DETERMINACIÓN DE LA ACTIVIDAD ANTIMICROBIANA IN VITRO

On the 9th August 2012, urine and DCD treatments were applied to the area within the appropriate gas sampling and soil sampling rings. The following treatments were used; control (no urine or DCD); urine (700 kg urine-N/ha); DCD (10 kg DCD/ha) and; urine plus DCD (700 kg urine-N/ha + 10 kg DCD/ha). These treatments are referred to as “control”, “urine-only”, “DCD-only” and “urine+DCD” respectively. Synthetic urine was used due to the large volume required (see Table 5.1 for composition). Herbage was cut by hand fortnightly or when required for ease of soil and gas sampling. The soil pH was monitoring weekly to ensure the pH was maintained at the correct level. On the 18th of September 2012 an additional application of HCl and NaOH was applied to the installed gas and soil sampling rings because the acidic and basic pH treatments had started to return to the natural soil pH.

Table 5.1 Synthetic urine composition based on Fraser et al. (1994). Compound Rate (g/L) Urea 14 Glycine 3.5 Potassium bicarbonate 16 Potassium chloride 3 Potassium sulphate 2

5.2.2

Soil sampling and analysis

For each soil sampling date two soil cores (52 cm3) were taken at a depth of 7.5 cm from each soil sampling ring. These were homogenised and subsamples were taken from each replicate to determine concentrations of DCD, NH4+ and NO3- concentrations, soil moisture and soil

pH.

DCD was extracted from5 g of soil using 25 mL of deionised water. Samples were shaken for 1 hour and centrifuged for 20 mins and then filtered using Whatman No. 41 filter paper. DCD concentration was analysed on a Shimadzu series High Performance Liquid Chromatography (Tokyo, Japan) using a cation-H guard column (Phenomenex, USA) and a 0.025 M sulphuric acid mobile phase at a flow rate of 0.6 ml/min by UV detection at a wavelength of 210 nm.

Extraction of NH4+ and NO3- was carried out through the addition of 25 mL of 2 M KCl to 5 g

of soil. Samples were shaken for 1 hour and centrifuged for 10 mins and the supernatant filtered using Whatman No. 41 filter paper. NH4+ and NO3- concentrations were analysed

using a flow injector analyser (FIA) (FOSS FIAstar 5000 triple channel analyser) with SoFIA software version 1.30 (Foss Tecator AB, Sweden).

Soil moisture was determined for each of the samples for each sampling date by weighing a subsample of soil (approximately 10 g), drying it at 105˚C for 24 hours and then reweighing it. Soil moisture was calculated using the following formula: ((wet soil (g) - dry soil (g))/dry soil (g)) x 100.

Soil pH was measured throughout the trial to ensure the pH was maintained <5 for the acidic treatment and >6.5 for the basic treatment. A 10 g subsample of field moist soil was taken and 25 mL of deionised water added (Blackmore et al. 1987). Samples were shaken briefly

and left overnight before the pH was read using a Mettler Toledo Seven Easy pH meter (Mettler Toledo, Switzerland).

5.2.3

Nitrous oxide sampling

Gas samples were taken for N2O flux calculation twice per week for the duration of the trial.

Samples were collected early-afternoon and samples were taken at time zero, 20 mins and 40 mins using a syringe through a rubber septum, and stored in 6 mL exetainers awaiting analysis. N2O concentrations were analysed on a gas chromatograph (SRO8610 linked to a

Filson 222XL autosampler) using an Electron Capture Detector (ECD) (SRI Instruments, USA) and quantified using stored ambient air samples. Elevated concentrations were achieved using ethylene and acetylene in stored air samples.

5.2.4

AOB and AOA assays

Soil subsamples were taken from the soil sampling rings at days 1, 7, 14, 33, 56 and 102 after the application of treatments to determine ammonia mono-oxygenase gene (amoA) copy numbers for AOB and AOA. Soil samples were stored at -80˚C prior to extraction.

DNA was extracted from frozen soil (0.25 g) using NucleoSpin® Soil Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. DNA was eluted with 100 µL of Buffer SE (Macherey-Nagel, Düren, Germany. LOT. PAF00456026) and stored at -20˚C before being analysed.

PCRs were set up using the CAS1200 Robotic Liquid Handling System (Corbett Life Science, Australia), and real-time PCR was performed on a Rotor-Gene™ 6000 (Corbett Life Science). 10-fold dilutions were used for the PCR. Bacterial and archaeal amoA genes were quantified using the primers amoA1F/amoA2R (Rotthauwe et al. 1997) and Arch-amoAF/Arch-amoAR (Francis et al. 2005) respectively, with SYBR® Premix Ex Taq™ (TaKaRa, Japan) using the thermal profiles as described in Di et al. (2009). The 20 µL reaction mixture contained 10 µL of SYBR® Premix Ex Taq™ including primers, and 1.5 µL of template DNA. To confirm PCR product specificity, a melting curve analysis was carried out, by measuring fluorescence continuously as the temperature increased from 50 to 99˚C. Data analysis was carried out using Rotor-Gene™ 6000 series software 1.7.

Standard curves for real-time PCR assays were developed using the following method. Briefly, the bacterial and archaeal amoA genes were PCR amplified from the extracted DNA with the aforementioned primers. The PCR products were purified using the PCR clean-up kit (Axygen) and cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and the resulting ligation mix transformed into Escherichia coli JM109 competent cells (Promega) following the manufacturer’s instructions. Plasmids which were used as standards for quantitative analyses were extracted from the correct insert cloners from each target gene and sent for sequencing. A Qubit™ fluorometer (Invitrogen NZ) was used to determine the plasmid DNA concentration and the copy numbers of target genes calculated. Tenfold serial dilutions of the known copy number of the plasmid DNA were then subjected to a real-time PCR assay in triplicate to generate an external standard curve and to ensure amplification efficiency.