5.1. Temperature and water
Both the overall denitrification rates and the proportions of N2O and dinitrogen gas produced by denitrifying microbes can vary depending on numerous environmental factors, such as pH, carbon, NO3, and NO2 availability, soil moisture, pore structure, aeration, temperature, freezing–
thawing, and drying–wetting events. Several of these are natural factors influenced by climatic conditions that cannot be managed. In addition, they are not constant, but show large variation over the vegetation period as well as between field sites. The estimated nitrogen losses are therefore highly variable in time and space. Emissions of N2O and dinitrogen show no consistent seasonal pattern. In some studies, the largest N2O emissions were recorded during spring (Kaiser and Heinemeyer, 1996; Parsonset al., 1991;
Ryden, 1985), in others during spring and autumn (Ambus and Christensen, 1995; De Klein and Van Logtestijn, 1994), or in summer (Bremner et al., 1980; Cates and Keeney, 1987). The difference in the results could not be related to environmental factors and management practices. A better under- standing of factors contributing to variability of denitrification activity would be helpful to improve estimations and modeling of nitrogen fluxes by denitrification.
Soil temperature and soil water content are known factors that affect gaseous nitrogen losses and the N2O/N2ratio. Under constant laboratory conditions, this ratio increased exponentially with increasing soil tempera- ture (Maag and Vinther, 1996). However, the ratio was strongly influenced by soil type, although these data could not be confirmed by field measure- ments. Whereas Bailey (1976) and McKeeneyet al.(1979) found a positive correlation between soil temperature and denitrification activity, others observed no relationship with temperature (Focht, 1974; Lensi and Chalamet, 1979). The reason might be the lower water content caused by increased plant transpiration rates at higher temperatures, which leads to a water deficiency. Under laboratory conditions, similar to the effects of increasing temperature, the overall denitrifying activity and N2/N2O ratio increased with increasing soil water content (Colbourne and Dowdell, 1984; Vinter, 1984). This was also confirmed in a pasture after harvest (Rudazet al., 1997).
Linked to soil water content is oxygen availability. Hochstein et al.
(1984) showed that soil oxygen concentrations below 5% resulted in deni- trification being the main microbial respiratory process when NO3 was available. In addition, at 10% oxygen concentration and moisture content between 40% and 60%, denitrification was the main source of emitted N2O.
Water content depends on the pore structure of the soil, which in turn is affected by soil type, organic matter content, and land use. Bakken et al.
(1987) demonstrated that the pore space structure appears to be the major factor explaining the difference in mean denitrification rates by comparing pasture and cropped soil. In the field, Bijay-Singhet al.(1989) found higher actual denitrification in cropped soil than in pasture, despite similar NO3 contents. They explained their results as the consequence of better drainage in the pasture soil, due to the higher porosity of this soil. Complementary measurements after the application of various amounts of water showed denitrification activity in pasture soil was higher than denitrification in cropped soil only at water suctions greater than 5.5 kPa (Bijay-Singh et al., 1989). In contrast, potential denitrification has often been reported to be higher in pasture than in cropped soil (Bijay-Singhet al., 1989; Lensi et al., 1995; Sotomayor and Rice, 1996).
5.2. Freeze–thaw cycles
5.2.1. Freeze–thaw effects on nitrous oxide emissions
Christensen and Tiedje (1990) were the first to report peak N2O emissions from arable soils in spring during thaw periods. Emissions of carbon dioxide and N2O and uptake of methane throughout the snow-covered period even at temperatures near 0C were later reported (Sommerfeld et al., 1993).
In order to decide whether N2O production can be attributed also to nonmicrobial processes in soil, emissions from a g-ray sterilized and a nonsterilized soil were compared in a laboratory experiment, where the freezing and thawing cycles were simulated. The results clearly indicated that microbial processes were responsible for N2O production in thawing and even frozen soils (Ro¨veret al., 1998). Therefore, efforts have been done to investigate the effects of freezing and thawing cycles on microbial denitrification, and to understand the mechanisms behind. Sehy et al.
(2003) first demonstrated the importance of denitrification for nitrogen losses during winter in arable soil. They separated the 12 months of investi- gation into the growing season (March to November) and the winter period (December to February). Independent of the amount of applied fertilizer, about 70% of the annual N2O amounts was emitted during the winter period. The temporal changes of the N2O emission rates were correlated to changes in soil temperature. Similarly, Do¨rsch et al. (2004) found persis- tently high N2O emissions in arable soil with peak emissions during mid- winter thawing, diurnal freezing–thawing, and spring thaw. Low and stable temperatures below the insulating snow or ice cover, in contrast, decreased N2O emissions. Several other field studies in the temperate regions also reported high N2O emissions from agricultural soils during freeze–thaw periods reaching 20–70% of the annual budget (Flessaet al., 1995; Nyborg et al., 1997; van Bochoveet al., 1996, 2000; Wagner-Riddle et al., 1997).
Nevertheless, a few studies have also reported that moderate freeze–thaw fluctuations had little impact on nitrogen dynamics and N2O emissions in soils (Groganet al., 2004; Neilsenet al., 2001).
There is considerable debate on which factors could be critical control- lers of winter N2O emissions from arable soils. However, most authors state that emissions during winter are related to the release of nutrients.
Christensen and Christensen (1991) could show that soluble carbon, applied as plant extract, was necessary to induce N2O production during freezing and thawing events. Therefore, plant residues from catch crops and green manure may play an important role in the regulation of N2O emissions in winter, since frost enhances the release of organic compounds from plant residues. Additionally, freeze–thaw events may result in transient pulses of carbon and nitrogen due to disruption of soil aggregates (Christensen and Christensen, 1991; Mu¨lleret al., 2002) and lysis of microorganisms (Schimel and Clein, 1996; Skoglandet al., 1988). Mu¨lleret al.(2002) showed that the increased ammonium and NO3 concentrations during freezing were asso- ciated to peak N2O emissions during the following thawing period.
