Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Use of NaCN to increase the growth of N2-fixing bacteria in a model spermosphere mimicked by sucrose and amino acids leached by seeds

In order to study the establishment of a spermosphere, the C₂H₂ reduction activity of N₂-fixing bacteria isolated from river sand was examined in a simulated spermosphere in the river sand which contained sucrose, an amino acid mixture, and CN^- released from plant seeds. The sand incubated with 10^-10 to 10^-9 mol CN^- 30 g sand^-1 exhibited higher C₂H₂ reduction activity than that without CN^-. The change in the most probable number of N₂ fixers with increasing quantities of CN^- roughly corresponded to that in C₂H₂ reduction activity. However, the most probable number of non-N₂-fixing bacteria decreased except for CN^--tolerant ones. Both C₂H₂ reduction activity and proliferation of the N₂ fixers isolated on a modified Burk's medium were almost similar to those in the bacteria in the sand. In contrast, the proliferation of some nonfixers decreased with an increasing CN^- concentration. C₂H₂ reduction activity of N₂ fixers cultured in combination with non-fixers exhibited a clear peak at 10^-7 M CN^- as for C₂H₂ reduction activity in the sand. We therefore speculate that cyanide evolved from seeds during a pregermination period may suppress the growth of general bacteria, but may promote the proliferation of N₂ fixers, thus contributing to the establishment of a spermosphere.     

Effect of an increasing carbon: nitrate-N ratio on the reliability of acetylene in blocking the N2O-reductase activity of denitrifying bacteria in soil

Summary In model experiments with a silty loam soil the effect of different C : NO _inf3 ^sup- -N ratios on the reliability of C₂H₂ (1% v/v) in blocking N₂O-reductase activity was examined. The soil was carefully mixed with different amounts of powdered lime leaves (Tilia vulgaris) to obtain organic C contents of about 1.8, 2.3, and 2.8%, and of NO _inf3 ^sup- solution to give C : NO _inf3 ^sup- -N ratios of 84, 107, 130, 156, 200, and 243. The soil samples were incubated in specially modified anaerobic jars (22 days, 25°C, 80% water-holding capacity, He atmosphere) and the atmosphere was analysed for N₂, N₂O, CO₂, and C₂H₂ by gas chromatography at regular intervals. Destruction jars were used to analyse soil NO _inf3 ^sup- , NH ₄ ⁺ and C. The results clearly showed that N₂O-reductase activity was completely blocked by 1% (v/v) C₂H₂ only as long as NO _inf3 ^sup- was present. In the presence of C₂H₂, NO _inf3 ^sup- was apparently entirely converted into N₂O. The C₂H₂ blockage of N₂O-reductase activity ceased earlier in soils with a wide C : NO _inf3 ^sup- -N ratio (156, 200, and 243) than in those with closer C : NO _inf3 ^sup- -N ratios (84, 107, and 130). As soon as NO _inf3 ^sup- was exhausted, N₂O was reduced to N₂ in spite of C₂H₂. The wider the C : NO _inf3 ^sup- -N ratio, the earlier the production of N₂ and the less the reliability of the C₂H₂ blockage. In the untreated control complete inhibition of N₂O-reductase activity by C₂H₂ lasted for 7–12 days. In the field, estimates of total denitrification losses by the C₂H₂ inhibition technique should be considered reliable only as long as NO _inf3 ^sup- is present. Consequently, NO _inf3 ^sup- monitoring in the field is essential, particularly in soils supplied with easily decomposable organic matter.     

Nitrogen fixation by biological soil crusts and heterotrophic bacteria in an intact Mojave Desert ecosystem with elevated CO2 and added soil carbon

