基于scale-down反应器的溶氧梯度及其对谷氨酸发酵的影响研究
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摘要
本文采用scale-down反应器模拟工业生产中的溶氧梯度,对scale-down方法的可行性进行了验证,同时以谷氨酸发酵为对象研究溶氧非均匀性对生理代谢的影响。该方法为放大过程研究提供了一种新的研究手段。
首先采用单室scale-down反应器系统模拟大规模反应器内的溶氧浓度梯度,重点研究溶氧波动范围0-30%,波动周期2h、4h、6h条件下,溶氧浓度梯度对谷氨酸棒杆菌生长及其代谢的影响。结果表明在溶氧周期性波动条件下,谷氨酸棒杆菌更倾向于利用葡萄糖产生菌体而非积累产物谷氨酸,同时周期性的厌氧环境导致副产物乳酸大量积累。
为了更好的模拟大规模反应器中溶氧非均匀环境的真实情况建立了双室scale-down反应器。通过实验测定与CFD模拟相结合的方法对双室scale-down反应器的流场特性进行研究,结果表明在反应器主体区与PFR部分之间形成了明显的溶氧差异,满足在实验室规模反应器内建立大规模反应器中溶氧非均匀环境的要求。
最后采用双室scale-down反应器进行谷氨酸发酵,结果表明在溶氧非均匀条件下,菌体生长缓慢,但生长期延长,导致最终菌浓升高,同时谷氨酸产量下降,副产物乳酸积累,这与单室反应器中得出的结论一致。同时实验结果证明在双室scale-down反应器内存在溶氧梯度,为scale-down方法的建立奠定了基础。
In this paper, the dissolved oxygen gradient occurred in industrial production was simulated in the scale-down reactors, and its results verified the feasibility of scale-down method. At the same time the effect of dissolved oxygen heterogeneity on physiology of Corynebacterium glutamincum for glutamic acid fermentation was studied. The results showed that the scale-down method provided a new research tool for the study of bioprocess scale-up.
Firstly, a laboratory scale one-compartment scale-down system was used to simulate dissolved oxygen (DO) gradients occurred in large-scale. The study was mainly focused on the influences of fluctuated DO (switch between 0% and 30% with cycle time ranging from 2h to 6h) on cell growth and metabolism of Corynebacterium glutamicum. It was shown that C. glutamicum preferred to use glucose for growth but not for product accumulation under oscillating DO condition, with much more byproduct, lactate, being produced at the same time.
In order to better simulate DO heterogeneous environment of large-scale bioreactor, two-compartment scale-down reactors was designed. The flow characteristics of two-compartment scale-down reactors were studied by experiment combined with CFD simulation. It was shown that DO is significantly different between the main bulk of the reactor and the PFR part. DO heterogeneity occurred in large-scale reactor was achieved in this system.
Finally, the glutamic acid fermentation was done in two-compartment scale-down reactors. It was found that DO heterogeneous conditions resulted in slower cell growth rate, longer growth period, and finally higher cell concentration. Meanwhile the glutamic acid production increased but lactate accumulation decreased. They were consistent with conclusions drawn in one-compartment scale-down reactors. Meanwhile it verified that the DO gradient occurred within the reactors, and provided the foundation for the establishment of scale-down method.
首先采用单室scale-down反应器系统模拟大规模反应器内的溶氧浓度梯度,重点研究溶氧波动范围0-30%,波动周期2h、4h、6h条件下,溶氧浓度梯度对谷氨酸棒杆菌生长及其代谢的影响。结果表明在溶氧周期性波动条件下,谷氨酸棒杆菌更倾向于利用葡萄糖产生菌体而非积累产物谷氨酸,同时周期性的厌氧环境导致副产物乳酸大量积累。
为了更好的模拟大规模反应器中溶氧非均匀环境的真实情况建立了双室scale-down反应器。通过实验测定与CFD模拟相结合的方法对双室scale-down反应器的流场特性进行研究,结果表明在反应器主体区与PFR部分之间形成了明显的溶氧差异,满足在实验室规模反应器内建立大规模反应器中溶氧非均匀环境的要求。
最后采用双室scale-down反应器进行谷氨酸发酵,结果表明在溶氧非均匀条件下,菌体生长缓慢,但生长期延长,导致最终菌浓升高,同时谷氨酸产量下降,副产物乳酸积累,这与单室反应器中得出的结论一致。同时实验结果证明在双室scale-down反应器内存在溶氧梯度,为scale-down方法的建立奠定了基础。
In this paper, the dissolved oxygen gradient occurred in industrial production was simulated in the scale-down reactors, and its results verified the feasibility of scale-down method. At the same time the effect of dissolved oxygen heterogeneity on physiology of Corynebacterium glutamincum for glutamic acid fermentation was studied. The results showed that the scale-down method provided a new research tool for the study of bioprocess scale-up.
