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Bioprocess and Biosystems Engineering

, Volume 42, Issue 6, pp 953–961 | Cite as

Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus

  • S. Lladó Maldonado
  • J. Krull
  • D. Rasch
  • P. Panjan
  • A. M. Sesay
  • M. P. C. Marques
  • N. Szita
  • R. KrullEmail author
Research Paper

Abstract

Bioreactors at the microliter scale offer a promising approach to accelerate bioprocess development. Advantages of such microbioreactors include a reduction in the use of expensive reagents. In this study, a chemostat operation mode of a cuvette-based microbubble column bioreactor made of polystyrene (working volume of 550 µL) was demonstrated. Aeration occurs through a nozzle (Ø ≤ 100 µm) and supports submerged whole-cell cultivation of Staphylococcus carnosus. Stationary concentrations of biomass and glucose were determined in the dilution rate regime ranging from 0.12 to 0.80 1/h with a glucose feed concentration of 1 g/L. For the first time, reaction kinetics of S. carnosus were estimated from data obtained from continuous cultivation. The maximal specific growth rate (µmax = 0.824 1/h), Monod constant (KS = 34 × 10− 3gS/L), substrate-related biomass yield coefficient (YX/S = 0.315 gCDW/gS), and maintenance coefficient (mS = 0.0035 gS/(gCDW·h)) were determined. These parameters are now available for further studies in the field of synthetic biology.

Keywords

Chemostat Reaction kinetics Microbioreactor Microbubble column bioreactor Staphylococcus carnosus 

Abbreviations

GOx

Glucose oxidase

MBR

Microbioreactor

PDMS

Polydimethylsiloxane

PMMA

Polymethyl methacrylate

µBC

Microbubble column bioreactor

List of symbols

CDW

Cell dry weight (g)

cS

Substrate concentration (g/L)

cCDW

Biomass concentration (g/L)

D

Dilution rate (1/h)

DO

Dissolved oxygen (%)

F

Flow rate (L/h)

kLa

Volumetric liquid-phase mass transfer coefficient (1/h)

KS

Monod constant (gS/L)

mS

Maintenance coefficient (gS/(gCDW·h))

OD

Optical density (-)

Pr

Biomass-related productivity (gCDW/(L·h))

qS

Specific substrate uptake rate (gS/(gCDW·h))

V

Reaction volume (L)

YX/S

Substrate-related biomass yield coefficient (gCDW/gS)

μ

Specific growth rate (1/h)

Notes

Acknowledgements

The authors thank Prof. Dr. Friedrich Götz, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Germany, for providing the Staphylococcus carnosus TM300 GFP strain. The authors gratefully acknowledge the financial support provided by the People Programme (Marie Curie Actions, Multi-ITN) of the European Union’s Seventh Framework Programme for research and technological development and demonstration within the project EUROMBREuropean network for innovative microbioreactor applications in bioprocess development (Project ID 608104).