Fixation of N by biological soil crusts and free-living heterotrophic soil microbes provides a significant proportion of ecosystem N in arid lands. To gain a better understanding of how elevated CO₂ may affect N₂-fixation in aridland ecosystems, we measured C₂H₂ reduction as a proxy for nitrogenase activity in biological soil crusts for 2 yr, and in soils either with or without dextrose-C additions for 1 yr, in an intact Mojave Desert ecosystem exposed to elevated CO₂. We also measured crust and soil δ¹⁵N and total N to assess changes in N sources, and δ¹³C of crusts to determine a functional shift in crust species, with elevated CO₂. The mean rate of C₂H₂ reduction by biological soil crusts was 76.9±5.6 μmol C₂H₄ m⁻² h⁻¹. There was no significant CO₂ effect, but crusts from plant interspaces showed high variability in nitrogenase activity with elevated CO₂. Additions of dextrose-C had a positive effect on rates of C₂H₂ reduction in soil. There was no elevated CO₂ effect on soil nitrogenase activity. Plant cover affected soil response to C addition, with the largest response in plant interspaces. The mean rate of C₂H₂ reduction in soils either with or without C additions were 8.5±3.6 μmol C₂H₄ m⁻² h⁻¹ and 4.8±2.1 μmol m⁻² h⁻¹, respectively. Crust and soil δ¹⁵N and δ¹³C values were not affected by CO₂ treatment, but did show an effect of cover type. Crust and soil samples in plant interspaces had the lowest values for both measurements. Analysis of soil and crust [N] and δ¹⁵N data with the Rayleigh distillation model suggests that any plant community changes with elevated CO₂ and concomitant changes in litter composition likely will overwhelm any physiological changes in N₂-fixation.     

Bacterial composition and N2-fixation of some Egyptian soils cultivated with wheat (Triticum aestivum L.)

The composition of the microflora, N₂-fixing bacteria particularly, in different soils cultivated with wheat in Egypt was investigated in some samples collected from the fields after applying the agricultural practices recommended for wheat cultivation and just before sowing. The influence of carbon sources, mineral nitrogen and water regimes on potential dinitrogen fixation (acetylene reduction assay) in soils was investigated. The bacterial population densities including-N₂-fixing organisms were related to a number of environmental factors such as organic matter content. Among diazotrophs, Azotobacter spp. and Azospirillum spp. were encountered in higher densities in comparison with clostridia. Unamended soils showed a lower acetylene-reducing activity (0.5–61.5 nmoles C₂H₄ g^?1 h^?1). Addition of glucose (1% w/w) greatly enhanced such activity being the highest (86.9–2846.5 nmoles C₂H₄ g^?1 h^?1) in the clay soil with the highest organic carbon content (1.42%). Glucose amendment had no significant influence on acetylene reduction in the saline soil. N₂-fixation in barley straw-amended (1%) soils was not much higher than in unamended soils. Concentrations of up to 70 ppm ammonium-nitrogen depressed N₂-fixation in soils that received barley straw. Acetylene reduction in submerged soil increased after addition of cellulose. Non-flooded conditions favoured N₂-fixation in the fertile clay soil amended with sucrose.     

Gaseous nitrogen losses from fertilized soil during nitrification and denitrification using the acetylene blockage technique

Summary A sandy soil amended with different forms and amounts of fertilizer nitrogen (urea, ammonium sulphate and potassium nitrate) was investigated in model experiments for N₂O emission, which may be evolved during both oxidation of ammonia to nitrate and anaerobic respiration of nitrate. Since C₂H₂ inhibits both nitrification and the reduction of N₂O to N₂ during denitrification, the amount of N₂O evolved in the presence and absence of C₂H₂ represents the nitrogen released through nitrification and denitrification.Results show that amounts of N₂O-N lost from soils incubated anaerobically with 0.1% C₂H₂ and treated with potassium nitrate (23.1 µg N-NO ₃ ^– /g dry soil) exceeded those from soils incubated in the presence of 20% oxygen and treated with even larger amounts of nitrogen as urea and ammonium sulphate. This indicates that nitrogen losses by denitrification may potentially be higher than those occurring through nitrification.     

Effects of soil type and nitrate concentration on denitrification products (N2O and N2) under flooded conditions in laboratory microcosms

Abstract

Denitrification products nitrous oxide ((N₂O) and nitrogen (N₂)) were measured in three flooded soils (paddy soil from Vietnam, PV; mangrove soil from Vietnam, MV; paddy soil from Japan, PJ) with different nitrate (NO₃^–) concentrations. Closed incubation experiments were conducted in 100-mL bottles for 7 d at 25°C. Each bottle contained 2 g of air-dried soil and 25 mL solution with NO₃^– (concentration 0, 5 or 10 mg N L^?1) with or without acetylene (C₂H₂). The N₂O + N₂ emissions were estimated by the C₂H₂ inhibition method. Results showed that N₂O + N₂ emissions for 7 d were positively correlated with those of NO₃^– removal from solution with C₂H₂ (R² = 0.9872), indicating that most removed NO₃^– was transformed to N₂O and N₂ by denitrification. In PJ soil, N₂O and N₂ emissions were increased significantly (P < 0.05) by the addition of greater NO₃^– concentrations. However, N₂O and N₂ emissions from PV and MV soils were increased by the addition of 0 to 5 mg N L^?1, but not by 5 to 10 mg N L^?1. At 10 mg N L^?1, N₂ emissions for 7 d were greater in PJ soil (pH 7.0) than in PV (pH 5.8) or MV (pH 4.3) soils, while N₂O emissions were higher in PV and MV soils than in PJ soil. In MV soil, N₂O was the main product throughout the experiment. In conclusion, NO₃^– concentration and soil pH affected N₂O and N₂ emissions from three flooded soils.     

