Continuous Culture

An-Ping Zeng; Jibin Sun

doi:10.1128/9781555816827.ch49

Chapter 49 : Continuous Culture

Authors: An-Ping Zeng, Jibin Sun

Content Type: Reference

Publication Year: 2010

Category: Applied and Industrial Microbiology

Book DOI: 10.1128/9781555816827

Chapter DOI: 10.1128/9781555816827.ch49

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:

Continuous Culture, Page 1 of 2

< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap49-1.gif /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap49-2.gif

Abstract:

The major applications of continuous culture are, however, still found in fundamental studies and process optimization at laboratory scale. This chapter provides a brief introduction to the general concept and theory of different types of continuous culture. The design and operation of equipment and experiments are discussed from an application point of view. Depending on the control parameter and the operation mode, continuous culture can be classified into four general types. The general concept and theory of four types of continuous culture are described in a detailed manner. A chemostat is usually started as a batch culture. The specific growth rate of a chemostat culture can be determined from a material balance for biomass: net increase in biomass = biomass in incoming medium + growth - output - death. Generally speaking, auxostats have the following advantages over conventional chemostats. First, auxostats permit stable operation in the “high-gain” areas near the maximum growth rate. Second, they reach steady state more rapidly at high dilution rates than the open-loop chemostat. Third, population selection pressures in an auxostat lead to cultures that grow rapidly. Finally, it is possible to design a dual set point auxostat that controls two growth parameters simultaneously. Nutrient reservoirs for continuous culture should have ports for feeding, addition and/or mixing of heat-labile nutrients and substrate, venting, and sparging of the medium. In particular, recent developments in implementing continuous culture in microfluidics or microdevices are worth mentioning and are briefly summarized in this chapter.

Citation: Zeng A, Sun J. 2010. Continuous Culture, p 685-699. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch49

Highlighted Text: Show | Hide

Full text loading...

Figures

Continuous Culture

[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555816827.ch49-1,webId=/content/author/10.1128/9781555816827.ch49-1,properties={foaf_givenname=An-Ping, foaf_name=An-Ping Zeng, foaf_surname=Zeng}], com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555816827.ch49-2,webId=/content/author/10.1128/9781555816827.ch49-2,properties={foaf_givenname=Jibin, foaf_name=Jibin Sun, foaf_surname=Sun}]]

/content/10.1128/9781555816827.ch49.ch49fig01

FIGURE 1

Schematic diagram of four types of continuous culture.

10.1128/9781555816827/f0686-01_thmb.gif

10.1128/9781555816827/f0686-01.gif

FIGURE 1

Schematic diagram of four types of continuous culture.

/content/10.1128/9781555816827.ch49.ch49fig02

FIGURE 2

Steady-state values of biomass and growth-limiting substrate concentration in a chemostat according to equations 9 and 10. Parameters: μmax = 1.0 h-1; Ks = 0.01 g/liter;

= 0.5 g/g.

10.1128/9781555816827/f0687-01_thmb.gif

10.1128/9781555816827/f0687-01.gif

FIGURE 2

Steady-state values of biomass and growth-limiting substrate concentration in a chemostat according to equations 9 and 10. Parameters: μmax = 1.0 h-1; Ks = 0.01 g/liter; = 0.5 g/g.

/content/10.1128/9781555816827.ch49.ch49fig03

FIGURE 3

Typical profiles and reasons for continuous cultures deviating from chemostat behavior. The biomass curve of a corresponding chemostat culture (see Fig. 2 ) is also shown for comparison. (A) Effect of wall growth. K is the amount of growing biomass attached to the reactor surface per unit volume of culture. (B) Effect of imperfect mixing. (C) Effect of maintenance metabolism. ms is the maintenance requirement for substrate (g/g • h). (D) Effect of growth inhibition by product(s) and/or substrate.

10.1128/9781555816827/f0688-01_thmb.gif

10.1128/9781555816827/f0688-01.gif

FIGURE 3

/content/10.1128/9781555816827.ch49.ch49fig04

FIGURE 4

Steady-state limiting-nutrient concentration, S̅, and dilution rate, D, as functions of biomass concentration in a turbidostat culture. Growth parameters are as given in the legend to Fig. 2 .

