Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from Streptomyces thioluteus

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N₂O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N₂O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.     

Distinguishing between Nitrification and Denitrification as Sources of Gaseous Nitrogen Production in Soil

The source of N₂O produced in soil is often uncertain because denitrification and nitrification can occur simultaneously in the same soil aggregate. A technique which exploits the differential sensitivity of these processes to C₂H₂ inhibition is proposed for distinguishing among gaseous N losses from soils. Denitrification N₂O was estimated from 24-h laboratory incubations in which nitrification was inhibited by 10-Pa C₂H₂. Nitrification N₂O was estimated from the difference between N₂O production under no C₂H₂ and that determined for denitrification. Denitrification N₂ was estimated from the difference between N₂O production under 10-kPa C₂H₂ and that under 10 Pa. Laboratory estimates of N₂O production were significantly correlated with in situ N₂O diffusion measurements made during a 10-month period in two forested watersheds. Nitrous oxide production from nitrification was most important on well-drained sites of a disturbed watershed where ambient NO₃⁻ was high. In contrast, denitrification N₂O was most important on poorly drained sites near the stream of the same watershed. Distinction between N₂O production from nitrification and denitrification was corroborated by correlations between denitrification N₂O and water-filled pore space and between nitrification N₂O and ambient NO₃⁻. This technique permits qualitative study of environmental parameters that regulate gaseous N losses via denitrification and nitrification.     

Nitrous Oxide Formation in the Colne Estuary, England: the Central Role of Nitrite

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N₂O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N₂O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N₂O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 μM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 μmol N · m⁻² · h⁻¹ with nitrite but only 180 μmol N · m⁻² · h⁻¹ with nitrate. The ratios of rates of nitrous oxide production and denitrification (N₂O/N₂ × 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N₂ formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N₂ via N₂O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N₂O as the end product. Consideration of free-energy changes during N₂O formation led to the conclusion that N₂O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N₂O in high-nutrient-load estuaries.     

The nitrogen cycle in shallow water sediment systems of rice fields. Part 1: The denitrification process

Denitrification causes important losses of N-fertilizer in rice-fields, where high temperature and high production of organic matter favour denitrification losses. Two techniques have been used to quantify the denitrification losses: the ¹⁵N technique, which can be used to quantify the amount finally incorporated, and the acetylyne inhibition technique which is a direct measure of the quantities lost.Both techniques were applied in enclosures (diameter = 44 cm) in the field while moreover bio-assays in 3 l glass beakers were carried out. In all experiments where nitrate was added we found a rapid decrease of nitrate; usually about 30–50% of the nitrate that disappeared was recovered as N₂O. As in one experiment, in which we measured the N₂O disappearance rate as well, the N₂O itself decreased at a rather constant rate of 20% per day, a correction must be made for this N₂O decrease in the calculations of the nitrate disappearance rate. Although we have only one series in which the decrease of N₂O was measured, the mathematical analysis indicates that as much as 80% of the N-fertilizer is actually lost. This figure is in full agreement with the ¹⁵N experiments; if the ¹⁵N was applied early only about 7% was recovered in soil and plants, while if it was applied later (after 7 weeks) about 20% was incorporated.Denitrification rate could be fitted on an negative exponential regression line; the rate constant increased during the summer. It is suggested that organic matter caused this increase.During denitrification considerable quantities of nitrite appear, which later on disappear again by processes still unknown; the nature of the available organic matter may be important for this nitrite production.With N-serve we tried to inhibit NH₃ oxidation. In this way we tried to prevent the considerable N losses and to demonstrate that the nitrite produced in our experiments was not derived from NH₃ oxidation. N-serve, however, had very little influence. It is probably inactivated by absorption onto the sediments.From these results it is suggested that the efficiency of N-application may be considerably increased by using low doses of N-fertilizer, but applied late in the growing season, e.g. 7 weeks after sowing. This favours environmental protection as well.     

