Applied Microbiology and Biotechnology

, Volume 90, Issue 1, pp 69–76 | Cite as

Growth and recombinant protein expression with Escherichia coli in different batch cultivation media

Biotechnological Products and Process Engineering
First Online:
Received:
Revised:
Accepted:

Abstract

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L−1. EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L−1. The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.

Keywords

Milliliter stirred-tank bioreactor Protein expression Optical sensors Alcohol dehydrogenase Formate dehydrogenase Batch cultivation 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The authors gratefully acknowledge Peter Neubauer (TU Berlin, Germany) and Antje Neubauer (BioSilta, Oulu, Finland) for helpful discussions with the EnBase experiments. Furthermore, the authors thank BioSilta for kindly providing the EnBase medium.

References

  1. Bräutigam S, Bringer-Meyer S, Weuster-Botz D (2007) Asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems by use of recombinant Escherichia coli with intracellular cofactor regeneration. Tetrahedron Asymmetr 18:1883–1887CrossRefGoogle Scholar
  2. Bräutigam S, Dennewald D, Schürmann M, Lutje-Spielberg J, Pintner WR, Weuster-Botz D (2009) Whole-cell biocatalysis: evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones. Enzyme Microb Technol 45:310–316CrossRefGoogle Scholar
  3. Hoefel T, Wittmann E, Reinecke L, Weuster-Botz D (2010) Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16. Appl Microbiol Biotechnol 88:477–484CrossRefGoogle Scholar
  4. Hortsch R, Weuster-Botz D (2010a) Power consumption and maximum energy dissipation in a milliliter-scale bioreactor. Biotechnol Prog 26:595–599Google Scholar
  5. Hortsch R, Weuster-Botz D (2010b) Milliliter-scale stirred tank reactors for the cultivation of microorganisms. Adv Appl Microbiol 73:59–80Google Scholar
  6. Hortsch R, Stratmann A, Weuster-Botz D (2010a) New milliliter-scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms. Biotechnol Bioeng 106:443–451Google Scholar
  7. Hortsch R, Krispin H, Weuster-Botz D (2010b) Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae. Bioprocess Biosyst Eng. doi: 10.1007/s00449-010-0471-1 Google Scholar
  8. Knorr B, Schlieker H, Hohmann HP, Weuster-Botz D (2007) Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis. Biochem Eng J 33:263–274CrossRefGoogle Scholar
  9. Krause M, Ukkonen K, Haataja T, Ruottinen M, Glumoff T, Neubauer A, Neubauer P, Vasala A (2010) A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 9:11CrossRefGoogle Scholar
  10. Kusterer A, Krause C, Kaufmann K, Arnold M, Weuster-Botz D (2008) Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations. Bioprocess Biosyst Eng 31:207–215CrossRefGoogle Scholar
  11. Nakamura K, Yamanaka R, Matsuda T, Harada T (2003) Recent developments in assymetric reduction of ketones with biocatalysts. Tetrahedron Asymmetr 14:2659–2681CrossRefGoogle Scholar
  12. Panula-Perälä J, Siurkus J, Vasala A, Wilmanowski R, Casteleijn MG, Neubauer P (2008) Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb Cell Fact 7:31CrossRefGoogle Scholar
  13. Phue JN, Noronha SB, Hattacharyya R, Wolfe AJ, Shiloach J (2005) Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses. Biotechnol Bioeng 90:805–820CrossRefGoogle Scholar
  14. Puskeiler R, Kaufmann K, Weuster-Botz D (2005a) Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD). Biotechnol Bioeng 89:512–523CrossRefGoogle Scholar
  15. Puskeiler R, Kusterer A, John GT, Weuster-Botz D (2005b) Miniature bioreactors for automated high-throughput bioprocess design (HTBD): reproducibility of parallel fed-batch cultivations with Escherichia coli. Biotechnol Appl Biochem 42:227–235CrossRefGoogle Scholar
  16. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  17. Siurkus J, Panula-Perälä J, Horn U, Kraft M, Rimseliene R, Neubauer P (2010) Novel approach of high cell density recombinant bioprocess development: optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures. Microb Cell Fact 9:35CrossRefGoogle Scholar
  18. Vester A, Hans M, Hohmann HP, Weuster-Botz D (2009) Discrimination of riboflavin producing Bacillus subtilis strains based on their fed-batch process performances on a millilitre scale. Appl Microbiol Biotechnol 84:71–76CrossRefGoogle Scholar
  19. Weuster-Botz D (2005) Parallel reactor systems for bioprocess development. Adv Biochem Eng Biotechnol 92:125–144Google Scholar
  20. Weuster-Botz D, Puskeiler R, Kusterer A, Kaufmann K, John GT, Arnold M (2005) Methods and milliliter scale devices for high-throughput bioprocess design. Bioprocess Biosyst Eng 28:109–119CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Lehrstuhl für BioverfahrenstechnikTechnische Universität MünchenGarchingGermany

Personalised recommendations