Nitrogen and oxygen isotope enrichment factors of nitrate at different denitrification rates in an agricultural soil

ABSTRACT

Quantitative evaluation of denitrification by the dual isotope approach, which measures the stable isotope ratios of nitrogen (δ¹⁵N) and oxygen (δ¹⁸O) in nitrate, has been hampered by the wide range of values reported for the ratio of enrichment factors for ¹⁵N and ¹⁸O (¹⁵ε and ¹⁸ε, respectively) during denitrification. The objectives of this study were to determine ¹⁵ε and ¹⁸ε values at different denitrification rates under controlled conditions, and to infer possible mechanisms by which the ¹⁸ε/¹⁵ε ratio is influenced under different conditions. Column experiments were conducted at 25, 15, and 10°C, which enabled determination of ¹⁵ε and ¹⁸ε at different denitrification rates, in the absence of nitrate replenishment from ammonium oxidation and other sources. The values of ¹⁵ε and ¹⁸ε ranged from ?11.8 to ?14.9‰ and from ?8.4 to ?15.9‰, respectively, with ¹⁵ε less sensitive to changes in the denitrification rates. The resultant ¹⁸ε/¹⁵ε ratio, ranging from 0.70 to 1.17, was close to the values reported for sediment incubations, and larger than those for groundwater systems. These results are consistent with the explanations that ¹⁸ε/¹⁵ε value itself is close to unity during denitrification, and that at smaller denitrification rates, concurrent reactions including re-oxidation of nitrite to nitrate lead to smaller apparent fractionation of ¹⁸O and smaller ¹⁸ε/¹⁵ε ratios. This suggests that while linear relationships between δ¹⁸O and δ¹⁵N give a strong evidence of denitrification, apparent ¹⁸ε/¹⁵ε values are site specific and depend on the ambient conditions. In evaluating denitrification in such systems, we suggest the use of ¹⁵ε in preference to ¹⁸ε because ¹⁵ε is less sensitive to denitrification rates.     

The influence of glucose and nitrate concentrations upon denitrification rates in sandy soils

Dentrification rates in two soils were assessed separately as a function of NO₃^? concentration while providing a constant initial glucose concentration, and as a function of glucose concentration while providing a constant initial NO₃^?-N concentration. Of the soils used, a Hanford sandy loam and a Coachella fine sand, the bacteria in the former produced higher rates of denitrification with a maximum loss of 1500 μg NO₃^?-N/ml day^?1 as compared to a loss of 150 μg NO₃^?-N/ml day^?1 from the latter. Rates of loss closely approximated Michaelis Menten kinetics in the Coachella sand, and K_m values for glucose-C and NO₃^?-N were 500 μg/ml and 170 μg/ml, respectively. Rates of loss of NO₃^?-N from the Hanford soil did not approximate Michaelis-Menten kinetics, and this was attributed to failure to saturate enzyme systems in the denitrifying bacteria with glucose and nitrogen when each was held constant. C/N ratios around 2 appeared to provide the greatest rates of denitrification. High C/N ratios or high glucose concentrations (1.8 per cent) retarded denitrification, with fungal growth and a subsequent drop in pH occuring. A Pseudomonas was incubated aerobically for 24 h followed by a 72 h anaerobic incubation with nitrate as the sole nitrogen source at 0, 10, 50, 100, 250 and 500mg N/ml concentrations. Assimilatory nitrate reduction never exceeded 75 mg N/ml, and it was concluded that this mode of nitrate reduction is insignificant at higher nitrate concentrations by comparison to dissimilatory nitrate reduction, i.e. denitrification.     

Nitrogen losses from agricultural soils through denitrification - a critical evaluation

The direct measurement of gaseous N losses through denitrification requires a complicated methodology. Mostly alternative approaches are used to estimate these losses for a particular arable or grassland site. Evaluation of the literature shows that N losses as a result of denitrification - determined by the ¹⁵N balance, by direct mass spectrometry of N₂ + N₂O and by the acetylene inhibition method, respectively - may reach about 10 % of the fertilizer input on agriculturally used sites without irrigation or organic manure addition. The estimation of denitrification losses can be improved by considering site-specific parameters affecting denitrification like total carbon content, pH, time of fertilizer application as well as soil temperature and soil moisture conditions.     

