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

Abstract

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting 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 them with SYTOX V R Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

“…The types of fluorescent dyes primary used in flow cytometry are: (i) fluorescent immune-conjugates and probes for fluorescence in situ hybridization; (ii) nucleic acid strains; and (iii) physiological probes to measure ions, membrane potential, enzymatic activity, viability, organelles, phagocytosis, cell development, and other cell properties (Haugland, 1994 ). The potential of flow cytometry to assess yeast cultures in food and beverage processing (e.g., bakery, wine industry, beer industry) is already shown (Herrero et al, 2006 ) as well as used to monitor the solid-state fermentation of basidiomycetes (Steudler et al, 2015 ). Flow cytometry is used in various studies to monitor bacterial inactivation (Luscher et al, 2004 ; Ananta et al, 2005 ; Berney et al, 2007 ; Mathys et al, 2007 ; Ananta and Knorr, 2009 ; Da Silveira and Abee, 2009 ; Joyce et al, 2011 ; Schenk et al, 2011 ; Fröhling et al, 2012b ; Tamburini et al, 2013 ; Bigoni et al, 2014 ).…”

Alterations in Mycelial Morphology and Flow Cytometry Assessment of Membrane Integrity of Ganoderma boninense Stressed by Phenolic Compounds

“…One mL of chloroform was added at the end of incubation and total leakage of mycelium was recorded. Electrolyte leakage was expressed as μ mhosg −1 fresh weight of mycelium [ 25 ].…”

Biosensors in Fermentation Applications

“…Examples of generalized biosensors include for instance: mass spectrometric measurements used in off-gas analyses in fermentation processes [10,11] and cytometry for fermentation process controls [12].…”