Role of Microorganisms in Emission of Nitrous Oxide and Methane in Pulse Cultivated Soil Under Laboratory Incubation Condition

Soil from a pulse cultivated farmers land of Odisha, India, have been subjected to incubation studies for 40 consecutive days, to establish the impact of various nitrogenous fertilizers and water filled pore space (WFPS) on green house gas emission (N₂O & CH₄). C₂H₂ inhibition technique was followed to have a comprehensive understanding about the individual contribution of nitrifiers and denitrifiers towards the emission of N₂O. Nevertheless, low concentration of C₂H₂ (5 ml: flow rate 0.1 kg/cm²) is hypothesized to partially impede the metabolic pathways of denitrifying bacterial population, thus reducing the overall N₂O emission rate. Different soil parameters of the experimental soil such as moisture, total organic carbon, ammonium content and nitrate–nitrogen contents were measured at regular intervals. Application of external N-sources under different WFPS conditions revealed the diverse role played by the indigenous soil microorganism towards green house gas emission. Isolation of heterotrophic microorganisms (Pseudomonas) from the soil samples, further supported the fact that denitrification might be prevailing during specific conditions thus contributing to N₂O emission. Statistical analysis showed that WFPS was the most influential parameter affecting N₂O formation in soil in absence of an inhibitor like C₂H₂.     

Dinitrogen and nitrous oxide produced by denitrification and nitrification in soil with and without barley plants

Summary To examine the effect of barley roots on denitrification, a pot experiment was designed to compare N₂O production and denitrification in soils with and without barley plants. Denitrification, N₂O resulting from denitrification and nitrification, and respiration were estimated by incubating pots with soil with and without intact plants in plastic bags at high moisture levels. C₂H₂-inhibition of nitrous oxide reductase (partial pressure of 10 kPa C₂H₂) was used to determine total denitrification rates while incubations with ambient air and with C₂H₂ at partial pressures of 2.5–5 Pa were used to estimate the amounts of N₂O released from autotrophic nitrification and from denitrification processes. Other sources of N₂O were presumed to be negligible. Potential denitrification, nitrification and root biomass were measured in subsamples collected from four soil depths. A positive correlation was found between denitrification rates and root biomass. N₂ was the predominant denitrification product found close to roots; N₂O formed by non autotrophic nitrifiers, assumed to be denitrifiers originated in soil not affected by growing roots. Apparently, roots promote denitrification because they consumed oxygen, thereby increasing the anaerobic volume of the soil. The ratio of actual to potential denitrification rates increased over time, especially in the presence of roots.     

Nitrous oxide production, its source and distribution in urine patches on grassland on peat soil

Urine patches are considered to be important sites for nitrous oxide (N₂O) production through nitrification and denitrification due to their high concentration of nitrogen (N). The aim of the present study was to determine the microbial source and size of production of N₂O in different zones of a urine patch on grassland on peat soil. Artificial urine was applied in elongated patches of 4.5 m. Four lateral zones were distinguished and sampled for four weeks using an intact soil core incubation method. Incubation of soil cores took place without any additions to the headspace to determine total N₂O production, with acetylene addition to determine total denitrification (N₂O+N₂), and with methyl fluoride to determine the N₂O produced through denitrification.Nitrous oxide production was largest in the centre and decreased towards the edge of the patch. Maximum N₂O production was about 50 mg N m^–2 d^–1 and maximum denitrification activity was 70 mg N m^–2 d^–1. Nitrification was the main N₂O producing process. Nitrous oxide production through denitrification was only of significance when denitrification activity was high. Total N loss through nitrification and denitrification over 31 days was 4.1 g N per patch which was 2.2% of the total applied urine-N.     

Acetylene Metabolism and Stimulation of Denitrification in an Agricultural Soil

The effects of C₂H₂ metabolism on N₂O production were examined in soil slurries. Enrichment of C₂H₂ consumption activity occurred only in aerobic incubations. Rapid disappearance of subsequent C₂H₂ additions, stimulation of CO₂ production, and most-probable-number enumerations of C₂H₂ utilizers indicated enrichment of the population responsible. During C₂H₂ consumption in slurries incubated statically under air, maximal rates of N₂O evolution were 19 times higher than those in anaerobic incubations. After 20 days of enrichment with C₂H₂, the production of N₂O by slurries supplemented with C₂H₂ and nitrate was 10 times higher than that in the unenriched controls. A Nocardia- or Arthrobacter-like bacterium was isolated that grew on C₂H₂ but did not denitrify. The behavior of soil inoculated with this bacterium became similar to that of C₂H₂-enriched soil incubated aerobically. Ethanol, acetate, and acetaldehyde were identified in enrichment experiments, and denitrification in soil slurries was stimulated by addition of the supernatant from a pure culture grown on mineral medium with C₂H₂. These results indicate that denitrification can be stimulated by the actions of an aerobic, nondenitrifying C₂H₂-metabolizing population. Utilization of intermediate metabolites by denitrifiers and enhanced O₂ consumption are two possible mechanisms for this stimulation.     