Compared effects of long-term pig slurry applications and mineral fertilization on soil denitrification and its end products (N2O, N2)

The long-term (9 years) effect of pig slurry applications vs mineral fertilization on denitrifying activity, N₂O production and soil organic carbon (C) (extractable C, microbial biomass C and total organic C) was compared at three soil depths of adjacent plots. The denitrifying activities were measured on undisturbed soil cores and on sieved soil samples with acetylene method to estimate denitrification rates under field or potential conditions. Pig slurry applications had a moderate impact on the C pools. Total organic C was increased by +6.5% and microbial biomass C by ≥25%. The potential denitrifying activity on soil suspension was stimulated (×1.8, P<0.05) 12 days after the last slurry application. This stimulation was still apparent, but not significant, 10 months later and, according to both methods of denitrifying activity measurement (r ²=0.916, P<0.01 on sieved soil; r ²=0.845, P<0.001 on soil cores), was associated with an increase in microbial biomass C above a threshold of about 105 mg kg⁻¹. The effect of pig slurry on denitrification and N₂O reduction rates was detected on the surface layer (0–20 cm) only. However, no pig slurry effect could be detected on soil cores at field conditions or after NO₃ ⁻ enrichments at 20°C. Although the potential denitrifying activity in sieved soil samples was stimulated, the N₂O production was lower (P<0.03) in the plot fertilized with pig slurry, indicating a lower N₂O/(N₂O + N₂) ratio of the released gases. The pig-slurry-fertilized plot also showed a higher N₂O reduction activity, which is coherent with the lower N₂O production in anaerobiosis.     

Nitrous oxide production at different soil moisture contents in an arable soil in China

Abstract

Laboratory incubations were conducted to investigate nitrous oxide (N₂O) production from a subtropical arable soil (Typic Plinthodults) incubated at different soil moisture contents (SMC) and with different nitrogen sources using a 10% (v/v) acetylene (C₂H₂) inhibitory technique at 25°C. The production of N₂O and CO₂ was monitored during the incubations and changes in the contents of KCl-extractable NO^? ₃-N and NH⁺ ₄-N were determined. The production of N₂O increased slightly with an increase in SMC from 40% water-holding capacity (WHC) to 70% WHC, but increased dramatically at 100% WHC. After incubation the NO^? ₃-N content increased even at a SMC of 100% WHC. At a SMC of 100% WHC, the addition of NH⁺ ₄-N promoted the production of N₂O and CO₂, whereas the addition of NO^? ₃-N decreased N₂O production. Compared with the incubation without C₂H₂, the presence of C₂H₂ increased NH⁺ ₄-N content, but decreased NO^? ₃-N content, and there was no significant difference in N₂O production. These results indicate that heterotrophic nitrification contributes to N₂O production in the soil.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.     

The effects on N2 fixation (C2H2 reduction), bacterial population and rice plant growth of two modes of straw application to a wetland rice field

Summary The effects of incorporation and surface application of straw to a wetland rice field on nitrogen fixation (C₂H₂ reduction), bacterial population and rice plant growth were studied. Rice straw (5 t ha^–1) was chopped (10- to 15-cm pieces) and applied to the field 2 weeks before transplanting IR42, a long-duration variety, and IR50, a short-duration variety. The acetylene-reducing activity (ARA) of IR42 and IR50 measured at heading stage for 3 consecutive days showed significantly higher ARA in IR42 as a result of the 2 straw application methods. Mostly up to 20 days after straw surface application and incorporation, the dark ARA in the soil, total and N₂-fixing heterotrophs, and photoorganotrophic purple nonsulphur bacteria (POPNS) in the soil and in association with degrading straw were stimulated. Higher bacterial populations were associated with straw on the surface than with straw incorporated. The POPNS counts, in particular, were increased hundreds fold in the surface-applied straw treatment. Straw applications also increased the root, shoot and total plant biomass at heading stage and the total dry matter yield at harvest in both varieties. The data show the potentials of straw as a source of substrate for the production of microbial biomass and for the non-symbiotic N₂ fixation to improve soil fertility and plant nutrition.     