References

  1. 1.
    Kirk TV, Szita N (2013) Oxygen transfer characteristics of miniaturized bioreactor systems. Biotechnol Bioeng 110:1005–1019.  https://doi.org/10.1002/bit.24824 CrossRefGoogle Scholar
  2. 2.
    Krull R, Lladó Maldonado S, Lorenz T et al (2016) Microbioreactors. In: Dietzel A (ed) Microsystems for pharmatechnology. Manipulation of fluids, particles, droplets, and cells. Springer International Publishing, Cham, pp 99–152CrossRefGoogle Scholar
  3. 3.
    Marques MPC, Szita N (2017) Bioprocess microfluidics: applying microfluidic devices for bioprocessing. Curr Opin Chem Eng 18:61–68.  https://doi.org/10.1016/j.coche.2017.09.004 CrossRefGoogle Scholar
  4. 4.
    Gruber P, Marques MPC, Szita N, Mayr T (2017) Integration and application of optical chemical sensors in microbioreactors. Lab Chip 17:2693–2712.  https://doi.org/10.1039/C7LC00538E CrossRefGoogle Scholar
  5. 5.
    Lasave LC, Borisov SM, Ehgartner J, Mayr T (2015) Quick and simple integration of optical oxygen sensors into glass-based microfluidic devices. RSC Adv 5:70808–70816.  https://doi.org/10.1039/C5RA15591F CrossRefGoogle Scholar
  6. 6.
    Ehgartner J, Sulzer P, Burger T et al (2016) Online analysis of oxygen inside silicon-glass microreactors with integrated optical sensors. Sensors Actuators B Chem 228:748–757.  https://doi.org/10.1016/j.snb.2016.01.050 CrossRefGoogle Scholar
  7. 7.
    Pfeiffer SA, Nagl S (2015) Microfluidic platforms employing integrated fluorescent or luminescent chemical sensors: a review of methods, scope and applications. Methods Appl Fluoresc 3:34003.  https://doi.org/10.1088/2050-6120/3/3/034003 CrossRefGoogle Scholar
  8. 8.
    Panjan P, Virtanen V, Sesay AM (2018) Towards microbioprocess control: an inexpensive 3D printed microbioreactor with integrated online real-time glucose monitoring. Analyst 143:3926–3933.  https://doi.org/10.1039/c8an00308d CrossRefGoogle Scholar
  9. 9.
    Bolic A, Larsson H, Hugelier S et al (2016) A flexible well-mixed milliliter-scale reactor with high oxygen transfer rate for microbial cultivations. Chem Eng J 303:655–666.  https://doi.org/10.1016/j.cej.2016.05.117 CrossRefGoogle Scholar
  10. 10.
    Peterat G, Schmolke H, Lorenz T et al (2014) Characterization of oxygen transfer in vertical microbubble columns for aerobic biotechnological processes. Biotechnol Bioeng 111:1809–1819.  https://doi.org/10.1002/bit.25243 CrossRefGoogle Scholar
  11. 11.
    Zhang Z, Szita N, Boccazzi P et al (2005) A well-mixed, polymer-based microbioreactor with integrated optical measurements. Biotechnol Bioeng 93:286–296.  https://doi.org/10.1002/bit.20678 CrossRefGoogle Scholar
  12. 12.
    Schäpper D, Stocks SM, Szita N et al (2010) Development of a single-use microbioreactor for cultivation of microorganisms. Chem Eng J 160:891–898.  https://doi.org/10.1016/j.cej.2010.02.038 CrossRefGoogle Scholar
  13. 13.
    Kostov Y, Harms P, Randers-Eichhorn L, Rao G (2001) Low-cost microbioreactor for high-throughput bioprocessing. Biotechnol Bioeng 72:346–352.  https://doi.org/10.1002/1097-0290(20010205)72:3%3C346::AID-BIT12%3E3.0.CO;2-X CrossRefGoogle Scholar
  14. 14.
    Zanzotto A, Szita N, Boccazzi P et al (2004) Membrane-aerated microbioreactor for high-throughput bioprocessing. Biotechnol Bioeng 87:243–254.  https://doi.org/10.1002/bit.20140 CrossRefGoogle Scholar
  15. 15.
    Kirk TV, Marques MPC, Radhakrishnan ANP, Szita N (2016) Quantification of the oxygen uptake rate in a dissolved oxygen controlled oscillating jet-driven microbioreactor. J Chem Technol Biotechnol 91:823–831.  https://doi.org/10.1002/jctb.4833 CrossRefGoogle Scholar
  16. 16.
    Marbà-Ardébol AM, Emmerich J, Muthig M et al (2018) Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy. Microb Cell Fact 17:73CrossRefGoogle Scholar
  17. 17.
    Klein T, Schneider K, Heinzle E (2013) A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas. Biotechnol Bioeng 110:535–542.  https://doi.org/10.1002/bit.24633 CrossRefGoogle Scholar
  18. 18.
    Krull R, Peterat G (2016) Analysis of reaction kinetics during chemostat cultivation of Saccharomyces cerevisiae using a multiphase microreactor. Biochem Eng J 105:220–229.  https://doi.org/10.1016/j.bej.2015.08.013 CrossRefGoogle Scholar
  19. 19.
