Linking processes to the underpinning microbiology

Understanding how changes in the size or in the diversity of microbial communities producing or reducing N2O in response to environmental conditions or agricultural practices are related to N2O fluxes is key to understanding controls on process rates and the microbial source of N2O. However, until recently, diversity and activity of microbial communities involved in N-cycling were most often investigated in separate studies, which resulted in an artificial dichotomy between research on microbial processes and microbial community ecology. This gap is now slowly being bridged by combining molecular-based analysis of microbial diversity with measurements of process rates.

Only a few studies have investigated the relationships between activity and diversity of nitrifiers with any focus on nitrification rates and nitrifier-N2O production being most often neglected (Kowalchuk et al, 2000; De Bie et al, 2001). In one of the most comprehensive studies, which used molecular and cultivation-based approaches of ammonia oxidizer diversity together with measurements of changes in nitrate and ammonium concentrations, Webster et al (2005) demonstrated a direct link between bacterial community structure and nitrification activity. More recently, Avrahami and Bohanann (2009) observed a significant relationship between diversity of AOB and N2O emission rates and suggested that N2O emissions may vary locally depending on local AOB community composition. However, other studies relating nitrifier-N2O production to the nitrifier community have concluded that any change in activity, for example following addition of N, or with differing water content, was probably not the result of a major change in the ammonia-oxidizing community but of a physiological response (Mendum et al, 1999; Avrahami et al, 2002; Bateman, 2005). Bateman (2005) found changes in the AOB community structure over time after application of NH4NO3 to soils of varying WFPS, but these changes occurred after the main period of flux activity (Figure 2.4).

Relationships between denitrifier community composition and N2O production have also been suggested by Cavigelli and Robertson (2000), who found that the enzymes involved in N2O production or reduction had different sensitivities to oxygen or pH in denitrifying communities from a conventionally tilled agricultural field and a never-tilled successional field. Further studies have both confirmed and contradicted such links between denitrifier community composition and N2O production. The addition of different types of artificial root exudates to soil microcosms resulted in differences in the N2O/N2 ratio, which were not related to differences in denitrifier community size or composition (Henry et al, 2008). Similarly, comparison of agricultural soil, riparian soil and creek sediment showed that denitrifier community composition and N2O production were uncoupled across these agroecosystems (Rich and Myrold, 2004). In contrast, within these same systems, Philippot et al (2009a) found a significant correlation between the distribution of the percentage of bacteria capable of reducing N2O within the total bacterial community and potential N2O emissions, both being also strongly correlated to soil pH (Plate 2.1).

Whilst major progress has been made in characterizing the soil microbial community, understanding the relationships between diversity and activity of

Figure 2.4 Nitrifier-15N-N2O production after application of fertilizer (20g Nm~2) to a silt loam soil at 20-70 per centWFPS, and the AOB community profiled by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene PCR products amplified from the soil at different WFPS before and following fertilizer application

Figure 2.4 Nitrifier-15N-N2O production after application of fertilizer (20g Nm~2) to a silt loam soil at 20-70 per centWFPS, and the AOB community profiled by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene PCR products amplified from the soil at different WFPS before and following fertilizer application

Note: L represents a control sample containing a recognized AOB from each cluster designation 1-4. Source: Adapted from Bateman (2005)

a functional community still remains a major challenge in microbial ecology (Philippot and Hallin, 2005). In the contradictory studies described above, the number of ecosystems/conditions studied is rather limited. However, relationships between microbial diversity and processes are probably very complex and interwoven with many parameters, and therefore a comprehensive understanding can only be achieved by the analysis of microbial diversity and activity under a much broader range of ecosystems or of environmental gradients using high-throughput approaches.

Alternatively, the development of approaches targeting the active fraction of functional communities such as stable isotope probing (SIP) provides an elegant means of distinguishing organisms contributing to the observed processes. 13C-SIP has successfully been applied to soil to identify methane-oxidizing bacteria (Bull et al, 2000; Morris et al, 2002; Radajewski et al, 2002)

and AOB (Whitby et al, 2001). Unfortunately the application of 13C-SIP to identify active denitrifiers is limited by the fact that denitrifiers can also assimilate the labelled C during respiratory processes other than denitrification. The identification of active denitrifiers using 15N-SIP is likely to remain elusive since N is dissimilated. However, there is the potential for links to be made between plant-derived C flow and denitrifiers by identifying heterotrophic microorganisms capable of denitrification and utilizing the plant-C, through 13C-CO2 pulsing of vegetation (Johnson et al, 2002; RangelCastro et al, 2005). Whilst issues about turnover and recovery of applied 13C, and cross-feeding of this C (Manefield et al, 2007) pose uncertainties for this approach, there are nevertheless some exciting opportunities for further characterization of active microorganisms through the use of stable isotopes to determine microbial sources.

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