Effects of acetylene on nitrification and denitrification in two soils during incubation with ammonium nitrate

A loam from the Frilsham and one from the Wickham Series were incubated at 50 and 90 per cent of their water contents at saturation with 100 μg NH₄NO₃-N_g^?1 soil in the presence and absence of C₂H₂ (0.5 per cent, v/v). Acetylene inhibited nitrification in both soils, but had no effect on mineralization of N. No denitrification (measured as the production of N₂O in the presence of C₂H₂) occurred during incubation at 50 per cent saturation. At 90 per cent saturation, denitrification resulted in a loss of 28.4 and 36.7 μg N_g^?1 after 48 h from the Frilsham and Wickham soils, respectively. The concurrent inhibition of nitrification had no effect on the extent of denitrification at this time. In the Wickham soil, NO₃^? was exhausted after 168 h incubation in the presence of C₂H₂ and denitrification was underestimated by 13 μg N_g^?. The data suggested that concurrent inhibition of nitrification during measurement of denitrification using the C₂H₂ inhibition technique is most likely to affect the estimate of denitrification loss when NO₃^?supply is limited by the inhibition of nitrification.     

Nitrogen fertilization and nitrate leaching into groundwater on arable sandy soils

Nitrate leaching depending on N fertilization and different crop rotations was studied at two sites with sandy soils in N Germany between 1995 and 2000. The leaching of NO was calculated by using a numerical soil‐water and N model and regularly measured N_min values as input data. Also the variability of N_min values on the sandy soils was determined along transects. They reveal the high variability of the N_min values and show that it is not possible to confirm a significant N_min difference between fertilizer treatments using the normal N_min‐sampling intensity. Nitrate‐leaching calculations of five leaching periods showed that even strongly reduced N‐fertilizer applications did not result in a substantially lower NO leaching into the groundwater. Strong yield reductions of even more than 50%, however, were immediately measured. Mean NO concentrations in the groundwater recharge are >50 mg L^–1 and are mainly due to mineralization from soil organic matter. Obviously, the adjustment of the N cycle in the soil to a new equilibrium and a reduced NO ‐leaching rate as a consequence of lower N inputs need a much longer time span. Catch crops are the most efficient way to reduce the NO concentrations in the groundwater recharge of sandy soils. Their success, however, strongly depends on the site‐specific development possibilities of the catch crop. Even with all possible measures implemented, it will be almost impossible to reach NO concentrations <50 mg L^–1 in sandy soils. The only way to realize this goal on a regional scale could be by increasing areas with lower nitrate concentrations in the groundwater recharge like grassland and forests.     

Nitrogen fertilizers promote denitrification

A laboratory study was conducted to compare the effects of different N fertilizers on emission of N₂ and N₂O during denitrification of NO₃ ^– in waterlogged soil. Field-moist samples of Drummer silty clay loam soil (fine-silty, mixed, mesic Typic Haplaquoll) were incubated under aerobic conditions for 0, 2, 4, 7, 14, 21, or 42 days with or without addition of unlabelled (NH₄)₂SO₄, urea, NH₄H₂PO₄, (NH₄)₂HPO₄, NH₄NO₃ (200 or 1000 mg N kg^–1 soil), or liquid anhydrous NH₃ (1000 mg N kg^–1 soil). The incubated soil samples were then treated with ¹⁵N-labelled KNO₃ (250 mg N kg^–1 soil, 73.7 atom% ¹⁵N), and incubation was carried out under waterlogged conditions for 5 days, followed by collection of atmospheric samples for ¹⁵N analyses to determine labelled N₂ and N₂O. Compared to samples incubated without addition of unlabelled N, all of the fertilizers promoted denitrification of ¹⁵NO₃ ^–. Emission of labelled N₂ and N₂O decreased in the order: Anhydrous NH₃>urea<$>\gg<$> (NH₄)₂HPO₄>(NH₄)₂SO₄≃NH₄NO₃≃NH₄H₂PO₄. The highest emissions observed with anhydrous NH₃ or urea coincided with the presence of NO₂ ^–, and ¹⁵N analyses indicated that these emissions originated from NO₂ ^– rather than NO₃ ^–. Emissions of labelled N₂ and N₂O were significantly correlated with fertilizer effects on soil pH and water-soluble organic C. Received: 17 January 1996     