Effectiveness of acetylene inhibition of N2O reduction for measuring denitrification in soils of varying wetness

Abstract

The use of acetylene (C₂H₂) in the inhibition of N₂O to N₂ is widely used for measuring denitrification. The objective of this study was to determine the effectiveness of acetylene inhibition of N₂O reduction for short‐term and prolonged incubation studies in soils of varying water saturation, and to find out the possible reasons for lower N₂O recovery in continuously sealed incubations. Two experiments carried out in the laboratory reconfirmed that acetylene was very effective in inhibiting the reduction of N₂O in denitrification even for the prolonged incubation period (up to 96 h) under moist to saturated soil water contents. With 90 and 120% water‐filled pore space (WFPS), the accumulated N₂O in containers kept sealed throughout the study period, was 28 to 41% less than total headspace N₂O produced in containers that were opened, flushed and fresh C₂H₂ added every 24 h. Interpretation of our results suggest the lower N₂O amount recovered from continuously sealed containers at high WFPS, as compared to short‐term incubations (flushed containers), resulted primarily from delayed N₂O release from soil and not greater N₂O dissolved in soil solution, lower rates of denitrification, or decomposition/loss of C₂H₂ during prolonged incubation. Reduction of N₂O diffusion from soil cores showed direct relationship with head space concentration‐of N₂O and soil WFPS. From these results it is concluded that to obtain quantitative recovery of N₂O produced via denitrification, especially from soil with high WFPS soil cores should be vigorously shaken before head‐space N₂O analysis.     

Effectiveness of acetylene inhibition of N2O reduction for measuring denitrification in soils of varying wetness

Abstract

The use of acetylene (C₂H₂) in the inhibition of N₂O to N₂ is widely used for measuring denitrification. The objective of this study was to determine the effectiveness of acetylene inhibition of N₂O reduction for short‐term and prolonged incubation studies in soils of varying water saturation, and to find out the possible reasons for lower N₂O recovery in continuously sealed incubations. Two experiments carried out in the laboratory reconfirmed that acetylene was very effective in inhibiting the reduction of N₂O in denitrification even for the prolonged incubation period (up to 96 h) under moist to saturated soil water contents. With 90 and 120% water‐filled pore space (WFPS), the accumulated N₂O in containers kept sealed throughout the study period, was 28 to 41% less than total headspace N₂O produced in containers that were opened, flushed and fresh C₂H₂ added every 24 h. Interpretation of our results suggest the lower N₂O amount recovered from continuously sealed containers at high WFPS, as compared to short‐term incubations (flushed containers), resulted primarily from delayed N₂O release from soil and not greater N₂O dissolved in soil solution, lower rates of denitrification, or decomposition/loss of C₂H₂ during prolonged incubation. Reduction of N₂O diffusion from soil cores showed direct relationship with headspace concentration of N₂O and soil WFPS. From these results it is concluded that to obtain quantitative recovery of N₂O produced via denitrification, especially from soil with high WFPS soil cores should be vigorously shaken before head‐space N₂O analysis.     

Relationships between soil moisture content and nitrous oxide production during nitrification and denitrification

Summary The effect of soil water content [60%–100% water-holding capacity (WHC)] on N₂O production during autotrophic nitrification and denitrification in a loam soil was studied in a laboratory experiment by selectively inhibiting nitrification with a low C₂H₂ concentration (2.1 Pa). Nitrifiers usually produced more N₂O than denitrifiers. During an initial experimental period of 0–6 days the nitrifiers produced more N₂O than the denitrifiers by a factor ranging from 1.4 to 16.5, depending on the water content and length of incubation. The highest N₂O production rate by nitrifiers was observed at 90% WHC, when the soil had become partly anaerobic, as indicated by the high denitrification rate. At 100% WHC there were large gaseous losses from denitrification, while nitrification losses were smaller except for the first period of measurement, when there was still some O₂ remaining in the soil. The use of 10 kPa C₂H₂ to inhibit reduction of N₂O to N₂ stimulated the denitrification process during prolonged incubation over several days; thus the method is unsuitable for long-term studies.     

