Biomass Measurement in Solid-State Fermentation of Basidiomycetes by Flow Cytometry

The solid-state fermentation (SSF) is a robust process which is perfectly suitable for the on-site cultivation of basidiomycetes, where fungi serve as enzyme producers for the treatment of lignocellulosics. The quantification of fungal biomass is essential for growth kinetics analysis. In SSF a direct biomass determination is not possible, because fungi grow into the substrate and use it as a nutrient source. Therefore, it is necessary to use indirect methods which are either very laborious and time-intensive, or can only refer to the biomass at certain growth periods. In the present study an alternative and rapid method was developed and optimized for the biomass determination during SSF by measuring the number of fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining their nuclei with SYTOX® Green, and subsequently counting the nuclei using a flow cytometer. It was possible to establish a calibration curve for the dry biomass and the concentration of the nuclei. Different buffer and disruption methods were tested. The results were compared with the method of ergosterol determination, a classical method of fungal biomass measurement in SSF. This approach allowed the calculation of fungal biomass at different scales ? from 15 mL up to the laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)).
Conclusion:
We could show that the biomass determination by flow cytometry is a good alternative technique for the determination of fungal biomass in SSF offering a lot of potential and advantages, like easy handling and rapidity. Based on the results of the optimization the Galbraith’s buffer and a disruption by ultra centrifugal mill will be used for all future experiments. Prior to measurement the flow cytometer was calibrated using fluorescent beads (AlignFlow 2.5 µm, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany).