Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Denitrification in San Francisco Bay Intertidal Sediments

The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO₃⁻ + NO₂⁻ concentrations (≤26 μM) were below the apparent K_m (50 μM) for nitrate. During the rainy season, when ambient NO₃⁻ + NO₂⁻ concentrations were higher (37 to 89 μM), an accurate estimate of the K_m could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N₂O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N₂O in the presence of C₂H₂ was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N₂O loss was caused by incomplete blockage of N₂O reductase by C₂H₂ at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N₂ m⁻² h⁻¹ (for undisturbed sediments) to 17 to 280 μmol of N₂ m⁻² h⁻¹ (for shaken sediment slurries).     

Capacity for Denitrification and Reduction of Nitrate to Ammonia in a Coastal Marine Sediment

The capacity for dissimilatory reduction of NO₃⁻ to N₂ (N₂O) and NH₄⁺ was measured in ¹⁵NO₃⁻-amended marine sediment. Incubation with acetylene (7 × 10⁻³ atmospheres [normal]) caused accumulation of N₂O in the sediment. The rate of N₂O production equaled the rate of N₂ production in samples without acetylene. Complete inhibition of the reduction of N₂O to N₂ suggests that the “acetylene blockage technique” is applicable to assays for denitrification in marine sediments. The capacity for reduction of NO₃⁻ by denitrification decreased rapidly with depth in the sediment, whereas the capacity for reduction of NO₃⁻ to NH₄⁺ was significant also in deeper layers. The data suggested that the latter process may be equally as significant as denitrification in the turnover of NO₃⁻ in marine sediments.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Denitrification Rates in the Low-Oxygen Waters of the Stratified Baltic Proper

Denitrification activity was shown in the deep, low-oxygen waters of the Baltic proper by both in vitro and in situ methods. The vertical distribution of NO₃⁻ in the water column showed nitrate consumption and NO₂⁻ and N₂O maxima in the deep waters when O₂ was below 0.2 ml liter⁻¹, which is suggestive evidence for denitrification. Direct in situ evidence for denitrification was obtained by finding an N₂ saturation of up to 108% in the deep waters. When these waters were incubated with ¹⁵NO₃⁻, ¹⁵N₂ was produced. Quantification of the denitrification rate done by the addition of C₂H₂ to water samples from the active depths showed a rate of about 0.10 μmol liter⁻¹ day⁻¹.     

Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Denitrification Associated with Periphyton Communities

Scrapings of decomposing Cladophora sp. mats (periphyton) covering stream bed rocks produced N₂O when incubated under N₂ plus 15% C₂H₂. Denitrification (N₂O formation) was enhanced by NO₃⁻ and was inhibited by autoclaving, Hg²⁺, and O₂. No N₂O was formed in the absence of C₂H₂ (air or N₂ atmosphere). Chloramphenicol did not block N₂O formation, indicating that the enzymes were constitutive. In field experiments, incubation of periphyton scrapings in the light inhibited denitrification because of algal photosynthetic O₂ production. The diurnal periphyton-associated denitrification rate was estimated to be 45.8 μmol of N₂O·m⁻²·day⁻¹, as determined by averaging light, aerobic plus dark, and anaerobic rates over a 24-h period.     

Temporal Change in Nitrous Oxide and Dinitrogen from Denitrification Following Onset of Anaerobiosis

Similar temporal patterns were found in three mineral soils for the composition of the gaseous products of denitrification following the onset of anaerobic conditions. During the early period of anaerobiosis (0 up to 1 to 3 h), N₂ was the dominant product of denitrification. The NO₃⁻ → N₂O activity then increased, but was not accompanied by a corresponding increase in N₂O-reducing activity. This resulted in a relatively extended period of time (1 to 3 up to 16 to 33 h) during which N₂O was a major product. Eventually (after 16 to 33 h), an increase in N₂O-reducing activity occurred without a comparable increase in the N₂O-producing activity. The increase in the rate of N₂O reduction did not occur in the presence of chloramphenicol and required the presence of N₂O or NO₃⁻ during the preceding anaerobic incubation. During the final period (16 to 33, up to 48 h), N₂ was generally the sole product of denitrification, since the rate of N₂O reduction exceeded the rate of N₂O production. A similar sequential pattern was also found for a culture of a denitrifying Flavobacterium sp. shifted to anaerobic growth. A staggered synthesis of the enzymes in the denitrification sequence apparently occurred in response to anoxia, which caused first a net production of N₂O followed by consumption of N₂O.     

Anaerobic Growth and Denitrification among Different Serogroups of Soybean Rhizobia

We screened soybean rhizobia originating from three germplasm collections for the ability to grow anaerobically in the presence of NO₃⁻ and for differences in final product formation from anaerobic NO₃⁻ metabolism. Denitrification abilities of selected strains as free-living bacteria and as bacteroids were compared. Anaerobic growth in the presence of NO₃⁻ was observed in 270 of 321 strains of soybean rhizobia. All strains belonging to the 135 serogroup did not grow anaerobically in the presence of NO₃⁻. An investigation with several strains indicated that bacteria not growing anaerobically in the presence of NO₃⁻ also did not utilize NO₃⁻ as the sole N source aerobically. An exception was strain USDA 33, which grew on NO₃⁻ but failed to denitrify. Dissimilation of NO₃⁻ by the free-living cultures proceeded without the significant release of intermediate products. Nitrous oxide reductase was inhibited by C₂H₂, but preceding steps of denitrification were not affected. Final products of denitrification were NO₂⁻, N₂O, or N₂; serogroups 31, 46, 76, and 94 predominantly liberated NO₂⁻, whereas evolution of N₂ was prevalent in serogroups 110 and 122, and all three were formed as final products by strains belonging to serogroups 6 and 123. Anaerobic metabolism of NO₃⁻ by bacteroid preparations of Bradyrhizobium japonicum proceeded without delay and was evident by NO₂⁻ accumulation irrespective of which final product was formed by the strain as free-living bacteria. Anaerobic C₂H₂ reduction in the presence of NO₃⁻ was observed in bacteroid preparations capable of NO₃⁻ respiration but was absent in bacteria that were determined to be deficient in dissimilatory nitrate reductase.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.     

