Experiment Overview

Repository ID: FR-FCM-ZZ9R Experiment name: Biomass Measurement in Solid-State Fermentation of Basidiomycetes by Flow Cytometry MIFlowCyt score: 59.25%
Primary researcher: Susanne Steudler PI/manager: Susanne Steudler Uploaded by: Susanne Steudler
Experiment dates: 2011-06-06 - 2013-06-28 Dataset uploaded: Tue Feb 04 07:16:52 -0500 2014 Last updated: Mon Feb 10 03:06:55 -0500 2014
Keywords: [flow cytometry] [Solid-state fermentation] [basidiomycetes] [biomass determination] [nuclei ] Manuscripts: Cytalogo
Organizations: Technische Universität Dresden, Institute of Food Technology and Bioprocess Engineering, Dresden, (Germany)
Purpose: 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.
Comments: None
Funding: Not disclosed
Quality control: Prior to measurement the flow cytometer was calibrated using fluorescent beads (AlignFlow 2.5 µm, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany).


Flow Sample/Specimen Details


buffer optimization-Arumuganathan&Earle.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with buffer of Arumuganathan and Earle (9.53 mM MgSO4 x 7 H2O, 47.67 mM KCl, 4.77 mM HEPES, 6.48 mM DTT, 0.25 % (v/v) Triton X-100, pH 8.0) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Baranyi-I.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with buffer of Baranyi I (100 mM citric acid monohydrate, 0.5 % (v/v) Triton X-100) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Baranyi-II.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with buffer of Baranyi II (400 mM Na2PO4 x 12 H2O, 10 mM sodium citrate, 25 mM sodium sulfate) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-GPB.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with GPB buffer (0.5 mM spermine x 4 HCl, 30 mM sodium citrate, 20 mM MOPS, 80 mM KCl, 20 mM NaCl, 0.5 % (v/v) Triton X-100, pH 7.0) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Gallbraith.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with Galbraith's buffer (45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 0.1 % (v/v) Triton X-100, pH 7.0) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-L01.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with LO1buffer (15 mM TRIS, 2 mM Na2EDTA, 0.5 mM spermine x 4 HCl, 80 mM KCl, 20 mM NaCl, 15 mM ß-mercaptoethanol, 0.1 % (v/v) Triton X-100, pH 7.5) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Marie's-Puffer.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with Marie's buffer (50 mM glucose, 15 mM KCl, 15 mM NaCl, 5 mM Na2EDTA, 50 mM sodium citrate, 0.5 % (v/v) Tween 20, 50 mM HEPES, 0.5 % (v/v) ß-mercaptoethanol, , pH 7.2) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Otto-I.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with Otto I buffer (100 mM citric acid monohydrate, 0.5 % (v/v) Tween 20 (pH approx. 2-3)) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Otto-II.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with Otto II buffer (400 mM Na2PO4 x 12 H2O (pH approx. 8-9)) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-Tris.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with TrisMgCl2 buffer (200 mM TRIS, 4 mM MgCl2 x 6 H2O, 0.5 % (v/v) Triton X-100, pH 7.5) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



buffer optimization-WPG.FCS :

Description buffer optimization: 200 mg pure fungal biomass were treated with WPG buffer (0.2 M TRIS x HCl, 4 mM MgCl2 x 6 H2O, 2 mM EDTA Na2 x 2 H2O, 86 mM NaCl, 10 mM sodium metabisulfite, 1.0 % (v/v) PVP-10, 1.0 % (v/v) Triton X-100, pH 7.5) and disrupted by Ultra-Turrax® (T-25 basic, IKA Staufen, Germany) at 11.000 min-1 for 40 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



disruption optimization-ultra centrifugal mill-18000rpm.FCS :

Description disruption optimization: Fungal biomass were disrupted by ultra cetrifugal mill at 18.000 rpm and treated with 10 mL Galbraith’s buffer. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



disruption optimization-ultrasonic bath-1min-ice.FCS :

Description disruption optimization: Fungal biomass were treated with 10 mL Galbraith’s buffer and disrupted by ultrasonic bath for 1 min at ice. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



disruption optimization-ultrasonic disintegrator-15s-ice.FCS :

Description disruption optimization: Fungal biomass were treated with 10 mL Galbraith’s buffer and disrupted by ultrasonic disintegrator for 15 s at ice. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



verification nuclei-blank-pine wood chips.FCS :

Description Pine wood chips were disrupted by ultra cetrifugal mill at 18.000 rpm and treated with 10 mL Galbraith’s buffer. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



verification nuclei-fungi T.h. and pine wood chips.FCS :

Description Pine wood chips and fungal biomass were disrupted by ultra cetrifugal mill at 18.000 rpm and treated with 10 mL Galbraith’s buffer. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other



disruption optimization-microwave-640W-5s.FCS :

Description disruption optimization: Fungal biomass were treated with 10 mL Galbraith’s buffer and disrupted by microwave at 640 W for 5 s. The resulting suspension was filtered through a 30 µm filter tissue (CellTrics®, Partec GmbH, Münster, Germany) for separating debris. Afterwards, 249 µL filtrated sample was incubated with 1 µL SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH, Darmstadt, Germany) for 10 min at room temperature in the dark. The tested disruption methods and buffers are shown in subchapter Optimization. Finally, 100 µL incubated sample were mixed with 900 µL bidistilled water and measured by flow cytometry (flow rate 0.9 µL/s).
Sample source:
Sample characteristic: n/a
Sample treatment: n/a
Staining: SYTOX Green
Characteristic measured Analyte Analyte detector Analyte reporter Clone Manufacturer Catalog number Other




Flow Cytometer Details

CyFlow SL (3 colors), Partec used for all 17 FCS files.

buffer optimization-Arumuganathan&Earle.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Baranyi-I.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Baranyi-II.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-GPB.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Gallbraith.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-L01.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Marie's-Puffer.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Otto-I.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Otto-II.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-Tris.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

buffer optimization-WPG.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

disruption optimization-ultra centrifugal mill-18000rpm.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

disruption optimization-ultrasonic bath-1min-ice.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

disruption optimization-ultrasonic disintegrator-15s-ice.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

verification nuclei-blank-pine wood chips.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

verification nuclei-fungi T.h. and pine wood chips.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008

disruption optimization-microwave-640W-5s.FCS
Manufacturer Partec Default instrument settings used.
Model CyFlow SL (3 colors)
Optical filter installation dates 2008


Data Analysis Details

No illustrations provided.