Oxygen inhibition of acetylene reduction (nitrogen fixation) in soil: Effect of glucose and oxygen concentrations

Sandy loam soil was amended with different concentrations of glucose and was incubated at different pO₂ levels. Under many conditions of incubation time and treatment, N₂ ase activity as determined by 1-h aerobic C₂H₂ reduction assay (flushed with Ar:O₂, 4:1 before assay) was significantly less than that determined by means of ambient assay (carried out at the pO₂ of incubation without flushing with Ar:O₂, 4:1 before assay). The difference between the N₂ase activity in aerobic assay and that in ambient assay increased with decreasing glucose and O₂ concentrations imposed during incubation. The inhition in aerobic assays was analogous to O₂-induced shut-off of N₂ase and amounted to 75 per cent inhibition after incubation at 0.06 atm pO₂ of samples amended with 0.75% glucose (w/w). Similar O₂ inhibition was observed after amendment with mannitol and with lactate. Times of incubation were chosen such that development of anaerobic N₂ase activity was either absent or too low to account for the observed effects of O₂ during assay. It was shown that 0.05 atm pC₂H₂ was adequate for routine 1-h assays of the soil system employed. Individual soil samples could be subjected to repeated 1-h assays (with removal of C₂H₂ and C₂H₄ by evacuation after each assay) thus avoiding side-effects of long exposure to C₂H₂.     

The effect of oxygen,pH and organic carbon on soil-layer specific denitrifying capacity in acid coniferous forest

Emissions of N₂O from acid coniferous forest soils are found to be low and considered to be due to nitrification rather than denitrification. Recently we have demonstrated soil-layer specific denitrification in a Scots pine forest in the Netherlands. N₂O production, in the presence of high concentrations of acetylene, was detected in the intact needle fraction but was absent in the fragmentation layer of this forest soil. To identify the factors regulating denitrification activity, in the present study the effects of oxygen, pH and organic carbon were investigated in the needle and fragmentation fraction of acid coniferous forest soils. Under natural circumstances denitrification in the Scots pine needles was higher than in Douglas fir needles and absent in fragmentation material. Under anaerobic conditions comparable N₂O production in the two soil types was found in needle suspensions of both forest types, indicating that differences in anaerobic microsites were responsible for different N₂O production under aerobic circumstances. Denitrifying capacity was absent in the fragmentation layer; under anaerobic circumstances little N₂O was produced. Neither an addition of available carbon (glucose and succinate) nor an increase in pH revealed a denitrifying capacity comparable to that observed in needles. The increase in pH, under anaerobic circumstances, was most effective on N₂O production in the fragmentation material. The denitrifying capacity in the fragmentation layer remained low during short-term incubation under optimal conditions. This indicates the presence of a low denitrifying population, most likely due to aerobic conditions, low pH and low available organic carbon. Although the significance of N₂O production under natural conditions remains speculative, this study seeks to clarify soil-layer specific denitrifying activity in acid coniferous forest soils.     

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon (C)-rich organic matter plays a vital role in sustaining soil fertility. However, it remains poorly understood whether dinitrogen (N₂) fixation occurs in support of soil heterotrophic respiration. In this study, ¹⁵N₂-tracing indicated that strong N₂ fixation occurred during heterotrophic respiration of carbon-rich glucose. Soil organic ¹⁵N increased from 0.37 atom% to 2.50 atom% under aerobic conditions and to 4.23 atom% under anaerobic conditions, while the concomitant CO₂ flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions. Soil N₂ fixation was completely absent in soils replete with inorganic N, although soil N bioavailability did not alter soil respiration. High-throughput sequencing of the 16S rRNA gene further indicated that: i) under aerobic conditions, only 15.2% of soil microbiome responded positively to glucose addition, and these responses were significantly associated with soil respiration and N₂ fixation and ii) under anaerobic conditions, the percentage of responses was even lower at 5.70%. Intriguingly, more than 95% of these responses were originally rare with < 0.5% relative abundance in background soils, including typical N₂-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N₂-fixing heterotrophs such as Sporosarcina, Agromyces, and Sedimentibacter. These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers. Our study indicates that N₂ fixation could be closely associated with microbial turnover of soil organic C when available in excess.     

