Use of a “least square” optimization procedure to estimate enzyme characteristics and substrate affinities in the denitrification reactions in soil

doi:10.1016/0038-0717(95)00064-L

Soil Biology and Biochemistry

Volume 27, Issue 10, October 1995, Pages 1261-1270

https://doi.org/10.1016/0038-0717(95)00064-L Get rights and content

Abstract

Predicting temporal variation in the production of gaseous forms of N generated in the denitrification process over a short time scale requires a quantification of the characteristics and kinetics of the denitrification enzymes. The treatments used soil aerobically incubated for 5 days, amended with or without C₂H₂ and a solution containing 3.4 mM KNO₃ with or without 3.7 mM chloramphenicol. Samples were then anaerobically incubated for 48 h at 25°C. Gas fluxes and mineral N pools were monitored periodically from 1 to 48 h. Experiments were set up to determine the initial concentrations of the reduction enzymes involved in denitrification and their rates of de novo synthesis when anaerobiosis was induced for a soil from a permanent pasture. The dynamics of the system were explored with the DETRAN model using an iterative curve-fitting procedure based on the “least square” criterion. The NO₃⁻ reduction to CO₂ production ratio was ca. 0.6 but increased to 0.8 when glucose was added. The link between the C mineralization and denitrification in the model was adjusted accordingly: “readily” decomposable organic material had a ratio of NO₃⁻ reduced to CO₂ produced of 1.17 while soil organic matter had a ratio of 0.6. Chloramphenicol did not inhibit the reduction of nitrate so de novo synthesis of nitrate reductase did not occur. The concentration of nitrate reductase could therefore be set at 1. Best fits (lowest residual sum of squares) for the simulation of the NO₂⁻ concentrations were obtained when the initial concentration of nitrite reductase, relative to the nitrate reductase concentration, was ca. 0.3, its affinity for NO₂⁻ was between 300 and 450, and its de novo synthesis occurred between ca. 4 and 8 h after the onset of anaerobiosis. Best fits for the simulation of the N₂O and N₂ concentrations were obtained when the relative initial concentration of nitrous oxide reductase was ca. 0.1, its affinity for N₂O was between 3 and 4, and its de novo synthesis occurred between 27 and 42 h after the onset of anaerobiosis. Chloramphenicol reduced the activity of the nitrite and nitrous oxide reductases; pointing at a possible inhibitory effect of the antibiotic. It was concluded that the iterative curve-fitting procedure enabled the estimation of the characteristics of the reduction enzymes involved in the denitrification process, i.e. initial concentrations and the rates of de novo synthesis, which could be used to simulate the kinetics of the denitrification process. The curve-fitting procedure can be used to test the validity of the concepts used to simulate the denitrification process and it can reveal anomalies, e.g. the inhibitory effect of chloramphenicol on the nitrite and nitrous oxide reductase.

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Cited by (20)