Growth, denitrification and nitrate ammonification of the rhizobial strain TNAU 14 in presence and absence of C2H4 and C2H2

Soil-N (NO₃ ^?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N₂-fixation. Organic N and ammonium oxidised to NO₃ ^? means oxygen depletion. Plants suffering under O₂ or infection stress start to excrete ethylene (C₂H₄). C₂H₄ widens the root intercellulars that O₂-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C₂H₄. Surplus nitrate and C₂H₄ inhibit nodulation of leguminous plants. Excess NO₃ ^? in the nodulesphere could be diminished by N₂-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C₂H₄ interferes with the potential of N₂-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO₃-mannitol-medium that was incubated under N₂-, 1% (v/v) N₂?C₂H₄-, and 1% (v/v) N₂?C₂H₂-atmosphere in the laboratory. C₂H₂ was included into the experiments because it is frequently used to quantify N₂-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 toRhizobium radiobacter andRhizobium tumefaciens. Strain TNAU 14 released N₂O into the bottle headspace in all treatments, surprisingly significantly less in presence of C₂H₂. Nitrate-ammonification was even completely blocked by C₂H₂. C₂H₄, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH₄ ⁺ during continuing incubation.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Denitrification Activity of a Remarkably Diverse Fen Denitrifier Community in Finnish Lapland Is N-Oxide Limited

Peatlands cover more than 30% of the Finnish land area and impact N₂O fluxes. Denitrifiers release N₂O as an intermediate or end product. In situ N₂O emissions of a near pH neutral pristine fen soil in Finnish Lapland were marginal during gas chamber measurements. However, nitrate and ammonium fertilization significantly stimulated in situ N₂O emissions. Stimulation with nitrate was stronger than with ammonium. N₂O was produced and subsequently consumed in gas chambers. In unsupplemented anoxic microcosms, fen soil produced N₂O only when acetylene was added to block nitrous oxide reductase, suggesting complete denitrification. Nitrate and nitrite stimulated denitrification in fen soil, and maximal reaction velocities (v_max) of nitrate or nitrite dependent denitrification where 18 and 52 nmol N₂O h^-1 g_DW ^-1, respectively. N₂O was below 30% of total produced N gases in fen soil when concentrations of nitrate and nitrite were <500 μM. v_max for N₂O consumption was up to 36 nmol N₂O h^-1 g_DW ^-1. Denitrifier diversity was assessed by analyses of narG, nirK/nirS, and nosZ (encoding nitrate-, nitrite-, and nitrous oxide reductases, respectively) by barcoded amplicon pyrosequencing. Analyses of ~14,000 quality filtered sequences indicated up to 25 species-level operational taxonomic units (OTUs), and up to 359 OTUs at 97% sequence similarity, suggesting diverse denitrifiers. Phylogenetic analyses revealed clusters distantly related to publicly available sequences, suggesting hitherto unknown denitrifiers. Representatives of species-level OTUs were affiliated with sequences of unknown soil bacteria and Actinobacterial, Alpha-, Beta-, Gamma-, and Delta-Proteobacterial sequences. Comparison of the 4 gene markers at 97% similarity indicated a higher diversity of narG than for the other gene markers based on Shannon indices and observed number of OTUs. The collective data indicate (i) a high denitrification and N₂O consumption potential, and (ii) a highly diverse, nitrate limited denitrifier community associated with potential N₂O fluxes in a pH-neutral fen soil.     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).     