Hydrogen oxidation in soil following rhizobial H2 production due to N2 fixation by a Vicia faba-Rhizobium leguminosarum symbiosis

Summary The rate of H₂ release from broad beans (Vicia faba) infected with Rhizobium leguminosarum Hup^- was much faster than from beans infected with the Hup⁺ strain. Acetylene reduction and H₂ release were abolished by cutting the plants down, by incubation in darkness, or after the addition of ammonium, indicating that the H₂ was released by N₂-fixing bacterial symbionts. In laboratory cultures using non-sterile soil, the bean plants released H₂ until an equilibrium between H₂ production and H₂ oxidation was reached. The H₂ equilibrium concentration was higher in Hup^--infected bean cultures (about 3 ppm H₂ in the gas phase) than in Hup⁺-infected cultures (0.3 ppm H₂) because of the higher H₂ production. The H₂ release from Hup^--infected bean cultures in sterile soil did not reach equilibrium. An equilibrium occurred, if Knallgas bacteria were added. However, the equilibrium value was higher (13 ppm H₂) than in non-sterile soil, which seemed to be more efficient at H₂ oxidation. The Knallgas bacteria exhibited a relatively high K _m for H₂ (> 1300 ppmv H₂); this activity was observed in unplanted non-sterile soil, and in nonsterile soil planted with Hup⁺-infected beans or planted with Hup^--infected beans which had been cut down before being assayed. All these soils also showed a second, low-K _m (<50 ppm) level of H₂ oxidation activity, which was presumably due to abiontic soil enzymes. In contrast, only one level of activity, which had an intermediate K _m (about 200 ppm H₂), was observed when the soil was planted with Hup^--infected beans. The origin of this activity, which was only observed in the presence of intact, H₂-producing beans, is still unknown.     

Incomplete Acetylene Inhibition of Nitrous Oxide Reduction in Potential Denitrification Assay as Revealed by using 15N-Nitrate Tracer

One lake sediment and three soils for rice production were used to test the effectiveness of inhibiting of nitrous oxide (N₂O) reduction to dinitrogen gas (N₂) by acetylene (C₂H₂) using ¹⁵N tracer. Regardless of the sources of the samples, results show that in presence of C₂H₂, significant isotopic enrichment of ¹⁵N of N₂ was found at end of a typical denitrification assay. The δ¹⁵N of N₂ value increased from 0‰ to 7.8–19.3‰ and 7.5–10.6‰ for the treatment with addition of 0.05 and 0.2 mg ¹⁵N nitrate, respectively. Such ¹⁵N enrichment can be interpreted as N₂ formation accounting for 15.3% and 2.5% of the total added N in these two treatments, respectively. Nitrous oxide accumulation in presence of C₂H₂ could not account for the total added N. The result indicates incomplete inhibition of N₂O reduction to N₂ by C₂H₂ in denitrification when N₂O reduction enzyme is developed.     

Verification of an in‐situ method for measuring denitr1f1cation

Abstract

The most common direct in‐situ method for the measurement of soil denitrification requires many acetylene (C₂H₂) supply probes and airflow lines to measure nitrous oxide (N₂O) flux from the soil under a sealed cover. A modification to this method simplified C₂H₂ supply by placing a single acetylene supply probe 30 cm deep into the soil and measured soil N₂O emission flux over a 0.11 m² area. Acetylene concentrations ranging from 0.1–10.0% were readily and predictably established by radial diffusion from the supply probe. Over 94% of the N₂O released into the enclosed air space of the soil cover was recovered at an air flow rate of 21 L/h. Recovery decreased rapidly with increased flow rates of 31 and 37 L/h.     

