Acroprep 0.2 m ghp membrane 96 well filter plates

Small scale simultaneous saccharification and fermentation (SSF) was conducted in 30 mL wide-necked glass vials containing 1 g (DM equivalent) of wet pretreated solid, made up to 17.9 mL with yeast nitrogen base (Formedium, Hunstanton, UK) at pH 5.0. The bottles were then autoclaved (121 °C, 15 min) to ensure sterility. The bottles were cooled to 25 °C, and 2 mL of yeast grown in Difco YM media (Fisher Scientific UK Ltd, Loughborough, UK), was added along with 100 µL Cellic^® CTec2 (Novozymes, Bagsvaerd, Denmark), 20 FPU/g substrate. The yeast inoculum used was a Saccharomyces cerevisiae strain—NCYC 2826—chosen from the National Collection of Yeast Cultures (UK), selected on the basis of its high ethanol tolerance (15–20 % v/v). The inoculum had a viable cell count of 9.87 × 10⁷ cells/mL. Bottles were incubated under continuous agitation (120 h, 25 °C) after which, a measured sample was boiled in gas tight screw cap tubes (Starlab Ltd, Milton Keynes, UK), centrifuged (13,000 rpm, 5 min) and supernatant filtered through AcroPrep™ 0.2 µm GHP Membrane 96 Well Filter Plates (VWR International Ltd, Lutterworth, UK) into a 96 deep well collection plate before analysis.

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.