Nitrate requirement for acetylene inhibition of nitrous oxide reduction in marine sediments

The inhibition of nitrous oxide (N₂O) reduction by acetylene (C₂H₂) in saltmarsh sediment was temporary; we investigated this phenomenon and possible causes. The reduction of N₂O in the presence of C₂H₂ was biological. N₂O consumption in the presence of C₂H₂ began when nitrate concentration became very low. The time course of N₂O consumption after periods of N₂O accumulation was unaffected by initial nitrate concentrations between 16 and 200M, or C₂H₂ concentrations between 10 and 100% of the gas phase. Sulfide had no effect on the kinetics of N₂O reduction in the presence of C₂H₂. In more dilute slurries of saltmarsh sediments and in estuarine sediment, N₂O persisted in the presence of C₂H₂ unless sufficient organic carbon was added to deplete nitrate. In saltmarsh sediments, the rate of N₂O consumption in the presence of C₂H₂ was not changed by preincubation with C₂H₂. Initial positive rates of N₂O production in the presence of C₂H₂ occurred only when the block was apparently effective (i.e., at nitrate concentrations greater than about 5–10M) and appeared to represent a valid estimate of denitrification. Conversely, and in agreement with previous studies, concentrations of NO₃ ^– below these levels resulted in reduced efficiency of C₂H₂ blockage of N₂O reductase.     

The effect of nitrate and nitrous oxide on hydrogen and methane accumulation in anaerobically-incubated soils

Summary The effect of KNO₃ and N₂O on the accumulation of CH₄, H₂ and denitrification products in two North Dakota soils during anaerobic incubation at 30°C was studied by means of gas chromatography. KNO₃ and N₂O (500 ppm N) reduced the rate of accumulation of CH₄ by a Tetonka soil regardless of whether the soil was in an air-dried condition or had been pre-incubated and actively producing CH₄ prior to the treatment application. Both KNO₃ and N₂O completely suppressed H₂ accumulation by the remoistened air-dried soil; no H₂ either in the presence or absence of added KNO₃ or N₂O was accumulated by the pre-incubated Tetonka soil subsequent to the treatment application. KNO₃ (250 ppm N) reduced the rate of accumulation of CH₄ by a Cavour loam during anaerobic incubation. No H₂ was accumulated by this soil during anaerobic incubation. At equivalent K⁺ concentrations, KNO₃ suppressed CH₄ accumulation by the Tetonka and Cavour soils to a greater extent than did KCl.     

Nitrate limitation of N2O production and denitrification from tropical pasture and rain forest soils

Nitrous oxide production was measured in intact cores taken from active pasture and old-growth forest Inceptisols in the Atlantic Lowlands of Costa Rica. Following additions of aqueous KNO₃ or glucose, or the two combined amendments, the cores were incubated in the laboratory to determine if N₂O production rates were either N-limited or C-limited in the two land use types. Differences in rates of denitrification (N2₂O + N₂ production) among amended forest and pasture soils were determined by addition of 10% C₂H₂.The forest soils were relatively insensitive to all amendment additions, including the acetylene block. Forest N₂O production rates among the treatments did not differ from the controls, and were consistently lower than those of the pasture soils. With the addition of glucose plus nitrate to the forest soils, production of N₂O was three times greater than the controls, although this increase was not statistically significant. On the other hand, the pasture soils were definitely nitrogen-limited since N₂O production rates were increased substantially beyond controls by all the amendments which contained nitrate, despite the very low N level (5 mg N kg^–1 soil) relative to typical fertilizer applications. With respect to the nitrate plus glucose plus acetylene treatment, denitrification was high in the pasture soils; N₂O production in the presence of C₂H₂ was 150% of the rate of N₂O production measured in the absence of the acetylene block. The results are discussed in relation to the effects of agricultural land use practices and subsequent impacts of disturbance on N₂O release.     

Aspects of nitrogen fixation and denitrification byAzospirillum

Summary Model experiments were performed to investigate the nitrogen fixation (C₂H₂ reduction) and denitrification (N₂O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C₂H₂ reduction or N₂O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C₂H₂ reduction and low N₂O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.     

