产生菌AS1．586的选育．微生物学报，1975，15(4)：325-329．
[26] 唐任天，郭永复，陈琦．L-缬氨酸生物合成的研究．氨基酸杂志，1986，1：12-17．
[27] 黄海华，李福德．发酵法生产L-缬氨酸的研究．微生物学杂志，1989(2)：1-8．
[28] 刁新民，周俊贤，徐大钢．微生物发酵合成L-缬氨酸-~(15)N．微生物学通报，1994，21(2)：75-78．
[29] 张伟国，钱和．L-缬氨酸高产菌选育及营养需求研究．沈阳药科大学学报，2001，18(6)：443-446．
[30] 来彩霞，刘堂生，陈炜．L-缬氨酸产生菌菌种选育和发酵条件考察．沈阳药科大学学报，1997，14(2)：107～110．
[31] 王子，何英，贺小伟．L-缬氨酸菌种的选育及其发酵条件．大连轻工业学院学报，2002，21(2)：116-119．
[32] 赵丽丽，陈宁，熊明勇，张克旭．原生质体紫外诱变选育L-缬氨酸生产菌的研究，食品与发酵工业，2002，28(10)：22-24
[33] 刘云，徐琪寿．氨基酸发酵生产的研究进展．氨基酸和生物资源，
[34] 浦军平，肖泉，林学军．一次性高糖发酵L-缬氨酸菌种的选育及发酵条件．大连轻工业学院学报，2001，20(4)：264-266．
[35] 蔡显鹏等．鸟苷酸发酵过程代谢流迁移的分析．生物工程学报，2002，18(5)：622～625．
[36] Bonarius HPJ,Schmid G.Tramper J. Flux analysis of underdetermined metabolic networks: the quest for the missing constraints. Trends Biotechnology, 1997,15:308-314.
[37] W.M.Van Gulik.J.J.Heijnen. A Metabolic Network Stoichiometry Analysis of Microbial Growth and Product Formation. Biotechnology and Bioengineering, 1995,48:681-698.
[38] Christophe H.Schilling,Jeremy S.Edwards B. Combining Pathway Analysis with Flux Balance Analysis for the Comprehensive Study of Metabolic Systems. Biotechnology and Bioengineering, 2001,71:286～306.
[39] Prit SJ. Measurement of K_La in absence of biomass. In: Principles of Microbe and cell cultivation. Blackwell Scientific Publications. Oxford. 1975:0-93.
[40] 王福荣．工业发酵分析，轻工业出版社，84—86，1997．
[41] 韩雅珊．现代有机化学实验技术导论，科学出版社，1985。
[42] 成都科技大学分析化学教研室，分析化学手册，化学工业出版社．
[43] 李志勇，郭祀远，李琳．藻类对微量元素的生物富集及其应用[J]．微生物学通报．1997，3：12-14．
[44] 苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究[J]．生物工程学报．2000，16(4)：509-513．
[45] 张磊．基因工程á-2b干扰素新型发酵技术及结构修饰与新制剂研究．天津大学博士学位论文，2003．
[46] Hirote Y., h. Sniai, Effect of oxygen on amino acids fermentation, Adv. Biotechnology, 1981 (1):465-503.
[47] Chin-Hang Shu et al. Optimization of L-phenylalanine production of Corynebacterium glutamicun under product feedback inhibition by Elevated Oxygen Transfer Rate. Biotechnol Bioeng,2002,77(2): 131-141.
[48] Debendra K et al. Effect of Oxygen Transfer on Glycerol Biosynthesis by an Osmophilic Yeast Candia magnoliae. Biotechnol Bioeng.2002,78(5):454-555.
[49] Kenichi Higashiyama.Katsushi murakami. Effect of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1s-4.
Biotechnol Bioeng, 1999, 63(4):442-448.
