Experimental determination of nitrogen kinetic isotope fractionation: Some principles; illustration for the denitrification and nitrification processes

Summary A few principles relative to the presentation and use of nitrogen stable isotopic data are briefly reviewed. Some classical relationships between the isotope composition of a substrate undergoing a single-step unidirectional reaction, are introduced. They are illustrated through controlled experiments on denitrification in a soil, and through nitrification by pure cultures ofNitrosomonas europaea. In the latter case, the isotope fractionation is calculated from the isotopic composition of the residual substrate, then of the product and the result is shown to be statistically the same for the two procedures. The isotopic enrichment factor for denitrification is −29.4±2.4‰ at 20°C, and −24.6±0.9‰ at 30°C; for nitrification this factor is −34.7±2.5‰ under the experimental conditions employed.     

Effect of organic loading on nitrification and denitrification in a marine sediment microcosm

Abstract The effects of organic additions on nitrification and dentrification were examined in sediment microcosms. The organic material, heat killed yeast, had a C/N ratio of 7.5 and was added to sieved, homogenized sediments. Four treatments were compared: no addition (control), 30 g dry weight (dw) m⁻² mixed throughout the 10 cm sediment column (30M), 100 g dw m⁻² mixed throughout sediments (100M), and 100 g dw m⁻² mixed into top 1 cm (100S). After the microcosms had been established for 7–11 days, depth of O₂ penetration, sediment-water fluxes and nitrification rates were measured. Nitrification rates were measured using three different techniques: N-serve and acetylene inhibition in intact cores, and nitrification potentials in slurris. Increased organic additions decreased O₂ penetration from 2.7 to 0.2 mm while increasing both O₂ consumption, from 30 to 70 mmol O₂ m⁻² d⁻¹, and NO₃⁻ flux into sediments. Nitrification rates in intact cores were similar for the two methods. Highest rates occurred in the 30M treatment, while the lowest rate was measured in the 100S treatment. Total denitrification rates (estimated from nitrification and nitrate fluxes) increased with increased organic addition, because of the high concentrations of NO₃⁻ (40 μM) in the overlaying water. The ratio of nitrification: denitrification was used as an indication of the importance of nitrification as the NO₃⁻ supply for denitrificaion. This ratio decreased from 1.55 to 0.05 iwth increase organic addition.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

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

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

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions

Abstract NO production and consumption rates as well as N₂O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N₂O. Under aerobic conditions untreated soil showed very low release of NO and N₂O but high consumption of NO. Fertilization with NH₄⁺ or urea stimulated both NO and N₂O production by nitrification. Addition of glucose at high soil moisture contents led to increased N₂ and N₂O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N₂O production by denitrification. The production of NO and N₂O was further stimulated at low moisture contents and after addition of glucose or NO₃⁻. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO₃⁻. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N₂O release.     

DNA-probing indicates the occurrence of denitrification and nitrogen fixation genes in Hyphomicrobium. Distribution of denitrifying and nitrogen fixing isolates of Hyphomicrobium in a sewage treatment plant

Abstract: Twenty-six Hyphomicrobium isolates from the sewage treatment plant and its receiving water body in Plön (Schleswig-Holstein, Germany) and two culture collection strains were screened for the occurrence of genes coding for denitrification enzymes (dissimilatory nitrate, nitrite and nitrous oxide reductases), for dinitrogen fixation (nitrogenase reductase) and for nitrification (ammonia monooxygenase catalyzing the first stage of this process) by DNA-probing. More than one half of the isolates had genes coding for denitrification enzymes. The DNA-DNA hybridization signals obtained with the gene segments correlated with enzyme activity measurements. The DNA of some isolates distinctly hybridized with the nif H probe indicating the occurrence of nitrogenase in the genus Hyphomicrobium . No signal was detected with the gene probe for nitrification. The results show that probes consisting of gene segments can be employed successfully to monitor the occurrence of genes which can show complex expression and in bacteria growing at low rates. The distribution pattern of the denitrification genes indicates that methylotrophic prosthecate bacteria of the sewage treatment plant and its receiving water body occupy different ecological niches.     