10.1128/9781555816827/f0689-01_thmb.gif

10.1128/9781555816827/f0689-01.gif

FIGURE 4

Steady-state limiting-nutrient concentration, S̅, and dilution rate, D, as functions of biomass concentration in a turbidostat culture. Growth parameters are as given in the legend to Fig. 2 .

/content/10.1128/9781555816827.ch49.ch49fig05

FIGURE 5

A typical setup of a bench-scale continuous-culture system. The feeding rate and the culture volume are controlled by a weight control unit.

10.1128/9781555816827/f0691-01_thmb.gif

10.1128/9781555816827/f0691-01.gif

FIGURE 5

A typical setup of a bench-scale continuous-culture system. The feeding rate and the culture volume are controlled by a weight control unit.

/content/10.1128/9781555816827.ch49.ch49fig06

FIGURE 6

Diagram of a drip tube.

10.1128/9781555816827/f0692-01_thmb.gif

10.1128/9781555816827/f0692-01.gif

FIGURE 6

Diagram of a drip tube.

/content/10.1128/9781555816827.ch49.ch49fig07

FIGURE 7

Control scheme for the reactor volume of a continuous culture that uses a liquid level controller backed up by an effluent gas system. (Modified from reference 66 .)

10.1128/9781555816827/f0692-02_thmb.gif

10.1128/9781555816827/f0692-02.gif

FIGURE 7

Control scheme for the reactor volume of a continuous culture that uses a liquid level controller backed up by an effluent gas system. (Modified from reference 66 .)

/content/10.1128/9781555816827.ch49.ch49fig08

FIGURE 8

Schematic diagram of a back-flushing configuration for a cell recycle reactor with membrane microfiltration. The permeate in the reservoir is back-flushed through the membrane module by using pressurized nitrogen gas or air and controlled by a timer.

10.1128/9781555816827/f0693-01_thmb.gif

10.1128/9781555816827/f0693-01.gif

FIGURE 8

/content/10.1128/9781555816827.ch49.ch49fig09

FIGURE 9

Transient behavior of a steady state after step changes in a continuous culture operated at a constant dilution rate. The culture is subjected to metabolic overflow and growth inhibition by substrate and products. The substrate concentration in the feed (y 0) is increased from (A) 0.334 to 0.550 (in dimensionless units), (B) 0.334 to 0.400, and (C) 0.334 to 0.335. The relative time is numerically equal to the number of reactor volume exchanges (residence times) ( 64 ).

10.1128/9781555816827/f0695-01_thmb.gif

10.1128/9781555816827/f0695-01.gif

FIGURE 9

/content/10.1128/9781555816827.ch49.ch49fig010

FIGURE 10

Steady-state concentrations of biomass (A), substrate in reactor (B), and product (C) in a continuous culture at a constant feed substrate concentration but varied dilution rates (dimensionless feed substrate concentration, y 0 = 0.2). This culture is subject to metabolic overflow and growth inhibition by product. Multiple steady states are found in a given range of dilution rates. Depending on the operation mode, different steady states can be obtained under identical operation conditions ( 64 ).

10.1128/9781555816827/f0696-01_thmb.gif

10.1128/9781555816827/f0696-01.gif

FIGURE 10

References

/content/book/10.1128/9781555816827.ch49

1. Axelsson, J. P.,, T. Münch, and, B. Sonnleitner. 1992. Multiple steady states in continuous cultivation of yeast, p. 383– 386. In M. N. Karim and, G. Stephanopoulos (ed.), Proceedings of ICCAFT5/IFAC Modeling and Control of Biotechnical Processes, Keystone, CO. Pergamon Press, Oxford, UK.

2. Bailey, J. E., and, D. F. Ollis. 1986. Biochemical Engineering Fundamentals. McGraw-Hill, New York, NY.

3. Bailey, J. E.,, A. Sburlati,, V. Hatzimanikatis,, K. Lee,, W. A. Renner, and, P. S. Tsai. 2002. Inverse metabolic engineering: a strategy for directed genetic engineering of useful phenotypes. Biotechnol. Bioeng. 79: 568– 579.