Aerobic nitrous oxide production through N-nitrosating hybrid formation in ammonia-oxidizing archaea

Soil emissions are largely responsible for the increase of the potent greenhouse gas nitrous oxide (N₂O) in the atmosphere and are generally attributed to the activity of nitrifying and denitrifying bacteria. However, the contribution of the recently discovered ammonia-oxidizing archaea (AOA) to N₂O production from soil is unclear as is the mechanism by which they produce it. Here we investigate the potential of Nitrososphaera viennensis, the first pure culture of AOA from soil, to produce N₂O and compare its activity with that of a marine AOA and an ammonia-oxidizing bacterium (AOB) from soil. N. viennensis produced N₂O at a maximum yield of 0.09% N₂O per molecule of nitrite under oxic growth conditions. N₂O production rates of 4.6±0.6 amol N₂O cell⁻¹ h⁻¹ and nitrification rates of 2.6±0.5 fmol NO₂⁻ cell⁻¹ h⁻¹ were in the same range as those of the AOB Nitrosospira multiformis and the marine AOA Nitrosopumilus maritimus grown under comparable conditions. In contrast to AOB, however, N₂O production of the two archaeal strains did not increase when the oxygen concentration was reduced, suggesting that they are not capable of denitrification. In ¹⁵N-labeling experiments we provide evidence that both ammonium and nitrite contribute equally via hybrid N₂O formation to the N₂O produced by N. viennensis under all conditions tested. Our results suggest that archaea may contribute to N₂O production in terrestrial ecosystems, however, they are not capable of nitrifier-denitrification and thus do not produce increasing amounts of the greenhouse gas when oxygen becomes limiting.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO₃⁻-N liter⁻¹. Traces of ¹⁵NO₃⁻ were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day⁻¹. Both assimilatory and dissimilatory reduction rates were estimated from analyses of ¹⁵N₂, ¹⁵N₂O, ¹⁵NH₄⁺, and ¹⁵N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N₂ and N₂O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO₃⁻ and NH₄⁺ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-³H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g⁻¹ day⁻¹. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N₂, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO₃⁻ was reduced to NH₄⁺. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month.     

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Most studies of bacterial denitrification have used nitrate (NO₃⁻) as the first electron acceptor, whereas relatively less is understood about nitrite (NO₂⁻) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO₂⁻ (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO₂⁻, as well as NO₃⁻. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO₂⁻ reductase containing copper that was involved in bacterial NO₂⁻ reduction. The strain denitrified up to 40 mM NO₂⁻ to dinitrogen under anaerobic conditions in which other denitrifiers or NO₃⁻ reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO₂⁻. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188^T proliferated even in the presence of higher levels of NO₂⁻ (100 mM), and both were considerably more resistant to acidic NO₂⁻ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO₃⁻) or nitrite (NO₂⁻) is reduced to gaseous nitrogen forms such as N₂ and nitrous oxide (N₂O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO₃⁻ reductase (Nar), NO₂⁻ reductase (Nir), nitric oxide (NO) reductase (Nor), and N₂O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO₃⁻ to generate NO₂⁻ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO₂⁻ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds (11), organophosphorus pesticides (45), and other hydrocarbons (38) and remove heavy metal ions such as chromium and cadmium (24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence (7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level (33). Some O. anthropi strains have been identified as denitrifiers (21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO₂⁻ (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO₂⁻ under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS.     

Environmental Parameters Regulating Gaseous Nitrogen Losses from Two Forested Ecosystems via Nitrification and Denitrification

Gaseous N losses from disturbed and reference forested watersheds at the Coweeta Hydrologic Laboratory in western North Carolina were studied by in situ N₂O diffusion measurements and laboratory incubations throughout a 10-month period. Soil temperature, percent base saturation, and water-filled pore space accounted for 43% of the variation in in situ N₂O diffusion measurements. Laboratory incubations distinguished the gaseous N products of nitrification and denitrification. Nitrifying activity, ambient NO₃⁻, and nitrification N₂O were positively correlated with percent base saturation. However, differences between watersheds in soil N substrate caused by presence of leguminous black locust in the disturbed watershed were confounded with differences in soil acidity. Denitrification was most strongly affected by soil moisture, which in turn was determined by precipitation events and slope position. Gaseous N losses from well-drained midslope and toeslope landscape positions appeared to be minor relative to other N transformations. Favorable conditions for denitrification occurred at a poorly drained site near the stream of the disturbed watershed. Laboratory incubations revealed high rates of NO₃⁻ reduction in these soils. We speculate that the riparian zone is a major site of depletion of NO₃⁻ from the soil solution via denitrification.     

Degradation of n-Hexadecane and Its Metabolites by Pseudomonas aeruginosa under Microaerobic and Anaerobic Denitrifying Conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O₂-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O₂-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO₂⁻-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO₂⁻-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO₃⁻-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N₂). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.