The urease (EC 3.5.1.5) activity in the soil was measured with the buffered method of Kandeler and
Bremner [20]. In this procedure, 0.5 mL of a solution of urea (0.48 %) and 4 mL of borate buffer (pH 10)
were added to 1 g of soil in hermetically sealed flasks, and then incubated for 2 h at 37 °C. The
ammonium content of the centrifuged extracts was colorimetrically determined at 578 nm by a modified
indophenol blue reaction. Controls were prepared without substrate to determine the ammonium
produced in the absence of added urea, and the activity was expressed as NH4
+ N mg/(g h).
After the soil amendment and incubation, ten grams of soil with six different levels was thoroughly
mixed with 90 mL distilled water, respectively. 1 mL of the obtained soil solution was further transferred
into 100 mL conical flask containing 30 mL nitrifying bacteria culture, and incubated for another 15 days
at 28 °C. Nitrifying activity of those soils was measured by the decrease of nitrite (%). Here, the
nitrifying bacteria culture consisted of 1 g NaNO2, 0.03 g MgSO4 ·7H2O, 0.01 g MnSO4·4H2O, 0.75 g
K2HPO4, 1 g Na2CO3, 0.25 g NaH2PO4 and 1g CaCO3 in 1 L distilled water with the natural pH value.