Fungal N2O production in an arable peat soil in Central Kalimantan,Indonesia

Abstract

To clarify the microbiological factors that explain high N₂O emission in an arable peat soil in Central Kalimantan, Indonesia, a substrate-induced respiration-inhibition experiment was conducted for N₂O production. The N₂O emission rate decreased by 31% with the addition of streptomycin, whereas it decreased by 81% with the addition of cycloheximide, compared with a non-antibiotic-added control. This result revealed a greater contribution of the fungal community than bacterial community to the production of N₂O in the soil. The population density of fungi in the soil, determined using the dilution plate method, was 5.5 log c.f.u. g^?1 soil and 4.9 log c.f.u. g^?1 soil in the non-selective medium (rose bengal) and the selective medium for Fusarium, respectively. The N₂O-producing potential was randomly examined in each of these isolates by inoculation onto Czapek agar medium (pH 4.3) and incubation at 28°C for 14 days. Significant N₂O-producing potential was found in six out of 19 strains and in five out of seven strains isolated from the non-selective and selective media, respectively. Twenty-three out of 26 strains produced more than 20% CO₂ during the 14-day incubation period, suggesting the presence of facultative fungi in the soil. These strains were identified to be Fusarium oxysporum and Neocosmospora vasinfecta based on the sequence of 18S rDNA, irrespective of the N₂O-producing potential and the growth potential in conditions of low O₂ concentration.     

Denitrification,N2-fixation and fermentation during anaerobic incubation of soils amended with glucose and nitrate

Nitrate and glucose additions were investigated for their role in the C and N dynamics during anaerobic incubation of soil. A gas-flow soil core method was used, in which the net production of N₂, N₂O, NO, CO₂, and CH₄ under a He atmosphere could be monitored both accurately and frequently. In all experiments clayey silt loam soil samples were incubated for 9 days at 25 °C. Addition of nitrate (50 mg KNO₃-N kg^-1 soil) had no effect on total denitrification and CO₂ production rates, while the N₂O/N₂ ratio was affected considerably. The cumulative N₂O production exceeded the cumulative N₂ production for 6 days in the treatment with nitrate addition, compared to 1.2 days in the unamended treatment. Glucose addition stimulated the microbial activity considerably. The denitrification rates were limited by the growth rate of the denitrifying population. During denitrification no significant differences were observed between the treatments with 700 mg glucose-C kg^-1 and 4200 mg glucose-C kg^-1, both in combination with 50 mg KNO₃-N kg^-1. The N₂ production rates were remarkably low, until NO _inf3 ^sup- exhaustion caused rapid reduction of N₂O to N₂ at day 2. During the denitrification period 15–18 mg N kg^-1 was immobilised in the growing biomass. After NO _inf3 ^sup- shortage, a second microbial population, capable of N₂-fixation, became increasingly important. This change was clearly reflected in the CO₂ production rates. Net volatile fatty acid (VFA) production was monitored during the net N₂-fixation period with acetate as the dominant product. N₂-fixation faded out, probably due to N₂ shortage, followed by increased VFA production. In the high C treatment butyrate became the most important VFA, while in the low C treatment acetate and butyrate were produced at equal rates. During denitrification no VFA accumulation occurred; this does not prove, however, that denitrification and fermentation appeared sequentially. The experiments illustrate clearly the interactions of C-availability, microbial population and nitrate availability as influencing factors on denitrification and fermentation.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday     

Assay system for measurement of denitrification-N loss from ornamental plants potted in peat substrate

An assay system was evaluated for denitrification measurement with potted ornamental plants cultivated in peat substrate (Pelargonium zonale, Euphorbia pulcherrima). Flow-through chambers only enclosing the pot of the plants were considered best for denitrification measurement. Loss of N₂O from the chambers by transport through the plant shoot was negligible with both species. To determine (N₂O + N₂)-N loss, C₂H₂ was applied to inhibit reduction of N₂O. Experiments were conducted with unplanted substrate in closed incubation systems to determine optimum C₂H₂ concentration and pre-treatment duration. Complete inhibition of N₂O reduction in peat substrate was achieved using 1 vol% C₂H₂. However, a concentration of 5 vol% C₂H₂ was chosen for further experiments because C₂H₂ concentrations in flow-through chambers varied. The duration of C₂H₂ pre-treatment (0, 2, 12, 24 h) showed no clear effect on (N₂O + N₂)-N accumulation. However, a pre-treatment duration of 2 h was chosen to guarantee immediate inhibition of N₂O reductase at the start of experiments. Exposure to C₂H₂ gas proved to affect plants of both species. During C₂H₂ exposition in flow-through chambers, the leaves of P. zonale became chlorotic (48 h) and necrotic (72 h). E. pulcherrima showed no chlorosis but did exhibit leaf epinasty (24 h) and wilting (96 h). Transpiration of P. zonale and C availability in the growing medium of both species were not affected by 52 h and 24 h treatments with C₂H₂, respectively. As N emissions usually ended within 38 h of C₂H₂ treatment, it was concluded that side effects of C₂H₂ did not affect denitrification measurements.