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Applied Microbiology and Biotechnology

, Volume 100, Issue 12, pp 5363–5373 | Cite as

At-line determination of spore inoculum quality in Penicillium chrysogenum bioprocesses

  • Daniela Ehgartner
  • Christoph Herwig
  • Lukas Neutsch
Biotechnological products and process engineering

Abstract

Spore inoculum quality in filamentous bioprocesses is a critical parameter influencing pellet morphology and, consequently, process performance. It is essential to determine the concentration of viable spores before inoculation, to implement quality control and decrease batch-to-batch variability. The ability to assess the spore physiologic status with close-to-real time resolution would offer interesting perspectives enhanced process analytical technology (PAT) and quality by design (QbD) strategies. Up to now, the parameters contributing to spore inoculum quality are not clearly defined. The state-of-the-art method to investigate this variable is colony-forming unit (CFU) determination, which assesses the number of growing spores. This procedure is tedious, associated with significant inherent bias, and not applicable in real time.

Here, a novel method is presented, based on the combination of viability staining (propidium iodide and fluorescein diacetate) and large-particle flow cytometry. It is compatible with the complex medium background often observed in filamentous bioprocesses and allows for a classification of the spores into different subpopulations. Next to viable spores with intact growth potential, dormant or inactive as well as physiologically compromised cells are accurately determined. Hence, a more holistic few on spore inoculum quality and early-phase biomass composition is provided, offering enhanced information content.

In an industrially relevant model bioprocess, good correlation to CFU counts was found. Morphological parameters (e.g. spore swelling) that are not accessible via standard monitoring tools were followed over the initial process phase with close temporal resolution.

Keywords

Filamentous fungi Flow cytometry Viability staining Spore quality Bioprocess development 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Funding

This study was funded by the Christian Doppler Gesellschaft (grant number 171).

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7319_MOESM1_ESM.docx (419 kb)
ESM 1 (DOCX 418 kb)

