Enzymes and Activities
Celluclast 1.5 L and Novozym 188 were obtained from Sigma Aldrich. Filter paper and carboxymethyl cellulose activities were used as a measure of cellulase activity. FPA 4.49 FPU/mL and CMCase to 20.6 U/mL were measured after 7 days of fermentation. p-Glucosidase activity on pNPG was measured as 4.77 U/mL and xylanase activity as 6.61 nkat/mL. Commercial cellulase (celluclast 1.5 L) showed 81.8 FPU/mL filter paper activity, p-glucosidase activity of 58.66 U/mL, and xylanase activity of 107.3 nkat/mL. pNPG activities ф-glucosidase) in A. saccharolyticus and in commercial enzyme Novozym 188 were 339.9 U/mL and 698.3 U/ mL, respectively.
Enzymatic Hydrolysis of Wet-Exploded Loblolly Pine (WELP)
Enzymatic hydrolysis assays were carried out in 125 mL shake flasks at 50 °C with agitation at 150 rpm at pH 5 in a shaking incubator. The total working volume was 50 mL and all the experiments were performed in quadruplicate with standard deviations less than ±2. WELP was mixed with citrate buffer (50 mM, pH 4.8) and appropriate amount of enzymes to achieve total solids 20 %. All the enzymatic hydrolysis assays were conducted at 20 % (w/w) total solids at the abovementioned conditions for 72 h. After 72 h, enzymatic hydrolysis was stopped. Samples were withdrawn periodically at 24, 48, and 72 h, centrifuged using benchtop centrifuge (Eppendorf, Model 5804, 8000 rpm, 10 min), and analyzed for sugars and sugar degradation compounds.
Constant amount of in-house produced cellulase (15 FPU/g cellulose from T. reesei RUT-C30 and 30 CBU/g cellulose from A. saccharolyticus) was used in all the experiment with varying amount of commercial enzyme supplementation, ranging between 1 and 100 % of in-house enzymes (FPU or CBU).
Two controls were used: control-1, in-house produced enzymes without any supplementation with loading of 15 FPU + 30 CBU per gram cellulose, and control- 2, commercial enzyme, Cellic®Ctec2, with loading of 45 FPU per gram cellulose. 45 FPU/g cellulose of Cellic®Ctec2 is representing the sum of both filter paper and P-glucosidase activities. For the supplementation assays, commercial cellulase, hemicellulase, and p-glucosidase were used. Cellic®Ctec2 and Cellic®Htec2 were used in a dosage ranging from 1 to 100 % of the total FPU (15 FPU) of the in-house enzymes. Novozym 188 was used with dosage ranging from 1 to 100 % of total CBU (30 CBU) of in-house enzymes.