Firstly, a laboratory scale one-compartment scale-down system was used to simulate dissolved oxygen (DO) gradients occurred in large-scale. The study was mainly focused on the influences of fluctuated DO (switch between 0% and 30% with cycle time ranging from 2h to 6h) on cell growth and metabolism of Corynebacterium glutamicum. It was shown that C. glutamicum preferred to use glucose for growth but not for product accumulation under oscillating DO condition, with much more byproduct, lactate, being produced at the same time.
In order to better simulate DO heterogeneous environment of large-scale bioreactor, two-compartment scale-down reactors was designed. The flow characteristics of two-compartment scale-down reactors were studied by experiment combined with CFD simulation. It was shown that DO is significantly different between the main bulk of the reactor and the PFR part. DO heterogeneity occurred in large-scale reactor was achieved in this system.
Finally, the glutamic acid fermentation was done in two-compartment scale-down reactors. It was found that DO heterogeneous conditions resulted in slower cell growth rate, longer growth period, and finally higher cell concentration. Meanwhile the glutamic acid production increased but lactate accumulation decreased. They were consistent with conclusions drawn in one-compartment scale-down reactors. Meanwhile it verified that the DO gradient occurred within the reactors, and provided the foundation for the establishment of scale-down method.
引文
[1]李玉英.发酵工程.2009,北京：中国农业大学出版社.
[2]刘勇,陈雅丽,张长铠等.L-异亮氨酸发酵对氧需求的研究.生物技术.1998,8(1)：26-28.
[3]张伟国,陈坚,伦世仪.供氧对L-异亮氨酸分批发酵的影响.无锡轻工大学学报.2001,20(5)：539-542.
[4]杨宁,王健,徐庆阳,陈宁,温廷益.基于途径分析的L-异亮氨酸发酵溶氧控制研究.中国生物工程杂志.2007,27(2)：70-75.
[5]马雷,秦永峰,徐庆阳,谢希贤.溶氧对L-缬氨酸发酵过程的影响.生物技术通讯.2010,21(4)：509-514.
[6]何向飞,张梁,石贵阳.利用溶氧控制策略进行高密度和高强度乙醇发酵的初步研究.食品与发酵工业.2008,34(1)：20-23.
[7]华璟薇.黄原胶发酵条件的研究.浙江食品工业.2000,2(2)：16-22.
[8]Femado Flores, Lugs G.Tores. Efect of the dissolved oxygen tension during cultivation of X. Campestris on the production and qualily of xanthan gum. Journal of Biotechnology.1994, 34:165-173.
[9]张莹,孙君社,张京声,罗岩,胡锦蓉,刘萍.溶氧对巨大芽孢杆菌发酵亚硝酸还原酶的影响.中国酿造.2011,6：44-47.
[10]杨利,张旭,谭文松.溶氧水平与搅拌转速对发酵生产透明质酸分子量的影响.华东理工大学学报.2008,34(5)：805-808.
[11]谢涛,方慧英,诸葛斌,诸葛健.溶氧对Candidaglycerinogenes产甘油发酵过程的影响.中国生物工程杂志.2008,28(5)：65-70.
[12]陈宁.氨基酸工艺学.2007,北京：中国轻工业出版社.
[13]Xiao Jie, Shi Zhongping, Gao Pei, etal.On-line optimization of glutamate production based on balanced metabolic control by RQ. Bioprocess Biosyst Eng.2006,29(2):109-117.
[14]Lara, Galindo, Ramirez, Palomares.Living with heterogeneities in bioreactors. Molecular Biotechnology.2006,34(3):355-381.