Nitrate reduction and denitrification in flooded soils

It has been well known that the utilization rate of ammonium sulfate fertilizer by lowland rice was as low as 40 percent of the applied ammonium. This low utilization rate was due to nitrogen loss from the paddy field. There are problems as to whether the loss of nitrogen from the flooded soil was caused by the nitrification of ammonium nitrogen and its subsequent denitrification, by the evaporation of ammonium, or by the leaching of ammonium-nitrogen with percolated water. Shioiri and his associatesll clarified that this loss of nitrogen resulted largely from denitrification through nitrate reduction in 1942. After the paddy soil is flooded with water, the oxygen in furrow slice is consumed by aerobic microorganisms, and then the soil becomes reductive. Conversely the oxigen is supplied to the soil through the water from the air, and from various kinds of algae and duckweeds, which produce oxygen through photosynthesis. At the early stage the reduction by oxygen consumption is superior to the oxidation by oxygen supply, the furrow slice is reductive, and is bluish gray in color due to the presence of certain ferrous compounds. After months flooding, the oxygen supply becomes superior to oxygen consumption and the uppermost layer of furrow slice becomes brown in color due to the presence of ferric compounds. This layer corresponds to an “oxidised layer” where microorganisms live aerobically. In this oxidized layer the nitrifying bacteria converts ammonium nitrogen into nitrate nitrogen which is percolated into the reduced layer, and lost through denitrification. A large amount of ammonium sulfate fertilizer is then dressed at the uppermost layer, after flooding, the loss of nitrogen through denitrification is serious.     

Availability of organic carbon for denitrification of nitrate in subsoils

Summary Previous work in our laboratory indicated that the slow rate of denitrification in Iowa subsoils is not due to a lack of denitrifying microorganisms, but to a lack of organic C that can be utilized by these microorganisms for reduction of NO ₃ ^– . This conclusion was supported by studies showing that drainage water from tile drains under agricultural research plots contained only trace amounts of organic C and had very little, if any, effect on denitrification in subsoils. Aqueous extracts of surface soils promoted denitrification when added to subsoils, and their ability to do so increased with increase in their organic C content. Amendment of surface soils with corn and soybean residues initially led to a marked increase in the amounts of organic C in aqueous extracts of these soils and in the ability of these extracts to promote denitrification in subsoils, but these effects were short-lived and could not be detected after incubation of residue-treated soils for a few days. We conclude from these observations that water-soluble organic C derived from plant residues is decomposed so rapidly in surface soils that very little of this C is leached into subsoils, and that this largely accounts for the slow rate of denitrification of nitrate in subsoils.     

