Measurements of Denitrification

The main products of heterotrophic denitrification are N2O and N2 and of nitrifier denitrification, they are NO and N2O. Emissions of NO and N2O are measured relatively easily. For terrestrial systems a small area of the soil surface is enclosed by a chamber (see Figure 1), or in the laboratory a small amount of soil is incubated in an airtight vessel, in order to allow the gases emitted to increase to measurable concentrations. N2O is a very stable gas and samples from the enclosures can be stored in syringes or vials until analysis by gas chromatography, using an electron-capture detector. NO is a highly reactive gas, with a half-life of less than 1 min and therefore can only be measured directly from the enclosed chamber by chemiluminescence. In aquatic systems, the concentration of N2O/NO in the water columns or in the headspace above the water column is analyzed.

The measurement of the final denitrification product N2 is not so straightforward. Direct measurements can only be made in an N2-free environment. This is difficult, as more than 80% of our atmosphere is N2. Contamination

Figure 1 This is a typical chamber to measure the fluxes of nitrous oxide from soil. The lid is closed for periods of 30-60 min, gas samples are collected from the chamber at the beginning and end of chamber closure through the sample port using a syringe. Samples are stored in vials or gas sample bags and are analyzed in the laboratory for nitrous oxide mostly by gas chromatography. Usually, information of the soil temperature, soil moisture, and soil mineral NO3 and NH4 are collected at the same time to provide a picture of the controlling variables.

can be a real problem, but direct measurements have been achieved by several scientific groups who replaced the atmosphere of soil columns with helium and then directly measured the N2 resulting from denitrification.

The traditional methods to measure total denitrification are by the acetylene block technique or by adding 1 N as a tracer. Acetylene at a partial pressure of 10 kPa blocks the final step of denitrification, so that the total product of denitrification is N2O. The main problem of this method is that it is not very easy to administer the inhibitor evenly throughout soils. The most active deni-trification zones in a soil are likely to be those into which diffusion is limited and have effectively managed to exclude O2.

In tracer experiments, nitrogen substrates are labeled with 15N and added to soils or waters. The appearance of this label in the denitrification products N2O and N2 can be measured by isotope-ratio mass spectrometry.

There are several methods that are used to distinguish between N2O production by autotrophic nitrifiers or het-erotrophic denitrifiers. (1) Acetylene at a partial pressure of 10 Pa inhibits N2O production during chemoauto-trophic nitrification, whereas a much higher partial pressure of 10 kPa is required to inhibit the reduction of N2O to N2 in heterotrophic denitrifiers. (2) The contribution ofnitrifiers and denitrifiers to N2O can be studied by differential labeling NH4 and NO3 with 15N and monitoring the occurrence and disappearance ofthe 15N label in the NH4, NO3, and N2O pools. (3) Inhibitors which specifically inhibit autotrophic nitrification (nitra-pyrin), denitrification (O2), or heterotrophic nitrification (cycloheximide) have been used to distinguish between these processes. (4) The use of natural abundance isotope ratios is a new approach, still in development. The frac-tionation of isotopic ratios 14/15N and 16/18O is different during nitrification and denitrification.

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