[15]Bylund F., Castan A., Mikkola R., etal.Influence of scale-up on the quality of recombinant human growth hormone.Biotechnology and Bioengineering.2000,69(2): 119-128.
[16]Oosterhuis, N. M. G., Kossen, N.W.F., Olivier, A.P. C., and Schenk E. S...Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans. Biotechnology and Bioenginering.1985,27(5):711-720.
[17]Amanullah A., McFarlane C., M.Emery A., N.Nienow A.W.. Scale-down model to simulate spatial pH variations in large-scale bioreactors. Biotechnology and Bioengineering.2001,73(5):390-399.
[18]Schilling B. M., Pfefferle W., Bachmann B., Leuchtenberger W., Deckwer W.. A special reactor design for investigations of mixing time effects in a scaled-down industrial L-lysine fed-batch fermentation process. Biotechnology and Bioengineering.1999,64(5):599-606.
[19]Swapnil Bhargaval, Kevin S., Wenger, Kishore Rane,, Vanessa Rising, Mark R. Marten 1.Effect of cycle time on fungal morphology,broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon sourse. Biotechnology and Bioengineering.2005,89(5):524-529.
[20]Varley J., Birch J..Reactor design for large scale suspension animal cell culture. Cytotechnology.1999,29(3):177-205.
[21]Osman J.J., Birch J., Varley J.. The response of GS-NSO myeloma cells to single and multiple pH perturbations. Biotechnology and Bioengineering.2002,79(4):398-407.
[22]Palomares L. A. and Ramirez O. T..Bioreactor Scale-Down, in The Encyclopedia of Cell Technology.2000, New York.
[23]Doran P.M..Design of bioreactors for plant cells and organs. In Advances in Biochemical Engineering/Biotechnology.1993, Berlin.
[24]Ozturk S.S..Engineering challenges in high cell density cell culture systems. Cytotechnology.1996,22:3-16.
[25]Vlaev D., Mann R., Lossev V., Vlaev, S.D., Zahradnik, J., Seichter P..Macro mixing and Streptomyces fradiae: Modelling oxygen and nutrient segregation in an industrial bioreactor. Chemical Engineering Research and Design.2000,78(3):354-362.
[26]Hristov H., Mann R., Lossev V., Vlaev S. D., Seichter P.. A 3-D analysis of gas-liquid mixing, mass transfer and bioreaction in a stirred bioreactor.Food and Bioproducts Processing. 2001,79(4):232-241.
[27]Amanullah A., Buckland B. C., and Nienow A.W..Mixing in the fermentation and cell culture industries, in Handbook of Industrial Mixing:Science and Practice (Paul, E. L., Atiemo-Obeng V. A., and Kresta S. M., eds.), John Wiley & Sons, Hoboken NJ.2004, pp:1071-1170.
[28]Reuss M., Schmalzriedt S., and Jenne, M..Structured Modelling of Bioreactors, in Advances in Bioprocess Engineering (Galindo E. and Ramirez, O.T., eds.), Kluwer Academic Press, Dordrecht, TheNetherlands,1994, pp:207-215.
[29]Langheinrich C., Nienow A.W., Stevenson N.C., Enery A. N., Clayton T. M., and Slater, N.K.H..Mixing and oxygen transfer in a large scale animal cell bioreactor, in Biochemical Engineering 3 (Schmid, R. D,. ed.), Universitat Stuttgart, Stuttgart,1995, pp:162-164.
[30]Oosterhuis N.M.G., Kossen N.W.F..Dissolved oxygen concentration profiles in a production cale bioreactor. Biotechnology and Bioengineering.1984,26(5):546-550.
[31]Hopkins D.J., Betenbaugh M. J., Dhurjati P..Effects of dissolved oxygen tension on the stability of recombinant Escherichia coli containing plasmid pKN401.Biotechnology and Bioengineering.1987,29:85-91.
[32]Yegneswara P.K., Gray M. R., Thompson B.G..Experimental simulation of dissolved oxygen fluctuations in large fermentors:effect on Streptomyces clavuligerus. Biotechnology and Bioengineering.1991,38(10):1203-1209.