[50] 姚汝华．微生物工程工艺原理．广州：华南理工大学出版社，1996
[51] 王沫然．MATALAB5．X与科学计算．北京：清华大学出版社，2000
[52] 许波，刘征．MatLab工程数学应用．北京：清华大学出版社，2000
[53] 周建华，黄燕．MATALAB5．3学习教程．北京：北京大学出版社，2000
[54] Matlab 4.2C with Notebook. Math Works, 1995
[55] Matlab 4.0 with Simulink1.2. Math Works, 1993
[56] Luedeking ,r.et al, J.Biochem.Microbio.Technol.Eng.,1959,1:393
[57] 李友荣，马辉文．发酵生理学，湖南：湖南科技出版社，1988
[58] 冯容保．生化工程．，1985，4(1)：48～53
[59] 李涛，贺勇军，刘志俭．MATALAB工具箱应用指南--应用数学篇，电子工业出版社，2000
[60] 唐军等．克鲁斯假丝酵母分批发酵生产甘油的代谢流分布．高校化学工程学报，2082，16(1)：59-63．
[61] 高红亮，丛威．杂交瘤细胞的代谢流分析．生物工程学报，2000，11：740-746．
[62] 林荣胜，朱涛，曹竹安．利用代谢网络分析提高长链二元酸的产率．南京化工大学学报，1999，21(3)：337-339．
[63] Soon Ho Hong. Metabolic flux analysis for Succinic acid production by recombinant Escherichia coli with amplified malic enzyme acitivity. Biotechnology and Bioengineering,2001,74(2):89-95.
[64] Joseph J. Metabolic flux distributions in corynebactrium glutamicum during growth and lysine overproduction. Biotechnology and bioengineering, 1992,41 (6):633-646.
[65] Sugimoto,S. Fructose metabolism and regulation of 1-phosphofructokinase and 6-phosphofructokinase in Brevibacterium flavum. Agric.Biol.Chem, 1989,53:2081-2087.
[66] Shiio, I. Toride. Production of lysine by pyrute dehydrogenase mutants of Brevibacterium flavum. Agric. Biol. Chem, 1984,48:3091-3098.
[67] Otsuka S, Miyajima R. comparative studies on the mechanisum of microbial
[68] Vallino, J.J. Identification of branch-point restrictions in microbial metabolism through metabolic flux analysis and local network perturbations, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, MA. 1991.
[69] Michiko Mori and Isamu Shio. Pyruvate formation and sugar metabolism in an amino acid-producing Bacterium, Brevibacterium flavum, 1986,51(1):129-138.
[70] Isamu Shio,Michiko Mori. Amino acid aminotransferases in an amino acid-producing Bacterium, Brevibacterium flavum, 1982,46(12):2967-2977.
[71] Tosaka.o.Morioka. The role of biotin dependent pyrute carboxylase in L-lysine production. Agric.Biol. Chem,1979, 43: 1513-1519.
[72] Isamu Shio, shin-ichi. Effect of carbon source sugars on the yield of amino acid production and sucrose metabolism in Brevibacterium flavum. Agri.Biol.Chem,1990, 54(6): 1513-1519.
[73] Shvinka, Yu.E., Viestur, U.E. Alternative pathways of oxidation in the respiratory chain of Brevibacterium flavum. Microbiol,1979,48:4-10.
[74] Sepil Takac. Metabolic flux distribution for the optimized production of L-glutamate. Enzyme and Microbial Technology,1998,23:286-300.
[75] 张星元，潘中明．代谢网络刚性与代谢工程．生物工程进展，1999，19(6)：38-42．
[76] 潘军华．次级代谢产物的代谢工程．中国抗生素杂志，2002，27(4)：251-255．
[77] 俞俊棠，唐孝宣．生物工艺学．上海：华东化工学院出版社，1991．
[78] 孙万儒，周铁锁等．普鲁兰的底物流加补料发酵研究．生物工程学报，1999，15(2)：183-187．
[79] Hisbullah, M. A. Hussain, K. B. Ramachandran. Comparative evaluation of various control schemes for fed-batch fermentation, bioprocess engineering, 2002, 24(5):309-318.
[80] Roberto Martínez-Campos, Mayra de la Torre. Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose. Biotechnology letters, 2002,24(6):427-431.
[81] A. Cabrera, A. Poznyak, T. Poznyak, J. Aranda. Identification of a fed-batch fermentation process: comparison of computational and laboratory experiments. bioprocess engineering,2002,24(5):319-327.
