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Cellulolytic activities in Cellic Ctec2 were determined with 1% carboxylmethyl cellulose sodium salt (CMC, Sigma-Aldrich, St. Louis, MO, USA) and 10 mM p-nitrophenyl-β-d-glucopyranoside (pNG, Sigma-Aldrich, St. Louis, MO, USA) as substrates for endo-glucanase and β-glucosidase, respectively [46 (link)]. One unit of enzyme activity refers to the enzyme amount that releases 1 micromole of the specified substrate per min under described protocol conditions [46 (link)]. The glucose content of hydrolyzed samples was quantified using a d-glucose GOPOD-format assay kit (Megazyme, Wicklow, Ireland). The soluble inhibitors of furfural, hydroxymethyfurfural (HMF), and acetic acid content were quantified by HPLC as described in our previous work [14 (link),15 (link)]. The HPLC system employed an Aminex HPX-87H ion exchange column (300 × 7.8 mm, Bio-Rad Laboratories Inc., Hercules, CA, USA) and was equipped with a refractive index detector.

To observe some possible soil biochemical changes, we studied acid phosphatase activity and it was measured using p-nitrophenyl phosphate (PNPP) (Sigma-Aldrich, St. Louis, MS, USA) as a substrate, according to the method described by Gianfreda, et al. [38 (link)]. The β-glucosidase activity was determined by detection of p-nitrophenol (PNP) released from p-nitrophenyl-β-d-glucopyranoside (PNG) (Sigma-Aldrich, St. Louis, MS, USA). In both assays, the amount of PNP formed was determined by spectrophotometry at 398 nm [39 (link)]. The hydrolysis of fluorescein diacetate (FDA) as an indicator of the total microbial activity of soil was determined according to Adam and Duncan [40 (link)] and expressed as μg fluorescein released per g of dry soil. The final concentration of FDA was measured as the absorbance at 490 nm.