Dissimilatory nitrate reduction to ammonium (DNRA), not denitrification dominates nitrate reduction in subtropical pasture soils upon rewetting
2018, Soil Biology and Biochemistry
Citation Excerpt :
Losses of N2 and N2O are tightly coupled to NO3− availability in the soil matrix, where denitrification competes with plant N uptake, dissimilatory NO3− reduction to ammonium (DNRA), and microbial N immobilisation for NO3− (Chen et al., 2015; Recous et al., 1990). Increased NO3− availability shifts the N2:N2O ratio towards N2O, as NO3− is favoured over N2O as electron acceptor during denitrification (Dendooven and Anderson, 1995). Despite their significance in determining both the magnitude and N2:N2O partitioning of denitrification, the relationship of gross NO3− production and consumption processes with denitrification remains poorly understood.
Soils under pasture are subjected to repeated wetting and drying cycles in response to rainfall, irrigation and evapotranspiration. The amplitude of these cycles is likely to increase under the predicted changes in rainfall variability, demanding a better quantitative understanding of the processes involved. The wetting of pasture soils triggers large pulses of N₂O emissions, predominantly produced via denitrification. Under anaerobic conditions in the soil matrix, denitrification and dissimilatory nitrate reduction to ammonia (DNRA) are thought to compete for available NO₃⁻. However, the relationship between gross NO₃⁻ production and consumption via denitrification (N₂ and N₂O) and DNRA remains poorly understood. This study combines the direct quantification of N₂ and N₂O with a numerical ¹⁵N tracing model to establish the relationship between denitrification and DNRA in pasture soils after wetting. Soil microcosms were fertilised with NH₄NO₃ (35 μg N g⁻¹ soil) using a triple ¹⁵N labelling approach, wetted to four different water-filled pore space (WFPS) levels and incubated over two days. The abrupt increase in soil moisture triggered a burst of N₂ and N₂O emissions, with peak fluxes of N₂ > 13.1 μg N g⁻¹ soil day⁻¹ at high soil moisture levels. At 95% and 80% WFPS, denitrification was dominated by N₂ emissions, with the N₂/(N₂+N₂O) ratio ranging from 0.5 to 0.9. At 60% and 40% WFPS, the N₂/(N₂+N₂O) ratio ranged from 0.2 to 0.3, showing N₂O as the main product of denitrification. The wetting of dry pasture soils resulted in increased DNRA rates across soils and WFPS. Both denitrification and DNRA increased exponentially with WFPS and responded to NO₃⁻ availability, demonstrating both processes as N-substrate driven. The labile C/NO₃⁻ ratio was not correlated to DNRA rates and as such did not explain NO₃⁻ partitioning between denitrification and DNRA, likely due to the high C availability in the pasture soils. Increasing labile C availability stimulated heterotrophic soil respiration, which had no effect on denitrification rates, but increased DNRA. Increased soil respiration is likely to have lowered the soil redox potential, promoting a shift of NO₃⁻ consumption from denitrification to DNRA, which implies the soil redox potential rather than the C/NO₃⁻ ratio as the key factor for NO₃⁻ partitioning between denitrification and DNRA in C rich pasture soils. Our findings suggest that the high labile C availability under perennial pastures, together with the increase of labile C upon rewetting, drives heterotrophic soil respiration, reduces the soil redox potential and ultimately shifts NO₃⁻ consumption from denitrification to DNRA. This shift limits denitrification losses and is therefore critical for limiting N loss and increasing N retention in subtropical pasture soils.
Evidence of carbon stimulated N transformations in grassland soil after slurry application
2003, Soil Biology and Biochemistry
High nitrification rates which convert ammonium (NH₄⁺) to the mobile ions NO₂⁻ and NO₃⁻ are of high ecological significance because they increase the potential for N losses via leaching and denitrification. Nitrification can be performed by chemoautotrophic or heterotrophic organisms and heterotrophic nitrifiers can oxidise either mineral (NH₄⁺) or organic N. Selective nitrification inhibitors and ¹⁵N tracer studies have been used in an attempt to separate heterotrophic and autotrophic nitrification. In a laboratory study we determined the effect of cattle slurry on the oxidation of mineral NH₄⁺-N and organic-N by labelling the NH₄⁺ or NO₃⁻ pools separately or both together with ¹⁵N. The size and enrichment of the mineral N pools were determined at intervals. To calculate gross N transformation rates a ¹⁵N tracing model was developed. This model consists of the three N-pools NH₄⁺, NO₃⁻ and organic N. Sub-models for decomposition of degradable carbon in the soil and the slurry were added to the model and linked to the N transformation rates. The model was set up in the software ModelMaker which contains non-linear optimization routines to determine model parameters. The application of cattle slurry increased the rate of nitrifcation by a factor of 20 compared with the control. The size and enrichment of the mineral N pools provided evidence that nitrification was due to the conversion of NH₄⁺ to NO₃⁻ and not the conversion of organic N to NO₃⁻. There was evidence that slurry-enhanced oxidation of NH₄⁺ to NO₃⁻ was due to a combination of autotrophic and heterotrophic transformations. Slurry application increased the mineralisation rate by approximately a factor of two compared with the control and the rate of immobilisation of NH₄⁺ by approximately a factor of three.
Comparison of denitrifying communities in organic soils: Kinetics of NO<inf>3</inf>/<sup>-</sup> and N<inf>2</inf>O reduction
2000, Soil Biology and Biochemistry
Our aim was to determine whether intrinsic differences in the denitrifier communities existed in farmed organic soils from three different sites (Germany, Sweden and Finland) on which field fluxes of N₂O had been measured continuously over 2 y. To estimate enzyme kinetic parameters (i.e. V_max and K_m) for N₂O reductase, NO⁻₃ was first removed by a combination of soil washing and anaerobic incubation. Then the samples were incubated anaerobically (as slurries) with or without added NO⁻₃, N₂O and C₂H₂. The estimated half saturation constants for N₂O reductase were similar for all soils, and very low (K_m=0.1–0.4 μM) compared to other investigations. In response to a prolonged anaerobic conditioning incubation (48 h), the K_m values increased significantly and similarly for all soils, suggesting a shift in the dominant members of the denitrifier communities. The ratio between the estimated V_max values for NO⁻₃ reduction-to-N₂O and N₂O reduction-to-N₂ was much lower for the Swedish than for the other two, indicating that the community of the Swedish soil would be more efficient in reducing NO⁻₃ all the way to N₂. This difference is congruent with differences in annual field fluxes. The results thus suggest that intrinsic differences in community composition of soils exist, with consequences for the emission of N₂O. Prolonged anaerobic incubation (48 h at 20°C) resulted in a convergence of the communities towards similar ratios between the two V_max values, suggesting that the apparent intrinsic differences may disappear in response to such severe treatment. Thus, the persistence of such patterns may depend on the drainage capacity of the soils. The results indicate that qualities of the denitrifying community must be taken into account when trying to understand and to model field fluxes of N₂O from soils. They also illustrate how global trace gas emissions can be affected by changes in the community compositions of soils.