[82] 刘勇，张长铠，曹光宇等．补料分批培养对L-异亮氨酸发酵的影响，工业微生物，2000，30(2)：26-29．
[83] 屈明波，龚建华，黄和容．L-缬氨酸发酵供氧与补料过程控制的研究．生物工程学报，1992，8(2)：184-191．
[84] 龚建华，陈琦．L-赖氨酸分批发酵连续补料的研究．生物工程学报，1987，3(4)：281-289．
[85] 曾祥群，周海滨．缬氨酸提取工艺研究．发酵科技通讯．2002，2(1)：22-24
[2] 欧阳平凯．生物化工产品．北京：化学工业出版社，1999．
[3] 李良铸，李明华．最新生化药物制备技术．中国药科技术出版社，2001．
[4] Iborra J L et al. Analysis of a laminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase. Biotechnol. Appl. Biochem., 1992, 15 (1): 22-30.
[5] 王平，何英等．L-缬氨酸菌种的选育及发酵条件．大连轻工业学院学报，2002，21(2)：116-119．
[6] 氨基酸发酵生产的研究进展．氨基酸和生物资源，1999，21(4)：19～23．
[7] Polanuer B M, Ivanov S V. High-performance liquid chromatography of amino acids in copper complex form: application to valine fermentation samples-valine analysis during fermentation by HPLC. J. Chromatogr. 1996,722:311-315.
[8] 朱曙东，赵昇皓．氨基酸的高效液相色谱分析．色谱，1994,12(1)：20-23．
[9] 蒋新宇，周春山．氨基酸的柱前衍生高效液相色谱分析述评．湖南化工，1999，29(4)：9-11．
[10] 张克旭，陈宁，张蓓等．代谢控制发酵．北京：中国轻工业出版社，1991．
[11] 屈明波，龚建华，黄和容．L-缬氨酸发酵供氧与补料过程控制的研究．生物工程学报，1992,8(2)：184-191．
[12] Ajinomoto. Enhanced L-valine production-by mutagenesis of Brevibacterium lactofermentum and corynebacterium glutamicum. EP287123,1988.
[13] Iborra J L et al. Analysis of a laminated enzyme membrane reactor for continuous resolution of amino acids-L-amino acid e.g. L-valine production by immobilized aminoacylase. Biotechnol. Appl. Biochem., 1992, 15 (1): 22-30.
[14] Tomota F et al. Methods for producing L-valine by fermentation. UP 5888783,1999.
[15] Mitsubishi-Petrochem et al. Production of L-valine using glucose-containing aqueous solution with biotin auxotrophic coryneform bacteria,e.g. Corynebacterium, Brevibacterium, etc.-Brevibacterium flavum fermentation in glucose culture medium. JP 094373, 1989.
[16] A. G. Khachatryan et al. Dependence of valine production by serratia marcescens on the ion composition of the fermentation. Microbiology, 1986,
22(4):554-556.
[17] Chattopadhyay et al. Production of valine by a Bacillus sp. Z.Allg. Microbiology, 1978,18(4):243-254.
[18] Katsurada et al. Process for producing L-valine by fermentation. UP 51889413, 1993.
[19] 徐旭东，肖灿鹏等．L-缬氨酸生产菌的选育．药物生物技术，1999，6(1)：24～27．
[20] 富田房男，横田笃等．L-缬氨酸和L-亮氨酸的生产方法．CN1162976，1997．
[21] Hoechst et al. New extrachromosomal element-containing aliphatic-transaminase gene-from Escherichia coli; alanine, valine and isoleucine over production. EP265852, 1986.
[22] Marquardt R,Then J.Cloning and application of the ilve transaminase gene. EP0265852,1986.
[23] Nakayama K, Kitada. L-valine production using microbial auxotroph. J. Gen.Appl.Microbiol. Tokyo, 1961,7:52-69.
[24] Kisumi M, S Komatsubara, and H Sahm. Vline accumulation by α-aminobutyric acid-resistant mutants of Serratia marcescens. J.Bacteriol, 1971,106:493-499.
