Denitrification activity of Bradyrhizobium sp. isolated from Argentine soybean cultivated soils

Two hundred and fifty strains, all of them representatives of native Bradyrhizobium sp., isolated from soils cultivated with soybean have been characterized by their denitrification activity. In addition, the denitrification potential of those soils was also measured by evaluating the most-probable-number (MPN) of denitrifying bacteria and the denitrification enzyme assay (DEA). Of the 250 isolates tested, 73 were scored as probable denitrifiers by a preliminary screening method. Only 41 were considered denitrifiers because they produced gas bubbles in Durham tubes, cultures reached an absorbance of more than 0.1 and NO³⁻ and NO²⁻ were not present. Ten of these 41 were selected to confirm denitrification and to study denitrification genes. According to N₂O production and cell protein concentration with NO³⁻, the isolates could be differentiated in three categories of denitrifiers. The presence of the napA, nirK, norC and nosZ genes was detected by production of a diagnostic PCR product using specific primers. RFLP from the 16S-23S rDNA spacer region (IGS) revealed that denitrifiers strains could be characterized as Bradyrhizobium japonicum and strains which were non-respiratory denitrifiers as B. elkanii.     

Denitrification and nitrous oxide emissions from riparian forests soils exposed to prolonged nitrogen runoff

Compared to upland forests, riparian forest soils have greater potential to remove nitrate (NO₃) from agricultural runoff through denitrification. It is unclear, however, whether prolonged exposure of riparian soils to nitrogen (N) loading will affect the rate of denitrification and its end products. This research assesses the rate of denitrification and nitrous oxide (N₂O) emissions from riparian forest soils exposed to prolonged nutrient runoff from plant nurseries and compares these to similar forest soils not exposed to nutrient runoff. Nursery runoff also contains high levels of phosphate (PO₄). Since there are conflicting reports on the impact of PO₄ on the activity of denitrifying microbes, the impact of PO₄ on such activity was also investigated. Bulk and intact soil cores were collected from N-exposed and non-exposed forests to determine denitrification and N₂O emission rates, whereas denitrification potential was determined using soil slurries. Compared to the non-amended treatment, denitrification rate increased 2.7- and 3.4-fold when soil cores collected from both N-exposed and non-exposed sites were amended with 30 and 60 μg NO₃-N g⁻¹ soil, respectively. Net N₂O emissions were 1.5 and 1.7 times higher from the N-exposed sites compared to the non-exposed sites at 30 and 60 μg NO₃-N g⁻¹ soil amendment rates, respectively. Similarly, denitrification potential increased 17 times in response to addition of 15 μg NO₃-N g⁻¹ in soil slurries. The addition of PO₄ (5 μg PO₄-P g⁻¹) to soil slurries and intact cores did not affect denitrification rates. These observations suggest that prolonged N loading did not affect the denitrification potential of the riparian forest soils; however, it did result in higher N₂O emissions compared to emission rates from non-exposed forest soils.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

“Hotspots” and “Hot Moments” of Denitrification in Urban Brownfield Wetlands

The influence of hydrology and soil properties on disproportionately high (“hot”) rates of nitrate (NO₃ ^?) removal via denitrification has been relatively well established. It is poorly understood, however, how the unique soil characteristics of brownfield wetlands contribute to or hinder denitrification. In this study, we examined drivers of “hot” denitrification rates over time (“hot moments”) and space (“hotspots”) in a watershed located on an unrestored brownfield in New Jersey, USA. We carried out measurements of denitrification over 9-day sequences during three seasons in sites with the same vegetation (Phragmites australis) but different soils (fill material, remnant marsh soils, flooded organic-rich soils). Denitrification rates above the 3rd quartile value of the data distribution were defined as “hot” and the most important drivers of these rates were determined using mixed models. Porosity and NO₃ ^? availability were the strongest spatial and temporal predictors, respectively, of high denitrification rates, with coarse-textured, unflooded fill materials unexpectedly supporting the highest rates. These results suggest that pore-scale hydrology is a more complex controller of wetland denitrification than previously thought. Course-textured, unflooded soils have high fractions of air-filled pores relative to flooded soils, leading to more endogenous NO₃ ^? production, and less diffusion constraints than fine-textured soils, leading to higher NO₃ ^? availability to denitrifiers in suboxic pores. Laboratory studies confirmed denitrifiers were limited by NO₃ ^? availability. However, denitrification rates in all soils matched or exceeded atmospheric NO₃ ^? deposition and stormwater NO₃ ^? loading at the site, suggesting that brownfields may play an important role in NO₃ ^? removal from urban stormwater.     