Failure to simulate C and N mineralization in soil using biomass C-to-N ratios as measured by the fumigation extraction method?
2000, Soil Biology and Biochemistry
A C and N mineralization model (DETRAN) was used to simulate C and N dynamics in an unfertilized soil (NIL plot) and a soil annually fertilized with organic (FYM plot) or inorganic fertilizer (NPK plot). The soils amended with or without rye, i.e. 500 mg C and 30 mg N kg⁻¹ dry soil (D.S.), were incubated for 180 d at 25°C and microbial biomass C and N, CO₂ production and inorganic N (NH₄⁺, NO₂⁻, NO₃⁻) were monitored. The production of CO₂ was greater in the NIL plot than in the NPK plot but three times lower than in the FYM plot. The soil microbial biomass C and N decreased in the FYM soil but not in the NIL and NPK plots. The N mineralization was 7.5 times greater in the FYM plot than in the NPK and NIL plots but the application of rye had no significant effect on it. In the unamended soil, an efficiency for C of 40%, i.e. the amount of C incorporated into the microbial biomass while the rest or 60% evolved as CO₂, had to be used to link the C and N dynamics in the NPK and NIL plots but the efficiency was only 26% in the FYM plot. The efficiency for C added with the rye was 45% in the NIL plot and 60% in the NPK plot but it was difficult to link C and N mineralization in the FYM plot.
We conclude that the dynamics of C and N as governed by the microbial biomass C and N were different for the FYM plot compared with the NPK and NIL plots. The difference, a result of long-term organic fertiliser application, could be due to a difference in efficiency for C, a difference in the C-to-N ratio of the microbial biomass; a difference not reflected in the measured values, or/and a difference in the dynamics of N.
Carbon and nitrogen dynamics in a grassland soil with varying pH: Effect of pH on the denitrification potential and dynamics of the reduction enzymes
1998, Soil Biology and Biochemistry
Soil pH is known to affect most soil biological processes, including denitrification. There are conflicting results as to the effect of pH on the denitrification process and the ability of denitrifiers to adapt to acid soil conditions. The denitrification potential and dynamics of the reduction enzymes involved were investigated for four soils of different pH values from the Park Grass Experiment at Rothamsted Experimental Station. Soils were incubated aerobically or anaerobically after being amended with 500 mg NO₃^--N kg^-1. Anaerobic treatments were with or without C₂H₂ or with or without chloramphenicol (known to inhibit protein synthesis). All samples were incubated for 48 h, while CO₂ and N₂O production and NO₃^-, NO₂^- and NH₄⁺ concentrations were monitored. CO₂ and N₂O production decreased with decreasing pH under both aerobic and anaerobic conditions. The addition of chloramphenicol under anaerobic conditions appeared to have no effect on CO₂ production, but decreased N₂O production in soils of pH_CaCl₂ greater than or equal to 5, but not in soils of pH_CaCl₂ less than or equal to 4, where N₂O production was generally slow. Investigations of the enzyme dynamics involved in denitrification using the DETRAN model, suggested that the initial concentrations of N reductase enzymes were small in all soils and the biomass of Park Grass was not adapted to anaerobiosis. Data also suggested that soils of pH_CaCl₂ greater than or equal to 4.9, NO₂^- could accumulate to high concentrations (15-30 mg N kg^-1) during anaerobic periods of 6-36 h. In the aerobic incubations nitrification was observed in all soils even at pH_CaCl₂ less than or equal to 3.9. Antecedent water regime of the soil appeared to be important in controlling denitrification activity and the dynamics of the reduction enzymes involved in the process. There were significant effects of pH on the number of microorganisms, the time to de novo enzyme synthesis and on the kinetics of the reduction enzymes involved in denitrification.
Denitrification potential and reduction enzymes dynamics in a Norway spruce plantation
1996, Soil Biology and Biochemistry
As part of a programme aimed at understanding controls of the N cycle and possible effects of increased N depositions in a soil under a Norway spruce plantation, the denitrification potential and the kinetics of NO₃⁻, NO₂⁻, N₂O and N₂ were investigated. Intact soil cores were conditioned for 14 days at 25°C, sieved and amended with 20 mg NO₃⁻N kg⁻¹. Treatments were with or without C₂H₂ and with or without chloramphenicol (found to inhibit de novo synthesis of reduction enzymes). Samples were purged of O₂ and anaerobically incubated at 25°C for 96 h while CO₂ and N₂O production and NO₃⁻ and NO₂⁻ concentrations were monitored. Chloramphenicol did not affect the CO₂ production which decreased by nearly 50% under anaerobic conditions. Differences in NO₃⁻ concentrations between chloramphenicol and unamended soil were only detectable after 48 h while differences in NO₂⁻ concentrations were only measurable after 70 h. Nitrous oxide production in the chloramphenicol-amended and unamended soils was comparable, 1.3 and 2.6 mg N kg⁻¹, respectively, for the first 38 h while no N₂ was produced in either treatment over the first 15 h. The production of N₂ was only 0.6 mg N kg⁻¹ after 96 h in the chloramphenicol-amended soil but it was the sole gaseous product of denitrification after 70 h in the unamended soil. The kinetics of NO₃⁻, NO₂⁻ N₂O and N₂ were satisfactorily described with a model, DETRAN, assuming a competitive Michaelis-Menten kinetic and an uptake of NO₃⁻ by micro organisms that was not directly matched by a reduction of NO₃⁻. The uptake of NO₃⁻ was higher (ca. 2 mg NO₃⁻N kg⁻¹) than the release of reduction products NO₂⁻ and N₂O for the entire incubation in the chloramphenicol-amended soil but only for the first 70 h in the unamended soil. Concentrations of NO₃⁻, NO₂⁻ and N₂O reductase appeared to increase by 4-fold between 20 and 30 h after the onset of anaerobiosis when the affinity for NO₃⁻ was set at 1 and the affinity for NO₂⁻ was between 120 and 160 and for N₂O between 1.2 and 2.6. Several factors of C and N dynamics in these experiments are compared with those made in soil from a poorly drained pasture. It is concluded that the microbial community in the well-drained forest soil described here was not well pre-adapted to periodic anaerobiosis.