    Edlich A, Magdanz V, Rasch D et al (2010) Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae. Biotechnol Prog 26:1259–1270.  https://doi.org/10.1002/btpr.449 CrossRefGoogle Scholar
  20. 20.
    Zhang Z, Boccazzi P, Choi H-G et al (2006) Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor. Lab Chip 6:906–913.  https://doi.org/10.1039/b518396k CrossRefGoogle Scholar
  21. 21.
    Lladó Maldonado S, Rasch D, Kasjanow A et al (2018) Multiphase microreactors with intensification of oxygen mass transfer rate and mixing performance for bioprocess development. Biochem Eng J 139:57–67.  https://doi.org/10.1016/j.bej.2018.07.023 CrossRefGoogle Scholar
  22. 22.
    Lladó Maldonado S, Panjan P, Sun S et al (2019) A fully online sensor-equipped, disposable multiphase microbioreactor as a screening platform for biotechnological applications. Biotechnol Bioeng 116:65–75.  https://doi.org/10.1002/bit.26831 CrossRefGoogle Scholar
  23. 23.
    Löfblom J, Rosenstein R, Nguyen MT et al (2017) Staphylococcus carnosus: from starter culture to protein engineering platform. Appl Microbiol Biotechnol 101:8293–8307.  https://doi.org/10.1007/s00253-017-8528-6 CrossRefGoogle Scholar
  24. 24.
    Davies MJ, Nesbeth DN, Szita N (2013) Development of a microbioreactor “cassette” for the cultivation of microorganisms in batch and chemostat mode. Chim Oggi Chem Today 31:46–49Google Scholar
  25. 25.
    Panjan P, Virtanen V, Sesay AM (2017) Determination of stability characteristics for electrochemical biosensors via thermally accelerated ageing. Talanta 170:331–336CrossRefGoogle Scholar
  26. 26.
    Mauthe M, Yu W, Krut O et al (2012) WIPI-1 positive autophagosome-like vesicles entrap pathogenic Staphylococcus aureus for lysosomal degradation. Int J Cell Biol 2012:179207.  https://doi.org/10.1155/2012/179207 CrossRefGoogle Scholar
  27. 27.
    Rosenstein R, Nerz C, Biswas L et al (2009) Genome analysis of the meat starter culture bacterium Staphylococcus carnosus TM300. Appl Environ Microbiol 75:811–822.  https://doi.org/10.1128/AEM.01982-08 CrossRefGoogle Scholar
  28. 28.
    Yu W, Götz F (2012) Cell wall antibiotics provoke accumulation of anchored mCherry in the cross wall of Staphylococcus aureus. PLoS One 7:e30076.  https://doi.org/10.1371/journal.pone.0030076 CrossRefGoogle Scholar
  29. 29.
    Dilsen S, Paul W, Herforth D et al (2001) Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus. J Biotechnol 88:77–84.  https://doi.org/10.1016/S0168-1656(01)00265-6 CrossRefGoogle Scholar
  30. 30.
    Atkinson B, Mavituna F (1991) Biochemical Engineering and Biotechnology Handbook, 2nd edn. Stockton Press, New YorkGoogle Scholar
  31. 31.
    Kovárová-Kovar K, Egli T (1998) Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62:646–666Google Scholar
  32. 32.
    Krull R, Hempel DC (1994) Biodegradation of naphthalenesulphonic acid-containing sewages in a two-stage treatment plant. Bioprocess Eng 10:229–234.  https://doi.org/10.1007/BF00369534 CrossRefGoogle Scholar
  33. 33.
    Rieger M, Käppeli O, Fiechter A (1983) The role of limited respiration in the incomplete oxidation of glucose by Saccharomyces cerevisiae. J Gen Microbiol 129:653–661.  https://doi.org/10.1099/00221287-129-3-653 Google Scholar
  34. 34.
    von Meyenburg HK (1969) Energetics of the budding cycle of Saccharomyces cerevisiae during glucose limited aerobic growth. Arch Mikrobiol 66:289–303.  https://doi.org/10.1007/BF00414585 CrossRefGoogle Scholar
  35. 35.
    von Meyenburg K (1969) Katabolit-Repression und der Sprossungszyklus von Saccharomyces cerevisiae. PhD thesis, ETH ZürichGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • S. Lladó Maldonado
    • 1
    • 2
  • J. Krull
    • 1
    • 2
  • D. Rasch
    • 1
    • 2
  • P. Panjan
    • 3
  • A. M. Sesay
    • 3
  • M. P. C. Marques
    • 4
  • N. Szita
    • 4
  • R. Krull
    • 1
    • 2
    Email author
  1. 1.Institute of Biochemical EngineeringTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Center of Pharmaceutical Engineering (PVZ)Technische Universität BraunschweigBraunschweigGermany
  3. 3.Measurement Technology Unit, CEMIS-OuluKajaani University Consortium, University of OuluKajaaniFinland
  4. 4.Department of Biochemical EngineeringUniversity College LondonLondonUK

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