OZONIZACIÓN

At 24 weeks, six plants per species in their vegetative growth stages (phenological growth stage determined by observation) were selected at random and the whole plant was cut just above the soil surface at noon, assuming peak physiological activity. The shoot of each plant was put in a labelled brown paper bag. The bagged shoots were oven dried at 65℃ (Contherm, ThermoTec 2000) for 24 h. Once dried, each sample was milled using a grinder and passed through a 1 mm sieve, then put in labelled resealable clear plastic bags (15 cm × 8 cm). All milled and sieved plant samples were stored in an airtight plastic container at room temperature. Dried plant powder of single plants was used for the following analyses.

6.3.2.1 Silicon

Silicon (Si) was extracted from the plant material (six replicates per plant) by digesting 0.1 g of ground plant material in 2.5 mL of 50% sodium hydroxide and 3 mL of 30% hydrogen peroxide with 5 drops of an antifoaming agent (octanol), made up to 50 mL with deionized water and autoclaved at 120℃ for 60 min (Snyder 2001). The digest was analysed for Si using the Varian 720 ICP-OES (Inductively Coupled Plasma Optical Emission Spectrophotometer) (Varian, Australia) with standard setting (axial torch power of 1.20 kW, plasma gas flow 15.0 L/min, aux 1.5 L/min and nebulizer 0.9 L/min). The concentration of Si in the sample was related to the intensity of lines in its optical spectrum. The emitted light was spectrally resolved by diffractive optics, and the intensity of light was measured with a detector (Nölte 2003), with complete wavelength coverage from 167-785 nm and a resolution of 7 rpm to capture all wavelengths in one simultaneous reading. Calibration standards and internal standards were serially diluted from Merck ICP standard solutions using MilliQ water (Barnstead). Calibration curves were generated using at least four standards and a standard blank.

6.3.2.2 Total carbon, nitrogen and carbon-nitrogen ratio

Total carbon (C) and nitrogen (N) were analysed using an Elementar Vario-Max CN Elemental Analyser. Samples (2 mg, six replicate per sample) were combusted at 900℃ in an oxygen atmosphere. The combustion process converts any elemental carbon and nitrogen into CO2, N2 and NOx. The NOx species were subsequently reduced to N2. These gases were then passed through a thermal conductivity (TC) cell to determine CO2 and N2 concentrations and the %C, %N and C/N ratios calculated from the sample weights.

6.3.2.3 Acid detergent fibre

Acid detergent fibre (ADF) was determined gravimetrically by extraction with a cetyl- trimethylammonium bromide and sulphuric acid solution. An acidified quaternary detergent solution was used to dissolve cell solubles, hemicellulose and soluble minerals leaving a residue of cellulose, lignin and heat damaged protein, plus a portion of cell wall protein and minerals (ash). ADF was determined as the residue remaining after extraction as follows: One gram per sample (six replicates) was dispensed into a 600 mL beaker and the sample weight recorded (sW). Samples were dispensed in duplicate, including one quality control per run. 50 mL of detergent solution (prepared by dissolving 20 g of technical grade cetyltrimethylammonium bromide to 28 mL of 95- 98% sulphuric acid and made up to a litre by adding reverse osmosis purified water) was added to each beaker. Beakers were fitted condensers that were brought to boil on a heating apparatus and refluxed for 1 h. When foaming of the sample settled, beakers were swirled and the sides of the beaker rinsed with a small quantity of ADF solution if required. Each sample was poured into a 60 mL Gooch crucible (porosity 1) to collect the fibre residue and the remaining beaker content was rinsed into the crucible with hot water. The detergent was drained using a Büchner apparatus for speed filtration. Samples were not fully drained between rinses to prevent blockage of the crucible. The residue was then washed about six times with hot water until all the detergent was removed and finally rinsed with acetone (Lab grade). Crucibles were placed in an oven and dried at 100℃ (± 5℃) for 12 hours. Subsequently, the crucibles were allowed to cool in a desiccator and weighed, with digested weights recorded as dW. The crucibles were then transferred to a furnace and the content ashed at 500℃ for 2 hours. The cool crucibles were placed in a desiccator and reweighed with sample weights recorded as aW.

ADF was calculated using the following formula;

% ADF (as is) = 100 ×�(dW_sW−aW)�

% ADF (DM basis) =�ADF × 100_rDM �

Where sW = sample weight, dW = digested weight, aW = ashed weight, and rDM = residual dry matter of the sample (determined on an independent sub-sample by standard methods).

6.3.2.4 Neutral detergent fibre

Neutral detergent fibre (NDF) was also determined gravimetrically by extraction with sodium lauryl sulphate, ammonium pentaborate and ethylenediaminetetraacetic acid (EDTA). A neutral

detergent solution was used to dissolve the easily digested pectins and plant cell contents (proteins, sugars and lipids), leaving a fibrous residue (NDF) that is primarily cell wall components of the plant (cellulose, hemicellulose and lignin). The detergent was used to solubilise the proteins and EDTA was used to chelate calcium and remove pectin at boiling temperatures.

One gram per sample (six replicates) was dispensed into 600 mL beakers with sample weights recorded as sW (samples were also dispensed in duplicate, including one quality control per run). 50 mL of detergent solution (prepared by dissolving 19 g of technical grade disodium-EDTA dihydrate, 27 g ammonium pentaborate decahydrate and 30 g of sodium lauryl sulphate in 1 L of reverse osmosis purified water) was added to each beaker. Samples were then refluxed, washed, rinsed, dried and ashed as in 6.2.6 above.

NDF was calculated as follows;

% NDF (as is) = 100 ×�(dW_sW−aW)�

% NDF (DM basis) =�NDF × 100_rDM �

Where sW = sample weight, dW = digested weight, aW = ashed weight, and rDM = residual dry matter of the sample (determined on an independent sub-sample by standard methods).