Short-term partitioning of <Superscript>14</Superscript>C-[U]-glucose in the soil microbial pool under varied aeration status

The effect of soil aeration status on carbon partitioning of a labelled organic substrate (¹⁴C-[U]-glucose) into CO₂, microbial biomass, and extra-cellular metabolites is described. The soil was incubated in a continuous flow incubation apparatus under four different aeration conditions: (1) permanently aerobic, (2) permanently anaerobic, (3) shifted from anaerobic to aerobic, and (4) shifted from aerobic to anaerobic. The soil was pre-incubated for 10 days either under aerobic or under anaerobic conditions. Afterwards, glucose was added (315 g C g^–1) and the soils were incubated for 72 h according to four treatments: aerobic or anaerobic conditions maintained, aerobic conditions shifted to anaerobic conditions and anaerobic conditions shifted to aerobic conditions. Carbon partitioning was measured 0, 8, 16, 24, 48 and 72 h after the glucose addition. In permanently aerobic conditions, the largest part of the consumed glucose was built into microbial biomass (72%), much less was mineralised to CO₂ (27%), and only a negligible portion was transformed to soluble extra-cellular metabolites. Microbial metabolism was strongly inhibited when aeration conditions were changed from aerobic to anaerobic, with only about 35% of the added glucose consumed during the incubation. The consumed glucose was transformed proportionally to microbial biomass and CO₂. In permanently anaerobic conditions, 42% of the consumed glucose was transformed into microbial biomass, 30% to CO₂, and 28% to extra-cellular metabolites. After a shift of anaerobic to aerobic conditions, microbial metabolism was not suppressed and the consumed glucose was transformed mainly to microbial biomass (75%) and CO₂ (23%). Concomitant mineralisation of soil organic carbon was always lower in anaerobic than in aerobic conditions.     

Kinetics of nitrogen fixation in a glucose-amended,anaerobically incubated soil

Nitrogen fixation and acetylene reduction activities were studied in a sandy loam soil amended with glucose (2% w/w) at field moisture content and incubated anaerobically. Optimum temperature for C₂H₂ reduction was about 37°C and the maximum was 45°C. Q₁₀ values were 1.6–3.7 in the range 10–35°C. Calculated activation energies were lower than those reported for Clostridium whole cells. Apparent K_m (C₂H₂) averaged 0.006 atm pC₂H₂ and the apparent K_m (N₂) was 0.095 atm pN₂. Low concentrations of C₂H₂ competed strongly with N₂ for the soil N₂ase (apparent K[ini] was 0.0003 atm pC₂H₂ with 0.8 atm pN₂). A relatively high concentration of ethylene (0.22 atm pC₂H₄) caused 30–40 per cent inhibition of N₂ase activity (measured as ¹⁵N₂ fixation) but the lower concentrations likely to be encountered in C₂H₂ assays had no significant effect. Conversion factors (C₂H₄/N₂ molar ratios) determined under various conditions ranged from 0.75 to 3.6. A value of 2.6 was obtained using the most favourable short-term C₂H₂ assays.     

Kinetics and electron transport of soil hydrogenases catalyzing the oxidation of atmospheric hydrogen

The soil hydrogenases of chernozem and eolian sand were different with respect to their kinetic properties. Increase of soil moisture above optimum moisture content or prior incubation of the soils under very high H₂-mixing ratios (i.e. 1%) resulted in a decrease of V_max or in an increase of the K_m of the H₂ oxidation reaction. Under anaerobic conditions, the K_m for H₂ was higher and V_max was lower than under aerobic conditions. The anaerobic H₂-oxidation activity of both soils was stimulated by the addition of artificial electron acceptors with redox potentials of at least 80 mV. Ferricyanide as the most efficient stimulator did not function as a final electron acceptor for anaerobic H₂-oxidation, but acted as a catalyst by bypassing a rate-limiting electron transport step. In eolian sand, the aerobic as well as the anaerobic activity for atmospheric H₂ oxidation decreased upon exposure to very high H₂-mixing ratios (i.e. 1%). A similar effect was observed after incubation with ferricyanide which enabled the inflow of excess electrons from soil reductants or added NADH into the electron transport system of the soil hydrogenase with anaerobic activity. The activity for atmospheric H₂ oxidation was regenerated during incubation in H₂-free atmospheres, especially in the presence of oxygen. Inhibition and regeneration were probably due to alterations in components of the soil hydrogenases caused by the extent of a maximal electron flow through the electron transport system of the soil hydrogenases. Two classes of hydrogenase activities were discerned in eolian sand: one predominantly active under aerobic and the other under anaerobic conditions.     