The response of denitrifiers in a sandy loam soil affected by a long-term fertilization to organic carbon and nitrate

The response of denitrifiers to carbon in the form of glucose (Glc-C) and nitrate (NO ₃ ⁻ -N) amendments was studied in four differently fertilized plots of sandy-loam soil. Two basically different characteristics of denitrification activity were determined: (1) potential denitrification measured as nitrous oxide production during 1-d incubation in the presence of acetylene, and (2) denitrifying enzyme activity determined in soil slurries as a N₂O production in the presence of acetylene and chloramphenicol during 1 h of incubation. Potential denitrification was strongly influenced by both Glc-C and NO ₃ ⁻ -N amendments in their various combinations, but was also affected by the fertilization practice. The response of denitrifiers to Glc-C and NO ₃ ⁻ -N was generally lower in unfertilized and surprisingly also in highly fertilized soils than in organically and moderately fertilized soils. Denitrifying enzyme activity was stimulated by the fertilization and was, in contrast to potential denitrification, the highest in highly fertilized soil. The results indicate that although active denitrifiers were present in a highly fertilized soil, their ability to develop under optimal conditions was decreased (being similar to that of denitrifiers in unfertilized soil). This suggests long-term changes in soil microbial community in a highly fertilized soil, presumably connected to changes in soil chemistry caused by fertilization.     

Biological denitrification by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> immobilized on microbial cellulose

This study demonstrated the feasibility of a biological denitrification process using immobilized Pseudomonas stutzeri. The microbial cellulose (MC) from Acetobacter xylinum was used as the support material for immobilization of the bacterium. Nitrate removal took place mainly in the anoxic system. The effects of various operating conditions such as the initial nitrate concentration, pH, and carbon source on biological denitrification were demonstrated experimentally. The system demonstrated a high capacity for reducing nitrate concentrations under optimum conditions. The denitrification rate increased up to a maximal value of 1.6 kg NO₃-N m⁻³ day⁻¹ with increasing nitrate loading rate. Because of its porosity and purity, MC may be considered as appropriate supports for adsorbed immobilized cells. The simplicity of immobilization and high efficiency in operation are the main advantages of such systems. To date, the immobilization of microorganisms onto MC has not been carried out. The results of this research shows that a pilot bioreactor containing P. stutzeri immobilized on MC exhibited efficient denitrification with a relatively low retention time.     

Effect of rice plants on nitrification-denitrification loss of nitrogen under greenhouse conditions

Summary Rice is unique among cereal crops in its ability to tolerate the anaerobic environment of waterlogged soils, but little is known about the influence of these plants on nitrogen loss by nitrification-denitrification. This problem was approached by loss of urea-N in cores with and without rice plants, using the acetylene inhibition method. Considerably greater denitrification was observed for surface-applied urea as compared to subsurface application in all cases. Regardless of the application point, however, the planted system yielded greater N₂O+N₂ accumulation in the first two days than from the nonplanted soil. After 4–6 days from fertilization no difference was observed in denitrification loss between planted and nonplanted systems. Inorganic NH ₄ ⁺ levels were observed to decrease rapidly in planted soils. Initial enhancement of gaseous N accumulation may occur because of the oxidized rice-root rhizosphere, however the appreciable denitrification in non-planted soil suggests that other N loss mechanisms are more important than the losses occurring in the root rhizosphere.     

Denitrification at pH 4 by a soil‐derived Rhodanobacter‐dominated community

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of N₂O produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO₃^‐ and N₂O reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of N₂O as denitrification end‐product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter‐like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils.     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Denitrification Potential and CO2 Emission in the Northern Forest Soils of the Yenisei Meridian (the Siberian IGBP Transect)

To estimate the probable contribution of northern forest soils to the global budget of greenhouse microgases, the cryogenic soils along the Yenisei meridian have been studied with respect to their potential denitrification and carbon mineralization activities. It is shown that the forest soils of the boreal zone have a high denitrification potential and, under conditions of a high nitrate nitrogen content, may be a source of nitrous oxide emission. A significant correlation is observed between N₂O and CO₂ emissions (r = 0.85, p < 0.001).     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Phosphate solubilization activity of rhizobia native to Iranian soils