[33]Buse R., Qazi G. N., Onken U..Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum. Journal of Biotechnology.1992,26(2-3):231-244.
[34]Amanullah A., Nienow A.W., Emery A.N., McFarlane C.M..The use of Bacillus subtilis as an oxygen sensitive culture to simulate dissolved oxygen cycling in large scale fermenters. Trans. Inst.Chem. Eng. Part C.1993,71:206-208.
[35]Trujillo-Roldan M.A., Pena C., Ramirez O.T., Galindo E. Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii. Biotechnoloy Progress.2001,17(6):1042-1048.
[36]Serrato J.A., Palomares, L.A., Meneses-Acosta A., Ramirez O.T..Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures. Biotechnology and Bioengineering.2004,88(2):176-188.
[37]Sandoval-Basurto E., Gosset G., Bol'var F., Ramirez O.T..Culture of Escherichia coli under dissolved oxygen gradients simulated in a two compartment scale-down system: metabolic response and production of recombinant protein.Biotechnology and Bioengineering.2005,89(4):453-463.
[38]Cortes G., Trujillo-Roldan M.A., Ramirez O.T.,, Galindo E..Production of beta-galactosidase by Kluyveromyces marxianus under oscillating dissolved oxygen tension. Process Biochemistry.2005,40(2):773-778.
[39]Lara A.R., Leal L., Flores N., Gosset G., Bolivar F., Ramirez O.T..Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.Biotechnology and Bioengineering.2006,93(2): 372-385.
[40]Garcia-Ochoa F., Gomez E.. Bioreactor scale-up and oxygen transfer rate in microbial processes:An overview. Biotechnology Advances.2009,27(2):153-176.
[41]戚以政,汪叔雄.生化反应动力学与反应器.1999,北京：化学工业出版社.
[42]De Leon-Rodriguez A., Hernandez V, Galindo E and Ramirez O.T..Effects of dissolved oxygen tension on the production of recombinant penicillin acylase in Escherichia coli. Enzyme Microb Technol 2003,33:689-697.
[43]De Leon-Rodriguez A., Galindo E and Ramirez O.T..Design and characterization of a one-compartment scale-down system for simulating dissolved oxygen tension gradients[J]. Journal of Chemical Technology and Biotechnology,2010,85(7):950-956.
[44]Oosterhuis N.M.G., Kossen N.M.F., Oliver A.P.C., Schenk E.S..Scale-down and optimization studies of gluconic acid fermentation by Gluconobacter oxydans.Biotechnology and Bioengineering.1985,27:711-721.
[45]云逢霖.氧的传递及供氧对谷氨酸发酵的影响(续).发酵科技通讯.1981.
[46]George S., Larsson G., Enfors S.O..A scale-down 2-compartment reactor with controlled substrate oscillations metabolic response of Saccharomyces cerevisiae. Bioprocess Engineering.1993,9:249-250.
[47]Hewitt C.J., Nebe-Von Caron G., Axelsson B., et al.Studies related to the scale-up of high-cell-density E-coli fed-batch fermentations using multiparameter flow cytometry: Effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration. Biotechnology and Bioengineering.2000,70(4):381-390.
[48]Larsson G..Use of microbial metabolism to predict and control the bioreactor performance. PhD thesis, Royal Institute of Technology, Stockholm, Sweden.
[2]刘勇,陈雅丽,张长铠等.L-异亮氨酸发酵对氧需求的研究.生物技术.1998,8(1)：26-28.
[3]张伟国,陈坚,伦世仪.供氧对L-异亮氨酸分批发酵的影响.无锡轻工大学学报.2001,20(5)：539-542.
[4]杨宁,王健,徐庆阳,陈宁,温廷益.基于途径分析的L-异亮氨酸发酵溶氧控制研究.中国生物工程杂志.2007,27(2)：70-75.
[5]马雷,秦永峰,徐庆阳,谢希贤.溶氧对L-缬氨酸发酵过程的影响.生物技术通讯.2010,21(4)：509-514.
[6]何向飞,张梁,石贵阳.利用溶氧控制策略进行高密度和高强度乙醇发酵的初步研究.食品与发酵工业.2008,34(1)：20-23.
[7]华璟薇.黄原胶发酵条件的研究.浙江食品工业.2000,2(2)：16-22.
