Nitrification

Ammonia-oxidizing bacteria (AOB) convert ammonia to nitrite in a two-step process with hydroxylamine as an intermediate (Figure 2.2). The membrane-bound ammonia mono-oxygenase enzyme (AMO) catalyses the oxidation of ammonia to hydroxylamine, and hydroxylamine is oxidized to nitrite by the periplasm-associated enzyme, hydroxylamine oxidoreductase (Hooper et al, 1997). The production of N2O occurs at this stage. Two of the four electrons derived from the oxidation of hydroxylamine to nitrite are required during the oxidation of ammonia to hydroxylamine, the other two are available for energy production and the reduction of oxygen to water (Colliver and Stephenson, 2000). All known bacterial autotrophic ammonia oxidizers in soils belong to the genus Nitrosomonas and Nitrosospira, which form a monophyletic cluster within the subclass of Proteobacteria. Recent findings have revolutionized the diversity of ammonia oxidizers by showing that archaea are also capable of oxidizing ammonia to nitrite and that they could dominate over AOB in soils (Prosser and Nicol, 2008). However their contribution to nitrification is still under debate and their role in N2O production is unknown.

The rate of ammonia oxidation is influenced by ammonia availability. This is closely linked to the protonation and lower availability of NH3 at low pH. Surprisingly, ammonia oxidizers seem to be able to survive under conditions of NH3 starvation although the ability to compete for NH3 and the ability to respond to NH3 after a period of starvation have been shown to vary between different AOB (Frijlink et al, 1992; Gerards et al, 1998; Bollmann et al, 2002). Furthermore, the theoretical sensitivity to low pH also varies between different AOB, with some possessing mechanisms to overcome this problem so that nitrification is not always restricted by low pH environments (De Boer and Kowalchuk, 2001). Much of the information on the physiology of AOB comes from studies on Nitrosomonas spp due to the relative ease of culturing this

Nitrification

Figure 2.2 The pathway of nitrification, showing stages at which N2O can be produced

Source: Adapted from Colliver and Stephenson (2000) and Wrage et al (2001)

Figure 2.2 The pathway of nitrification, showing stages at which N2O can be produced

Source: Adapted from Colliver and Stephenson (2000) and Wrage et al (2001)

microorganism in the laboratory. However, molecular investigations into AOB communities have shown that the more predominant AOB in the soil, especially soils treated with ammonia-based fertilizers, are members of the genus Nitrosospira (Kowalchuk and Stephen, 2001; Avrahami et al, 2002).

Nitrifier-N2O production has been measured in cultures of nitrifiers under reduced O2 potential (Goreau et al, 1980), and there is recent evidence to suggest that ammonia oxidation can significantly contribute to net N2O emission from soil (e.g. Abbasi and Adams, 2000; Bateman and Baggs, 2005; Avrahami and Bohannan, 2009; Wan et al, 2009). Bateman and Baggs (2005) showed nitrification to be the predominant N2O-producing process in a silt loam soil held at 35-60 per cent WFPS, accounting for up to 81 per cent of N2O emitted at 60 per cent WFPS, indicating the significance of this process for global warming, despite its role having often been underplayed compared to that of denitrification.

Nitrification can also be carried out by a wide range of heterotrophic microbes (bacteria and fungi) using organic substrates such as urea as well as NH3 (Papen et al, 1989). Several different pathways of heterotrophic nitrification have been proposed, including an inorganic pathway similar to that of the autotrophic nitrifiers (Killham, 1986), and this capacity has been demonstrated in pure-culture studies of a range of heterotrophic microbes. For example, many heterotrophs isolated from soil (including Absidia cylindro-spora, Pseudomonas putida and Paracoccus denitrificans) have the ability to nitrify NH3 in culture (Stroo et al, 1986; Moir et al, 1996a; Daum et al, 1998). Brierley and Wood (2001) isolated heterotrophic bacteria and fungi from an acid forest soil capable of nitrifying both inorganic (ammonium acetate) and organic (b-alanine, peptone) N in pure culture and in inoculated soil solution. The potential for heterotrophic nitrification is further supported by the characterization of enzymes (ammonia and hydroxylamine reductases) capable of catalysing oxidation reactions typical of the autotrophic pathway in hetero-trophs (Moir et al, 1996a, 1996b). The exact mechanisms of both organic and mineral heterotrophic nitrification pathways remain to be clarified, and the evidence seems to suggest that a combination of an appropriate N source and suitable soil environmental conditions combine to dictate whether the process occurs (Killham, 1986).

The regulation and magnitude of N2O production during these pathways are less well constrained, although the production of N2O by heterotrophic nitrifiers has been demonstrated in culture (Papen et al, 1989), and it has been shown under aerobic culture conditions that some heterotrophic nitrifiers, such as Alcaligenes faecalis, can produce much more N2O per cell than the autotrophic nitrifier Nitrosomonas europaea (Papen et al, 1989; Anderson et al, 1993). The oxygen concentration at which most N2O was produced was higher for the heterotroph Alcaligenes faecalis than the autotroph Nitrosomonas europaea (Anderson et al, 1993). To our knowledge the only direct investigation of N2O production in soil by heterotrophic nitrification was undertaken by Bateman and Baggs (2005), using C2H2 to inhibit ammonia oxidation. However, due to high variability, there was no conclusive evidence for heterotrophic contribution in their arable soil.

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