Effects of nitrification inhibitors on denitrification of nitrate in soil

Summary The influence of 28 nitrification inhibitors on denitrification of nitrate in soil was studied by determining the effects of different amounts of each inhibitor on the amounts of nitrate lost and the amounts of nitrite, N₂O and N₂ produced when soil samples were incubated anaerobically after treatment with nitrate or with nitrate and mannitol. The inhibitors used included nitrapyrin (N-Serve), etridiazole (Dwell), potassium azide, 2-amino-4-chloro-6-methylpyrimidine (AM), sulfathiazole (ST), 4-amino-1,2,4-triazole(ATC),2,4-diamino-6-trichloromethyl-s-triazine (CL-1580), potassium ethylxanthate, guanylthiourea (ASU), 4-nitrobenzotrichloride, 4-mesylbenzotrichloride, sodium thiocarbonate (STC), phenylmercuric acetate (PMA), and dicyandiamide (DCD).Only one of the nitrification inhibitors studied (potassium azide) retarded denitrification when applied at the rate of 10 g g^–1 soil, and only two (potassium azide and 2,4-diamino-6-trichloromethyl-s-triazine) inhibited denitrification when applied at the rate of 50 g g^–1 soil. The other inhibitors either had no appreciable effect on denitrification, or enhanced denitrification, when applied at the rate of 10 or 50 g g^–1 soil, enhancement being most marked with 3-mercapto-1,2,4-triazole. Seven of the inhibitors (potassium azide, sulfathiazole, potassium ethylxanthate, sodium isopropylxanthate, 4-nitrobenzotrichloride, sodium thiocarbonate, and phenylmercuric acetate) retarded denitrification when applied at the rate of 50 g g^–1 soil to soil that had been amended with mannitol to promote microbial activity.Reports that nitrapyrin (N-Serve) and etridiazole (Dwell) inhibit denitrification when applied at rates as low as 0.5 g g^–1 soil could not be confirmed. No inhibition of denitrification was observed when these compounds were applied at the rate of 10 g g^–1 soil, and enhancement of denitrification was observed when they were applied at the rate of 50 or 100 g g^–1 soil.     

Influence of tillage system on denitrification in maize-cropped soils

Denitrification losses show an irregular pattern through the year, often being caused by climatic conditions and management practices. The objectives of the present work were to quantify denitrification losses and to determine the influence of tillage system on the factors that control denitrification in fertilized soils. The modal profile of the soil was an Vertic Argiudoll, clay loam texture, located in Buenos Aires province, Argentina. The treatments were: (a) fertilized, (b) incorporated fertilization and (c) without fertilization for both no tillage and conventional tillage systems. Chambers were placed in the field to measure denitrification. In this clayish soil the estimated mean values of accumulated denitrification during the crop cycle (90 days) were 0.190kgNha^–1 for conventional tillage and 0.350kgNha^–1 for no tillage. In treatments with no tillage, losses by denitrification were approximately twice those of conventional tillage. These differences were also evidenced by the number of microorganisms, which were significantly higher (P<>;5%) for no tillage on all dates, except for at flowering. The increase at flowering coincided with the period of highest rainfall and consequently the highest water contents in the soil. The highest denitrification losses, except for sowing, were measured when soil moisture content was more than 30% (v/v). Denitrification increased in conjunction with an increase in the availability of carbon that is consumed by the heterotrophic microorganisms (including the denitrifiers). Received: 30 July 1996     

Relationships between denitrification gene expression,dissimilatory nitrate reduction to ammonium and nitrous oxide and dinitrogen production in montane grassland soils

The montane grassland soils of Europe store significant amounts of nitrogen (N), and climate change might drive their volatilization due to the stimulation of gaseous nitrous oxide (N₂O) and dinitrogen (N₂) losses. Hence, a thorough, mechanistic understanding of the processes responsible for N loss and retention such as denitrification and dissimilatory nitrate reduction to ammonium (DNRA) in these soils is urgently needed. Here we aimed to explore the relationships between denitrifier gene abundance and expression with N₂ and N₂O production and the importance of DNRA versus denitrification in nitrate consumption and N₂O production for typical montane grassland soils of Southern Germany. In a laboratory incubation experiment with glucose and nitrate addition, we combined direct measurements of N₂O and N₂ production with a molecular analysis of the denitrifier communities involved in nitrite, nitric oxide (NO) and N₂O reduction and with the quantification of DNRA. The soils originated from a space-for-time climate change experiment, where intact plant-soil mesocosms were exposed for three years either to ambient conditions at a high elevation site (“HE” control treatment) or to predicted climate change conditions (warming, reduced summer precipitation and reduced winter snow cover) by translocation to lower elevation (“LE” climate change treatment).The abundance (DNA) of cnorB genes was significantly reduced in LE soils, whereas the abundance of nosZ genes did not differ between the HE and LE soils. However, the decreased abundance of cnorB genes unexpectedly resulted in slightly increased rather than decreased potential N₂O emissions. This effect could be explained by the increased levels of cnorB mRNA and, therefore, the higher physiological activity of the NO reducers in the LE soils. In contrast with the DNA levels, the dynamics of the cnorB mRNA levels followed N₂O emission patterns, whereas the nosZ expression was strongly correlated with the N₂ emission (R² = 0.83). The potential rates of DNRA were approximately one-third of the rates of denitrification, and DNRA was not a source for N₂O.We conclude that DNRA significantly competes with denitrification in these soils, thus contributing to N conservation. This work demonstrates that the molecular analysis of nosZ gene expression has great potential to contribute to solving the enigmatic problem of understanding N₂ loss from soil.     