4. Baloo, S., and, D. Ramkrishna. 1991. Metabolic regulation in bacterial continuous cultures. I. Biotechnol. Bioeng. 38: 1337– 1352.

5. Banik, G. G., and, C. A. Heath. 1995. Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture. Biotechnol. Bioeng. 48: 289– 300.

6. Barford, J. P.,, J. H. Johnston, and, P. K. Mwesigye. 1995. Continuous culture study of transient behaviour of Saccharomyces cerevisiae growing on sucrose and fructose. J. Ferment. Bioeng. 79: 158– 162.

7. Benthin, S.,, J. Nielsen, and, J. Villadsen. 1993. Two uptake systems for fructose in Lactococcus lactis subsp. cremoris FD1 produce glycolytic and gluconeogenic fructose phosphates and induce oscillations in growth and lactic acid formation. Appl. Environ. Microbiol. 59: 3206– 3211.

8. Bentley, W., and, D. S. Kompala. 1990. Stability in continuous cultures of recombinant bacteria: a metabolic approach. Biotechnol. Lett. 12: 329– 334.

9. Biebl, H. 1991. Glycerol fermentation to 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat. Appl. Microbiol. Biotechnol. 35: 701– 705.

10. Bro, C, and, J. Nielsen. 2004. Impact of ’ome’ analyses on inverse metabolic engineering. Metab. Eng. 6: 204– 211.

11. Brown, D. E.,, D. J. Halsted, and, C. G. Sinclair. 1979. Application of an aerated mixing model to a continuous culture system. Biotechnol. Lett. 1: 159– 164.

12. Byun, T. G.,, A.-P. Zeng, and, W.-D. Deckwer. 1994. Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate. Bioprocess Eng. 11: 167– 175.

13. Calcott, P. H. 1981. Continuous Culture of Cells, vol. 1 and vol. 2. CRC Press, Inc., Boca Raton, FL.

14. Charati, S., and, S. Stern. 1998. Diffusion of gases in silicone polymers: molecular dynamics simulation. Macro-molecules 31: 5529– 5535.

15. Cooney, C. L.,, H. M. Koplove, and, M. Häggstrom. 1981. Transient phenomena in continuous culture, p. 143– 168. In P. H. Calcott (ed.), Continuous Culture of Cells, vol. 2. CRC Press, Inc., Boca Raton, FL.

16. Daran-Lapujade, P.,, J. M. Daran,, A. J. van Maris,, J. H. de Winde, and, J. T. Pronk. 2009. Chemostat-based micro-array analysis in baker’s yeast. Adv. Microb. Physiol. 54: 257– 311.

17. Deckwer, W.-D.,, R. Luttmann,, H. G. Reng, and, S. Yon-sel. 1987. Bioreaktoren: Ein Leitfaden für Anwender. GBF Texte 4, Braunschweig-Stöckheim, Germany.

18. Dunn, I. J., and, J. R. Mor. 1975. Variable volume continuous cultivation. Biotechnol. Bioeng. 17: 1805– 1822.

19. Dykhuizen, D. E., and, D. L. Hard. 1983. Selection in chemostats. Microbiol. Rev. 47: 150– 168.

20. El-Ali, J.,, P. K. Sorger, and, K. F. Jensen. 2006. Cells on chips. Nature 442: 403– 411.

21. Ferenci, T. 2008. Bacterial physiology, regulation and mutational adaptation in a chemostat environment. Adv. Microb. Physiol. 53: 169– 229.

22. Fiechter, A. 1981. Batch and continuous culture of microbial, plant and animal cells, p. 454– 505. In H. J. Rehm and, G Reed (ed.), Biotechnology: a Comprehensive Treatise, vol. 1. Verlag Chemie, Weinheim, Germany.

23. Frame, K. K., and, W.-S. Hu. 1991. Kinetic study of hybridoma cell growth in continuous culture. II. Behavior of producers and comparison to non-producers. Biotechnol. Bioeng. 38: 1020– 1028.

24. Frame, K. K., and, W.-S. Hu. 1991. Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells. Biotechnol. Bioeng. 37: 55– 64.

25. Gostomski, P.,, M. Miihlemann,, Y.-H. Lin,, R. Mormino, and, H. Bungay. 1994. Auxostats for continuous culture research. J. Biotechnol. 37: 167– 177.