Effects of transient anaerobic conditions in the presence of acetylene on subsequent aerobic respiration and N2O emission by soil aggregates

Our objective was to assess the effect of anaerobic conditioning in the presence of acetylene on subsequent aerobic respiration and N₂O emission at the scale of soil aggregates. Nitrous oxide production was measured in intact soil aggregates Δ (compacted aggregates without visible porosity) and Γ (aggregates with visible porosity) incubated under oxic conditions, with or without anaerobic conditioning for 6 d. N₂O emissions were much higher in aggregates that had been submitted to anaerobic conditioning than in aggregates that did not experience this conditioning, although very little NO₃⁻ remained in soil after the anaerobic period. ¹⁵N isotope tracing technique was used to check whether N₂O came from nitrification or denitrification. The results showed that denitrification was the major process responsible for N₂O emissions. The aerobic CO₂ production rate was also measured in intact soil aggregates. It was greater in aggregates submitted to anaerobic conditioning than in those that were not, suggesting that the anaerobic conditioning lead to an accumulation of small compounds including fatty acids that are readily available for microbial decomposition in aerobic conditions. This process increases the aerobic CO₂ production and favours the N₂O emissions through denitrification.     

Nitrous oxide emission as affected by alternate anaerobic and aerobic conditions from soil suspensions enriched with ammonium sulfate

The effect of several anaerobic and aerobic cycles of varying duration on N₂O emission and labelled N loss was investigated in (¹⁵NH₄)₂SO₄ amended soil suspensions. No N₂O was evolved from the continuously-anaerobic treatment. The continuously-aerobic treatment produced approximately 0.8 μg N₂O-N g^?1 dry soil in 56 days. Alternate anaerobic-aerobic cycles increased the net N₂O evolution with 7.2 μg N₂O-N g^?1 dry soil produced in 56 days from the 7-day anaerobic, 7-day aerobic treatment. The net N₂O evolution increased further when the duration of the anaerobic and aerobic periods was increased from 7-7 days to 14-14 days (15.7μg N₂O-N g^?1 dry soil in 56 days), although the total ¹⁵N loss from the system was approximately the same for the two treatments. The results of this study show that N₂O evolution from soils is likely to be greater under fluctuating moisture conditions than under either continuously well-aerated conditions, or continuously excess-moisture conditions.     

Enhancement of aerobic nitrogenase activity (acetylene reduction assay) by phenol in soils and the rhizosphere of cereals

By addition of phenol at concentrations between 0.1 and 10 mmol·l^?1, nitrogenase activity (acetylene reduction assay) is enhanced by a factor of 5 in the rhizosphere of Pennisetum glaucum (pearl millet) incubated under 20% O₂. No increase is found under microaerobic conditions. This enhancement effect is also noticed in a soil amended with a sucrose concentration of 20 mmol·l^?1. Under those conditions, however, an enhancement is found under aerobic as well as under microaerobic conditions and a further increase of the phenol added reduces the activity to almost zero. A 4-fold increase of N₂-fixation by phenol addition under aerobic conditions was determined with homogenous sediments from a fresh water lake while anaerobic N₂-fixation was already slightly reduced by the same concentration added. Excised roots of Sorghum nutans CSH 5 failed to show any phenol enhancement of nitrogenase activity. After a preincubation of 6h, inhibition of nitrogenase activity under air by addition of 1 mmol·l^?1 was much more pronounced than under microaerobic conditions.     