Agricultural soils in Iran are predominantly calcareous with very low plant available phosphorus (P) content. In addition to their beneficial N₂-fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. Isolates from different cross-inoculation groups of rhizobia, obtained from Iranian soils were tested for their ability to dissolve inorganic and organic phosphate. From a total of 446 rhizobial isolates tested for P solubilization by the formation of visible dissolution halos on agar plates, 198 (44%) and 341(76%) of the isolates, solubilized Ca₃(PO₄)₂ (TCP) and inositol hexaphosphate (IHP), respectively. In the liquid Sperber TCP medium, phosphate-solubilizing bacteria (Bacillus sp. and Pseudomonas fluorescens) used as positive controls released an average of 268.6 mg L⁻¹ of P after 360 h incubation. This amount was significantly (P < 0.05) higher than those observed with all rhizobia tested. The group of Rhizobium leguminosarum bv. viciae mobilized in liquid TCP Sperber medium significantly (P < 0.05) more P (197.1 mg L⁻¹ in 360 h) than other rhizobia tested,. This group also showed the highest dissolution halo on the TCP solid Sperber medium. The release of soluble P was significantly correlated with a drop in the pH of the culture filtrates indicating the importance of acid production in the mobilization process. None of the 70 bradyrhizobial isolates tested was able to solubilize TCP. These results indicate that many rhizobia isolated from soils in Iran are able to mobilize P from organic and inorganic sources and this beneficial effect should be tested with crops grown in Iran.     

Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification,primarily due to acidification of the soils

China is the world's largest producer and consumer of fertilizer N, and decades of overuse has caused nitrate leaching and possibly soil acidification. We hypothesized that this would enhance the soils' propensity to emit N₂O from denitrification by reducing the expression of the enzyme N₂O reductase. We investigated this by standardized oxic/anoxic incubations of soils from five long‐term fertilization experiments in different regions of China. After adjusting the nitrate concentration to 2 mM, we measured oxic respiration (R), potential denitrification (D), substrate‐induced denitrification, and the denitrification product stoichiometry (NO, N₂O, N₂). Soils with a history of high fertilizer N levels had high N₂O/(N₂O+N₂) ratios, but only in those field experiments where soil pH had been lowered by N fertilization. By comparing all soils, we found a strong negative correlation between pH and the N₂O/(N₂O+N₂) product ratio (r² = 0.759, P < 0.001). In contrast, the potential denitrification (D) was found to be a linear function of oxic respiration (R), and the ratio D/R was largely unaffected by soil pH. The immediate effect of liming acidified soils was lowered N₂O/(N₂O+N₂) ratios. The results provide evidence that soil pH has a marginal direct effect on potential denitrification, but that it is the master variable controlling the percentage of denitrified N emitted as N₂O. It has been known for long that low pH may result in high N₂O/(N₂O+N₂) product ratios of denitrification, but our documentation of a pervasive pH‐control of this ratio across soil types and management practices is new. The results are in good agreement with new understanding of how pH may interfere with the expression of N₂O reductase. We argue that the management of soil pH should be high on the agenda for mitigating N₂O emissions in the future, particularly for countries where ongoing intensification of plant production is likely to acidify the soils.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

Nitrification and Denitrification in High-Strength Ammonium by <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

Alcaligenes faecalis sp. No. 4, that has the ability of heterotrophic nitrification and aerobic denitrification in high-strength ammonium at about 1200 mg-N/l, converted about one-half of removed NH ₄⁺-N to intracellular nitrogen and nitrified only 3% of the removed NH₄⁺. From the nitrogen balance, 40–50% of removed NH₄⁺-N was estimated to be denitrified. Production of N₂ was confirmed by GC-MS and 90% of denitrified products was N₂. The maximum ammonium removal rate, 29 mg-N/l h and its denitrification rate in aerated batch experiments, were 5–40 times higher than those of other bacteria with the same ability.     

Chemical soil conditions in pristineNothofagus forests of New Zealand as compared to German forests

In many German forest soils low base saturation of CEC in deeper soil layers was reported and acidic deposition is seen as the major cause of these findings. To test this hypothesis we sampled 5 New Zealand forest soils from pristine beech (Nothofagus fusca, N. menziesii, N. solandri) sites under climatic and geological conditions comparable to higher elevations in Germany. The soils developed from granite and greywacke. Soil samples were analyzed for pH and the exchangeable cations were extracted with 1M NH₄Cl. The base saturation of all soil profiles was very low, even in deeper layers and was thus similar to the patterns found in many German forest soils. The pH was generally higher in the New Zealand soils as compared to Germany. The reason for the depletion of base cations in deeper soil layers of New Zealand forest soils is most likely the leaching of base cations with HCO₃ ^- resulting from the dissociation of carbonic acid in connection with high amounts of seepage. Thus, under high rainfall conditions, the low base saturation found in deeper layers of forest soils cannot exclusively be attributed to the effects of acidic depositions and land use. ei]Section editor: R F Huettl     

