VALORACIÓN DE CRITERIOS DE EVALUACIÓN PARA WCF

3.4.1 Experimental design

An incubation study investigated the effect that different cow urine application rate, DCD application rates, and biochar had on soil nitrification rates and ammonia oxidisers under dairy winter forage grazing conditions.

The Balmoral stony silt loam soil was collected from the Lincoln University Ashley Dene Farm. Prior to starting, 100% water holding capacity (field capacity) of the soil was calculated. This determined whether the soil needed to be wetted or dried prior to treatment application and allowed the soil moisture content to be maintained at field capacity throughout the trial by adding deionised water. A randomised block design was chosen to reduce the effect of airflow and temperature variation within the incubator. Ten different treatments were blocked in four replicates (Table 3.9). Plastic honey pots referred to as ‘pottles’ were used. They had a volume of 1000 ml and were chosen as they allowed enough soil for the duration of the trial.

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Table 3.9 Treatments applied to the soil for the incubation experiment.

Treatment Urine (kg N ha-1) DCD (kg DCD ha-1) Biochar (tonnes biochar ha-1) Control 0 0 0 DCD 0 20 0 Urine 500 500 0 0 Urine 700 700 0 0 Urine 500 + DCD 10 500 10 0 Urine 500 + DCD 20 500 20 0 Urine 700 + DCD 10 700 10 0 Urine 700 + DCD 20 700 20 0 Urine + biochar 700 0 1.75 Urine + DCD + biochar 700 20 1.75

3.4.2 Trial setup

On December 12th 2011, 20 kg of Balmoral stony silt loam soil was collected from the top 10 cm of the soil profile from the Lincoln University Ashley Dene Farm. At the time of collection, the soil was sieved into large 20 L buckets using a 5 mm sieve. This removed all stones from the soil and resulted in a uniform soil. The soil was then transported back to Lincoln University where the buckets of soil were stored at 4oC.

On January 20th 2012, 500 g of dry soil was weighed out for each pottle. It was important to ensure an accurate weight of soil was measured as the moisture content could have been affected if not. Biochar was applied to the pottles requiring it at this time. Two grams of biochar (equivalent to 1.75 tonnes biochar ha-1) was uniformly mixed into the soil by placing both the soil and biochar into a plastic bag and shaking well. This low biochar application rate was used for economic reasons. The biochar used was made from Pinus radiata pyrolysis at 550oC (Pacific Pyrolysis, NSW, Australia). The characteristics of biochar are shown in Table 3.4. Once all soil pottles were filled with soil (and biochar), each pottle was tapped on the bench twice to settle the soil. A lid with two aeration holes (0.5 cm diameter) was then placed on top of each pottle. The pottles were placed into an incubator set at 10oC for one week so that pre-incubation equilibrium could occur (incubation prior to the remainder of the treatments being applied) (Figure 3.12). During this period, the soil moisture content was maintained at field capacity. To

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maintain the moisture content, deionised water was added to the individual pottles by weight. The soil was then well mixed ensuring the structure of the soil was maintained.

Figure 3.12 Pottles in the incubator.

The treatments (Table 3.9) were assigned in a random block design to the pottles. Biochar was applied on January 20th _{2012 while urine and DCD were applied on the January 31}st _{2012. The}

urine used was fresh dairy cow urine from grass-fed lactating cows. This was collected from Lincoln University Research Dairy Farm two days prior to treatment application. Following the protocols outlined in Section 3.2.4.1, the urine-N content was measured and corrected to 500 kg N ha-1 and 700 kg N ha-1. The urine was applied by pouring 30 ml (500 kg N ha-1) or 42 ml (700 kg N ha-1) of urine evenly over the soil surface to those pottles which require it. The two different DCD concentrations were made up by adding 83.3 mg of DCD to 100 ml deionised water for 10 kg DCD ha-1 and by adding 166.6 mg of DCD to 100 ml deionised water for 20 kg DCD ha-1. Using an auto-pipette 5 ml of DCD in solution was applied to the relevant pottles after urine addition.