References

  1. Allen PJ (1965) Metabolic aspects of spore germination in fungi. Annu Rev Phytopathol 3:313–342CrossRefGoogle Scholar
  2. Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu Rev Microbiol 22:87–108CrossRefPubMedGoogle Scholar
  3. Berney M, Hammes F, Bosshard F, Weilenmann HU, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290. doi: 10.1128/AEM.02750-06 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Binder U, Chu M, Read ND, Marx F (2010) The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa. Eukaryot Cell 9:1374–1382. doi: 10.1128/EC.00050-10 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Breeuwer P, Abee T (2000) Assessment of viability of microorganisms employing fluorescence techniques. Int J Food Microbiol 55:193–200CrossRefPubMedGoogle Scholar
  6. Broger T, Odermatt RP, Huber P, Sonnleitner B (2011) Real-time on-line flow cytometry for bioprocess monitoring. J Biotechnol 154:240–247. doi: 10.1016/j.jbiotec.2011.05.003 CrossRefPubMedGoogle Scholar
  7. Brul S, Nussbaum J, Dielbandhoesing SK (1997) Fluorescent probes for wall porosity and membrane integrity in filamentous fungi. J Microbiol Methods 28:169–178CrossRefGoogle Scholar
  8. Budde BB, Rasch M (2001) A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins. Int J Food Microbiol 63:65–72CrossRefPubMedGoogle Scholar
  9. Bunthof CJ, van den Braak S, Breeuwer P, Rombouts FM, Abee T (1999) Rapid fluorescence assessment of the viability of stressed Lactococcus lactis. Appl Environ Microbiol 65:3681–3689PubMedPubMedCentralGoogle Scholar
  10. d’Enfert C (1997) Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa. Fungal Genet Biol 21:163–172CrossRefGoogle Scholar
  11. Davis C (2014) Enumeration of probiotic strains: review of culture-dependent and alternative techniques to quantify viable bacteria. J Microbiol Methods 103:9–17. doi: 10.1016/j.mimet.2014.04.012 CrossRefPubMedGoogle Scholar
  12. Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D (1998) Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast 14:147–160. doi: 10.1002/(SICI)1097-0061(19980130)14:2<147::AID-YEA207>3.0.CO;2-L CrossRefPubMedGoogle Scholar
  13. Díaz M, Herrero M, García LA, Quirós C (2010) Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 48:385–407CrossRefGoogle Scholar
  14. Ehgartner D, Sagmeister P, Herwig C, Wechselberger P (2015) A novel real-time method to estimate volumetric mass biodensity based on the combination of dielectric spectroscopy and soft-sensors. J Chem Technol Biotechnol 90:262–272CrossRefGoogle Scholar
  15. Fletcher J, Morton G (1970) Physiology of germination of Penicillium griseofulvum conidia. Trans Brit Mycol Soc 54:65–81CrossRefGoogle Scholar
  16. Gottlieb D (1950) The physiology of spore germination in fungi. Bot Rev 16:229–257CrossRefGoogle Scholar
  17. Hashemi N, Erickson JS, Golden JP, Jackson KM, Ligler FS (2011) Microflow cytometer for optical analysis of phytoplankton. Biosens Bioelectron 26:4263–4269. doi: 10.1016/j.bios.2011.03.042 CrossRefPubMedGoogle Scholar
  18. Hua SS, Brandl MT, Hernlem B, Eng JG, Sarreal SB (2011) Fluorescent viability stains to probe the metabolic status of aflatoxigenic fungus in dual culture of Aspergillus flavus and Pichia anomala. Mycopathologia 171:133–138. doi: 10.1007/s11046-010-9352-z CrossRefPubMedGoogle Scholar
  19. Hyka P, Züllig T, Ruth C, Looser V, Meier C, Klein J, Melzoch K, Meyer HP, Glieder A, Kovar K (2010) Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures. Appl Environ Microbiol 76:4486–4496. doi: 10.1128/AEM.02475-09 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hyka P, Lickova S, Pribyl P, Melzoch K, Kovar K (2013) Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol Adv 31:2–16. doi: 10.1016/j.biotechadv.2012.04.007 CrossRefPubMedGoogle Scholar
  21. Jones KH, Senft JA (1985) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide. J Histochem Cytochem 33:77–79CrossRefPubMedGoogle Scholar
  22. Kell DB, Kaprelyants AS, Weichart DH, Harwood CR, Barer MR (1998) Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Van Leeuwenhoek 73:169–187CrossRefPubMedGoogle Scholar
  23. Kumar N, Borth N (2012) Flow-cytometry and cell sorting: an efficient approach to investigate productivity and cell physiology in mammalian cell factories. Methods 56:366–374. doi: 10.1016/j.ymeth.2012.03.004 CrossRefPubMedGoogle Scholar
  24. Langemann T, Koller VJ, Muhammad A, Kudela P, Mayr UB, Lubitz W (2010) The bacterial ghost platform system: production and applications. Bioengineered bugs 1:326–336. doi: 10.4161/bbug.1.5.12540 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lein J (1986) The Panlabs penicillin strain improvement program. In: Vanek Z, Hostalek Z (eds) Overproduction of microbial metabolites. Butterworths, Boston, pp 105–139Google Scholar