High-Resolution Denitrification Kinetics in Pasture Soils Link N2O Emissions to pH,and Denitrification to C Mineralization

Denitrification in pasture soils is mediated by microbial and physicochemical processes leading to nitrogen loss through the emission of N₂O and N₂. It is known that N₂O reduction to N₂ is impaired by low soil pH yet controversy remains as inconsistent use of soil pH measurement methods by researchers, and differences in analytical methods between studies, undermine direct comparison of results. In addition, the link between denitrification and N₂O emissions in response to carbon (C) mineralization and pH in different pasture soils is still not well described. We hypothesized that potential denitrification rate and aerobic respiration rate would be positively associated with soils. This relationship was predicted to be more robust when a high resolution analysis is performed as opposed to a single time point comparison. We tested this by characterizing 13 different temperate pasture soils from northern and southern hemispheres sites (Ireland and New Zealand) using a fully automated-high-resolution GC detection system that allowed us to detect a wide range of gas emissions simultaneously. We also compared the impact of using different extractants for determining pH on our conclusions. In all pH measurements, soil pH was strongly and negatively associated with both N₂O production index (IN₂O) and N₂O/(N₂O+N₂) product ratio. Furthermore, emission kinetics across all soils revealed that the denitrification rates under anoxic conditions (NO+N₂O+N₂ μmol N/h/vial) were significantly associated with C mineralization (CO₂ μmol/h/vial) measured both under oxic (r² = 0.62, p = 0.0015) and anoxic (r² = 0.89, p<0.0001) conditions.     

Zinc phytoavailability after remediation in soils contaminated by sphalerite-containing pyritic sludge

Zinc can be toxic to plants growing on soils in areas of the Guadiamar River valley (southwestern Spain) affected by the spillage of pyritic sludge in April 1998. The shoots and the soil around the roots of two wild plants (viz. Amaranthus blitoides S. Wats., November 2000; and Xanthium strumarium L., June 2001) growing in the sludge-affected areas were sampled with the purpose of relating Zn phytoavailability to soil properties. The soils were calcareous and non-calcareous Entisols and Inceptisols which, after remediation, contained ploughed-in residual sludge and unevenly distributed industrial lime. Chemical extracts from the soils suggested that much of the sphalerite (ZnS) originally present in the sludge had weathered and Zn was partly bound to carbonates and Fe oxides, the total Zn concentration ranging from 37 to 2407 mg kg ^–1. To identify the soil properties that influenced Zn phytoavailability under controlled conditions, the soil samples (n=63) were homogenized and oilseed rape (Brassica napus var. Karola) was pot-grown on them in a growth chamber. The concentrations of Zn in oilseed rape shoots and roots were below phytotoxic levels, with mean ± standard deviation values of 142 ± 128 and 244 ± 328 mg kg ^–1 dry matter, respectively. Citrate/bicarbonate-extractable Zn in soil (Zn _cb) was found to be the best predictor for the Zn concentration in both shoots and roots. Also, the Zn _cb/Olsen P ratio exhibited a high predictive power for Zn in shoots as the likely result of the Zn-P interaction in soil. The shoot Zn concentration in the wild plants, generally lay below phytotoxic levels (the mean ± standard deviation values were 261 ± 255 and 200 ± 228 mg kg ^–1 dry matter for Amaranthus blitoides and Xanthium strumarium, respectively) and was not correlated with soil properties – by exception, there was slight correlation between the Zn concentration in Amaranthus blitoides and Zn _cb/Olsen P. Such a lack of correlation can be ascribed to the local small-scale soil heterogeneity caused by remediation practices. The Zn concentration in wild plants growing on CaCO ₃-poor soils was weakly correlated with Zn _cb/Olsen P; no similar correlation was found in CaCO ₃-rich soils, however. The wild plants growing on CaCO ₃-poor and CaCO ₃-rich soils differed little in Zn concentration; this suggests that further addition of lime to reduce Zn phytoavailability may be unjustified.     

Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans

Summary A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO ₄ ⁼ /l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO ₂ ^– /l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: _max=0.11 h^–1, K _S=0.2 mg NO ₃ ^– /l, maximum denitrification rate=0.78 g NO ₃ ^– /g cells·h, g cells/g NO ₃ ^– , g cells/g S₂O ₃ ⁼ . Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N₂. The concentrations of N₂O and NO were below 1 ppm.