Enhanced oxygen consumption during degradation of plant residues com- bined with a high water content of the thawing soil increases the anaerobic volume, thus enhancing denitrification. The freeze–thaw-induced emission of N2O could thus be a straightforward result of enhanced denitrification.
N2O may also be produced by microorganisms in unfrozen water films on the soil matrix during freezing. Several authors showed that an ice layer covering the unfrozen water film could be a diffusion barrier, which reduces oxygen supply to the microorganisms and partly prevents the release of N2O to the air (Burton and Beauchamp, 1994; Goodroad and Keeney, 1984; Teepeet al., 2001).
Nitrification could also be of significance for N2O emissions during winter. It has been demonstrated that freeze–thaw cycles enhances nitrogen mineralization, which results in the release of substrate for ammonia- oxidizing bacteria (Delucaet al., 1992). Lowered oxygen availability during freeze–thaw-induced respiration could also induce higher N2O emissions from nitrifiers, since the N2O/(NO3 þNO2) ratio of nitrification increases sharply in response to oxygen limitation (Davidson, 1991; Dundee and Hopkins, 2001; Goreau, 1980). However, it has been demonstrated that only a few percent of the measured N2O originate from nitrification.
Denitrification was the main N2O source at various oxygen concentrations investigated in freeze–thaw-affected soil (Ludwiget al., 2004; Mrkvedet al., 2006).
5.2.2. Freeze–thaw effects on denitrifier communities
Although microbial denitrification is believed to be the major source of N2O during freeze–thaw events, few have analyzed the denitrifier commu- nities involved. Actually, little is known about the significance of the
denitrifier community composition for N2O emissions in general, since most of the work conducted has focused on gas and soil analysis. Freeze–
thawing effects on total bacterial community structure are contradictory.
Erikssonet al.(2001) observed a change in ribosomal internal spacer analysis patterns during freeze–thaw events, whereas Koponen et al. (2006) con- cluded that neither microbial biomass nor community structure was affected in boreal soils.
It has been postulated that the relative activity of N2O reductase can be lowered at near-freezing temperatures (Holtan-Hartwiget al., 2002b; Melin and No¨mmik, 1983), possibly resulting in high N2O/(N2þN2O) ratios in soil during thawing. A high N2O/(N2 þ N2O) ratio could also be a
‘‘postfreezing trauma’’; the N2O reductase appears to be more vulnerable to perturbations than the other denitrification enzymes, and if this holds for frost damages, it would result in a higher proportion of produced N2O to total denitrification after freezing (Do¨rsch and Bakken, 2004; Holtan- Hartwiget al., 2002; Melin and No¨mmik, 1983). Nevertheless, how specific enzymes involved in denitrification are influenced by freezing and thawing is still not answered.
Sharmaet al. (2006) investigated the mRNA levels of genes encoding the periplasmic NO3 reductase gene (napA) and cytochrome cd1 NO2 reductase (nirS) in the upper horizon of a grassland soil during thawing in a laboratory experiment. By using a MPN-based reverse transcriptase PCR approach they could show that high transcript levels occurred for both genes 2 days after thawing had begun, followed by a decrease. The peak of N2O production coincided with the peak fornapAandnirStranscripts, and it timely shifted after 2 days. In the same study, the napA and nirS genotype diversity was analyzed. Interestingly, DNA-based profiles showed no change in banding patterns, whereas those derived from cDNA showed a clear succession of the genotypes, with the most diverse community structure at the time point of the highest gene expression.
5.3. Dry–wet cycles
Similar to freeze–thaw cycles in soil, dry–wet cycles can enhance N2O emissions. Prieme´ and Christensen (2001) compared the effects of drying–
wetting and freezing–thawing cycles on the emission of N2O, carbon dioxide, and methane from intact soil cores from farmed organic soils.
During the first week, following wetting or thawing, up to a 1000-fold increase in N2O emission rates were recorded from the cores. The total N2O emission ranged between 3 and 140 mg N–N2O m2, and between 13 and 340 mg N–N2O m2due to the first wetting and thawing event, respec- tively. Nevertheless, the emission rates declined after two successive freeze–
thaw events. Many other studies have also documented differences in the rate of denitrification following wetting (Ambus and Lowrance, 1991;
Gilliam et al., 1978; Groffman and Tiedje, 1989; Rice and Tiedje, 1982;
Robertson and Tiedje, 1985, 1988; Sexstoneet al., 1986). Some studies have also noted denitrification differences between the wet-up and dry-down phases of soil moisture following rainfall events (Gilliamet al., 1978).
Bergsma et al. (2002) showed that a short wet-treatment significantly decreased the relative amount of N2O emitted from cropped soil compared with a long wet-treatment, while no effect of moisture history was seen in a successional agrosystem. The authors hypothesized that these differences in N2O production were due to selection of denitrifiers with enhanced capac- ity for enzyme maintenance at lower levels of NO3, such as found in the successional soil. Others later confirmed differences in denitrifier commu- nity composition in the successional and cropped soil at this site (Streset al., 2004). Denitrification enzymes were also more sensitive to oxygen in the cropped soil and N2O activity was higher in the successional soil (Cavigelli and Robertson, 2000). Soil moisture history seems to be important for denitrification. If denitrification enzymes are induced differentially in response to wetting, then both the overall rate of denitrification as well as the relative amount of N2O will differ substantially among ecosystems.