Nitrogen removal in a wastewater treatment plant through biofilters: nitrous oxide emissions during nitrification and denitrification

In order to estimate N₂O emissions from immersed biofilters during nitrogen removal in tertiary treatments at urban wastewater treatment plants (WWTPs), a fixed culture from the WWTP of “Seine Centre” (Paris conurbation) was subjected to lab-scale batch experiments under various conditions of oxygenation and a gradient of methanol addition. The results show that during nitrification, N₂O emissions are positively related to oxygenation (R ² = 0.99). However, compared to the rates of ammonium oxidation, the percentage of emitted N₂O is greater when oxygenation is low (0.5–1 mgO₂ L⁻¹), representing up to 1% of the oxidized ammonium (0.4% on average). During denitrification, the N₂O emission reaches a significant peak when the quantity of methanol allows denitrification of between 66% and 88%. When methanol concentrations lead to a denitrification of close to 100%, the flows of N₂O are much lower and represent on average 0.2% of the reduced nitrate. By considering these results, we can estimate, the emissions of N₂O during nitrogen removal, at the “Seine Centre” WWTP, to approximately 38 kgN-N₂O day⁻¹.     

Molecular analysis of the nitrogen cycle in deep-sea microorganisms from the Nankai Trough: genes for nitrification and denitrification from deep-sea environmental DNA

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.     

Cultivation of nitrifying bacteria in the retentostat, a simple fermenter with internal biomass retention

Abstract: The retentostat was developed for long-term continuous, axenic cultivation of microorganisms at those low growth rates which prevail in most natural habitats and which cannot be established properly in chemostats. How a microbial population approaches 'zero-growth' was studied in axenic cultures of Nitrosomonas europaea with complete biomass retention at 25°C and constant input of a nutrient solution containing ammonium (0.57 mM) as energy source. Since only cell-free filtrate left the reactor, biomass accumulated until a stable maximum of 2.7 × 10⁹ cells ml⁻¹ (398 mg l⁻¹ dry matter) was reached after about 5 weeks. In this state, growth rate approached zero, and the ammonium input just met the substrate demand required for maintenance energy (1.43 μmol NH₃–N mg dm⁻¹ h⁻¹). The potential of the retentostat for studying interactions between different microorganisms was demonstrated with a cascade of cultures of Nitrosomonas, Nitrobacter , and a denitrifying Pseudomonas . Thereby the ammonia was completely eliminated from artificial wastewater.     

开放式空气CO2浓度增高试验中的NO和NO2地-气交换观测研究

介绍了农田FACE(free airCO2 enrichment)试验中的NO和NO2 地 气交换观测方法 ,即静态暗箱采样—NO和NO2 化学发光分析法 ,并对观测结果进行了分析讨论 .此观测方法简单、易于操作 ,并可获得可靠的NO和NO2 净交换通量观测结果 .在稻麦轮作农田的旱地阶段 ,无论FACE还是对照处理 ,NO主要表现为地面净排放 ,NO2 主要表现为地面净吸收 .逐日的NO净排放不依赖于土壤温度 ,但却与土壤含水量呈线性负相关 (R2 =0 .82 ,P <0 .0 0 1) .NO2 净吸收具有明显的季节变化特征 ,逐日的净吸收通量随土壤温度和土壤含水量的变化可分别用抛物线方程拟合 (温度 :R2 =0 .74 ,P <0 .0 0 1;含水量 :R2 =0 .6 9,P <0 .0 0 1) .大气CO2 浓度升高 2 0 0± 4 0 μmol·mol-1使NO净排放减弱 19% (t 检验P =0 .0 96 ) ,NO2 净吸收减弱 10 % (t 检验P =0 .2 6 ) ,这主要是植物生长受到促进的缘故 .     

Maintenance energy demand and starvation recovery dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi cultivated in a retentostat with complete biomass retention.

Nitrosomonas europaea and Nitrobacter winogradskyi (strain "Engel") were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25 degrees C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 micromol of O2 mg-1 of biomass-C x h-1 for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 microg x ml-1) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514-1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Effects of N-serve (2-chloro-6-(trichloromethyl)pyridine) formulations on nitrification and on loss of nitrate in sand culture experiments

