Denitrification, the reduction of the nitrogen (N) oxides, nitrate (NO3-) and nitrite (NO2-), to the gases nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2), is important to primary production, water quality, and the chemistry and physics of the atmosphere at ecosystem, landscape, regional, and global scales. Unfortunately, this process is very difficult to measure, and existing methods are problematic for different reasons in different places at different times. In this paper, we review the major approaches that have been taken to measure denitrification in terrestrial and aquatic environments and discuss the strengths, weaknesses, and future prospects for the different methods. Methodological approaches covered include (1) acetylene-based methods, (2) 15N tracers, (3) direct N2 quantification, (4) N2:Ar ratio quantification, (5) mass balance approaches, (6) stoichiometric approaches, (7) methods based on stable isotopes, (8) in situ gradients with atmospheric environmental tracers, and (9) molecular approaches. Our review makes it clear that the prospects for improved quantification of denitrification vary greatly in different environments and at different scales. While current methodology allows for the production of accurate estimates of denitrification at scales relevant to water and air quality and ecosystem fertility questions in some systems (e.g., aquatic sediments, well-defined aquifers), methodology for other systems, especially upland terrestrial areas, still needs development. Comparison of mass balance and stoichiometric approaches that constrain estimates of denitrification at large scales with point measurements (made using multiple methods), in multiple systems, is likely to propel more improvement in denitrification methods over the next few years.

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Methods for measuring denitrification: diverse approaches to a difficult problem

Publisher Summary This chapter highlights the physiology of autotrophic ammonia- and nitrite-oxidizing bacteria. Several aspects of the physiology of nitrifiers of relevance to their growth and activity in natural environments are considered. The chapter describes the basic features of the biochemistry of ammonia and nitrite oxidation and discusses the growth limiting factors and activity of these organisms. The influence of oxygen concentration, pH value, and inhibitors on their physiology is also described. Nitrification plays a central role in the nitrogen cycle of terrestrial and aquatic ecosystems, converting the most reduced form of nitrogen, NH3, to the most oxidized form, NO3. Nitrifying bacteria occupy niches in many ecosystems and compete successfully with faster and more efficiently growing organisms for oxygen, ammonia, and carbon dioxide. Nitrifiers are capable of reversing the nitrification process, carrying out denitrification and producing nitrite, ammonia, nitrous and nitric oxides, and gaseous nitrogen. Ammonia oxidizers can metabolize urea and can assimilate carbon from methane while nitrite oxidizers can grow anaerobically in the presence of organic compounds and nitrate.

Autotrophic nitrification in bacteria

Nitrification in acid soils was first reported in the beginning of the 20th century. Although this finding has been well substantiated by countless studies since then, it has until recently remained unclear which micro-organisms were responsible for nitrate production at low pH. Substantial evidence now supports the role of chemolitho–autotrophic bacteria as the main nitrifying agents in most acid soils. Heterotrophs may make some contribution to nitrification in acid soils, but this is difficult to demonstrate conclusively. Current insights in the phylogenetic position of autotrophic nitrifying bacteria in acid soils and the mechanisms that may enable them to be active at low pH are presented. In addition, the spatial variability of their activity and their contribution to the flux of the greenhouse gas N2O is discussed.

Nitrification in acid soils: micro-organisms and mechanisms

In situ Identification of Ammonia-oxidizing Bacteria

To predict emissions of nitrous oxide (N 2 O) and nitric oxide (NO) from forest soils, we have developed a process-oriented model by integrating several new features with three existing models, PnET, Denitrification-Decomposition (DNDC), and a nitrification model. In the new model, two components were established to predict (1) the effects of ecological drivers (e.g., climate, soil, vegetation, and anthropogenic activity) on soil environmental factors (e.g., temperature, moisture, pH, redox potential, and substrates concentrations), and (2) effects of the soil environmental factors on the biochemical or geochemical reactions which govern NO and N 2 O production and consumption. The first component consists of three submodels for predicting soil climate, forest growth, and turnover of soil organic matter. The second component contains two submodels for nitrification and denitrification. A kinetic scheme, a so-called anaerobic balloon,' was developed to calculate the anaerobic status of the soil and divide the soil into aerobic and anaerobic fractions. Nitrification is only allowed to occur in the aerobic fraction, while denitrification occurs only in the anaerobic fraction. The size of the anaerobic balloon is defined by the simulated oxygen partial pressure which is calculated based on oxygen diffusion and consumption rates in the soil. As the balloon swells or shrinks, the model dynamically allocates substrates (e.g., dissolved organic carbon, ammonium, nitrate, etc.) into the aerobic and anaerobic fractions. With this approach, the model is able to predict both nitrification and denitrification in the same soil at the same time. This feature is important for soils where aerobic and anaerobic microsites often exist simultaneously. With the kinetic framework as well as its interacting functions, the PnET-N-DNDC model links ecological drivers to trace gas emissions. Tests for validating the new model are published in a companion paper [Stange et al., this issue].

https://agupubs.onlinelibrary.wiley.com/doi/pdf/10.1029/1999JD900949

A process-oriented model of N2O and NO emissions from forest soils: 1. Model development

Inhibition by acetylene of ammonia oxidation in Nitrosomonas europaea

The oxidation of NH4+ by Nitrosomonas europaea was insensitive to 10 mM NaClO3 (sodium chlorate) but was strongly inhibited by NaClO2 (sodium chlorite; Ki, 2 μM). The oxidation of NO2− by Nitrobacter winogradskyi was inhibited by both ClO3− and ClO2− (Ki for ClO2−, 100 μM). N. winogradskyi reduced ClO3− to ClO2− under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO2− was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH4+ and NO2− was inhibited in the presence of 10 mM ClO3− after a 2-h lag period, despite the fact that, under these conditions, ClO2− was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH4+ oxidation is inhibited by ClO2− produced by reduction of ClO3− by the NO2− oxidizer. The use of ClO3− inhibition of NO2− oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH4+ is not affected.

Inhibition of chemoautotrophic nitrification by sodium chlorate and sodium chlorite: a reexamination.

The discovery that acetylene inhibited ammonia oxidation by Nitrosomonas europeae came about by studying linkages of microbial Nitrogen cycling processes in laboratory soil columns. Continuous addition of ammonium chloride (25 μg N/ml) to the soil columns resulted in steady state nitrification of all of the ammonia to nitrate. Acetylene (10.1 kPa) was introduced to detect nitrous oxide, a product of denitrification of nitrate, however, nitrification was inhibited and washout and disappearance of nitrate in the column effluent was detected. Concomitantly, ammonium levels in the effluent rose. Nitrification of nitrite was not affected by acetylene. Summary Acetylene inhibited the metalloenzyme, ammonia monooxygenase in the obligate chemoautotrophic nitrifier, Nitrosomonas europeae.

Russell K. Hynes

Papers

Inhibition by acetylene of ammonia oxidation in Nitrosomonas europaea

Inhibition of chemoautotrophic nitrification by sodium chlorate and sodium chlorite: a reexamination.

Commentary on 'Inhibition by acetylene of ammonia oxidation in Nitrosomonas europaea'.