[8]Femado Flores, Lugs G.Tores. Efect of the dissolved oxygen tension during cultivation of X. Campestris on the production and qualily of xanthan gum. Journal of Biotechnology.1994, 34:165-173.
[9]张莹,孙君社,张京声,罗岩,胡锦蓉,刘萍.溶氧对巨大芽孢杆菌发酵亚硝酸还原酶的影响.中国酿造.2011,6：44-47.
[10]杨利,张旭,谭文松.溶氧水平与搅拌转速对发酵生产透明质酸分子量的影响.华东理工大学学报.2008,34(5)：805-808.
[11]谢涛,方慧英,诸葛斌,诸葛健.溶氧对Candidaglycerinogenes产甘油发酵过程的影响.中国生物工程杂志.2008,28(5)：65-70.
[12]陈宁.氨基酸工艺学.2007,北京：中国轻工业出版社.
[13]Xiao Jie, Shi Zhongping, Gao Pei, etal.On-line optimization of glutamate production based on balanced metabolic control by RQ. Bioprocess Biosyst Eng.2006,29(2):109-117.
[14]Lara, Galindo, Ramirez, Palomares.Living with heterogeneities in bioreactors. Molecular Biotechnology.2006,34(3):355-381.
[15]Bylund F., Castan A., Mikkola R., etal.Influence of scale-up on the quality of recombinant human growth hormone.Biotechnology and Bioengineering.2000,69(2): 119-128.
[16]Oosterhuis, N. M. G., Kossen, N.W.F., Olivier, A.P. C., and Schenk E. S...Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans. Biotechnology and Bioenginering.1985,27(5):711-720.
[17]Amanullah A., McFarlane C., M.Emery A., N.Nienow A.W.. Scale-down model to simulate spatial pH variations in large-scale bioreactors. Biotechnology and Bioengineering.2001,73(5):390-399.
[18]Schilling B. M., Pfefferle W., Bachmann B., Leuchtenberger W., Deckwer W.. A special reactor design for investigations of mixing time effects in a scaled-down industrial L-lysine fed-batch fermentation process. Biotechnology and Bioengineering.1999,64(5):599-606.
[19]Swapnil Bhargaval, Kevin S., Wenger, Kishore Rane,, Vanessa Rising, Mark R. Marten 1.Effect of cycle time on fungal morphology,broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon sourse. Biotechnology and Bioengineering.2005,89(5):524-529.
[20]Varley J., Birch J..Reactor design for large scale suspension animal cell culture. Cytotechnology.1999,29(3):177-205.
[21]Osman J.J., Birch J., Varley J.. The response of GS-NSO myeloma cells to single and multiple pH perturbations. Biotechnology and Bioengineering.2002,79(4):398-407.
[22]Palomares L. A. and Ramirez O. T..Bioreactor Scale-Down, in The Encyclopedia of Cell Technology.2000, New York.
[23]Doran P.M..Design of bioreactors for plant cells and organs. In Advances in Biochemical Engineering/Biotechnology.1993, Berlin.
[24]Ozturk S.S..Engineering challenges in high cell density cell culture systems. Cytotechnology.1996,22:3-16.
[25]Vlaev D., Mann R., Lossev V., Vlaev, S.D., Zahradnik, J., Seichter P..Macro mixing and Streptomyces fradiae: Modelling oxygen and nutrient segregation in an industrial bioreactor. Chemical Engineering Research and Design.2000,78(3):354-362.
[26]Hristov H., Mann R., Lossev V., Vlaev S. D., Seichter P.. A 3-D analysis of gas-liquid mixing, mass transfer and bioreaction in a stirred bioreactor.Food and Bioproducts Processing. 2001,79(4):232-241.
[27]Amanullah A., Buckland B. C., and Nienow A.W..Mixing in the fermentation and cell culture industries, in Handbook of Industrial Mixing:Science and Practice (Paul, E. L., Atiemo-Obeng V. A., and Kresta S. M., eds.), John Wiley & Sons, Hoboken NJ.2004, pp:1071-1170.
