Covalent Immobilization of Microbial Cells on Microchannel Surfaces

  • Gorazd Stojkovič
  • Polona Žnidaršič-PlazlEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)


Microfluidic devices with integrated biological material have found many applications in analytics (e.g., protein and DNA analysis), biochemistry (e.g., PCR), and medical diagnostics (e.g., ELISA test). Recently they are also considered as promising tools for bioprocess development and intensification. In order to enable long-term biocatalyst use and to facilitate its separation from the product, immobilization within the microreactor is often preferred over the use of free enzymes or cells. Surface immobilization is frequently selected due to the very high surface-to-volume ratio of microfluidic devices that offers the possibility for high biocatalyst load and at the same time good biocatalyst accessibility. Moreover, such reactor design prevents the increase in backpressure, often encountered in packed-bed or monolithic microreactors.

Microbial cells are beneficial over the isolated enzymes in many biotransformations, especially in multistep syntheses and in cofactor-dependent reactions. Their immobilization within microreactors, especially made from disposable polymers, is of a big interest for analytical and synthetic applications.

This chapter describes procedure for immobilization of eukaryotic and prokaryotic cells onto inner surfaces of microreactors made from various polymeric materials and glass. Cells could be immobilized in high densities and remain stably attached over several days of continuous microreactor operation.

Key words

Microreactors Microfluidics Microorganisms Immobilization Biocatalysis Whole-cell biotransformations Silanization 


  1. 1.
    Wohlgemuth R, Plazl I, Žnidaršič-Plazl P, Gernaey KV, Woodley JM (2015) Microscale technology and biocatalytic processes: opportunities and challenges for synthesis. Trends Biotechnol 33:302–314CrossRefGoogle Scholar
  2. 2.
    Žnidaršič-Plazl P (2017) Biotransformations in microflow systems: bridging the gap between academia and industry. J Flow Chem 7(3–4):111–117CrossRefGoogle Scholar
  3. 3.
    Zydney AL (2015) Perspectives on integrated continuous bioprocessing—opportunities and challenges. Curr Opin Chem Eng 10:8–13CrossRefGoogle Scholar
  4. 4.
    Polakovic M, Švitel J, Bučko M, Filip J, Nedĕla V, Ansorge-Schumacher MB, Gemeiner P (2017) Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications. Biotechnol Lett 39:667–683CrossRefGoogle Scholar
  5. 5.
    Buchholz K, Kasche V, Bornscheuer UT (2012) Biocatalysts and enzyme technology, 2nd edn. Wiley-VCH Verlag & Co, WeinheimGoogle Scholar
  6. 6.
    Bajić M, Žnidaršič-Plazl P, Kingston M, Hessel V (2018) General aspects of immobilized biocatalysts and their applications in flow. In: Nielsen MB (ed) Science of synthesis: knowledge updates 2018/1. Georg Thieme, Stuttgart, pp 397–443Google Scholar
  7. 7.
    Stojkovič G, Žnidaršič-Plazl P (2012) Continuous synthesis of L-malic acid using whole-cell microreactor. Process Biochem 47:1102–1107CrossRefGoogle Scholar
  8. 8.
    Stojkovič G, Krivec M, Vesel A, Marinšek M, Žnidaršič-Plazl P (2014) Surface cell immobilization within perfluoroalkoxy microchannels. Appl Surf Sci 320:810–817CrossRefGoogle Scholar
  9. 9.
    Miložič N, Stojkovič G, Vogel A, Bouwes D, Žnidaršič Plazl P (2018) Development of microreactors with surface-immobilized biocatalysts for continuous transamination. New Biotechnol 47:18–24. Scholar
  10. 10.
    Andrade LH, Kroutil W, Jamison TF (2014) Continuous flow synthesis of chiral amines in organic solvents: immobilization of E. coli cells containing both omega-transaminase and PLP. Org Lett 16:6092–6095CrossRefGoogle Scholar
  11. 11.
    De Vitis V, Dall’Oglio F, Pinto A, De Micheli C, Molinari F, Conti P, Romano D, Tamborini L (2017) Chemoenzymatic synthesis in flow reactors: a rapid and convenient preparation of captopril. ChemistryOpen 6:668–673CrossRefGoogle Scholar
  12. 12.
    Jovanovic GN, Schilke KF, Loeb C, Atadana F, Weymann D (2017) Bio-lamina bioreactors and methods of making and using the same. Oregon State University: Corvallis, OR WO 2017087535 A1Google Scholar
  13. 13.
    Akay G, Erhan E, Keskinler B (2005) Bioprocess intensification in flow-through monolithic microbioreactors with immobilized bacteria. Biotechnol Bioeng 90:180–190CrossRefGoogle Scholar
  14. 14.
    Karande R, Schmid A, Buehler K (2016) Applications of multiphasic microreactors for biocatalytic reactions. Org Proc Res Dev 20:361–370CrossRefGoogle Scholar
  15. 15.
    Stojkovič G, Marinšek M, Drobne D, Žnidaršič-Plazl P (2015) Whole-cell biotransformation within magnetic field-assisted microreactor using magnetic nanoparticles. In: BIOTRANS 2015: July 26th–30th, 2015, Vienna, Austria. Gumpoldskirchen: Book-of-Abstracts. p. 555Google Scholar
  16. 16.
    Shriver-Lake LC, Gammeter WB, Bang SS, Pazirandeh M (2002) Covalent binding of genetically engineered microorganisms to porous glass beads. Anal Chim Acta 470:71–78CrossRefGoogle Scholar
  17. 17.
    Stojkovič G, Žnidaršič-Plazl P (2010) Immobilisation of yeast cells within microchannels of different materials. Acta Chim Slov 57:144–149PubMedGoogle Scholar
  18. 18.
    Betancor L, Lopez-Gallego F, Hidalgo A, Alonso-Morales N, Dellamora-Ortiz G, Mateo C, Fernandez-Lafuente R, Guisan JM (2006) Different mechanisms of protein immobilization on glutaraldehyde activated supports: effect of support activation and immobilization conditions. Enzyme Microb Technol 39:877–882CrossRefGoogle Scholar
  19. 19.
    Howarter JA, Youngblood JP (2007) Surface modification of polymers with 3-aminopropyltriethoxysilane as a general pretreatment for controlled wettability. Macromolecules 40:1128–1132CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Medical Biochemistry and BiophysicsUmeå UniversityUmeåSweden
  2. 2.Faculty of Chemistry and Chemical TechnologyUniversity of LjubljanaLjubljanaSlovenia

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