2.4 Procedures for SOM and density fractionation
The soil samples obtained for the determination of the SOM and its densimetric fractions were air-dried and passed through a 2-mm sieve. The total carbon content was determined by oxidation with potassium dichromate and colorimetric determination (Cantarella et al., 2001).
The SOM was fractionated into the free light fraction (FLF), occluded light fraction (OLF) and heavy fraction (HF) by following the methodology proposed by Elliott and Cambardella (1991). Briefly, 35 mL of sodium polytungstate solution (PTS) with a density of 1.85 g cm-3 was added to 10 g of soil contained in a 50-mL centrifuge tube. The tube was closed and inverted manually and slowly five times to release the FLF without breaking the aggregates. The suspension was centrifuged at 4100 g for 60 min, and the supernatant containing the FLF was vacuum-filtered on a pre-weighed AP40 glass fiber filter. To remove the excessive PTS, the filters and FLF contained in the sample were washed twice with distilled water and dried at 60 °C for 24 hours. The mass of the filter plus the FLF was quantified. To separate the OLF, the PTS solution was returned to the tube containing the pellet, and two glass balls with a diameter of 7 mm were added. The tubes were slowly dispersed in a shaker at a rate of 75 oscillations per min for 18 hours. After dispersion, the suspension was centrifuged again (4100 g for 60 min), and the OLF was obtained by filtration using the same protocol as that used for the FLF. The mass of the HF was obtained as the difference between the SOM and the mass of FLF + OLF.