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References (32)

Ratios of microbial biomass carbon to total organic carbon in arable soils

Soil Biology & Biochemistry

Inhibitory effect of nitrate on reduction of N₂O to N₂ by soil micro-organisms

Soil Biology & Biochemistry

Relationships between the denitrification capacity of soils and total, water soluble and readily decomposable soil organic matter

Soil Biology & Biochemistry

Dynamics of reduction enzymes involved in the denitrification process in pasture

Soil Biology & Biochemistry

Kinetics of the denitrification process in a soil under permanent pasture

Soil Biology & Biochemistry

Denitrification in north temperate forest soils: spatial and temporal patterns at the landscape and seasonal scales

Soil Biology & Biochemistry

Carbon and nitrogen mineralization from two soils of contrasting texture and microaggregate stability: influence of sequential fumigation, drying and storage

Soil Biology & Biochemistry

Turnover of carbon and nitrogen through the microbial biomass in a sandy loam and a clay soil incubated with [¹⁴C(U)]glucose and [¹⁵N](NH₄)₂SO₄ under different moisture regimes

Soil Biology & Biochemistry

Hydrological consequences of artificial drainage of grassland

Hydrological Processes

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus

Applied and Environmental Microbiology

Decomposition of lysine and leucine in soil aggregates: adsorption and compartmentalisation

Soil Biology & Biochemistry

Kinetic explanations for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification

Applied and Environmental Microbiology

Physiology and biochemistry of denitrification

Kinetic formulation of the denitrification process in soil

Canadian Journal of Soil Science

Spatial variation in denitrification dependency of activity centres on the soil environment

Temporal patterns of soil denitrification their stability and causes

Cited by (20)

Dissimilatory nitrate reduction to ammonium (DNRA), not denitrification dominates nitrate reduction in subtropical pasture soils upon rewetting

Evidence of carbon stimulated N transformations in grassland soil after slurry application

Comparison of denitrifying communities in organic soils: Kinetics of NO<inf>3</inf>/<sup>-</sup> and N<inf>2</inf>O reduction

Failure to simulate C and N mineralization in soil using biomass C-to-N ratios as measured by the fumigation extraction method?

Carbon and nitrogen dynamics in a grassland soil with varying pH: Effect of pH on the denitrification potential and dynamics of the reduction enzymes

Denitrification potential and reduction enzymes dynamics in a Norway spruce plantation