Supplementary Materials
Methods of biomass measurement – detailed description of the analytical methods
Ergosterol content was measured by mixing a 400 mg subsample of SSF culture with 2 g
KOH, 10 mL methanol and 5 mL ethanol (Roth, Karlsruhe, Germany) and then saponified for
30 min at 70 °C. After cooling to room temperature, 5 mL of distilled water was added and
the ergosterol was extracted with n-hexane (Roth, Karlsruhe, Germany). The n-hexane
fraction was separated and vacuum-evaporated at 40 °C in a rotavapor (Büchi, Flawil,
Switzerland). The dried residue was dissolved in 5 mL methanol. After centrifugation (7200
g) for 5 min and filtration (0.45 µm, A-Z Analytik, Langen, Germany) the ergosterol content
of the sample was measured by HPLC (Knauer, Berlin, Germany) using a LiChrospher® 100
RP-18 (5 µm) LiChroChart® 125-4 column (Merck Millipore, Darmstadt, Germany) with
detection by a UV detector (GAT GmbH, Bremerhaven, Germany) at 282 nm. The sample
was eluted with 95 % (v/v) methanol at a flow rate of 1 mL/min and a column temperature of
35 °C.
To determine glucosamine content, 400 mg subsample of SSF culture was incubated with 5
mL 72 % (v/v) sulphuric acid for 1 h at 28 °C on a rotary shaker at 150 rpm. The resulting
suspension was diluted with 54 ml of distilled water and hydrolysed by autoclaving for 2 h at
121 °C. After cooling to room temperature, the hydrolysate was neutralized with 10 M
sodium hydroxide solution. 1 mL of neutralized solution was mixed with 1 mL 5 % (w/v)
sodium nitrite and 1 mL 5 % (w/v) potassium hydrogen sulfate and then incubated for 15 min
on an overhead rotator. After centrifugation (10 000 g) for 15 min, 1 mL of supernatant was
mixed with 300 µL 12.5 % (w/v) ammonium sulfamate and incubated for 5 min with
occasional shaking. 300 µL 0.5 % (w/v) MBTH (3-methyl-2-benzo-thiazolinin hydrazone)
was added and then the mixture boiled for 3 min. After cooling to room temperature, 300 µL
0.5 % (w/v) iron-(III)-chloride and 5 mL of distilled water were added to the sample solution.
After incubation for 30 min, the glucosamine content of the sample was measured
photometrically at 650 nm.
For enumerating fungal nuclei [28], the fungal cells in a sample of the SSF culture were
disrupted at 18 000 rpm using a ZM 200 ultra-centrifugal mill (Retsch, Haan, Germany)
equipped with a 12-tooth rotor (Part No. 02.608.0041) and a 0.5 mm ring sieve (Part No.
03.647.0257). After milling, 600 mg of each sample was treated with 10 mL of Galbraith’s
buffer [22]. The resulting suspension was filtered through a 30 µm CellTrics® mesh filter
(Partec GmbH, Münster, Germany) to remove debris, then 249 µL of the filtrate was
incubated with 1 µL of SYTOX® Green (0.5 mM, InvitrogenTM, Life Technologies GmbH,
Darmstadt, Germany) for 10 min, in the dark at room temperature. Finally, 100 µL of
incubated sample was mixed with 900 µL of bidistilled water and analysed by flow cytometry
(flow rate: 0.9 µL/s) using a CyFlow® SL Blue instrument (Partec GmbH, Münster, Germany,
manufactured in 2008, Ser.-No 081260417) equipped with a 20 mW 488 nm solid-state laser.
The nucleic acid content was determined photometrically after extraction with perchloric
acid. For this, 5 g of SSF material was mixed with 5 mL 0.25 M perchloric acid and incubated
for 15 min at 4 °C. After centrifugation (7200 g) for 5 min, the supernatant was discarded and
the procedure was repeated. After the second centrifugation, 5 mL 0.5 M perchloric acid was
added to the pellet, incubated for 15 min at 90 °C and centrifuged (7200 g) for 5 min. The
supernatant was collected and the procedure was repeated. After the second centrifugation,
200 µL of each supernatant were combined and diluted with 1.6 mL of distilled water. The
absorbance of the mixture was measured photometrically at 260 nm.
To determine protein contents, 250 mg of SSF culture was mixed with 5 mL of distilled
water and boiled for 5 min. After centrifugation (7200 g) for 5 min, 200 µL of the supernatant
was added to 200 µL 10 % (m/v) trichloroacetic acid and incubated for 1 h at 4 °C with
occasional shaking. After centrifugation (20 000g) for 25 min, the supernatant was discarded
and the pellet resuspended in 200 µL of reagent A (reagent A: 20 g/L sodium carbonate and 4
g/L sodium hydroxide). 10 µL of the resulting solution was pipetted into a microtiter plate (96
wells) and mixed with 100 µL of reagent C (reagent C: 1 mL copper sulphate-pentahydrate
(10 g/L), 1 mL sodium potassium tartrate (20 g/L) and 100 mL of reagent A). After
incubation for 10 min, 10 µL of 1 N phenol reagent was added and incubated for 30 min
again. The absorbance of the mixture was measured photometrically at 750 nm (Tecan
GENios, Maennedorf, Switzerland).
