2.4. Soil enzymes activities
Soil catalase activity was determined using the potassium permanganate titration (Guan, 1986; Li et al., 2014). 40 ml of distilled water and 5 ml of hydrogen peroxide solution (3%) were added to 2 g of soil, which was shaken for 30 min and then filtered. We then took 25 ml of the filtrate and titrated it to pink with 0.1 M potassium permanganate.
The urease activities were analyzed according to the methods used by Guan and Yin (Guan,1986; Yin et al, 2014). The soil (2 g) was treated with 10 ml urea (10%), 20 ml citrate buffer (1 M, pH 6.7), and 1 ml of methylbenzene and stored at room temperature for 15 min. The sample was then shaken at 37℃ for 24 h. The solution was filtered, and 1 ml of the filtrate was mixed with 20 ml of distilled water, 4 ml of sodium phenolate hydroxide, and 3 ml of sodium hypochlorite. The NH4+-N was analyzed 20 minutes later using a spectrophotometer at 578 nm. Urease activity was expressed in milligrams of NH4+-N per gram of dry soil released in 24 h.
The invertase activity and amylase enzyme activities were analyzed according to the methods used by Guan (Guan, 1986), using 3, 5-dinitrosalicylic acid, the invertase activity and amylase activities were measured using sucrose solution and soluble starch as respective substrates, respectively. The invertase activity was expressed as the mass (mg) of glucose in 1 g of soil after 24 h; the amylase activity was expressed as the mass (mg) of maltose in 1 g of soil after 24 h.