Rhodanese activity of flooded and nonflooded soils

Rhodanese activity (RA) was studied in 4 soils, incubated under flooded and nonflooded (60% water-holding capacity) conditions. RA in 3 soils including an acid sulphate soil pokkali increased 2.5–6.0-fold (over respective nonflooded soils), while activity of the enzyme decreased markedly in flooded alluvial soil. Similarly, anaerobic incubation of nonflooded soils under N₂ decreased RA in an alluvial soil, but increased it in pokkali soil. RA was negligible in soils, that had been reduced by flooding for 30 days and then sterilized by autoclaving. Rice rhizosphere soil exhibited significantly higher RA than the nonrhizosphere soil samples under flooded or nonflooded conditions. RA in aerobic soils was related to the microbial oxidation of S° to SO^2?₄. But, no relationship could be established between RA and S-oxidation in flooded soils and in rhizosphere soil suspensions of flooded rice plants.     

Effect of cellulose and straw incorporation in soil on total denitrification and nitrogen immobilization at initially aerobic and permanent anaerobic conditions

Summary Laboratory experiments were used to examine the influence of cellulose and straw on denitrification and N immobilization in a sandy loam soil. The soil was mixed with 300 g nitrate-N/g and incubated in a special vessel under conditions that changed from aerobic to anaerobic or in the permanent absence of O₂. Gases (O₂, CO₂, N₂, N₂O, NO and CH₄) were analysed by gas chromatography at regular intervals and the soil was examined for nitrate, nitrite, ammonium and cellulose. Compared with controls, the application of straw and cellulose (0.5% and 1.0%, respectively) enhanced nitrate immobilization and decreased denitrification, under both anaerobic and originally aerobic (PO₂ = 20 vol%) conditions. However, a comparison of results from the aerobic and the anaerobic incubations shows that an increase in denitrification and N immobilization was apparent at an original O₂ concentration of 20 vol%. N₂ was the major product of denitrification in all experiments. Free methane was apparent as soon as nitrate was respired. The stimulating effect of O₂ on total denitrification in the presence of relatively high amounts of easily decomposable cellulose is ascribed to a higher turnover and an intensified mineralization rate (CO₂ production), which increased the total demand for electron acceptors.     

Nitrogenase activity in relation to season,carbohydrates and organic acids in a temperate zone root association

Nitrogenase activity associated with grasses is commonly reported to be maximal during reproductive growth. For Spartina alterniflora, a perennial grass growing in salt marshes, seasonal changes in potential (excised root) N₂ase activity followed, by about 2 weeks, seasonal changes in ethanol soluble carbohydrate concentrations in roots and rhizomes. Both varied by about 3-fold during the year, declined rapidly with the onset of vegetative growth, and increased to previous levels during reproductive growth. N₂-ase activity measured on intact systems in situ was also maximal during reproductive growth, but the absolute rates were much lower than the potential N₂ase activities.     

Anaerobic Eury- and Crenarchaeota inhabit ectomycorrhizas of boreal forest Scots pine

Members of the euryarchaeotal genera Methanolobus and Halobacterium as well as group 1.1c Crenarchaeota were enriched from ectomycorrhizal samples and cultured under anaerobic conditions. 16S rRNA gene sequences of Methanolobus were obtained in a H₂ + CO₂ atmosphere and autofluorescent putatively methanogenic microbial cells were detected by epifluorescence microscopy of the anaerobic methane-producing enrichment cultures. Halobacterium and group 1.1c Crenarchaeota grew anaerobically when either H₂ or CH₄ was added to the atmosphere. Group 1.1c Crenarchaeota were also enriched under aerobic conditions on mineral media, but only when methane or methanol was added as carbon sources. The 16S rRNA gene sequences of 1.1c Crenarchaeota grown under both anaerobic and aerobic conditions were highly similar. Our study demonstrates the growth of group 1.1c Crenarchaeota and Halobacteria derived from non-extreme soil environment in non-saline enrichments under anaerobic conditions. The results suggest that 1.1c Crenarchaeota may play a role in the cycling of C-1 substrates in the boreal forest soil ecosystem.     