Following treatment application, the soil moisture content was adjusted to field capacity by adding deionised water. Soil within each pottle was then thoroughly mixed while maintaining the soil structure. The pottles were then placed back into the incubator which remained at 10oC. Throughout the trial period, the soil moisture content was maintained at field capacity. This was achieved by checking the weight of each pottle twice weekly and adjusting to the desired weight with deionised water.

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3.4.3 Field capacity of the soil

The field capacity of the soil was measured prior to treatment application. This was determined by saturating (with water) three soil cores with a volume of 302 g cm-1 (packed to a bulk density of 1.0 g cm-1) on a tension table set at -10 kPa and allowing them to reach equilibrium over a three day period. The moisture content of the soil samples was then measured gravimetrically. The soil was weighed and dried (at 105oC for 24 hours) and weighed again to calculate the field capacity gravimetrically (wet-basis).

3.4.4 Soil extractions

Subsamples of soil were collected on day 1, 7, 14, 21, 28, 42, 56, 70, 86, and 112 after treatment application. On the same day before sample collection the soil moisture content of each pottle was adjusted to 100% water holding capacity and mixed. A subsample of soil was removed from each pottle and placed into a small vial for molecular analysis. These samples were kept at -80oC until analysis. For NO3--N and NH4+-N analysis the protocols outlined in Section 3.2.8.2 were

followed. For DCD analysis the protocols outlined in Section 3.2.8.3 were followed.

3.4.5 Soil moisture

At each sampling occasion, the soil moisture content was determined following the protocols outlined in Section 3.2.8.4.

3.4.6 Chemical analysis

The soil extracts were analysed following the protocols outlined in Section 3.2.9.

3.4.7 Soil AOB and AOA gene abundance

On day 1, 7, 14, 21, 28, 42, 56, 70, 86, and 112 after treatment application, DNA was extracted using the MO BIOTM PowerSoil® Total DNA Isolation Kit (MO BIO Laboratories, GeneWorks Pty Ltd, South Australia, Australia) following the manufacturer’s instructions (Section 3.2.10.1) and the AOB and AOA abundance was determined using a real-time qPCR (Section 3.2.10.2).

3.4.8 Soil AOB and AOA gene expression

On day 56, a 1 g soil sample was collected to determine the AOB and AOA activity (amoA

transcript abundance). The soil was stored at -80oC until use. RNA was extracted using the MO BIOTM RNA PowerSoil® Total RNA Isolation Kit (MO BIO Laboratories, GeneWorks Pty Ltd, South

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Australia, Australia), following the manufacturer’s instructions (Section 3.2.11.1). To remove residual amounts of genomic DNA, the extracted RNA samples were treated with TURBO DNA- free Kit (Ambion®, Life Technologies, Auckland, New Zealand) (Section 3.2.11.2). First-stand cDNA was produced using Superscript III reverse transcriptase (RT) (Life Technologies, Auckland, New Zealand) with a random hexamer primer following the manufacturer’s recommendations (Section 3.2.11.3) before a real-time PCR was conducted (Section 3.2.10.2). RNA analysis was only done on samples from a single sampling date and not on other samples due to resource constraints.

3.4.9 Statistical analysis

A regression analysis was performed on the ammonium oxidation rate versus the incubation period. The following first order degradation equation (Equation 3.1) was used to describe the ammonium oxidation within the soil:

_{Equation 3.1}

Where y is the ammonium concentration within the soil (mg NH4+-N kg-1 soil), t is the period of

incubation (days), k is the ammonium oxidation rate constant, and a is a constant. The ammonium oxidation rate constants and half-lives were estimated based on the regression.

Mean values and standard errors of the means for NO3--N concentrations, DCD concentrations,

and ammonia oxidiser populations were calculated based on the four replicates for each treatment using Microsoft Excel 2010 (Microsoft Corporation, USA). Two different factorial designs were used, the first a 2 x 3 design, and the second 2 x 2 + 1 design. P-values, main effects, and interactions were calculated following analysis of variance (ANOVA) using Genstat© (Version 15.1, VSN International Ltd, U.K.).

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