4.4. Soil Chemical Analysis

Urease activity was measured following the method described in [64]. Five grams of soil was incubated with 10 mL of citrate phosphate buffer (pH 6.7) and 5 mL of 10 % urea solution at 38 °C for 3 h. Activity was determined by measuring the released NH₄⁺ with a spectrophotometer at 578 nm. Acid phosphatase activity was analyzed with nitrophenyl phosphate disodium (PhOH mg g¹, 37 °C, 24 h), and catalase with KMnO₄ (0.1 mol L⁻¹ KMnO₄ ug g⁻¹, 30 °C, 20 h) [65]. Sucrase activity was determined by the method of [66]. For sucrose, the air-dried soil (5 g) was incubated with 15 mL sucrose. Five microliters (5 mL) of phosphate buffer (pH 5.5) and five drops of toluene at 37 °C for 24 h, and the reaction solution was filtered through the quantitative filter paper as rapidly as possible after incubation. Filtrate (1 mL) was mixed with 3 mL salicylic acid at 100 °C for 5 min in the water bath, and the mixture was adjusted to 50 mL and cooled with deionized water. Sucrase activity was determined spectrophotometrically at 508 nm. The protease activity was determined by ninhydrin colorimetry, expressed in milligrams of amino nitrogen in 1 g of soil cultured for 24 h in a 37 °C incubator [67].

SOC was determined by the hydrated potassium thermo-dichromate oxidation method. TN was determined using the CN elemental analyzer, while the colorimetric method of the molybdenum–antimony solution with royal acid was used to determine TP. The flame photometer method was used to determine TK. AN was analyzed by the Kjeldahl method [68]; AP was determined by the diacid extraction spectrophotometric colorimetry method [69], and AK was determined using a flame photometer method by ammonium acetate extraction [70]. Detailed information about the total and available nutrients was also mentioned in our published paper [6].