Quantifying denitrification

It is important to quantify denitrification rates to determine the conversion of reactive N to non-reactive N2 in a range of ecosystems and in technological approaches that enhance denitrification for water treatment (riparian buffers, constructed wetlands, denitrifying bioreactors etc.) to remove N from a range of different sources (groundwater, streams, drains, wastewater etc). However, the high background concentration of N2 in the environment makes direct measurement of the main end product (N2) of denitrification very difficult (Groffman et al. 2006).

The following range of techniques has been used to measure denitrification rates in terrestrial and aquatic environments in the past.

a) Nitrate removal

Quantifying the denitrification rate by measuring the decrease in NO3--N concentration is often unsatisfactory because NO3- can be removed by various mechanisms such as DNRA, assimilation, anammox, precipitation and absorption. Furthermore, varying sources of NO3--N (groundwater, multiple surface water inlets) and varying NO3--N inlet concentrations over time can cause incorrect estimations of NO3--N removal data. When NO3--N removal is calculated as the

difference in NO3--N concentration at the inlet and outlet, the NO3- measured in examined water at the outlet likely represents the inlet water at some time before the sampling date (Schipper et al., 2010a). However, some studies have assumed that denitrification was the mechanism responsible for the NO3--N removal. Consequently these studies suggested that the NO3--N removal rate was identical with the denitrification rate (Robertson et al., 2000, Greenan et al., 2009; Moorman et al., 2010).

b) Changes in natural abundance of 15N (δ15_{Ν) in NO}

3- and N2

Both biotic and abiotic mechanisms alter the natural 15N/14N ratio (expressed as δ15_{N) in ecosystems and different N conversion mechanisms result in distinct δ}15_N signatures in these environments (Bedard-Haughn et al., 2003). Many studies have used these signatures to determine the source of N or to track N within an ecosystem (Kreitler and Browning, 1983; Wassenaar, 1995; Karr et al., 2001), even though the variability of δ15_{N due to ongoing fractionation of} 15_N/14_{N make} this kind of study difficult (Robinson, 2001). Denitrification increases δ15_N-NO

3-

and decreases δ15_N-N

2 due to discrimination against 15N during denitrification (Mariotti et al., 1981). This natural fractionation of 15N/14N of the NO3- is described by a fractionation factor. After reviewing 15 different studies, Bedard- Haughn et al. (2003) calculated a median fractionation factor of 1.0185 for denitrification equivalent to an enrichment of 15N-NO3- by 18.5 ‰. There was also the successful attempt from Erler and Eyre (2010) to estimate denitrification rates using the natural fractionation of 15N-NO3- in a constructed wetland.

c) Acetylene block

The most commonly used technique to measure denitrification rates is the acetylene inhibition method (Groffman et al., 2006). Acetylene inhibits the reduction of the nitrous oxide during denitrification. Accumulation of N2O can be easily measured via gas chromatography, because background concentration of N2O is relatively low (Tiedje et al., 1989). However, there are a range of problems arising from the acetylene inhibition technique for quantifying denitrification rates. Acetylene can act as a carbon source for microorganisms (Kanner and Bartha, 1979; De Bont and Peck, 1980; Tam et al., 1983; Schink, 1985) and inhibits nitrifiers, which lead to a lack of NO3- production constraining NO3- supply to denitrifiers (Mosier et al., 1980; Walter et al., 1979). Furthermore, the acetylene-inhibition technique is generally performed in laboratory, which creates artifical conditions (e.g., constant temperature, shaking), that encourage denitrifiers and do not reflect the in situ denitrification rate. In situ measurements of denitrification rates using acetylene and soil chambers were evaluated by Ryden and Dawson (1982), but this approach is very labour intensive and is also subject to some problems described before. Therefore denitrification measurements via acetylene inhibition technique are not always accurate, but are most useful for comparing denitrification activity between different sites and seasons (Groffman et al., 1992; Groffman et al., 2006).

d) 15_{N enrichment}

Addition of 15N is used to track N in ecosystems and to determine the rate of the N-cycle processes (Nadelhoffer and Fry, 1994). Hauck and Melsted (1956)

directly quantified denitrification in soils following addition of 15N-labelled NO3- and measurement of the production of 15N-N2 and oxides of 15N in a closed system. Hauck and Melsted (1956) recovered between 89 and 100% of N in the system using this 15N enrichment technique. More recently, enrichment with 15N has been used to determine denitrification rates in water-saturated environments using the push-pull method, where a solution of 15N-labelled NO3- is injected into the area of interest and subsequently pulled out during an incubation period (Addy et al., 2002). The denitrification rate can then be determined by measuring the decrease of 15N-NO3- or the increase of 15N-N2 relative to the conservative tracer (Hauck and Melsted, 1956; Addy et al., 2002; Baker and Vervier, 2004). Moorman et al. (2010) used this approach in a woodchip bioreactor and measured the decline of 15N-NO3- concentration. The decline in 15N-NO3- was attributed to denitrification, DNRA, anammox and/or biological uptake (immobilization). In general, the push-pull test is most appropriate in environments where NO3- is not limiting denitrification, otherwise adding NO3- may increase the denitrification rate (Groffman et al., 2006). When bromide is used as conservative tracer plant uptake has to be considered (Schnabel et al., 1995). For accurate measurements, the increase in denitrification products (15_N-N

2 and 15N-N2O) should be measured (Groffman et al., 2006).

e) Direct N2 measurement

The direct measurement of N2 emission would likely be the most accurate measurement approach, but is generally not possible due to the high background concentrations of N2 in most environments. Butterbach-Bahl et al. (2002) successfully measured N production from soils in N -free boxes, which was

extremely time consuming and was still prone to atmospheric N2 contamination. In aquatic environments, denitrification rates can be calculated from the increase in dissolved N2 concentrations (Blicher-Mathiesen et al., 1998) or the ratio of dissolved N2/Argon (Laursen and Seitzinger, 2002). Blicher-Mathiesen et al. (1998) successfully measured the denitrification rate in the groundwater samples of a riparian wetland. They sampled groundwater along the groundwater-flow path in sealable vials and introduced helium gas headspaces, which were analysed for N2 after headspace equilibrium of the dissolved gases. Similarly, Laursen and Seitzinger (2002) estimated denitrification rates of three small rivers using membrane inlet mass spectrometry (MIMS) that measured increases in dissolved N2/Argon of water down the length of the river.

This range of approaches for measuring denitrification should be tested in a denitrification bed to determine the denitrification rates of the bed and to compare the usefulness of each method for application in denitrification beds.