Switchgrass Biomass Sugar Release Assay

The stems from the above-ground R1 stage transgenic switchgrass were harvested by removing the inflorescence, leaf blades, sheaths, internode 1 (I1) and top of the tiller following the standardized protocol [106 (link)]. After air-drying in the greenhouse for 3 weeks, the stem of transgenic switchgrass was ground to pass through a 20-mesh (0.841 mm) screen using a Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ). The milled material was then tested for total sugar release through a high-throughput method that combines hot-water pretreatment with enzymatic hydrolysis [116 (link)]. Briefly, 5 mg ground biomass was weighed in sample replicates into random individual wells on 96-well Hastelloy plates; ultrapure water (18.3 MΩ cm) from a MilliQ filter system was added. The plates were sealed with Teflon tape, clamped, and subjected to hot-water pretreatment at 180 °C for 17.5 min. The subsequent enzymatic saccharification was carried out by adding buffer to each well in the plate, mixing, and using Novozymes CTec2 at loadings of 70 mg enzyme/g biomass with incubation at 40 °C for 70 h. The sugar release was measured using a glucose oxidase–peroxidase (GOPOD) assay for glucose and a xylose dehydrogenase (XDH) assay for xylose absorbances versus standard curves [82 (link)].