Series 200 lc instrument

The concentration of reducing sugars released from the substrates was quantified using a scaled dinitrosalicylic acid (DNS) method [23 (link)]. Glucose concentrations were quantified using a glucose-specific kit (GOPOD, Megazyme International Ireland, Bray, Ireland). Substrate and enzyme controls were included wherever necessary.
Terminated fermentations, after centrifugation and filtration through a 0.2 μm filter, were loaded directly onto a Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector. The analyses were carried out using an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with matching guard columns operating at 65 °C with ultrapure water as mobile phase at a flow rate of 0.6 mL/min as described [22 (link)].

A sample of each pretreated substrate was suspended in 10 mL solution with a final concentration of 5 % substrate in nitrogen base (Formedium, Hunstanton, UK) and held in 20mL screw-topped glass vials. Both cellulases (36 FPU cellulase/g substrate) and a concentrated yeast inoculum were added to each vial and incubated for 96 h, 40 °C.
The yeast inoculum used was a robust thermo-tolerant yeast (Saccharomyces cerevisiae, strain NCYC 2826, National Collection of Yeast Cultures, Norwich, UK) grown from a slope culture, inoculating 1 L of yeast mould (YM) broth (3 d, 25 °C) before centrifuging, discarding the supernatant and partially reconstituting the yeast in nitrogen base. The final solutions contained 3.83 × 10⁷ viable cells/mL when inoculated—assayed using a NucleoCounter® YC-100™ (ChemoMetec, Denmark). SSFs were conducted as three independent replicates, and the ethanol released from a cellulase + yeast control was subtracted from each sample.
Ethanol concentrations were quantified using HPLC using a Series 200 LC instrument (PerkinElmer, UK) equipped with an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The mobile phase used was ultrapure water (0.6 mL/min) and concentration quantified using a refractive index (RI) detector, comparing absorbance to a set of ethanol standards.

Freeze-dried solids were acid hydrolysed (72 % (w/w) H₂SO₄, 3 h, RT followed by dilution to 98 g/L H₂SO₄, and heating for 2.5 h, 100 °C) to convert polymeric sugars into their monomeric constituents [20 ] . The hydrolysed samples were cooled on ice (>10 min) and then centrifuged (2500 rpm, 2 min). To a 1 mL aliquot from each hydrolysed sample 100 µL ribose internal standard (30 mg/mL) was added. Samples were neutralised with CaCO₃ (2.5 mL, 2 mol/L). The precipitated salt was removed by centrifugation (3000 rpm, 10 min). Filter plates (AcroPrep™ 0.2 µm GHP Membrane 96 Well Filter Plates, VWR International Ltd, Lutterworth, UK) were used to filter portions of each sample (1 mL) prior to HPLC by centrifugation at 500 rpm for 10 min. Deep well collection plates were sealed with pierceable lids (Starlab (UK) Ltd, Milton Keynes, UK) and loaded directly onto Series 200 LC instrument (Perkin Elmer, Seer Green, UK) equipped with a refractive index detector and employing an Aminex HPX-87P carbohydrate analysis column (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with matching guard columns.