Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Denitrification at pH 4 by a soil‐derived Rhodanobacter‐dominated community

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of N₂O produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO₃^‐ and N₂O reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of N₂O as denitrification end‐product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter‐like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils.     

Warming-induced enhancement of soil N2O efflux linked to distinct response times of genes driving N2O production and consumption

Temperature responses of denitrifying microbes likely play a governing role in the production and consumption of N₂O. We investigated temperature effects on denitrifier communities and their potential to produce N₂O and N₂ by incubating grassland soils collected in multiple seasons at four temperatures with ¹⁵N-enriched NO₃ ^? for ~24 h. We quantified [N₂O] concentration across time, estimated its production and reduction to N₂, and quantified relative abundance of genes responsible for N₂O production (cnorB) and reduction (nosZ). In all seasons, net N₂O production was positively linked to incubation temperature, with highest estimates of net and gross N₂O production in late spring soils. N₂O dynamics were tightly coupled to changes in denitrifier community structure, which occurred on both seasonal and incubation time scales. We observed increases in nosZ abundance with increasing incubation temperature after 24 h, and relatively larger increases in cnorB abundance from winter to late June. The difference between incubation and in situ temperature was a robust predictor of cnorB:nosZ. These data provide convincing evidence that short-term increases in temperature can induce remarkably rapid changes in community structure that increase the potential for reduction of N₂O to N₂, and that seasonal adaptation of denitrifying communities is linked to seasonal changes in potential N₂O production, with warmer seasons linked to large increases in N₂O production potential. This work helps explain observations of high spatial and temporal variation in N₂O effluxes, and highlights the importance of temperature as an influence on denitrification enzyme kinetics, denitrifier physiology and community adaptations, and associated N₂O efflux and reduction.     

Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil

Analyses of the complete genomes of sequenced denitrifying bacteria revealed that approximately 1/3 have a truncated denitrification pathway, lacking the nosZ gene encoding the nitrous oxide reductase. We investigated whether the number of denitrifiers lacking the genetic ability to synthesize the nitrous oxide reductase in soils is important for the proportion of N₂O emitted by denitrification. Serial dilutions of the denitrifying strain Agrobacterium tumefaciens C58 lacking the nosZ gene were inoculated into three different soils to modify the proportion of denitrifiers having the nitrous oxide reductase genes. The potential denitrification and N₂O emissions increased when the size of inoculated C58 population in the soils was in the same range as the indigenous nosZ community. However, in two of the three soils, the increase in potential denitrification in inoculated microcosms compared with the noninoculated microcosms was higher than the increase in N₂O emissions. This suggests that the indigenous denitrifier community was capable of acting as a sink for the N₂O produced by A. tumefaciens. The relative amount of N₂O emitted also increased in two soils with the number of inoculated C58 cells, establishing a direct causal link between the denitrifier community composition and potential N₂O emissions by manipulating the proportion of denitrifiers having the nosZ gene. However, the number of denitrifiers which do not possess a nitrous oxide reductase might not be as important for N₂O emissions in soils having a high N₂O uptake capacity compared with those with lower. In conclusion, we provide a proof of principle that the inability of some denitrifiers to synthesize the nitrous oxide reductase can influence the nature of the denitrification end products, indicating that the extent of the reduction of N₂O to N₂ by the denitrifying community can have a genetic basis.     

