Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes

Area of Science:

• Biotechnology
• Microbiology
• Bioprocess Engineering

Background:

• Solid-state fermentation (SSF) is crucial for cultivating basidiomycetes that produce lignocellulosic enzymes.
• Accurate fungal biomass quantification is vital for analyzing growth kinetics in SSF.
• Existing biomass determination methods for SSF are often laborious, time-consuming, or limited in applicability.

Purpose of the Study:

• To develop and optimize a rapid, reliable method for fungal biomass determination during SSF.
• To establish a flow cytometry-based approach for quantifying fungal nuclei.
• To validate the new method against established techniques like ergosterol determination.

Main Methods:

• Fungal biomass was cultivated using SSF on an organic substrate.
• Fungal nuclei were isolated after cell disruption.
• Nuclei were stained with SYTOX® Green and quantified using flow cytometry.
• A calibration curve correlating dry biomass to nuclei concentration was generated.

Main Results:

• The developed flow cytometry method provides rapid fungal biomass measurements.
• The method was optimized by testing various buffers and disruption techniques.
• Results showed good correlation with the ergosterol determination method.
• The technique is scalable from small cultures (15 mL) to larger reactors (10 L).

Conclusions:

• Flow cytometry-based fungal nuclei counting is a rapid and effective method for biomass determination in SSF.
• This approach overcomes limitations of traditional methods, offering broader applicability.
• The technique supports efficient monitoring and optimization of SSF processes for enzyme production.