10a series chromatograph

Products of the saccharification assays were analyzed by high performance liquid chromatography (HPLC) with a Shimadzu series 10A chromatograph. The HPLC was equipped with an Aminex HPX-87P column (300 x 7.8 mm, BioRad, Hercules, CA, USA) and a refractive index detector (Shimadzu Corporation, Kyoto, Japan). The column was eluted with water at a flow rate of 0.6 mL/min at 80°C.

The crude enzymatic extracts produced by C. cubensis and com- mercial cellulase (Multifect® CL) were applied in a biomass saccharification experiment. The C. cubensis enzymatic extract were concentrated 5-fold before the experiment using an Amicon Ultra- filtration system (Millipore Co. – Billerica, MA, USA) and an YM-10 (Cut-off Mr 10,000 Da) membrane filter. Enzymatic saccharification of alkali-treated sugarcane bagasse was performed in 2 mL sample tubes at an initial solid concentration of 2% dry matter (w/v) in 1.5 mL of 50 mM sodium acetate buffer at pH 4.5. Enzyme loading was specified as 10 FPase units per gram of biomass with the addition of sodium azide (10 mM) and tetracycline (40 μg mL⁻¹) to the reaction mixture to inhibit microbial contamination. The reaction was carried out in an orbital shaker at 250 rpm and 50 °C for different time intervals up to 72 h. These samples were immediately heated to 100 °C to denature the enzymes, cooled and then centrifuged for 5 min at 15,000 g. Products of the saccharification assays were analyzed by high performance liquid chromatography (HPLC) with a Shimadzu series 10 A chromatograph. The HPLC was equipped with an Aminex HPX-87P column (300 × 7.8 mm) and refractive index detectors. The column was eluted with water at a flow rate of 0.6 mL min⁻¹ and 80 °C.