  26. Liao RS, Rennie RP, Talbot JA (1999) Assessment of the effect of amphotericin B on the vitality of Candida albicans. Antimicrob Agents Chemother 43:1034–1041PubMedPubMedCentralGoogle Scholar
  27. Martin JF, Nicolas G, Villanueva JR (1973) Chemical changes in the cell walls of conidia of Penicillium notatum during germination. Can J Microbiol 19:789–796CrossRefPubMedGoogle Scholar
  28. Mesquita N, Portugal A, Pinar G, Loureiro J, Coutinho AP, Trovao J, Nunes I, Botelho ML, Freitas H (2013) Flow cytometry as a tool to assess the effects of gamma radiation on the viability, growth and metabolic activity of fungal spores. Int Biodeter Biodegr 84:250–257CrossRefGoogle Scholar
  29. Metz B, Kossen NWF (1977) The growth of molds in the form of pellets—a literature review. Biotechnol Bioeng 19:781–799CrossRefGoogle Scholar
  30. Meyerhoff J, Bellgardt K (1995) A morphology-based model for fed-batch cultivations of Penicillium chrysogenum growing in pellet form. J Biotechnol 38:201–217CrossRefGoogle Scholar
  31. Nielsen J (1992) Modelling the growth of filamentous fungi. Adv Biochem Eng Biotechnol 46:187–223PubMedGoogle Scholar
  32. O’Brien MC, Bolton WE (1995) Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19:243–255. doi: 10.1002/cyto.990190308 CrossRefPubMedGoogle Scholar
  33. Paul GC, Kent CA, Thomas CR (1993) Viability testing and characterization of germination of fungal spores by automatic image analysis. Biotechnol Bioeng 42:11–23CrossRefPubMedGoogle Scholar
  34. Posch AE, Herwig C (2014) Physiological description of multivariate interdependencies between process parameters, morphology and physiology during fed-batch penicillin production. Biotechnol Prog 30:689–699. doi: 10.1002/btpr.1901 CrossRefPubMedGoogle Scholar
  35. Posch AE, Spadiut O, Herwig C (2012) Switching industrial production processes from complex to defined media: method development and case study using the example of Penicillium chrysogenum. Microb Cell Fact 11:88. doi: 10.1186/1475-2859-11-88 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Prigione V, Filipello Marchisio V (2004) Methods to maximise the staining of fungal propagules with fluorescent dyes. J Microbiol Methods 59:371–379. doi: 10.1016/j.mimet.2004.07.016 CrossRefPubMedGoogle Scholar
  37. Quiros C, Herrero M, Garcia LA, Diaz M (2007) Application of flow cytometry to segregated kinetic modeling based on the physiological states of microorganisms. Appl Env Microbiol 73:3993–4000. doi: 10.1128/AEM.00171-07 CrossRefGoogle Scholar
  38. Rieseberg M, Kasper C, Reardon KF, Scheper T (2001) Flow cytometry in biotechnology. Appl Microbiol Biotechnol 56:350–360CrossRefPubMedGoogle Scholar
  39. Rotman B, Papermaster BW (1966) Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci U S A 55:134–141CrossRefPubMedPubMedCentralGoogle Scholar
  40. Schnürer J, Rosswall T (1982) Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. Appl Env Microbiol 43:1256–1261Google Scholar
  41. Sundstrom H, Wallberg F, Ledung E, Norrman B, Hewitt CJ, Enfors SO (2004) Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes. Biotechnol Lett 26:1533–1539. doi: 10.1023/B:BILE.0000044458.29147.75 CrossRefPubMedGoogle Scholar
  42. Ueckert J, Breeuwer P, Abee T, Stephens P, von Caron GN, ter Steeg PF (1995) Flow cytometry applications in physiological study and detection of foodborne microorganisms. Int J Food Microbiol 28:317–326CrossRefPubMedGoogle Scholar
  43. Veal DA, Deere D, Ferrari B, Piper J, Attfield PV (2000) Fluorescence staining and flow cytometry for monitoring microbial cells. J Immunol Methods 243:191–210CrossRefPubMedGoogle Scholar
  44. Vermes I, Haanen C, Reutelingsperger C (2000) Flow cytometry of apoptotic cell death. J Immunol Methods 243:167–190CrossRefPubMedGoogle Scholar
  45. Walther TC, Brickner JH, Aguilar PS, Bernales S, Pantoja C, Walter P (2006) Eisosomes mark static sites of endocytosis. Nature 439:998–1003. doi: 10.1038/nature04472 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Daniela Ehgartner
    • 1
    • 2
  • Christoph Herwig
    • 1
    • 2
  • Lukas Neutsch
    • 2
  1. 1.CD Laboratory on Mechanistic and Physiological Methods for Improved BioprocessesVienna University of TechnologyViennaAustria
  2. 2.Research Area Biochemical Engineering, Institute of Chemical EngineeringVienna University of TechnologyViennaAustria

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