Gerente General

On 27 August 2012, all the lysimeters were destructively sampled. Nine cores, 2.5 cm diameter were taken to a depth of 70 cm (the total depth of the lysimeters). Six soil cores were designated for laboratory analysis and were cut and separated into depths of 0-5, 5- 10, 10-15, 15-30, and 30-70 cm. The soil samples were stored, field moist, in plastic bags overnight in a chiller (< 4˚C). The following morning, in preparation for laboratory analyses, these soils were passed through a 4 mm sieve, and any herbage material was removed.

The remaining three cores were designated for root and stubble analysis. The stubble was removed and stored separately, while the soil from the entire length of the three cores was

bulked together for root analysis. All soil samples were stored, field moist in plastic bags in a chiller (< 4˚C) overnight.

3.2.7.1 Gravimetric soil moisture

Sub-samples of 5-10 g field moist sieved soil were weighed into small metal cups of a known weight, and oven-dried at 105˚C for 24 hours. The samples were cooled in a dessicator, then re-weighed and the gravimetric water content was calculated as a percentage of the dry soil mass using Equation 3.5 (Topp and Ferré, 2002): 𝜃𝜃𝑔𝑔 = 100 × �𝑀𝑀_𝑀𝑀𝑤𝑤

𝑠𝑠�

3.5

Where θg = gravimetric soil moisture content (g water per g oven dry soil); Mw= mass of

water (g) (mass of field moist soil (g) – mass of oven dry soil (g)); and Ms = mass of oven

dry soil (g).

3.2.7.2 Soil mineral N

The field moist equivalent of 10 g oven dry soil (approximately 15 g field moist soil) was extracted with 50 mL 0.5 M potassium sulphate (K2SO4) at 20°C on a reciprocating shaker (Mulvaney, 1996). The extractant was then filtered through fluted 12 cm

Whatman #42 filter papers into 50 mL screw cap specimen vials and frozen until analysis. Blanks consisting of filtered K2SO4 were also prepared. The extracts were analysed for ammonium (NH4+-N), and nitrate + nitrite (NO3--N + NO2--N) on the Skalar SAN++ segmented flow analyser (Skalar Analytical B.V., Breda, Netherlands). Equation 3.6 was used to determine the inorganic N concentrations in the soil:

𝑁𝑁𝑆𝑆 = 𝑁𝑁𝑒𝑒_𝑀𝑀 ×𝑉𝑉

𝑠𝑠 3.6

Where Ns = inorganic N content (mg kg-1 dry soil); Ne = inorganic N concentration of soil

extract (mg L-1); V = volume of solution (K2SO4 extract + soil moisture) (L); and Ms =

mass of oven dry soil (kg).

3.2.7.3 Soil microbial biomass N

The chloroform fumigation extraction method (Brookes et al., 1985) was used to

determine soil microbial biomass N. This method involved duplicate sub-samples of field moist soil being weighed out, one for fumigation, and the other non-fumigated sub-

prepared for soil mineral N analysis were also suitable “non-fumigated” extracts, therefore only the fumigated extracts required preparation.

Chloroform was first purified because commercial grade chloroform contains ethanol as a stabiliser, which is a C source (Jenkinson et al., 2004). The purification was carried out by shaking 100 mL of chloroform (Analar grade) with 200 mL of DI water in a 500 mL separating funnel. The water layer was discarded and the process was repeated twice more. The purified chloroform was stored in a stoppered glass bottle with anhydrous Na2SO4. Field moist soil (equivalent of 10 g oven dry soil) was weighed into aluminium cups. In a fume cupboard, the aluminium cups were positioned inside a dessicator along with a small conical flask containing 25 mL purified chloroform and ≈5 boiling chips. The lid was secured and the dessicator was evacuated using a vacuum pump until the chloroform was boiling – indicated by vigorous bubbling, at which point the dessicator was sealed and the vacuum pump removed. The dessicator was left inside a dark

cupboard and the samples fumigated for at least 12 h. After this time, the dessicator was brought back to atmospheric pressure and the flask with any remaining liquid chloroform was removed. The dessicator was then re-evacuated and flushed three times with fresh air and left open inside the fume cupboard for 0.5 h to remove any remaining chloroform vapour. The soil samples were removed from the dessicator and re-weighed (as the fumigation process can slightly dry out the samples), after which they underwent extraction with 0.5 M K2SO4 as described in Section 3.2.7.2. The fumigated and non- fumigated extracts were analysed for total N by “oxidative combustion-

chemiluminescence” using a Shimadzu TOC-VCSH/TNM-1 analyser and the fumigated samples were analysed for mineral N (NH4+-N and NO2--N + NO3--N) on a Skalar SAN++ segmented flow analyser (Skalar Analytical B.V., Breda, Netherlands). Organic N was calculated as the difference between total N and inorganic N. The microbial biomass N was calculated as the flush of organic N extracted from a fumigated soil less that

extracted from a non-fumigated soil. The microbial biomass N was adjusted by a kEN factor of 0.54 (Brookes et al., 1985; Joergensen and Mueller, 1996), which accounts for the efficiency of extraction of organic microbial N after fumigation.

3.2.7.4 Soil total N and 15_N

All soils were oven-dried at 50˚C for 48 hours. A representative sub-sample was

removed by riffling the dried soil. The sub-samples were then ground using a RockLabs orbital soil grinding machine CH-4 (Gilco Products, Albany, Auckland, New Zealand)

and analysed for total N and 15N enrichment on an Isotope Ratio Mass Spectrometer (EA- CF/IRMS; PDZ Europa GSL / 20-20). A detailed description of 15N analysis by IRMS is given in Section 3.2.8. Samples from lysimeters that did not receive 15N fertiliser

treatments were riffled and ground first to minimise the risk of cross contamination of 15_{N. All equipment was also thoroughly cleaned between treatments.}

The 15N recovered from the soil pool as a percentage of the total 15N applied, was then calculated using Equation 3.2.

3.2.7.5 Roots and stubble

The soil was rinsed off the root and stubble samples and they were oven dried at 55˚C, weighed, recorded, and then finely ground using the Cyclone Sample Mill with 1 mm screen (UDY Corporation, USA). The ground samples were then analysed for total N and 15_{N enrichment using an Isotope Ratio Mass Spectrometer (EA-CF/IRMS; PDZ Europa} GSL / 20-20). A detailed description of 15N analysis by IRMS is given in Section 3.2.8. The 15N recovered from the roots and stubble as a percentage of the total 15N applied, was calculated using Equation 3.2.