Laboratory analyses
Moreover, after 3 years of vegetation in October 2011 samples of
surface soils (0–25 cm) were taken and kept at 4 °C in October 2011
after 3 years of vegetation. Enzymatic activity analysis was done in
7 days. Dehydrogenase activity determination was based on the estimation
of 2,3,5-triphenyltetrasolium chloride (TTC) reduction rate to
triphenylformazan (TPF) in soils after incubation at 30 °C for 24 h as described
by Thalmann (1968). Acid phosphatase activity was assayed as
described by Tabatabai and Bremner (1969) using p-nitrophenyl phosphate
(pNPP, 0.115 M) as the substrate. These assays are based on the
release and detection of p-nitrophenol (pNP). 4 cm3 of 0.1 M MUB
(modified universal buffer) with pH 6.5 and 1 cm3 of substrate were
added to 1 g soil sample and incubated at 37 °C for 1 h. After stopping
the reaction the amount of pNP released for the enzyme was determined
spectrophotometrically at 400 nm. Urease activity was assayed
as described by Zantua and Bremner (1975) using urea as the substrate.
Afterincubationat37 °Cfor2 h, theN-NH4+formedwasextractedwith
1 M KCl and subsequently measured. Protease activity was assayed as
described by Ladd and Butler (1972) using 2% solution of sodium caseinate
in Tris (hydroxymethyl) buffer with pH 8.1 as the substrate.
After incubation in a water bath at 50 °C for 1 h, the level of activity of
the enzyme was determined according to the amount of amino acids
produced colorimetrically with Folin reagents. The color intensity was
measured in a spectrophotometer at wavelength of 700 nm.