Measurement of enzyme activities in soil microcosms

The dehydrogenase activity of soil microcosm was assessed by a modified protocol as suggested by Singh and Singh (2005). 5 g soil sample was taken from each microcosm and subsequently placed in a sterile 50-mL polypropylene tube. Then, the soil was mixed with 5 mL of 0.5% 2,3,5-triphenyl tetrazolium chloride (TTC)-Tris solution. Tubes were then incubated at 30 °C for 24 h in dark. After the incubation got over, TTC was reduced to triphenyl formazan (TPF) which was then extracted with 100 mL methanol (Merck). The concentration of TPF was then determined spectrophotometrically by recording the absorbance at 485 nm.

Fluorescein diacetate hydrolysis assay (FDAH) was performed in soil microcosms by following the modified protocol described by Adam and Duncan (2001). 5 g soil sample was taken from each microcosm and mixed with 15 mL of sterile sodium phosphate buffer (Merck) (pH 7.6) in a 50-mL Erlenmeyer flask. After that, 0.2 mL solution of fluorescein diacetate (1 mg mL⁻¹) in acetone (Merck) was added to each sample. Flasks were then incubated for 20 min at 30 °C on a rotary shaker at 200 rpm. After the incubation got over, fluorescein was extracted from the soil with chloroform–methanol (2:1) mixture. Fluorescein concentration was thereafter measured spectrophotometrically by recording the absorbance at 490 nm.