[25] 中国科学院微生物研究所氨基酸组．L-缬氨酸产生菌AS1．586的选育．微生物学报，1975，15(4)：325-329．
[26] 唐任天，郭永复，陈琦．L-缬氨酸生物合成的研究．氨基酸杂志，1986，1：12-17．
[27] 黄海华，李福德．发酵法生产L-缬氨酸的研究．微生物学杂志，1989(2)：1-8．
[28] 刁新民，周俊贤，徐大钢．微生物发酵合成L-缬氨酸-~(15)N．微生物学通报，1994，21(2)：75-78．
[29] 张伟国，钱和．L-缬氨酸高产菌选育及营养需求研究．沈阳药科大学学报，2001，18(6)：443-446．
[30] 来彩霞，刘堂生，陈炜．L-缬氨酸产生菌菌种选育和发酵条件考察．沈阳药科大学学报，1997，14(2)：107～110．
[31] 王子，何英，贺小伟．L-缬氨酸菌种的选育及其发酵条件．大连轻工业学院学报，2002，21(2)：116-119．
[32] 赵丽丽，陈宁，熊明勇，张克旭．原生质体紫外诱变选育L-缬氨酸生产菌的研究，食品与发酵工业，2002，28(10)：22-24
[33] 刘云，徐琪寿．氨基酸发酵生产的研究进展．氨基酸和生物资源，
[34] 浦军平，肖泉，林学军．一次性高糖发酵L-缬氨酸菌种的选育及发酵条件．大连轻工业学院学报，2001，20(4)：264-266．
[35] 蔡显鹏等．鸟苷酸发酵过程代谢流迁移的分析．生物工程学报，2002，18(5)：622～625．
[36] Bonarius HPJ,Schmid G.Tramper J. Flux analysis of underdetermined metabolic networks: the quest for the missing constraints. Trends Biotechnology, 1997,15:308-314.
[37] W.M.Van Gulik.J.J.Heijnen. A Metabolic Network Stoichiometry Analysis of Microbial Growth and Product Formation. Biotechnology and Bioengineering, 1995,48:681-698.
[38] Christophe H.Schilling,Jeremy S.Edwards B. Combining Pathway Analysis with Flux Balance Analysis for the Comprehensive Study of Metabolic Systems. Biotechnology and Bioengineering, 2001,71:286～306.
[39] Prit SJ. Measurement of K_La in absence of biomass. In: Principles of Microbe and cell cultivation. Blackwell Scientific Publications. Oxford. 1975:0-93.
[40] 王福荣．工业发酵分析，轻工业出版社，84—86，1997．
[41] 韩雅珊．现代有机化学实验技术导论，科学出版社，1985。
[42] 成都科技大学分析化学教研室，分析化学手册，化学工业出版社．
[43] 李志勇，郭祀远，李琳．藻类对微量元素的生物富集及其应用[J]．微生物学通报．1997，3：12-14．
[44] 苗志奇，未作君，元英进．水杨酸在紫杉醇生物合成中诱导作用的研究[J]．生物工程学报．2000，16(4)：509-513．
[45] 张磊．基因工程á-2b干扰素新型发酵技术及结构修饰与新制剂研究．天津大学博士学位论文，2003．
[46] Hirote Y., h. Sniai, Effect of oxygen on amino acids fermentation, Adv. Biotechnology, 1981 (1):465-503.
[47] Chin-Hang Shu et al. Optimization of L-phenylalanine production of Corynebacterium glutamicun under product feedback inhibition by Elevated Oxygen Transfer Rate. Biotechnol Bioeng,2002,77(2): 131-141.
[48] Debendra K et al. Effect of Oxygen Transfer on Glycerol Biosynthesis by an Osmophilic Yeast Candia magnoliae. Biotechnol Bioeng.2002,78(5):454-555.
[49] Kenichi Higashiyama.Katsushi murakami. Effect of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1s-4.
Biotechnol Bioeng, 1999, 63(4):442-448.
[50] 姚汝华．微生物工程工艺原理．广州：华南理工大学出版社，1996
[51] 王沫然．MATALAB5．X与科学计算．北京：清华大学出版社，2000
[52] 许波，刘征．MatLab工程数学应用．北京：清华大学出版社，2000
[53] 周建华，黄燕．MATALAB5．3学习教程．北京：北京大学出版社，2000
[54] Matlab 4.2C with Notebook. Math Works, 1995
[55] Matlab 4.0 with Simulink1.2. Math Works, 1993
[56] Luedeking ,r.et al, J.Biochem.Microbio.Technol.Eng.,1959,1:393
[57] 李友荣，马辉文．发酵生理学，湖南：湖南科技出版社，1988
[58] 冯容保．生化工程．，1985，4(1)：48～53
[59] 李涛，贺勇军，刘志俭．MATALAB工具箱应用指南--应用数学篇，电子工业出版社，2000
[60] 唐军等．克鲁斯假丝酵母分批发酵生产甘油的代谢流分布．高校化学工程学报，2082，16(1)：59-63．
[61] 高红亮，丛威．杂交瘤细胞的代谢流分析．生物工程学报，2000，11：740-746．
[62] 林荣胜，朱涛，曹竹安．利用代谢网络分析提高长链二元酸的产率．南京化工大学学报，1999，21(3)：337-339．
[63] Soon Ho Hong. Metabolic flux analysis for Succinic acid production by recombinant Escherichia coli with amplified malic enzyme acitivity. Biotechnology and Bioengineering,2001,74(2):89-95.
