Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM⁻¹, and there was 95% inhibition at ~30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM⁻¹; 95% inhibition at ~70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM⁻¹, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ~50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N₂ production in marine sediments and the use of ¹⁵N isotope methods for their quantification.     

Denitrification measurements in aquatic sediments: A comparison of three methods

Measurements of denitrification using the acetylene inhibition,¹⁵N isotope tracer, and N₂ flux methods were carried out concurrently using sediment cores from Vilhelmsborg sø, Denmark, in an attempt to clarify some of the limitations of each technique. Three experimental treatments of overlying water were used: control, nitrate enriched, and ammonia enriched water. The N₂ flux and¹⁵N tracer experiments showed high rates of coupled nitrification/denitrification in the sediments. The acetylene inhibition method did not capture any coupled nitrification/denitrification. This could be explained by acetylene inhibition of nitrification. A combined¹⁵N tracer/acetylene inhibition experiment demonstrated that acetylene inhibition of N₂O reduction was incomplete and the method, therefore, only measured approximately 50% of the denitrification due to nitrate from the overlying water. Similar rates of denitrification due to nitrate in the overlying water were measured by the N₂ flux method and the acetylene inhibition method, after correcting for the 50% efficiency of acetylene inhibition. Rates of denitrification due to nitrate from the overlying water measured by the¹⁵N tracer method, however, were only approximately 35% or less of those measured by the acetylene inhibition or N₂ flux methods.     

The production of nitric oxide by marine ammonia‐oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger

Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia‐oxidizing archaea (AOA) to nitrification. However, quantification of the relative contributions of AOA and ammonia‐oxidizing bacteria (AOB) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide (NO) by Nitrosopumilus maritimus SCM1. Activity of SCM1 was always associated with the release of NO with quasi‐steady state concentrations between 0.05 and 0.08 μM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2‐phenyl‐4,4,5,5,‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO). Comparison of marine and terrestrial AOB strains with SCM1 and the recently isolated marine AOA strain HCA1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea (ATU). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub‐micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU. These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism.     

Effects of allylthiourea,salinity, and pH on ammonia/ammonium-oxidizing prokaryotes in mangrove sediment incubated in laboratory microcosms

Anaerobic ammonium-oxidizing (anammox) bacteria, aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB) are three groups of ammonia/ammonium-oxidizing prokaryotes (AOPs) involved in the biochemical nitrogen cycling. In this study, the effects of allylthiourea (ATU), pH, and salinity on these three groups from mangrove sediment were investigated through microcosm incubation in laboratory. ATU treatments (50, 100, and 500 mg L^?1) obviously affected the community structure of anammox bacteria and AOB, but only slightly for AOA. ATU began to inhibit anammox bacteria growth slightly from day 10, but had an obvious inhibition on AOA growth from the starting of the study. At 100 mg L^?1 of ATU or higher, AOB growth was inhibited, but only lasted for 5 days. The pH treatments showed that acidic condition (pH 5) had a slight effect on the community structure of anammox bacteria and AOA, but an obvious effect on AOB. Acidic condition promoted the growth of all groups of AOPs in different extent, but alkaline condition (pH 9) had a weak effect on AOB community structure and a strong effect on both anammox bacteria and AOA. Alkaline condition obviously inhibited anammox bacteria growth, slightly promoted AOA, and slightly promoted AOB in the first 20 days, but inhibited afterward. Salinity treatment showed that higher salinity (20 and 40?‰) resulted in higher anammox bacteria diversity, and both AOA and AOB might have species specificity to salinity. High salinity promoted the growth of both anammox bacteria and AOB, inhibited AOA between 5 and 10 days, but promoted afterward. The results help to understand the role of these microbial groups in biogeochemical nitrogen cycling and their responses to the changing environments.     

Nitrogen removal with the anaerobic ammonium oxidation process

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium to N₂ with nitrite as the terminal electron acceptor in the absence of O₂. Nitritation–anammox bioreactors provide a cost-effective and environment-friendly alternative to conventional nitrification/denitrification nitrogen removal systems. Currently, this process is only applied for ammonium removal from wastewater with high ammonium load and temperature. Nevertheless, recent results obtained with laboratory-scale bioreactors suggest new possible routes of application of the Nitritation–anammox technology including (1) municipal wastewater treatment, removal of (2) methane in combination with nitrite-reducing methane-oxidizing bacteria, (3) nitrate coupled to organic acid oxidation and (4) nitrogen oxides. The current review summarizes the state-of-the-art of the application of Nitritation–anammox systems and discusses the possibilities of utilizing these recent results for wastewater treatment.     

Measurement of Denitrification in Two Freshwater Sediments by an In Situ Acetylene Inhibition Method

An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N m⁻²h⁻¹, respectively. Acetylene reduction at an average rate of 0.07 mmol of C₂H₄ formed m⁻²h⁻¹ was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N m⁻²h⁻¹ in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.     