26. Gottschal, J. C. 1992. Continuous culture, p. 559– 572. In J. Lederberg (ed.), Encyclopedia of Microbiology, vol. 1. Academic Press, Inc., New York, NY.

27. Gregory, M. E.,, M. Bulmer,, I. D. L. Bogle, and, N. Titcherner-Hooker. 1996. Optimising enzyme production by bakers yeast in continuous culture: physiological knowledge useful for process design and control. Bioprocess Eng. 15: 239– 245.

28. Harrison, D. E. F., and, H. H. Topiwala. 1974. Transient and oscillatory states of continuous culture, p. 168– 219. In T. H. Ghose and, A. Fiechter (ed.), Advanced Biochemical Engineering, vol. 3. Springer-Verlag, Berlin, Germany.

29. Hellmuth, K.,, D. J. Korz,, E. A. Sanders, and, W.-D. Deckwer. 1994. Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1. J. Biotechnol. 32: 289– 298.

30. Herbert, D.,, R. Elsworth, and, R. C. Telling. 1956. The continuous culture of bacteria; a theoretical and experimental study. J. Gen. Microbiol. 14: 601– 622.

31. Hiller, G. W.,, D. S. Clark, and, H. W. Blanch. 1993. Cell retention-chemostat studies of hybridoma cells: analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium. Biotechnol. Bioeng. 42: 185– 195.

32. Hiller, G. W.,, D. S. Clark, and, H. W. Blanch. 1994. Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations. Biotechnol. Bioeng. 44: 303– 321.

33. Hoskisson, P. A., and, G. Hobbs. 2005. Continuous culture—making a comeback? Microbiology 151( Pt. 10): 3153– 3159.

34. Hung, P. J.,, P. J. Lee,, P. Sabounchi,, N. Aghdam,, R. Lin, and, L. P. Lee. 2005. A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array. Lab Chip 5: 44– 48.

35. Hung, P. J.,, P. J. Lee,, P. Sabounchi,, R. Lin, and, L. P. Lee. 2005. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol. Bioeng. 89: 1– 8.

36. Imanaka, T.,, T. Kaieda,, K. Sato, and, H. Taguchi. 1972. Optimization of a-galactosidase production by mold. I. a-Galactosidase production in batch and continuous culture and a kinetic model for enzyme production. J. Ferment. Technol. 50: 633– 646.

37. Jang, K.,, K. Sato,, K. Igawa,, U. I. Chung, and, T. Kitamori. 2008. Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening. Anal. Bioanal. Chem. 390: 825– 832.

38. Kiss, R. D., and, G. Stephanopoulos. 1992. Culture instability of auxotrophic amino acid producers. Biotechnol. Bioeng. 40: 75– 85.

39. Kubitschek, H. E. 1970. Introduction to Research with Continuous Culture. Prentice Hall, Englewood Cliffs, NJ.

40. Leclerc, E.,, Y. Sakai, and, T. Fujii. 2004. Perfusion culture of fetal human hepatocytes in microfluidic environments. Biochem. Eng. J. 20: 143– 148.

41. Lee, C. W., and, H. N. Chang. 1987. Kinetics of etha-nol fermentations in membrane cell recycle fermentors. Biotechnol. Bioeng. 29: 1105– 1112.

42. Lee, P. J.,, P. J. Hung,, V. M. Rao, and, L. P. Lee. 2006. Nanoliter scale microbioreactor array for quantitative cell biology. Biotechnol. Bioeng. 94: 5– 14.

43. Linz, M.,, A.-P. Zeng,, R. Wagner, and, W.-D. Deckwer. 1997. Stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous cultures. Biotechnol. Prog. 13: 453– 463.

44. Marr, A. G. 1991. Growth rate of Escherichia coli. Microbiol. Rev. 55: 316– 333.

45. Menzel, K.,, A.-P. Zeng,, H. Biebl, and, W.-D. Deckwer. 1996. Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture. Part I. The phenomena and characterization of oscillation and hysteresis. Biotechnol. Bioeng. 52: 549– 560.

