Composition analyses

For measurement of reducing sugars, 3 mL of the dinitrosalicylic acid reagent (DNS) was added to 1.5 mL of appropriately diluted sample, and the mixture was placed in boiling water for 5 min (Miller, 1959). After cooling and adding 16 mL of distilled water, absorbance was read at 550 nm in order to determine the concentration of RS. Glucose was used to prepare standard curve.

3.8.1.2 Extracellular protein content

Extracellular protein content was determined by Bradford method (Bradford, 1976) using Coomassie Blue G250 reagent. Calibration curve was prepared with Bovine Serum Albumin.

3.8.1.3 Cell mass determination

For cell mass determination 100 mL fermentation broth obtained from fermenter experiments was vacuum-filtered onto a dry and preweighed filter cloth and washed with distilled water. After drying at 105°C for 6 hours the filter cloth containing a filter cake was weighed again. For further analysis 0.5 g homogenous sample from each filter cake was subjected to a two step sulfuric acid hydrolysis to release N-acetyl-glucosamine and glucosamine from fungal cell wall (building units of chitin and chitosan): the sample was incubated with 60% (w/v) sulfuric acid at room temperature for 24 hours with subsequent dilution to 1 N and hydrolyzed at elevated temperature (121°C for 1 hour)

(Sakurai et al., 1977). This two-step hydrolysis results in complete deacetylation, thus N-acetyl-glucosamine was also converted to glucosamine (Zamani et al., 2008). The room temperature mixture was neutralized by addition of 1 N solution of NaOH and the total volume was measured. A portion was centrifuged at 3400 g for 5 minutes and supernatants were analyzed for glucosamine concentration by the method of Blix (Blix, 1948) as described by Bussari et al. (2008). Glucosamine content was calculated according to a calibration curve prepared with reagent grade D-glucosamine.HCl (Sigma- Aldrich). Cell mass is expressed as mg glucosamine/mL fermentation broth.

3.8.1.4 Determination of biomass composition

Glucan and xylan contents of raw materials before and after pretreatments were determined using a two-step sulfuric acid hydrolysis based on the method described by Hägglund (1951). A modified method was performed in which 0.5 g raw material was dispersed in 2.5 mL 72% sulfuric acid and maintained at room temperature for 2 h with occasional stirring. Next, 75 mL of distilled water was added and autoclaved for 1 h at 121°C. The reaction mixture was separated on preweighed, dried G4 glass filtering crucibles by applying vacuum.

The filtrate was analyzed for carbohydrates by HPLC. The filter cake was washed with hot distilled water and dried at 105°C. This residue was defined as lignin content, and it was corrected for ash content, which was determined after incineration at 550°C for 6 h.

For starch content determination a slightly modified method of NREL (NREL, 1996) was applied. A dry mass of 0.5 g of ground wheat grain was suspended in a mixture of 25 mL distilled water and 10 mL 2 M NaOH. It was then incubated at 90°C for 20 min, followed by cooling to a temperature below 50°C. The sample was neutralized through the addition of 10 mL 2 M HCl.

Then 10 mL of 0.1 M Na-acetate buffer (pH 4.2) and AMG 300L (Novozymes A/S, Bagsvaerd, Denmark) amyloglucosidase of A. niger at 600 U/g solid were added for a 12-h hydrolysis at 40°C. This mixture was centrifuged at 3,400g for 5 min, and the supernatant was analyzed for glucose via HPLC. Measured glucose concentrations were reduced by the glucose content of AMG 300L.

Initial sugar content of sweet sorghum juice was determined by ’Sucrose, D- fructose and D-glucose’ kit according to the attached description (Megazyme International Ltd., Ireland).

3.8.1.5 HPLC analyses

Samples for HPLC analysis were prepared by filtering through a regenerated cellulose syringe filter with 0.45 μm pore size (ProFill, Langerwehe, Germany).

The glucose, cellobiose, xylose and ethanol contents of different samples were separated on an Aminex ion exclusion HPX-87H cation-exchange column (BioRad, Hercules, CA, USA) running at 65°C with 5 mM sulfuric acid as mobile phase at a flow rate of 0.5 mL/min. After separation, compounds were detected by a Shimadzu RID-I0A refractive index detector (Shimadzu, Kyoto, Japan) as presented on Figure 6. and in Table 10. (Paper I, Paper II, Paper III and hydrolysis samples in Paper IV). Based on many years of gathered laboratory experiences in the research group and also supported by Bonn and Bobleter (1984) this HPLC system with the respective parameters is convenient to measure sugars and ethanol simultaneously in fermentation samples. The inhibitor content of liquid fraction in Paper III was also measured by this system.

Figure 6. – Chromatogram of the calibration mixture by the HPLC system described above. Peaks from the left: cellobiose, glucose, xylose, arabinose, (acetonitrile) and ethanol.

Table 10. – Properties of compound identification by the HPLC system described above. Concentrations assumed to exceed the calibration range were measured after dilution with distilled water.

Compound Retention time min

Calibration range g/L

Cellobiose 9.0 0.2-5.0

Glucose 11.0 0.3-10.0

Xylose 11.8 0.3-10.0

Arabinose 12.8 0.2-5.0

Ethanol 26.2 0.2-7.0

Total sugars (including both monomers and oligomers) of the liquid fraction used in Paper IV were determined after mild acid hydrolysis (4% (v/v) H2SO4, 120°C and 30 min). Monomeric and total sugar concentrations in liquid fractions were measured by HPLC (Waters, Mildford, MA, USA) with a 2414 refractive index detector (Waters, Mildford, MA, USA). A Transgenomic CarboSep CHO-682 carbohydrate analysis column was employed for the separation, and operating at 80°C with deionized water as mobile phase (0.5 mL/min).

The inhibitor content of liquid fraction in Paper IV was analyzed as follows.

Furfural, HMF, vanillin, 4-hydroxybenzaldehyde, coumaric acid, and ferulic acid were analyzed on an Aminex HPX-87H column at 65°C. For the mobile phase, 89% 5 mM H₂SO₄ and 11% acetonitrile, at a flow rate of 0.7 mL/min, were used. For detection, a 1050 photodiode-array detector (Agilent, Waldbronn, Germany) was employed. Acetic and formic acid were also quantified by HPLC (Waters, Milford, MA, USA) with a 2414 refractive index detector and an Aminex HPX-87H column maintained at 65°C with a 5 mM sulfuric acid mobile phase at a flow rate of 0.6 mL/min.

3.8.2 Enzyme activity assays