Material and methods

5.2.1. Study area and soil sampling

The soil samples were collected from three Chilean Andisols (UFRO experimental site) in Southern Chile in La Araucania and Los Rios regions: Pemehue (PEH) (39°04` S and 072°10` W), Puerto Fonck (41° 28` S and 72° 59` O) and Piedras Negras (40° 19` S and 72° 56` O). In the past, all three sites were characterized by a forest land cover, today the actual land use are, for Puerto Fonck and Piedras Negras, permanent grassland (or graze), while for Pemehue annual cropping (wheat) under conventional tillage. In each site, three replicate samples were taken from the top layers (0-20 cm), air-dried and sieved at <2 mm mesh prior to analyses.

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5.2.2 Dry sieved fractionation

All dry samples were sieved and separated into 4 size of aggregates: > 2 mm, 2 to 0.25 mm, 0.25 to 0.053 mm and < 0.053 mm. The sieving was performed by Sieve Shaker instrument (Retsch AS 200) equipped with a tower of three sieves placed, from the top to the bottom, in the followed order of mesh size: 2 mm, 0.250 mm and the finest of 0.053 mm. Each sample was sieved for 3 minutes at 150 rpm. This method allow us to separate the micro- (< 250 µm) and macro-aggregates (>250 µm), and in our case the size classes were 0.025-0.053 and <0.053 mm, >2 and 2-0.025 mm micro- and macroaggregates respectively.

After the sieving, all the separated fractions were weighed and it was calculated the % mass recovery:

% 100

Where ai was the mass of a specific fraction and X was the total mass. 5.2.3. Hydrogen peroxide oxidation

The soil aggregates were oxidized by hydrogen peroxide. This oxidation is never complete and it has been used as the oxidation step in methods of mineralogical analyses (Kunze & Dixon, 1986). The organic matter resisting peroxide treatment consisted of organic interlayer complexes (Righi et al., 1995; Theng et al., 1992), so this treatment could be considered analogous to biological mineralization over the long term (Plante et al., 2004). In particular, 500 mg of > 2mm fraction and 200 mg of other three size dimensional fractions (2 -0.25 mm, 0.25-0.053, < 0.053 mm) were oxidized with 30% H2O2 adding in a flask respectively 37,5 ml and 15 ml and 75 ml and 30 ml of acid water solution at pH 2 (water acidified at pH 2 by HCl 0.1 M) with H2O2:suspension ratio of 1:2. The reaction was performed at the constant temperature of 60 °C for 16 h and on a magnetic stirrer. The mixing and the introduction in each flask of little glass bolls ensure homogeneous exposure of the organic matter to the oxidant and the breakdown of soil aggregates. At the end of reaction period, the

176 suspension was neutralized by NaOH 2 M and dialyzed against deionized water, to

remove excess of H2O2 and salt. The dialysis was performed transferring

quantitatively each sample in a dialysis bag of 1000 kDa membrane by a pipette and placing each one in deionized water until the achievement of constant value of water electrical conductivity. Each sample was later transferred into little plastic jars, frozen and then lyophilized. The dried samples were weighed and it was calculated the removed mass (%), with the purpose to evaluate the efficiency of OM oxidation for each fraction:

% 100

Where ai1 was the mass fraction before oxidation, and ai2 the mass fraction after

oxidation.

5.2.4. Organic C and total N

The soil fractions were analyzed for total carbon and nitrogen content, using the flash combustion method performed by Elemental Analyzer EuroEA3000 series instrument. Prior the analyses, the samples were grounded in a agata mortar and sieving in a sieve of 160 µm mesh. About 2,5 mg and 1,5 mg respectively for < 2 mm and smaller fractions, were weighed in tin caps by analytical balance. To determine the C and N concentration in each sample, a calibration curve was performed using L-Cystine as standard and a soil certified reference material (C 4,40%, N 0,262%).

5.2.4. Total P content

The total phosphorus content was determined by hypobromite alkaline oxidation, using sodium hypobromite (NaBrO) (Dick & Tabatabai, 1977). Briefly, 100 mg of crushed sample were weighed in flask and added with 3 ml of NaBrO; the suspension was heated at 260-280°C (by hot-plate with sand bath) until the solution was totally dried, then from this time, they were continue heating for more 1 h. When the samples were cold, 4 mL of distillated water, 1 mL of formic acid (HCOOH) and 25

177 mL of 0,5 M H2SO4 were added. The samples were left overnight to the complete Br removal, and the next day, the total P was determined by the molybdenum blue method: 2,5 ml of each samples was added with 500 μl of NaOH 2N and 10 ml of molybdenum blue, the samples were left for 1 h until the spectrophotometric analyses by UV/visible spectrophotometer, and then the absorbance of each samples was measured at 750-800 nm (at typical molybdenum blue wavelength absorbance). The calibration standards were prepared at the same way.

5.2.4. Laser Scanning Confocal Microscope images

All the dry fractions, in one replicate for each experimental site, were observed at confocal microscope. The technique allows an image analyses in which the distribution of organic matter through soil fraction can be estimated thanks to the organic matter autofluorescence. The fluorescence is the property of some atoms and molecules to emit light at longer wavelengths after absorbing light of particular and shorter wavelengths (Herman, 1998). The absorption of a photon of energy, cause the excitation of the electron of a fluorescent molecule from the ground state to a higher electronic energy and vibrational state; the energized electron then returns to the ground energy state with a loss of vibrational energy to the environment and a photon of longer wavelength is emitted (Li et al., 2004). Some molecules are autofluorescent and emit fluorescence when excited (primary fluorescence, i.e. protein, nucleic acid, lipids). The image was taken by Laser Scanning Confocal Microscope (CLSN, fluorview 1000 CLSM), the light emitted by an excitation source is firstly absorbed by the sample and then emitted, in this way the autofluorescent OM is showed in light blue or green, and the color is related to different wavelength selected for the analyses and to chemical functional group in organic matter. Images were taken on one replicate for each Andisol.

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5.2.5. Electrophoretic mobility

Electrophoretic mobility measurements were made using a Zetasizer Nano ZS apparatus (Malvern Instruments). The analyses was performed only for Piedra Negras site and for <2mm and <0.053 mm aggregates, before and after the H2O2 oxidation. Briefly, 1 mg of sample was suspended in 1 mL of 0.001 M NaCl by immersion in an ultrasonic bath for 5 min. Measurements were carried out over a range of pH values (between 3 and 10), adjusted by careful addition of either 0.01 M HCl or 0.01 M NaOH (Calabi-Floody et al., 2011)

5.2.6. Data analysis

Statistical analysis was performed using SigmaPlot 12.0 software. The differences among soil fraction and among experimental sites were tested by One way ANOVA followed by Student-Newman-Keuls test (significance level P< 0.05).

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