Fate of nitrogen and carbon in the vadose zone: in situ and laboratory measurements of seasonal variations in aerobic respiratory and denitrifying activities

In situ and laboratory measurements of aerobic respiratory and denitrifying activities were studied in the vadose zone (almost 2.5 m thick) of a fluvic hypercalcaric cambisol characterized by transitory anaerobic conditions. A field experiment was conducted in a bare soil, over a 7-month period starting just after maize harvest and incorporation of maize crop residues. Weather variables (air and soil temperature, rainfall), soil water content, soil solutes (NO₃⁻ and dissolved organic carbon) and soil gases (CO₂ and N₂O), were recorded throughout the experiment. Four soil layers were defined. Bacterial counts were performed in each layer using the most probable number (MPN) method. Aerobic respiratory and denitrifying activities were estimated from laboratory measurements. In situ microbial activity, as revealed by CO₂ and N₂O measurements in the soil atmosphere, was strongly influenced by weather. Laboratory measurements showed that potential aerobic respiratory activity (ARA) occurred throughout the soil profile, whereas semi-potential denitrifying activities SPDA (i.e. measured under organic-C limiting condition) occurred mainly in the top 30 cm soil layer. In the soil profile, the CO₂ concentration gradient was stronger than the N₂O concentration gradient. Seasonal variations in microbial activities increased with depth, whereas DOC concentrations, and variations in those concentrations, decreased with depth, suggesting that DOC quality investigations are necessary in the deep vadose zone to understand microbial activities seasonal variations. Laboratory measurements of potential activities agreed well with in situ microbial activity in natural environmental conditions. NO₃⁻ was a stronger limiting factor for SPDA than was denitrifier density in the soil profile.     

Effect of the herbicide glyphosate on respiration and hydrogen consumption in soil

The effect of glyphosate on soil respiration and Hz oxidation in an agricultural soil was investigated. The effects of the pure herbicide and commercial formulation, Roundup® (Monsanto Company), were compared in soil under both aerobic and anaerobic conditions. Both formulations stimulated O₂ uptake as well as aerobic and anaerobic CO₂ evolution. Roundup caused more stimulation than glyphosate under aerobic incubation conditions; the formulations had an equal effect on anaerobic CO₂ evolution. Hydrogen oxidation was inhibited by both formulations in aerobic and anaerobic soil. Aerobic H₂ oxidation was inhibited to the same extent by both formulations; Roundup had a stronger inhibitory effect on anaerobic H₂ oxidation than did glyphosate.     

Microbiological characterization and nitrate reduction in subsurface soils

Summary Two borings (20 m depth) were performed in a sandy-clayey soil over a limestone bed and in a sandy soil with lumps of clay in some depths. Bacteria were found in the deeper soil layers of both profiles. The methods used to detect bacteria were those normally used for topsoil layers, plate counts of bacteria, ATP content, and direct microscopy. Measurements of CO₂ evolution showed that the potential for bacterial activity was present in all depths of the two profiles. However, the activity was strongly dependent on the presence of easily available organic C. An indication of the denitrification potential was obtained by measuring the N₂O evolution. Under aerobic incubation without the addition of glucose, N₂O was detected only in the topsoil. When glucose was added to the soil samples, N₂O was found at a low level in the deeper soil layers. Under anaerobic incubation, N₂O was detected in all deeper layers, and increased markedly when glucose was added to the soil samples.     

Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: Results from N tracer experiments

Recent research has proven soil nitrite to be a key element in understanding N-gas production (NO, N₂O, N₂) in soils. NO is widely accepted to be an obligatory intermediate of N₂O formation in the denitrification pathway. However, studies with native soils could not confirm NO as a N₂O precursor, and field experiments mainly revealed ammonium nitrification as the source of NO. The hypothesis was constructed, that the limited diffusion of NO in soil is the reason for this contradiction. To test this diffusion limitation hypothesis and to verify nitrite and NO as free intermediates in native soils we conducted through-flow (He/O₂ atmosphere) ¹⁵N tracer experiments using black earth soil in an experimental set up free of diffusion limitation. All of the three relevant inorganic N soil pools (ammonium, nitrite, nitrate) were ¹⁵N labelled in separate incubation experiments lasting 81 h based on the kinetic isotope method. During the experiments the partial pressure of O₂ was decreased in four steps from 20% to about 0%. The net NO emission increased up to 3.7 μg N kg⁻¹ h⁻¹ with decreasing O₂ partial pressure. Due to the special experimental set up with little to no obstructions of gas diffusion, only very low N₂O emission could be observed. As expected the content of the substrates ammonium, nitrate and nitrite remained almost constant over the incubation time. The ¹⁵N abundance of nitrite revealed high turnover rates. The contribution of nitrification of ammonium to the total nitrite production was approx. 88% under strong aerobic soil conditions but quickly decreased to zero with declining O₂ partial pressure. It is remarkable that already under the high partial pressure of 20% O₂ 12 % of nitrite is generated by nitrate denitrification, and under strict anaerobic conditions it increases to 100%. Nitrite is present in two separate endogenous pools at least, each one fed by the nitrification of ammonium or the denitrification of nitrate. The experiments clearly revealed that nitrite is almost 100% the direct precursor of NO formation under anaerobic as well as aerobic conditions. Emitted N₂O only originated to about 100% from NO under strict anaerobic conditions (0-0.2% O₂), providing evidence that NO is a free intermediate of N₂O formation by denitrification. To the best of our knowledge this is the first time that NO has been detected in a native soil as a free intermediate product of N₂O formation at denitrification. These results clearly verify the “diffusion limitation” hypothesis.     