46. Merten, O. W.,, D. Moeurs,, H. Keller,, M. Leno,, G. E. Palfi,, L. Cabanie, and, E. Couve. 1994. Modified monoclonal antibody production kinetics, kappa/gamma mRNA levels, and metabolic activities in a murine hybridoma selected by continuous culture. Biotechnol. Bioeng. 44: 753– 764.

47. Meyvantsson, I.,, J. W. Warrick,, S. Hayes,, A. Skoien, and, D. J. Beebe. 2008. Automated cell culture in high density tube less microfluidic device arrays. Lab Chip 8: 717– 724.

48. Minkevich, I. G.,, A. Yu Krynitskaya, and, V. K. Eroshin. 1989. Bistat: a novel method of continuous cultivation. Biotechnol. Bioeng. 33: 1157– 1161.

49. Mohan, S. B.,, S. R. Chohan,, J. Eade, and, A. Lyddiatt. 1993. Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery. Biotechnol. Bioeng. 42: 974– 986.

50. Monod, J. 1950. La technique de culture continue: theorie et application. Ann. Inst. Pasteur (Paris) 79: 390.

51. Moser, A. 1985. Continuous cultivation, p. 285– 309. In H. J. Rehm and, G. Reed (ed.), Biotechnology: a Comprehensive Treatise, vol. 2. Verlag Chemie, Weinheim, Germany.

52. Nancib, N., and, J. Boudrant. 1992. Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture of Escherichia coli in a non-selective medium. Biotechnol. Lett. 14: 643– 648.

53. Novick, A., and, L. Szilard. 1950. Description of the che-mostat. Science 112: 715– 716.

54. Park, T. H., and, M. L. Shuler. 2003. Integration of cell culture and microfabrication technology. Biotechnol. Prog. 19: 243– 253.

55. Petrik, M.,, O. Käppeli, and, A. Fiechter. 1983. An expanded concept for the glucose effect in the yeast Sac-charomyces uvarum: involvement of short- and long-term regulation. J. Gen. Microbiol. 129: 43– 49.

56. Pirt, S. J. 1975. Principles of Microbe and Cell Cultivation. Blackwell Scientific Publications, Oxford, UK.

57. Pirt, S. J. 1965. The maintenance energy of bacteria in growing cultures. Proc. R. Soc. Lond. Ser. B 163: 305– 314.

58. Postma, E.,, C. Verduyn,, W. A. Scheffers, and, J. P. van Dijken. 1989. Enzymic analysis of the Crabtree effect in glucose-limited chemostat cultures of Saccharomyces cere-visiae. Appl. Environ. Microbiol. 55: 468– 477.

59. Powers, M. J.,, K. Domansky,, M. R. Kaazempur-Mofrad,, A. Kalezi,, A. Capitano,, A. Upadhyaya,, P. Kurzawski,, K. E. Wack,, D. B. Stolz,, R. Kamm, and, L. G. Griffith. 2002. A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 78: 257– 269.

60. Rice, C. W., and, W. P. Hempfling. 1985. Nutrient-limited continuous culture in the pH-auxostat. Biotechnol. Bioeng. 27: 187– 191.

61. Sanchez, O.,, H. van Gemerden, and, J. Mas. 1996. Description of a redox-controlled sulfidostat for the growth of sulfide-oxidizing phototrophs. Appl. Environ. Microbiol. 62: 3640– 3645.

62. Sauer, U. 2001. Evolutionary engineering of industrially important microbial phenotypes. Adv. Biochem. Eng. Biotechnol. 73: 129– 169.

63. Schügerl, K. 1991. Common instruments for process analysis and control, p. 5– 25. In H. J. Rehm and, G. Reed (ed.), Biotechnology: a Comprehensive Treatise, vol. 4. Verlag Chemie, Weinheim, Germany.

64. Shackman, J. G.,, G. M. Dahlgren,, J. L. Peters, and, R. T. Kennedy. 2005. Perfusion and chemical monitoring of living cells on a microfluidic chip. Lab Chip 5: 56– 63.

65. Shoham, Y., and, A. L. Demain. 1991. Kinetics of loss of a recombinant plasmid in Bacillus subtilis. Biotechnol. Bioeng. 37: 927– 935.