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

The objective of this study was to investigate how changes in soil pH affect the N₂O and N₂ emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H₂SO₄ solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N₂O and N₂ emissions using the ¹⁵N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N₂O/(N₂O + N₂) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N₂O production were observed. Denitrification activity and N₂O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N₂O/(N₂O + N₂) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.Denitrification is the microbial reduction of NO₃⁻ via NO₂⁻ to gaseous NO, N₂O, and N₂, which are then lost into the atmosphere (36). It therefore results in considerable loss of nitrogen, one of the most limiting nutrients for crop production in agriculture (20). Denitrification is also of environmental concern since, together with nitrification, it is the main biological process responsible for N₂O emissions (7). N₂O is a potent greenhouse gas which has a global warming potential about 320 times greater than that of CO₂ and has a lifetime of approximately 120 years (32). In the stratosphere, N₂O can also react with O₂ to produce NO, which induces the destruction of stratospheric ozone (8). N₂O can be released into the atmosphere by incomplete denitrification due to the effect of environmental conditions on the regulation of the different denitrification reductases (14, 41, 51), but it has recently been suggested that it could also be due to lack of nitrous oxide reductase in some denitrifiers (19, 41). Since N₂O is an intermediate in the denitrification pathway, both the amount of N₂O produced and the N₂O/(N₂O + N₂) ratio are important in understanding and predicting N₂O fluxes from soils.The main environmental factors known to influence the N₂O/(N₂O + N₂) ratio are pH, organic carbon and NO₃⁻ availability, water content, and O₂ partial pressure (50). Soil pH is one of the most important factors influencing both denitrification and N₂O production (43). In general, the denitrification rate increases with increasing pH values (up to the optimum pH) while, in contrast, the N₂O/(N₂O + N₂) ratio decreases (50). This relationship has been characterized in laboratory experiments (9, 45), but it is not clear whether the same relationships exist in the field because of methodological limitations of in situ measurement of N₂ emissions (16). Nevertheless, ¹⁵N tracing experiments based on the addition of a labeled denitrification substrate to soil offer a useful tool to quantify emissions of both N₂O and N₂ in situ (47, 49). Soil pH is also an important factor influencing denitrifier community composition (35, 39), which can be an important driver of denitrification activity and N₂O emissions (5, 21). A recent study reported a negative relationship between the proportion of bacteria genetically capable of reducing N₂O within the total bacterial community and the N₂O/(N₂O + N₂) ratio, with both being strongly correlated with soil pH (38).The objective of the present study was to explore the effect of changes in soil pH on in situ N₂O and N₂ emissions, denitrifying enzyme activity (DEA), and potential N₂O production. In addition, we also investigated whether differences in N fluxes could be related to changes in the size of the microbial community possessing the different denitrification genes. A field experiment was conducted using replicated grassland plots in which the soil pH was modified by addition of either acid or hydroxide to the soil. A ¹⁵N tracer method was used to provide information on N emissions. In addition to measuring potential denitrification activity, the size of the denitrifier community was determined by real-time PCR quantification of the denitrification genes.     

Denitrification activity of Bradyrhizobium sp. isolated from Argentine soybean cultivated soils

Two hundred and fifty strains, all of them representatives of native Bradyrhizobium sp., isolated from soils cultivated with soybean have been characterized by their denitrification activity. In addition, the denitrification potential of those soils was also measured by evaluating the most-probable-number (MPN) of denitrifying bacteria and the denitrification enzyme assay (DEA). Of the 250 isolates tested, 73 were scored as probable denitrifiers by a preliminary screening method. Only 41 were considered denitrifiers because they produced gas bubbles in Durham tubes, cultures reached an absorbance of more than 0.1 and NO³⁻ and NO²⁻ were not present. Ten of these 41 were selected to confirm denitrification and to study denitrification genes. According to N₂O production and cell protein concentration with NO³⁻, the isolates could be differentiated in three categories of denitrifiers. The presence of the napA, nirK, norC and nosZ genes was detected by production of a diagnostic PCR product using specific primers. RFLP from the 16S-23S rDNA spacer region (IGS) revealed that denitrifiers strains could be characterized as Bradyrhizobium japonicum and strains which were non-respiratory denitrifiers as B. elkanii.     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Effects of drought and N-fertilization on N cycling in two grassland soils