[28]Reuss M., Schmalzriedt S., and Jenne, M..Structured Modelling of Bioreactors, in Advances in Bioprocess Engineering (Galindo E. and Ramirez, O.T., eds.), Kluwer Academic Press, Dordrecht, TheNetherlands,1994, pp:207-215.
[29]Langheinrich C., Nienow A.W., Stevenson N.C., Enery A. N., Clayton T. M., and Slater, N.K.H..Mixing and oxygen transfer in a large scale animal cell bioreactor, in Biochemical Engineering 3 (Schmid, R. D,. ed.), Universitat Stuttgart, Stuttgart,1995, pp:162-164.
[30]Oosterhuis N.M.G., Kossen N.W.F..Dissolved oxygen concentration profiles in a production cale bioreactor. Biotechnology and Bioengineering.1984,26(5):546-550.
[31]Hopkins D.J., Betenbaugh M. J., Dhurjati P..Effects of dissolved oxygen tension on the stability of recombinant Escherichia coli containing plasmid pKN401.Biotechnology and Bioengineering.1987,29:85-91.
[32]Yegneswara P.K., Gray M. R., Thompson B.G..Experimental simulation of dissolved oxygen fluctuations in large fermentors:effect on Streptomyces clavuligerus. Biotechnology and Bioengineering.1991,38(10):1203-1209.
[33]Buse R., Qazi G. N., Onken U..Influence of constant and oscillating dissolved oxygen concentrations on keto acid production by Gluconobacter oxydans subsps. melanogenum. Journal of Biotechnology.1992,26(2-3):231-244.
[34]Amanullah A., Nienow A.W., Emery A.N., McFarlane C.M..The use of Bacillus subtilis as an oxygen sensitive culture to simulate dissolved oxygen cycling in large scale fermenters. Trans. Inst.Chem. Eng. Part C.1993,71:206-208.
[35]Trujillo-Roldan M.A., Pena C., Ramirez O.T., Galindo E. Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii. Biotechnoloy Progress.2001,17(6):1042-1048.
[36]Serrato J.A., Palomares, L.A., Meneses-Acosta A., Ramirez O.T..Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures. Biotechnology and Bioengineering.2004,88(2):176-188.
[37]Sandoval-Basurto E., Gosset G., Bol'var F., Ramirez O.T..Culture of Escherichia coli under dissolved oxygen gradients simulated in a two compartment scale-down system: metabolic response and production of recombinant protein.Biotechnology and Bioengineering.2005,89(4):453-463.
[38]Cortes G., Trujillo-Roldan M.A., Ramirez O.T.,, Galindo E..Production of beta-galactosidase by Kluyveromyces marxianus under oscillating dissolved oxygen tension. Process Biochemistry.2005,40(2):773-778.
[39]Lara A.R., Leal L., Flores N., Gosset G., Bolivar F., Ramirez O.T..Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.Biotechnology and Bioengineering.2006,93(2): 372-385.
[40]Garcia-Ochoa F., Gomez E.. Bioreactor scale-up and oxygen transfer rate in microbial processes:An overview. Biotechnology Advances.2009,27(2):153-176.
[41]戚以政,汪叔雄.生化反应动力学与反应器.1999,北京：化学工业出版社.
[42]De Leon-Rodriguez A., Hernandez V, Galindo E and Ramirez O.T..Effects of dissolved oxygen tension on the production of recombinant penicillin acylase in Escherichia coli. Enzyme Microb Technol 2003,33:689-697.
[43]De Leon-Rodriguez A., Galindo E and Ramirez O.T..Design and characterization of a one-compartment scale-down system for simulating dissolved oxygen tension gradients[J]. Journal of Chemical Technology and Biotechnology,2010,85(7):950-956.
[44]Oosterhuis N.M.G., Kossen N.M.F., Oliver A.P.C., Schenk E.S..Scale-down and optimization studies of gluconic acid fermentation by Gluconobacter oxydans.Biotechnology and Bioengineering.1985,27:711-721.
[45]云逢霖.氧的传递及供氧对谷氨酸发酵的影响(续).发酵科技通讯.1981.
[46]George S., Larsson G., Enfors S.O..A scale-down 2-compartment reactor with controlled substrate oscillations metabolic response of Saccharomyces cerevisiae. Bioprocess Engineering.1993,9:249-250.
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