66. Stafford, K. 1986. Continuous fermentation, p. 137– 151. In A. L. Demain and, N. A. Solomon (ed.), Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology, Washington, DC.

67. Tempest, D. W., and, O. M. Neijssel. 1992. Physiological and energetic aspects of bacterial metabolite overproduction. FEMS Microbiol. Lett. 79: 169– 176.

68. Thompson, D. M.,, K. R. King,, K. J. Wieder,, M. Toner,, M. L. Yarmush, and, A. Jayaraman. 2004. Dynamic gene expression profiling using a microfabricated living cell array. Anal. Chem. 76: 4098– 4103.

69. Toda, K. 2003. Theoretical and methodological studies of continuous microbial bioreactors. J. Gen. Appl. Microbiol. 49: 219– 233.

70. Togna, A. P.,, J. Fu, and, M. L. Shuler. 1993. Use of a simple mathematical model to predict the behavior of Escherichia coli overproducing β-lactamase within continuous single- and two-stage reactor systems. Biotechnol. Bioeng. 42: 557– 570.

71. Topiwala, H. H., and, G. Hamer. 1971. Effect of wall growth in steady-state continuous cultures. Biotechnol. Bioeng. 13: 919– 922.

72. Vierheller, C.,, A. Goel,, M. Peterson,, M. M. Domach, and, M. M. Ataai. 1995. Sustained and constitutive high levels of protein production in continuous cultures of Bacillussubtilis. Biotechnol. Bioeng. 47: 520– 524.

73. Walker, G. M.,, M. S. Ozers, and, D. J. Beebe. 2002. Insect cell culture in microfluidic channels. Biomed. Microdevices 4: 161– 166.

74. Walker, G. M.,, H. C. Zeringue, and, D. J. Beebe. 2004. Microenvironment design considerations for cellular scale studies. Lab Chip 4: 91– 97.

75. Werner, R. G.,, R. Walz,, W. Noe, and, A. Konrad. 1992. Safety and economic aspects of continuous mammalian cell culture. J. Biotechnol. 22: 51– 68.

76. Weusthuis, R. A.,, J. T. Pronk,, P. J. van den Broek, and, J. P. van Dijken. 1994. Chemostat cultivation as a tool for studies on sugar transport in yeasts. Microbiol. Rev. 58: 616– 630.

77. Xiu, Z.-L.,, A.-P. Zeng, and, W.-D. Deckwer. 1998. Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition. Biotechnol. Bioeng. 57: 251– 261.

78. Yang, R. D., and, A. E. Humphrey. 1975. Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures. Biotechnol. Bioeng. 17: 1211– 1235.

79. Zeng, A.-P., and, W.-D. Deckwer. 1995. A kinetic model for substrate and energy consumption of microbial growth under substrate-sufficient conditions. Biotechnol. Prog. 11: 71– 79.

80. Zeng, A.-P., and, W.-D. Deckwer. 1995. Mathematical modeling and analysis of glucose and glutamine utilization and regulation in cultures of continuous mammalian cells. Biotechnol. Bioeng. 47: 334– 346.

81. Zeng, A.-P.,, H. Biebl, and, W.-D. Deckwer. 1990. 2,3-Butanediol production by Enterobacter aerogenes in continuous culture: role of oxygen supply. Appl. Microbiol. Biotechnol. 33: 264– 268.

82. Zeng, A.-P.,, H. Biebl, and, W.-D. Deckwer. 1991. Production of 2,3-butanediol in a membrane bioreactor. Appl. Microbiol. Biotechnol. 34: 463– 468.

83. Zeng, A.-P., and, W.-D. Deckwer. 1996. Bioreaction techniques under low oxygen tension and oxygen limitation: from molecular level to pilot plant reactor. Chem. Eng. Sci. 51: 2305– 2314.

84. Zhang, Z.,, P. Boccazzi,, H. G. Choi,, G. Perozziello,, A. J. Sinskey, and, K. F. Jensen. 2006. Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor. Lab Chip 6: 906– 913.

85. Zhu, X.,, C. L. Yi,, B. H. Chueh,, M. Shen,, B. Hazarika,, N. Phadke, and, S. Takayama. 2004. Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation. Analyst 129: 1026– 1031.