Changes in frequency and intensity of drought events are anticipated in many areas of the world. In pasture, drought effects on soil nitrogen (N) cycling are spatially and temporally heterogeneous due to N redistribution by grazers. We studied soil N cycling responses to simulated summer drought and N deposition by grazers in a 3-year field experiment replicated in two grasslands differing in climate and management. Cattle urine and NH₄NO₃ application increased soil NH₄ ⁺ and NO₃ ^? concentrations, and more so under drought due to reduced plant uptake and reduced nitrification and denitrification. Drought effects were, however, reflected to a minor extent only in potential nitrification, denitrifying enzyme activity (DEA), and the abundance of functional genes characteristic of nitrifying (bacterial and archaeal amoA) and denitrifying (narG, nirS, nirK, nosZ) micro-organisms. N₂O emissions, however, were much reduced under drought, suggesting that this effect was driven by environmental limitations rather than by changes in the activity potential or the size of the respective microbial communities. Cattle urine stimulated nitrification and, to a lesser extent, also DEA, but more so in the absence of drought. In contrast, NH₄NO₃ reduced the activity of nitrifiers and denitrifiers due to top-soil acidification. In summary, our data demonstrate that complex interactions between drought, mineral N availability, soil acidification, and plant nutrient uptake control soil N cycling and associated N₂O emissions. These interactive effects differed between processes of the soil N cycle, suggesting that the spatial heterogeneity in pastures needs to be taken into account when predicting changes in N cycling and associated N₂O emissions in a changing climate.     

Differentiated free-living and sediment-attached bacterial community structure inside and outside denitrification hotspots in the river–groundwater interface

This study assessed the functional significance of attached and free-living bacterial communities involved in the process of denitrification in a shallow aquifer of a riparian zone (Garonne River, SW France). Denitrification enzyme activity (DEA), bacterial density (BD) and bacterial community composition (BCC) were measured in two aquifer compartments: the groundwater and the sandy fraction of the sediment deposit. Samples were collected in wells located inside (IHD) and outside (OHD) identified hotspots of denitrification. Despite high BD values (up to 1.14 × 10¹² cells m⁻³), DEA was not detected in the water compartment (< 0.32 mg N–N₂O m⁻³ d⁻¹). The sandy fraction showed detectable DEA (up to 1,389 mg N–N₂O m⁻³ d⁻¹) and, consistent with BD pattern, higher DEA values were measured in IHD zones than in OHD zones. The BCC assessed by 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) partly supported this result: attached and free-living communities were significantly different (< 30% similarity) but patterns of BCC did not cluster according to IHD and OHD zones. Targeting the denitrifying communities by means of a culture enrichment step prior to 16S rDNA PCR-DGGE showed that the free-living and sediment attached communities differed. Most sequences obtained from DGGE profiles of denitrifying communities were affiliated to Proteobacteria and showed low genetic distance with taxa that have already been detected in aquifers (e.g., Azoarcus sp., Acidovorax sp. and Pseudomonas spp.). This study confirms that in the aquifer the sediment-attached fraction exhibits different functions (DEA) from free-living communities and suggests that this functional difference is related to the communities’ structure.     

Denitrification in the vadose zone and in surficial groundwater of a sandy entisol with citrus production

A portion of nitrate (NO ₃ ⁻ ), a final breakdown product of nitrogen (N) fertilizers, applied to soils and/or that produced upon decomposition of organic residues in soils may leach into groundwater. Nitrate levels in water excess of 10 mg L⁻¹ (NO₃–N) are undesirable as per drinking water quality standards. Nitrate concentrations in surficial groundwater can vary substantially within an area of citrus grove which receives uniform N rate and irrigation management practice. Therefore, differences in localized conditions which can contribute to variations in gaseous loss of NO ₃ ⁻ in the vadose zone and in the surficial aquifer can affect differential concentrations of NO₃–N in the groundwater at different points of sampling. The denitrification capacity and potential in a shallow vadose zone soil and in surficial groundwater were studied in two large blocks of a citrus grove of ‘Valencia’ orange trees (Citrus sinensis (L.) Obs.) on Rough lemon rootstock ( Citrus jambhiri (L.)) under a uniform N rate and irrigation program. The NO₃–N concentration in the surficial groundwater sampled from four monitoring wells (MW) within each block varied from 5.5- to 6.6-fold. Soil samples were collected from 0 to 30, 30 to 90, or 90 to 150 cm depths, and from the soil/groundwater interface (SGWI). Groundwater samples from the monitoring wells (MW) were collected prior to purging (stagnant water) and after purging five well volumes. Without the addition of either C or N, the denitrification capacity ranged from 0.5 to 1.53, and from 0.0 to 2.25 mg N₂O–N kg⁻¹ soil at the surface soil and at the soil/groundwater interface, respectively. The denitrification potential increased by 100-fold with the addition of 200 mg kg⁻¹ each of N and C. The denitrification potential in the groundwater also followed a pattern similar to that for the soil samples. Denitrification potential in the soil or in the groundwater was greatest near the monitor well with shallow depth of vadose zone (MW3). Cumulative N₂O–N emission (denitrification capacity) from the SGWI soil samples and from stagnant water samples strongly correlated to microbial most probable number (MPN) counts (r² = 0.84 – 0.89), and dissolved organic C (DOC) (r² = 0.96 – 0.97). Denitrification capacity of the SGWI samples moderately correlated to water-filled pore space (WFPS) (r² = 0.52). However, extractable NO₃-N content of the SGWI soil samples poorly (negative) correlated to denitrification capacity (r² = 0.35). However, addition C, N or both to the soil or water samples resulted in significant increase in cumulative N₂O emission. This study demonstrated that variation in denitrification capacity, as a result of differences in denitrifier population, and the amount of readily available carbon source significantly (at 95% probability level) influenced the variation in NO₃–N concentrations in the surficial groundwater samples collected from different monitoring wells within an area with uniform N management. This revised version was published online in June 2006 with corrections to the Cover Date.     

