2.4. Biochemical properties of soil
The soil biological activity measurements included enzymatic activity and soil respiration. Dehydrogenase activity (DhA) was determined by the method in Thalmann (1968) using triphenyltetrazolium chloride (TTC) as the electron acceptor. Samples were incubated at 37±2 °C for 24 hours. The enzymatic activity of the dehydrogenases was determined by colourimetry using a Backman DU 640 spectrophotometer (Beckman Coulter, Woonsocket, USA) at a wavelength of 546 nm.
Urease activity (Ure) was determined by the Zantua and Bremner (1975) method, with urea as a substrate, after a 18 hour incubation at 37 °C. The enzymatic activity of ureases was determined by colourimetry using a Backman DU 640 spectrophotometer at a wavelength of 470 nm.
Basal respiration (BR) was determined according to the ISO 16072 (International Organization for Standardization, 2002) method. Moist soil subsamples (20 g) were incubated at 20±2 °C for 24 hours. The released CO2 was absorbed in a solution of 0.05 M NaOH and precipitated as barium carbonate with the addition of a 0.5 M BaCl2 solution. The unconsumed sodium hydroxide was titrated with 0.1 M HCl in the presence of phenolphthalein as an indicator, and the amount of CO2 was calculated (ISO 16072: 2002). Substrate-inducted respiration (SIR) was determined by the quantification of carbon dioxide evolution shortly (6 hours) after glucose addition (aqueous solution equivalent to 10 g glucose per kg of soil) (ISO 14240-1: 1997). The respiratory-activation quotient (QR) was calculated by dividing the BR rate by the SIR rate according to ISO 17155 (2012).