Total denitrification and the ratio between N2O and N2 during the growth of spring barley

Summary Total denitrification (N₂O+N₂) and nitrous oxide emission were measured on intact soil cores using the acetylene inhibition technique.Total denitrification from the depth 0–8 cm during the growth period from April to August was 7 kg N/ha from plots supplied with 30 kg N/ha and 19 kg N/ha from plots supplied with 120 kg N/ha. The amounts of precipitation, plant growth, and N application were found to affect the denitrification rate. These factors also affected the ratio (N₂O+N₂)/N₂O, which varied from 1.0 to 7.2. Plant growth and precipitation increased the proportion of N₂ produced, whereas a high nitrate content increased the proportion of N₂O.     

Nitrogen gas (N2+N2O) flux from urea applied to lowland rice as affected by green manure

A field study was conducted on a clay soil (Andaqueptic Haplaquoll) in the Philippines to directly measure the evolution of (N₂+N₂O)−¹⁵N from 98 atom %¹⁵N-labeled urea broadcast at 29 kg N ha⁻¹ into 0.05-m-deep floodwater at 15 days after transplanting (DT) rice. The flux of (N₂+N₂O)−¹⁵N during the 19 days following urea application never exceeded 28 g N ha⁻¹ day⁻¹. The total recovery of (N₂+N₂O)−¹⁵N evolved from the field was only 0.51% of the applied N, whereas total gaseous¹⁵N loss estimated from unrecovered¹⁵N in the¹⁵N balance was 41% of the applied N. Floodwater (nitrate+nitrite)−N in the 5 days following urea application never exceeded 0.14 g N m⁻³ or 0.3% of the applied N. Prior cropping of cowpea [Vigna unguiculata (L.) Walp.] to flowering with subsequent incorporation of the green manure (dry matter=2.5 Mg ha⁻¹, C/N=15) at 15 days before rice transplanting had no effect on fate of urea applied to rice at 15 DT. The recovery of (N₂+N₂O)−¹⁵N and total¹⁵N loss during the 19 days following urea application were 0.46 and 40%, respectively. Direct recovery of evolved (N₂+N₂O)−¹⁵N and total¹⁵N loss from 27 kg applied nitrate-N ha⁻¹ were 20% and 53% during the same 19-day period. The failure of directly-recovered (N₂+N₂O)−¹⁵N to match total¹⁵N loss from added nitrate-¹⁵N might be due to entrapment of denitrification end products in soil or transport of gaseous end products to the atmosphere through rice plants. The rapid conversion of added nitrate-N to (N₂+N₂O)−N, the apparently sufficient water soluble soil organic C for denitrification (101 μg C g⁻¹ in the top 0.15-m soil layer), and the low floodwater nitrate following urea application suggested that denitrification loss from urea was controlled by supply of nitrate rather than by availability of organic C.     

Impacts of grazing intensity on denitrification and N<Subscript>2</Subscript>O production in a semi-arid grassland ecosystem

N₂O production from denitrification in soils contributes to the enhanced greenhouse effect and the destruction of the stratospheric ozone. Ungulate grazing affects denitrification and the production of N₂O. The short-term effect of grazing on denitrification and N₂O production has been examined in several grassland ecosystems. However, the effects of long-term grazing have rarely been studied. We measured denitrification and N₂O production during the 2005 and 2006 growing seasons in a long-term (17 years) experiment that had five grazing intensities (GI; 0.00, 1.33, 2.67, 4.00 and 5.33 sheep ha⁻¹). We found that denitrification and N₂O production rates were seasonally variable during the measurement period, with higher values observed in summer and lower values found in spring and autumn. The grazed treatments resulted in decreased denitrification and N₂O production, primarily due to the reduced soil nitrate concentration and organic N content under the long-term grazing. This supported our hypothesis that long-term over-grazing suppresses denitrification and N₂O production. Although significant differences in denitrification and N₂O production were not found between the four GI, there was a general trend that cumulative denitrification and N₂O production decreased as grazing intensity increased, especially in 2006. Lower N losses via denitrification and N₂O production in the grazed plots, to some extent, may contribute to the mitigation of greenhouse gas emission and help to preserve soil N and ameliorate the negative impacts of grazing on plant growth, productivity, and ecological restoration processes in the temperate steppe in northern China.     

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.