Nitrogen fixation in paddy soils

The 4 long-term experimental plots (Umbric haplaquept) with different fertilizer treatment at Cent. Agric. Exp. Sta. in Konosu City, Saitama Prefecture, were used for the sites of investigation. The 4 plots were NF (applied with no fertilizer), IF (applied with inorganic fertilizers), GM (applied with green manure and CaCO₂), and OM (applied with manure and inorganic fertilizers). Flooded water, floating weed, upper (0-2cm) and lower (2-10cm) parts of Apg horizon and rhizosphere were collected from each plot before flooding, during flooding, and after drainage. These samples were analyzed for N₂-fixing activity by acetylene reduction method, pH, Eh, and contents of Fe²⁺, NH₄ ⁺, chlorophyll-type compounds, and water-soluble carbohydrates.

The N₂-fixing activity of all samples showed almost the same pattern of change with time: very low before flooding, rapidly increased after flooding, the maximum value at the maximum tillering stage of rice plant, declined afterwards and reached a very low value after drainage.

Rough estimation of the “N₂-fixing capacity” of each part of the paddy field revealed that the most important site of the N₂ fixation was the reduced Apg horizon, that the importance of flooded water and/or the oxidized layer in the N₂ fixation was rather low except in infertile soil, and that the role of rhizosphere in the N₂ fixation could not be neglected also in Japan.

Reduced condition and content of easily decomposable organic substances were judged to be main factors which control the N₂-fixing activity in the flooded soil on the basis of correlations between the Nt-fixing activity and several analytical data of the paddy soils.     

Nitrous oxide production and denitrification in Scottish arable soils

Nitrous oxide (N₂O) emissions and concentrations in the soil atmosphere were measured at a number of sites of differing soil type in south-east Scotland between 1985 and 1988. Concentrations followed log-normal distributions and were significantly affected by soil type, tillage treatment, and nitrate application rate. The shape of the profiles suggested significant consumption in the upper 5 cm, making calculations of emission rates using Fick's Law unsatisfactory. Emission rates measured using closed flux chambers were at least one order of magnitude smaller from heavier-textured arable soils than from lighter ones. Denitrification fluxes measured by field application of the acetylene inhibition technique were lowest in a clay loam, and highest in an alluvial sandy loam; this was attributed to a failure to achieve a satisfactory distribution of acetylene in the heavier soil. Denitrification rates in soil cores generally exceeded measured surface fluxes; incubation at decreased oxygen concentrations typical of those measured in the field produced a further significant increase. Core incubation should be used as an alternative to in situ field measurement only if the oxygen concentration in the incubation vessels is adjusted to mimic that in the field; otherwise denitrification rates may be significantly underestimated.     

