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 HerwigEmail author
  • Lukas Neutsch
Biotechnological products and process engineering


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.


Filamentous fungi Flow cytometry Viability staining Spore quality Bioprocess development 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


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)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Daniela Ehgartner
    • 1
    • 2
  • Christoph Herwig
    • 1
    • 2
    Email author
  • 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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