Carbon monoxide as an inhibitor of N2ase activity(C2H2) in control measurements of endogenous formation of ethylene by forest soils

Two problems with the recently suggested method to measure endogenous formation of C₂H₄ in an atmosphere enriched with C₂H₂ and CO in studies of N₂ase activity (C₂H₂) in forest soils were analysed, namely the effect of consumption of CO during incubation and the effect of water-saturated conditions.After an initial addition of 100 ml C₂H₂ and 20 ml CO 1^?1 to soil incubation vessels, CO was gradually consumed and followed by a recovery of N₂ase activity when the concentration of CO was lower than about 10 ml 1^?1. The shortest period within which this concentration was achieved was 1 day when incubating fresh soil cores at 15°C, and it was concluded that longer incubations should be avoided.The inhibition of N₂ase activity by CO was strongly suppressed when all soil pores were filled with water. Dissolved inorganic N (0.1% of dry mass soil) was much more efficient in inhibiting N₂ase activity under such conditions.     

Increase in nitrous oxide production in soil induced by ammonium and organic carbon

We observed that soil cores collected in the field containing relatively high NH _inf4 ^sup+ and C substrate levels produced relatively large quantities of N₂O. A series of laboratory experiments confirmed that the addition of NH _inf4 ^sup+ and glucose to soil increase N₂O production under aerobic conditions. Denitrifying enzyme activity was also increased by the addition of NH _inf4 ^sup+ and glucose. Furthermore, NH _inf4 ^sup+ and glocose additions increased the production of N₂O in the presence of C₂H₂. Therefore, we concluded that denitrification was the most likely source of N₂O production. Denitrification was not, however, directly affected by NH _inf4 ^sup+ in anaerobic soil slurries, although the use of C substrate increased. In the presence of a high substrate C concentration, N₂O production by denitrifiers may be affected by NO _inf3 ^sup- supplied from NH _inf4 ^sup+ through nitrification. Alternatively, N₂O may be produced during mixotrophic and heterotrophic growth of nitrifiers. The results indicated that the NH _inf4 ^sup+ concentration, in addition to NO _inf3 ^sup- , C substrate, and O₂ concentrations, is important for predicting N₂O production and denitrification under field conditions.     

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

The relationships between the denitrification capacities of 17 surface soils and the amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon in these soils were investigated. The soils used differed markedly in pH, texture, and organic-matter content. Denitrification capacity was assessed by determining the N evolved as N₂ and N₂O on anaerobic incubation of nitrate-treated soil at 20°C for 7 days, and mineralizable carbon was assessed by determining the C evolved as CO₂ on aerobic incubation of soil at 20°C for 7 days. The denitrification capacities of the soils studied were significantly correlated (r = 0·7771) with total organic carbon and very highly correlated (r = 0·9971) with water-soluble organic carbon or mineralizable carbon. The amount of nitrate N lost on anaerobic incubation of nitrate-treated soils for 7 days was very closely related (r = 0·99971) to the amount of N evolved as N₂ and N₂O.The work reported indicates that denitrification in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter and that analysis of soils for mineralizable carbon or water-soluble organic carbon provides a good index of their capacity for denitrification of nitrate.