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

Cryoturbated peat circles (that is, bare surface soil mixed by frost action; pH 3–4) in the Russian discontinuous permafrost tundra are nitrate-rich ‘hotspots'' of nitrous oxide (N₂O) emissions in arctic ecosystems, whereas adjacent unturbated peat areas are not. N₂O was produced and subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated but not in those with unturbated peat soil. Nitrate, nitrite and acetylene stimulated net N₂O production of both soils in anoxic microcosms, indicating denitrification as the source of N₂O. Up to 500 and 10 μ nitrate stimulated denitrification in cryoturbated and unturbated peat soils, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification were 28 and 18 nmol N₂O g_DW⁻¹ h⁻¹, for cryoturbated and unturbated peat soils, respectively. Barcoded amplicon pyrosequencing of narG, nirK/nirS and nosZ (encoding nitrate, nitrite and N₂O reductases, respectively) yielded ≈49 000 quality-filtered sequences with an average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were detected per soil and gene, many of which were distantly related to cultured denitrifiers or environmental sequences. Denitrification-associated gene diversity in cryoturbated and in unturbated peat soils differed. Quantitative PCR (inhibition-corrected per DNA extract) revealed higher copy numbers of narG in cryoturbated than in unturbated peat soil. Copy numbers of nirS were up to 1000 × higher than those of nirK in both soils, and nirS nirK⁻¹ copy number ratios in cryoturbated and unturbated peat soils differed. The collective data indicate that the contrasting N₂O emission patterns of cryoturbated and unturbated peat soils are associated with contrasting denitrifier communities.     

Impacts of varying durations of passive oxygen exposure on freshwater denitrifier community structure and function

Fertilizer use has dramatically increased the availability of nitrate (NO₃ ^?) in aquatic systems. Microbe-mediated denitrification is one of the predominant means of NO₃ ^? removal from freshwaters, yet oxygenation (O₂)-induced disruptions—e.g., extreme precipitation events—can occur, resulting in a disproportional increase in nitrous oxide (N₂O) production and efflux as facultative anaerobic bacterial populations use of O₂ as a terminal electron acceptor increases. We examined the effects of 12- and 24-h passive O₂ exposure on previously anaerobic bacterial communities focusing on denitrification enzyme activity (DEA), N₂O production, and bacterial community 16S rRNA and nitrous oxide reductase gene (nosZ) profiles after 12, 24, and 48 h of anaerobic recovery. Treatments experiencing 24-h O₂ exposure had significantly higher DEA 12 h into anaerobic recovery than treatments undergoing 12-h O₂ exposure. Initial N₂O emissions were significantly lower in the 24-h O₂ exposure treatments although by 24 h a dramatic spike (tenfold relative to the 12-h O₂ exposure treatments) in N₂O concentrations was observed. However, within 6 h (30-h anaerobic recovery) these differences were gone. Community nosZ profiles experiencing 24-h O₂ exposure exhibited reduced diversity after 24-h recovery, which corresponded with an increase in N₂O emissions. However, after 48 h of anaerobic recovery, nosZ diversity had recovered. These observations highlight the effects of short-term aerobic disruption on denitrification, as well as the effects on the denitrifier community profile. Together, these data suggest that recovery to ambient N cycling is exacerbated by disturbance length due to increased lag time and subsequent loss of denitrifier community diversity.     