[64] Joseph J. Metabolic flux distributions in corynebactrium glutamicum during growth and lysine overproduction. Biotechnology and bioengineering, 1992,41 (6):633-646.
[65] Sugimoto,S. Fructose metabolism and regulation of 1-phosphofructokinase and 6-phosphofructokinase in Brevibacterium flavum. Agric.Biol.Chem, 1989,53:2081-2087.
[66] Shiio, I. Toride. Production of lysine by pyrute dehydrogenase mutants of Brevibacterium flavum. Agric. Biol. Chem, 1984,48:3091-3098.
[67] Otsuka S, Miyajima R. comparative studies on the mechanisum of microbial
[68] Vallino, J.J. Identification of branch-point restrictions in microbial metabolism through metabolic flux analysis and local network perturbations, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, MA. 1991.
[69] Michiko Mori and Isamu Shio. Pyruvate formation and sugar metabolism in an amino acid-producing Bacterium, Brevibacterium flavum, 1986,51(1):129-138.
[70] Isamu Shio,Michiko Mori. Amino acid aminotransferases in an amino acid-producing Bacterium, Brevibacterium flavum, 1982,46(12):2967-2977.
[71] Tosaka.o.Morioka. The role of biotin dependent pyrute carboxylase in L-lysine production. Agric.Biol. Chem,1979, 43: 1513-1519.
[72] Isamu Shio, shin-ichi. Effect of carbon source sugars on the yield of amino acid production and sucrose metabolism in Brevibacterium flavum. Agri.Biol.Chem,1990, 54(6): 1513-1519.
[73] Shvinka, Yu.E., Viestur, U.E. Alternative pathways of oxidation in the respiratory chain of Brevibacterium flavum. Microbiol,1979,48:4-10.
[74] Sepil Takac. Metabolic flux distribution for the optimized production of L-glutamate. Enzyme and Microbial Technology,1998,23:286-300.
[75] 张星元，潘中明．代谢网络刚性与代谢工程．生物工程进展，1999，19(6)：38-42．
[76] 潘军华．次级代谢产物的代谢工程．中国抗生素杂志，2002，27(4)：251-255．
[77] 俞俊棠，唐孝宣．生物工艺学．上海：华东化工学院出版社，1991．
[78] 孙万儒，周铁锁等．普鲁兰的底物流加补料发酵研究．生物工程学报，1999，15(2)：183-187．
[79] Hisbullah, M. A. Hussain, K. B. Ramachandran. Comparative evaluation of various control schemes for fed-batch fermentation, bioprocess engineering, 2002, 24(5):309-318.
[80] Roberto Martínez-Campos, Mayra de la Torre. Production of propionate by fed-batch fermentation of Propionibacterium acidipropionici using mixed feed of lactate and glucose. Biotechnology letters, 2002,24(6):427-431.
[81] A. Cabrera, A. Poznyak, T. Poznyak, J. Aranda. Identification of a fed-batch fermentation process: comparison of computational and laboratory experiments. bioprocess engineering,2002,24(5):319-327.
[82] 刘勇，张长铠，曹光宇等．补料分批培养对L-异亮氨酸发酵的影响，工业微生物，2000，30(2)：26-29．
[83] 屈明波，龚建华，黄和容．L-缬氨酸发酵供氧与补料过程控制的研究．生物工程学报，1992，8(2)：184-191．
[84] 龚建华，陈琦．L-赖氨酸分批发酵连续补料的研究．生物工程学报，1987，3(4)：281-289．
[85] 曾祥群，周海滨．缬氨酸提取工艺研究．发酵科技通讯．2002，2(1)：22-24