Steady-state denitrification in aggregated soils: a mathematical model

A model is presented which calculates steady-state denitrification rates as a function of more readily-measured soil parameters: the soil moisture characteristic, the probability distributions of aggregate size and oxygen reduction potential, the nitrate concentration, and the moisture tension. The model does not depend on curve-fitting. It indicates that aggregates of intermediate size may be more efficient denitrifiers than very large ones. It provides a theoretical explanation for the reported observation of proportionality between denitrification rate and calculated anaerobic fraction of incubated soil cores. This proportionality extends over some five orders of magnitude and appears to be independent of moisture tension. Calculated whole-soil denitrification rates are affected principally by soil texture, structure and moisture tension, and less so by nitrate concentration. In addition to predicting denitrification rates, the model may be extended to predict the fraction of the gaseous products of denitrification emitted as nitrous oxide.     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

Temporal denitrification patterns in different horizons of two riparian soils

The dynamics of biological denitrification in riparian soil is still poorly understood. We studied the spring‐time pattern of denitrifying enzyme activity (DEA) and the rate of denitrification (DNT) in two hydromorphic riparian soils, one a mollic Gleysol and the other a terric Histosol. The average DEA ranged from 73 to 1232 ng N g^?1 hour^?1, and DNT ranged from 4 to 36 ng N g^?1 hour^?1. Both DEA and DNT diminished with increasing depth in both soil types. This decrease corresponded to a decrease in total and K₂SO₄‐extractable organic carbon and K₂SO₄‐extractable mineral nitrogen. The DEA and DNT differed in their dynamics. The former had no evident pattern in subsurface horizons but increased with temperature at the end of spring in surface and structural horizons. The DNT diminished as the soil dried in the mollic Gleysol when the water table fell. In the terric Histosol, the water table was still too high at the end of spring to affect the DNT. The results suggest that the vertical pattern of denitrification is related to that of organic carbon content. This organic carbon content determines biological activity and the supply of carbon and nitrous oxides. In biologically active horizons temperature drives the dynamics of DEA, whereas soil moisture drives the dynamics of DNT. Our results show the importance of the dynamic soil–water relationship in controlling denitrification within the riparian zone.     

地下水硝酸盐去除中反硝化微生物的研究进展

地下水硝酸盐污染已经成为一个全球问题,由于饮用高硝酸盐含量的地下水会增加高铁血红蛋白症和癌症风险,地下水硝酸盐污染受到越来越多的关注。反硝化脱氮是地下水硝酸盐脱氮的主要途径之一。本文就参与地下水硝酸盐去除的反硝化微生物种类、反硝化机理、碳源类型以及地下水污染中微生物作用的国内外研究现状进行了较全面系统的评述。在此基础上,提出了该类研究中存在的不足,包括实验室研究较多但野外研究较少,野外原位应用中对特定微生物特性方面研究缺乏,碳源利用率低和硝酸盐去除速度慢,去除过程中有效微生物的代谢途径仍不清楚等问题。针对这些问题,本文认为以后的研究应该进一步开发野外原位应用中反硝化微生物资源,并借助先进的分子方法和功能基因鉴定此类特殊微生物的种类、功能及其生态学行为,选择最佳碳源,完整深入地了解地下水硝酸盐去除中微生物的代谢过程,识别反硝化过程中氮的来源与去向,为寻找提高处理效率的方法提供理论依据,真正将理论和实践结合起来。     

Nitrogen mineralization and nitrate reduction in forests

The mineralization of soil nitrogen was studied in four forests growing on krasnozem soils. Soils from Silver Wattle (Acacia dealbata Link.) and Mountain Ash (Eucalyptus regnans F. Muell.) forests showed considerable nitrification in laboratory incubations. Messmate (Eucalyptus obliqua L'Herit) and Monterey Pine (Pinus radiata D. Don) forest soils were predominantly ammonifiers. Forests having significant soil nitrification were found to have considerable nitrate reductase activity (NRA) in root or leaf tissue or both. NRA may therefore be useful as an indication of soil nitrification in natural ecosystems. The occurrence of nitrification in Australian forests appears to be predominantly related to the amount of N present and its rate of turnover rather than to inhibitory effects.