Genomic DNA was extracted from 20 mg of SSF material using a NucleoSpin® Plant II Mini
extraction kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), following the
manufacturer’s protocol [25], but with a modification to the cell lysis step. The sample was
mixed with 300 µL of buffer PL1 and 15 µL of RNaseA, incubated for 30 min at 65 °C, then
300 µL of chloroform was added and the sample was mixed thoroughly. After removing the
chloroform by centrifugation (7200 g) for 10 min, the extraction proceeded as per the
manufacturer’s protocol. Finally, genomic DNA content was quantified photometrically at
260 nm.
The carbon dioxide and oxygen contents of the gas phase were determined by pumping
samples of the phase into a circuit using a master flex pump (flow rate: 5 mL/min) and
measuring the gases by BlueSens gas sensors (CO2, 0-10 vol.%, Part No. BCPCO20106AA
PS0813PA2E SGL45S-2N1C3G; O2, 0.1-25 vol.%, Part No. BCPO20256AA PS0813PA2E
SGL45S-2N1C3G: GmbH, Herten, Germany).
In the liquid phase, the determination of the glucose content was performed by mixing 100
µL of the sample with 900 µL 50 mM citrate buffer and pipetting 200 µL of the resulting
solution into a deep well plate (96 wells). After addition of 600 µL dinitrosalicylic acid
solution (standard: 4 g/L glucose solution), the mixture was boiled for 5 min and then cooled
to room temperature. 57 µL of the boiled solution was pipetted into a microtiter plate (96
wells) and mixed with 150 µL of distilled water. Finally, the absorbance of the solution was
quantified photometrically at 545 nm (Tecan GENios, Maennedorf, Switzerland).
To determine the protein content of the liguid phase, 200 µL of the sample was added to 200
µL 10 % (m/v) trichloroacetic acid and incubated for 1 h at 4 °C with occasional shaking.
After centrifugation (20 000g) for 25 min, the supernatant was discarded and the pellet
resuspended in 200 µL of reagent A (reagent A: 20 g/L sodium carbonate and 4 g/L sodium
hydroxide). 10 µL of the resulting solution was pipetted into a microtiter plate (96 wells) and
mixed with 100 µL of reagent C (reagent C: 1 mL copper sulphate-pentahydrate (10 g/L), 1
mL sodium potassium tartrate (20 g/L) and 100 mL of reagent A). After incubation for 10
min, 10 µL of 1 N phenol reagent was added and incubated for 30 min again. The absorbance
of the mixture was measured photometrically at 750 nm (Tecan GENios, Maennedorf,
Switzerland).
Lignolytic enzyme activities were determined photometrically at 420 nm for laccases and at
436 nm for peroxidases. To determine laccase activity, 500 µL 2 mM ABTS (2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) was mixed with 500-x µL of sample solution and x µL
0.1 M sodium malonate buffer (pH 5). The activity was calculated from the increase in
absorbance in 1 min at 420 nm using an extinction coefficient of 36 mM-1 cm-1. For
measurement of the peroxidase activity, 500 µL 20 mM guaiacol solution was mixed with 20
µL 0.03 % (v/v) hydrogen peroxide solution, 480-x µL of sample solution and x µL 0.1 M
sodium malonate buffer (pH 5). The activity was calculated from the increase in absorbance
in 1 min at 436 nm using an extinction coefficient of 25.5 mM-1 cm-1.
The cellulolytic activity of cellulases was determined using AZO-carboxymethyl cellulose
(AZO-CMC) as the substrate and a kit (both supplied by Megazyme, Bray, Ireland),
according to the manufacturer’s protocol [27]. For this, 100 µL of the sample was mixed with
100 µL AZO-CMC solution (10 g/L AZO-CMC in 5 % (v/v) sodium acetate buffer (100 mM,
pH 4.6)) and incubated for 10 min at 40 °C and 350 rpm on a thermomixer (Thermomixer
comfort®, eppendorf, Hamburg, Germany). The resulting suspension was mixed with 500 µL
of precipitation solution (40 g/L sodium acetate trihydrate, 4 g/L zinc acetate, 200 µL of
distilled water and 800 mL 95 % (v/v) ethanol, pH 5) and incubated for 10 min at room
temperature. After centrifugation (7200 g) for 10 min, the absorbance of the supernatant was
measured photometrically at 590 nm. For xylanases, xylan from beechwood (Sigma-Aldrich,
Taufkirchen, Germany) was used as the substrate. To determine the cellulolytic activity, 100
µL of sample was mixed with 100 µL of 50 mM citrate buffer and 800 µL of 2.5% (w/v)
xylan solution and incubated for 20 min at 50 °C. The reducing sugars produced were
quantified by the method of Miller [26]. Therefore, 200 µL of the incubated sample was
pipetted into a deep well plate (96 wells). After addition of 600 µL dinitrosalicylic acid
solution (standard: 1 g/L xylose solution), the mixture was boiled for 5 min and then cooled to
room temperature. 57 µL of the boiled solution was pipetted into a microtiter plate (96 wells)
and mixed with 150 µL of distilled water. Finally, the absorbance of the solution was
quantified photometrically at 545 nm (Tecan GENios, Maennedorf, Switzerland) and used to
calculate the enzyme activity.