Deciphering the oxygen isotope composition of nitrous oxide produced by nitrification

The ability to use δ¹⁸O values of nitrous oxide (N₂O) to apportion environmental emissions is currently hindered by a poor understanding of the controls on δ¹⁸O–N₂O from nitrification (hydroxylamine oxidation to N₂O and nitrite reduction to N₂O). In this study fertilized agricultural soils and unfertilized temperate forest soils were aerobically incubated with different ¹⁸O/¹⁶O waters, and conceptual and mathematical models were developed to systematically explain the δ¹⁸O–N₂O formed by nitrification. Modeling exercises used a set of defined input parameters to emulate the measured soil δ¹⁸O–N₂O data (Monte Carlo approach). The Monte Carlo simulations implied that abiotic oxygen (O) exchange between nitrite (NO₂^?) and H₂O is important in all soils, but that biological, enzyme‐controlled O‐exchange does not occur during the reduction of NO₂^? to N₂O (nitrifier‐denitrification). Similarly, the results of the model simulations indicated that N₂O consumption is not characteristic of aerobic N₂O formation. The results of this study and a synthesis of the published literature data indicate that δ¹⁸O–N₂O formed in aerobic environments is constrained between +13‰ and +35‰ relative to Vienna Standard Mean Ocean Water (VSMOW). N₂O formed via hydroxylamine oxidation and nitrifier‐denitrification cannot be separated using δ¹⁸O unless ¹⁸O tracers are employed. The natural range of nitrifier δ¹⁸O–N₂O is discussed and explained in terms of our conceptual model, and the major and minor controls that define aerobically produced δ¹⁸O–N₂O are identified. Despite the highly complex nature of δ¹⁸O–N₂O produced by nitrification this δ¹⁸O range is narrow. As a result, in many situations δ¹⁸O values may be used in conjunction with δ¹⁵N–N₂O data to apportion nitrifier‐ and denitrifier‐derived N₂O. However, when biological O‐exchange during denitrification is high and N₂O consumption is low, there may be too much overlap in δ¹⁸O values to distinguish N₂O formed by these pathways.     

Production of N2O in soil during decomposition of dead yeast cells with different spatial distributions

Production and sources of N₂O were determined in soil columns amended with autoclaved yeast cells either mixed into or added as 0.5 cm³ lumps to the soil in combination with no or 200 g NO₃ ^--N g^-1. At four occasions over a two-week study period, subsets of cores were measured for N₂O production during 4-hour incubations under atmospheres of ambient air, 10 Pa of C₂H₂, and N₂, respectively. Denitrification enzyme activity (DEA) was assessed in subsamples of cores that had been incubated continuously under air.Autoclaved yeast provided a C-source readily available for denitrifying bacteria in the soil. Nitrous oxide production was negligible in unamended columns whereas accumulated N₂O losses in the presence of yeast material were substantial, varying between 15 to 49 ng N₂O-N g^-1 h^-1. Mixing yeast into the soil caused the highest production of N₂O followed by the yeast lump and no yeast treatments. Incubation in the presence of 10 Pa C₂H₂ indicated that denitrification was the sole source of N₂O, in accordance with an increase in DEA. Nitrous oxide production and DEA peaked after 4–7 days of incubation, and both were unaffected by additional NO₃ ^-. Two-to four-fold responses to anaerobiosis and accumulation of NO₃ ^- and NH₄ ⁺ in proximity of the lumps indicated that N₂O production here was limited by relatively low C-availability. In contrast, 10- to 12-fold responses to anaerobiosis and no accumulation of inorganic N suggested a higher C-availability where yeast was mixed into the soil.     

Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification,primarily due to acidification of the soils

China is the world's largest producer and consumer of fertilizer N, and decades of overuse has caused nitrate leaching and possibly soil acidification. We hypothesized that this would enhance the soils' propensity to emit N₂O from denitrification by reducing the expression of the enzyme N₂O reductase. We investigated this by standardized oxic/anoxic incubations of soils from five long‐term fertilization experiments in different regions of China. After adjusting the nitrate concentration to 2 mM, we measured oxic respiration (R), potential denitrification (D), substrate‐induced denitrification, and the denitrification product stoichiometry (NO, N₂O, N₂). Soils with a history of high fertilizer N levels had high N₂O/(N₂O+N₂) ratios, but only in those field experiments where soil pH had been lowered by N fertilization. By comparing all soils, we found a strong negative correlation between pH and the N₂O/(N₂O+N₂) product ratio (r² = 0.759, P < 0.001). In contrast, the potential denitrification (D) was found to be a linear function of oxic respiration (R), and the ratio D/R was largely unaffected by soil pH. The immediate effect of liming acidified soils was lowered N₂O/(N₂O+N₂) ratios. The results provide evidence that soil pH has a marginal direct effect on potential denitrification, but that it is the master variable controlling the percentage of denitrified N emitted as N₂O. It has been known for long that low pH may result in high N₂O/(N₂O+N₂) product ratios of denitrification, but our documentation of a pervasive pH‐control of this ratio across soil types and management practices is new. The results are in good agreement with new understanding of how pH may interfere with the expression of N₂O reductase. We argue that the management of soil pH should be high on the agenda for mitigating N₂O emissions in the future, particularly for countries where ongoing intensification of plant production is likely to acidify the soils.     

Control of denitrification enzyme activity in a streamside soil

Progress curve analysis of NO₃⁻ and NO₂⁻ reduction in surface soil samples from a streamside soil gave K_m values of 4.24 and 6.33 μM, and V_max values of 2.16 and 1.83 μmol l⁻¹ min⁻¹, respectively. Recoveries of reduced NO₃⁻ and NO₂⁻ as gaseous N averaged 82 and 108%. The unrecovered NO₃⁻-N was presumably dissimilated to NH₄⁺-N. The idenitification enzyme activity (DEA) was examined throughout a year and showed seasonal and spatial variabilities of only 10% to 26%, suggesting a high persistency of denitrifying enzymes. Soil moisture and DEA correlated significantly (r = 0.7671; P<0.01). The DEA in saturated subsoil also showed a relatively little variation, with spatial variabilities of between 28 and 38%. Amendment with NO₃⁻ rarely enhanced the acctivity more than two-fold at either depth. Addition of glucose increased the activity 2.3 and 2.5 times in the surface soil and suboil respectively, indicating a moderate carbon limitation of denitrification. The activation energy of DEA was found to be 64.9 kJ mol⁻¹ and Q₁₀ values for the 2–12°C and 12–22°C temperature ranges were 2.71 and 2.53, respectively. Extrapolation suggested there would be a 4.4-fold increase in DEA if the temperature was changed from 0 to 15°C. Substrate diffusion limited the denitrification 10 to 25 fold.Thus, under anaerobic moist conditions it appears that changes in denitrification might primarily be due to varying diffusion of substrates into the anaerobic soil centers. Over a year, fluctuations in DEA, temperature changes and fluctuations of electron-acceptor and -donor supply will only have a minor effect on natural denitrification activity.     

Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen

Wetlands are sources of denitrification-derived nitrous oxide (N₂O). Thus, the denitrifier community of an N₂O-emitting fen (pH 4.7 to 5.2) was investigated. N₂O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10¹¹ cells·g_DW⁻¹ (where DW is dry weight) and 10⁸ cells·g_DW⁻¹, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v_max) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N₂O h⁻¹· g_DW⁻¹, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N₂O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N₂O at in situ pH.Nitrous oxide (N₂O) is a potent greenhouse gas with a global warming potential that is 300-fold higher than that of CO₂, and its concentration increased from 270 ppb in 1750 to 319 ppb in 2005 (17). N₂O can be produced in soils during denitrification, nitrification, the dissimilatory reduction of nitrate to nitrite and/or ammonium (hereafter referred to as dissimilatory nitrate reduction), or the chemical transformation of nitrite or hydroxylamine (5, 7, 49). The percentage of N₂O produced in any of these processes is variable, depending mainly on the redox potential, pH, and C/N ratio (49). In anoxic ecosystems such as waterlogged soils, most of the N₂O is considered to be denitrification derived (7, 9). Complete denitrification is the sequential reduction of nitrate to dinitrogen (N₂) via nitrite, nitric oxide (NO), and N₂O (75). The main product of denitrification varies with the organism and in situ conditions and is usually either N₂O or N₂ (68). N₂O can occur as a by-product during dissimilatory nitrate reduction when accumulated nitrite interacts with nitrate reductase to form N₂O (59). The production of N₂O by dissimilatory nitrate reducers is favored in environments with large amounts of readily available organic carbon (65). Thus, their contribution to nitrate-dependent production of N₂O in soils is likely insignificant compared to that of denitrifiers.The oxidoreductases involved in denitrification are termed dissimilatory nitrate reductase (Nar, encoded by narGHJI, or Nap, encoded by napEDABC), nitrite reductase (Nir, encoded by nirK and nirS), NO reductase (cNor and qNor, encoded by norBC and norB, respectively), and N₂O reductase (Nos, encoded by nosZ) (75). Nitrate reductase is also found in dissimilatory nitrate reducers (60). narG can therefore be used as a molecular marker to assess both denitrifiers and dissimilatory nitrate reducers, whereas nosZ is specific for the assessment of denitrifiers (25, 43, 48).Denitrification in soils is regulated by temperature, pH, substrate (i.e., carbon) availability, and water content (10, 24, 66). Although denitrification increases with increasing temperature, it can still occur at temperatures below 0°C (10, 24). Low temperatures appear to limit the activity of N₂O reductase more severely than other enzymes involved in denitrification and thus yield higher relative amounts of denitrification-derived N₂O (24). Although denitrification activity usually decreases under acidic conditions, the relative percentage of N₂O to total denitrification-derived nitrogenous gases increases with increasing acidity, a result attributed to the sensitivity of N₂O reductase to low pH (27, 70). However, denitrifier communities can be adapted to the in situ pH of the system (40, 58, 73).Wetlands are ecosystems in which denitrification is likely a dominant source of emitted N₂O (7, 44, 45). The identification and analysis of main drivers for N₂O production (i.e., the microbiota catalyzing N₂O production and consumption) is thus of major concern in such environments. Fens are specialized wetlands characterized by soil acidity (67). However, information on acid-tolerant denitrifier communities of such wetlands is scarce. It is hypothesized that fens harbor a diverse, hitherto unknown, denitrifier community that is adapted to in situ conditions and associated with N₂O fluxes (i.e., fen denitrifiers are acid tolerant and have a high affinity for nitrate and N₂O). Thus, the main objectives of the present study were to evaluate the capacities of denitrifier communities of an N₂O-emitting fen (20) to produce or consume N₂O and to determine if a novel and diverse denitrifier community was associated with these capacities.     

硝态氮异化还原机制及其主导因素研究进展

硝态氮(NO_3~-)异化还原过程通常包含反硝化和异化还原为铵(DNRA)两个方面,是土壤氮素转化的重要途径,其强度大小直接影响着硝态氮的利用和环境效应(如淋溶和氮氧化物气体排放)。反硝化和DNRA过程在反应条件、产物和影响因素等方面常会呈现出协同与竞争的交互作用机制。综述了反硝化和DNRA过程的研究进展及其二者协同竞争的作用机理,并阐述了在NO_3~-、pH、有效C、氧化还原电位(Eh)等环境条件和土壤微生物对其发生强度和产物的影响,提出了今后应在产生机理、土壤环境因素、微生物学过程以及与其他氮素转化过程耦联作用等方面亟需深入研究,以期增进对氮素循环过程的认识以及为加强氮素管理利用提供依据。