Presence and activity of anammox and denitrification process in low ammonium-fed bioreactors

A combination of anammox and denitrification process was studied for 300 days in low ammonium-fed bioreactors under the support of organic carbon. Nutrient profiles, (15)N-labelling techniques and qualitative fluorescence in situ hybridization (FISH) probes were used to confirm the nitrogen removal pathways and intercompetition among different bacteria populations. About 80% of nitrogen removal was achieved throughout the study period. The results confirmed that anammox bacteria were absent in the bioreactor inoculated with anaerobic granules only but they were present and active in the central anoxic parts of biopellets in the bioreactor inoculated with mixed microbial consortium from activated sludge and anaerobic granules. It also showed that the anammox bacteria were successfully enriched in the low ammonium-fed bioreactors. Results of this study clearly demonstrated that anammox and denitrification processes could coexist in same environment and anammox bacteria were less competitive than denitrifying bacteria.     

Production of N(2) through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments

In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N(2), thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N(2) production in marine sediments. Incubations with (15)N-labeled nitrate or ammonium demonstrated that during this process, N(2) is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N(2) production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N(2) production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N(2) production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N(2), anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.     

Mechanism of nitrogenase switch-off by oxygen.

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiella pneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strains AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O2 concentration of 0.37 microM. Cyanide (90 microM), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O2 concentration that caused 50% inhibition of nitrogenase activity to 2.9 microM. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but its nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 microM O2. Inhibition by CN- of 40 to 50% of the O2 uptake in the mutant shifted the O2 concentration that caused 50% inhibition of nitrogenase to 1.58 microM. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO2-. Lower concentrations of NO2- were required to inhibit the activity in NO3- -grown cells, which have higher activities of nitrite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tanganyika)

Here we provide the first direct evidence for the anammox process (anaerobic ammonium oxidation) in a lacustrine system, Lake Tanganyika, the second largest lake in the world. Incubations with (15)N labelled nitrate showed that anammox occurred in the suboxic water layer at 100-110 m water depth. Anammox rates up to 10 nM N(2) h(-1) are comparable to those reported for the marine water column. Up to approximately 13% of produced N(2) could be attributed to the anammox process whereas the remainder was related to denitrification. Typical lipid biomarkers characteristic of anammox bacteria were found in filtered water from the depths where anammox occurred, thus supporting the presence of anammox bacteria. Further evidence is provided by fluorescence in situ hybridization (FISH), revealing up to 13 000 anammox bacteria cells per ml or 1.4% of all DAPI (4'-6-Diamidino-2-phenylindole)-stained cells. Phylogenetic analyses of partial 16S rRNA genes indicated the presence of sequences most closely related to the known anammox bacterium Candidatus "Scalindua brodae" (95.7% similarity). Using the incubation results, a total loss of 0.2 Tg N(2) per year linked to anammox was estimated for the Northern basin of Lake Tanganyika.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus.

Suspensions of denitrifying cells of Pseudomonas perfectomarinus reduced nitrate and nitrate as expected to dinitrogen; but, in the presence of acetylene, nitrous oxide accumulated when nitrate or nitrate was reduced. When supplied at the outset in place of nitrate and nitrate, nitrous oxide was rapidly reduced to dinitrogen by cells incubated in anaerobic vessels in the absence of acetylene. In the presence of 0.01 atmospheres of acetylene, however, nitrous oxide was not reduced. Ethylene was not produced, nor did it influence the rate of nitrous oxide reduction when provided instead of acetylene. Cells exposed to 0.01 atmospheres of acetylene for as long as 400 min were able to reduce nitrous oxide after removal of acetylene at a rate comparable to that of cells not exposed to acetylene. Acetylene did not affect the production or functioning of assimilatory nitrate or nitrite reductase in axenic cultures of Enterobacter aerogenes or Trichoderma uride. While exposed to acetylene, bacteria in marine sediment slurries produced measurable quantities of nitrous oxide from glucose- or acetate-dependent reduction of added nitrate. Possible use of acetylene blockage for measurement of denitrification in unamended marine sediments is discussed.     

Induced cooperation between marine nitrifiers and anaerobic ammonium-oxidizing bacteria by incremental exposure to oxygen

In oxygen-limited marine ecosystems cooperation between marine nitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria is of importance to nitrogen cycling. Strong evidence for cooperation between anammox bacteria and nitrifiers has been provided by environmental studies but little is known about the development of such communities, the effects of environmental parameters and the physiological traits of their constituents. In this study, a marine laboratory model system was developed. Cooperation between marine nitrifiers and anammox bacteria was induced by incremental exposure of a marine anammox community dominated by Scalindua species to oxygen in a bioreactor set-up under high ammonium (40 mM influent) conditions. Changes in the activities of the relevant functional groups (anammox bacteria, aerobic ammonia oxidizers and nitrite oxidizers) were monitored by batch tests. Changes in community composition were followed by Fluorescence in situ Hybridization (FISH) and by amplification and sequencing of 16S rRNA and amoA genes. A co-culture of Scalindua sp., an aerobic ammonia-oxidizing Nitrosomonas-like species, and an aerobic (most likely Nitrospira sp.) nitrite oxidizer was obtained. Aerobic ammonia oxidizers became active immediately upon exposure to oxygen and their numbers increased 60-fold. Crenarchaea closely related to the ammonia-oxidizer Candidatus 'Nitrosopumilus maritimus' were detected in very low numbers and their contribution to nitrification was assumed negligible. Activity of anammox bacteria was not inhibited by the increased oxygen availability. The developed marine model system proved an effective tool to study the interactions between marine anammox bacteria and nitrifiers and their responses to changes in environmentally relevant conditions.     