Summary N-serve (2-chloro-6-(trichloromethyl)pyridine) was tested as an inhibitor of nitrification of ammonium or urea in sand cultures. Nitrification was reduced but not prevented by N-Serve present at between 5 and 20 ppm in solution or by weight of sand. In the presence of root debris and acetone, used in some experiments at 2–4 ml/l of nutrient to convey N-Serve, denitrification was stimulated under the same conditions and resulted in loss of a large proportion of nitrate, probably mainly as gaseous products and some nitrite. These losses were greater when N-serve was also present. There was also conversion of nitrate to an insoluble form in the sand. A smaller proportional loss of nitrate occurred in other treatments in the presence of root debris when N-Serve was added without acetone, either as the commercial formulation 24E or as a solid. Thus, using N-Serve to inhibit nitrification may encourage denitrifying organisms especially in the presence of carbon sources including root debris or acetone. Large decreases of nitrate reductase activity in plants produced by using N-Serve in the presence of ammonium or urea were caused as much by losses of nitrate in the presence of acetone as by prevention of nitrate formation. Other N-Serve treatments (solid or 24E) decreased enzyme induction by between 50 and 90 per cent as a result mainly of reduced nitrification.     

Effect of agitation rate on biomass and protease production by a marine bacterium Vibrio harveyi cultured in a fermentor

A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell-based medium, supplemented with skim milk under different hydrodynamic conditions, namely agitation and aeration rates. The addition of skim milk to Zobell medium enhanced fivefold the extracellular enzyme production. Protease production seemed to take place after maximum luminescence had been produced. Specific growth rate increased as a consequence of increasing agitation rates. The maximum activity of 4.28 units mg^–1 protein were formed with 700 rev min^–1 and 0.5 v/v/m. Protease activity detected has a molecular weight of 34 kDa. Another minor band of protease activity was found at 40 kDa.     

Reduction of nitrous oxide by a soil Cytophaga in the presence of acetylene and sulfide

Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S²⁻), nitrous oxide (N₂O), and acetylene (C₂H₂). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N₂O rapidly even in the presence of a normally inhibitory concentration of C₂H₂ (4 kPa) providing S²⁻ was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C₂H₂ inhibition of the reduction of N₂O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments.     

Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition

The synthesis of nitrate, nitrite, and nitrous oxide reductases is highly enhanced by the addition of nitrate during growth of Rhodobacter sphaeroides forma sp. denitrificans. Contrary to what is observed in many denitrifiers, the synthesis of these enzymes is not repressed by oxygen at concentrations as high as 37% air saturation. When oxygen concentration is increased up to 100% air saturation, the synthesis of nitrite and nitrous oxide reductases is repressed while the nitrate reductase is still synthesized. Two proteins, one periplasmic (35kDa) and the other cytoplasmic (32kDa), are also induced by nitrate, but not by trimethylamine-N-oxide or oxygen. Although their function is not yet known, these two proteins appear to be specifically linked to the denitrification pathway. The amino acid sequences of tryptic peptides and of the N-terminal ends of these proteins indicate no significant similarity with the sequences in the Swiss Prot Data Bank. However, a very good alignment is obtained between the amino acid sequences of the periplasmic nitrate reductase of Alcaligenes eutrophus H16 and those of various tryptic peptides of the nitrate reductase of R. sphaeroides forma sp. denitrificans.Abbreviations 2D Two-dimensional - DTT Dithiotreitol - PAGE Polyacrylamide gel electrophoresis - TMAO Trimethylamine-N-oxide - DMSO Dimethylsulfoxide - TMPD N,N,N,N tetramethyl-p-phenylenediamine     

Biomass production and nitrogen utilization by Alnus incana when grown on N2 or NH 4 + made available at the same rate

A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The plants were either inoculated with Frankia and fixed atmospheric nitrogen or were left uninoculated but received ammonium at the same rate as the first group fixed their nitrogen. Nitrogen fixation was calculated from frequenct measurements of acetylene reduction and hydrogen evolution. The diurnal variation of acetylene reduction was also taken into account. The relative efficiency of nitrogenase could be used in the calculations of fixed nitrogen since the Frankia used did not show any detectable hydrogenase activity. Alders fixing nitrogen developed more biomass, longer shoots, larger leaf areas and contained more nitrogen than alders receiving ammonium. In one experiment, almost all ammonium given to the non-nodulated alders was taken up and 15% of the nitrogen taken up was excreted. In the other experiment, 34% of the ammonium was left in the nutrient solution and 8% of the nitrogen taken up was excreted. Alders inoculated with Frankia did not excrete any detectable amount of nitrogen. It seems that the energy demand for nitrogen fixation is not so high that biomass production in alders is retarded. The symbiotic system of A. incana and Frankia seems to be more efficient in utilizing its nitrogen than non-symbiotic A. incana receiving ammonium.