Measurement of nitrification rates in lake sediments: Comparison of the nitrification inhibitors nitrapyrin and allylthiourea

A method for measuring rates of nitrification in intact marine sediment cores has been modified and adapted for use in freshwater sediments. The technique involves subsampling a sediment core into minicores. Half of these cores are treated with an inhibitor of chemolithotrophic nitrification and, after incubation, differences in ammonia and nitrate concentration between inhibited and uninhibited systems are calculated. The within-treatment variability of ammonia and nitrate concentrations could be reduced by storing the cores overnight prior to subsampling. Estimates of the nitrification rate using the difference in ammonia concentrations between the inhibited and uninhibited mini-cores were always greater than the rate estimate using the difference in nitrate concentrations. Comparison between the results using the nitrification inhibitors allylthiourea (ATU) and nitrapyrin (N-Serve) indicated that the former appeared to give larger values for the nitrification rate than did the latter. Differences in the efficiency of these inhibitors in the control of nitrification under the conditions used partly explain these results. Data are also presented on the effect of N-Serve and ATU on some other nitrogen transformations affecting ammonia and nitrate concentrations.     

Seasonal oxygen,nitrogen and phosphorus benthic cycling along an impacted Baltic Sea estuary: regulation and spatial patterns

The regulatory roles of temperature, eutrophication and oxygen availability on benthic nitrogen (N) cycling and the stoichiometry of regenerated nitrogen and phosphorus (P) were explored along a Baltic Sea estuary affected by treated sewage discharge. Rates of sediment denitrification, anammox, dissimilatory nitrate reduction to ammonium (DNRA), nutrient exchange, oxygen (O₂) uptake and penetration were measured seasonally. Sediments not affected by the nutrient plume released by the sewage treatment plant (STP) showed a strong seasonality in rates of O₂ uptake and coupled nitrification–denitrification, with anammox never accounting for more than 20 % of the total dinitrogen (N₂) production. N cycling in sediments close to the STP was highly dependent on oxygen availability, which masked temperature-related effects. These sediments switched from low N loss and high ammonium (NH₄ ⁺) efflux under hypoxic conditions in the fall, to a major N loss system in the winter when the sediment surface was oxidized. In the fall DNRA outcompeted denitrification as the main nitrate (NO₃ ^?) reduction pathway, resulting in N recycling and potential spreading of eutrophication. A comparison with historical records of nutrient discharge and denitrification indicated that the total N loss in the estuary has been tightly coupled to the total amount of nutrient discharge from the STP. Changes in dissolved inorganic nitrogen (DIN) released from the STP agreed well with variations in sedimentary N₂ removal. This indicates that denitrification and anammox efficiently counterbalance N loading in the estuary across the range of historical and present-day anthropogenic nutrient discharge. Overall low N/P ratios of the regenerated nutrient fluxes impose strong N limitation for the pelagic system and generate a high potential for nuisance cyanobacterial blooms.     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans

Nitrogen stable-isotope compositions (delta15N) can help track denitrification and N2O production in the environment, as can knowledge of the isotopic discrimination, or isotope effect, inherent to denitrification. However, the isotope effects associated with denitrification as a function of dissolved-oxygen concentration and their influence on the isotopic composition of N2O are not known. We developed a simple steady-state reactor to allow the measurement of denitrification isotope effects in Paracoccus denitrificans. With [dO2] between 0 and 1.2 microM, the N stable-isotope effects of NO3- and N2O reduction were constant at 28.6 per thousand +/- 1.9 per thousand and 12.9 per thousand +/- 2.6 per thousand, respectively (mean +/- standard error, n = 5). This estimate of the isotope effect of N2O reduction is the first in an axenic denitrifying culture and places the delta15N of denitrification-produced N2O midway between those of the nitrogenous oxide substrates and the product N2 in steady-state systems. Application of both isotope effects to N2O cycling studies is discussed.     

Effect of oxygen concentration on nitrification and denitrification in single activated sludge flocs

Simultaneous nitrification and denitrification (SND) was investigated in the single aeration tank of a municipal wastewater treatment plant. Microelectrode measurements and batch experiments were performed to test for the presence of SND. Microelectrodes recorded the presence of O(2) concentration gradients in individual activated sludge flocs. When the O(2) concentration in the bulk liquid was <45 microM, anoxic zones were detected within flocs with a larger diameter (approximately 3000 microm). The O(2) penetration depth in the floc was found to be dependent on the O(2) concentration in the bulk liquid. Nitrification was restricted to the oxic zones, whereas denitrification occurred mainly in the anoxic zones. The nitrification rate of the activated sludge increased with increasing O(2) concentration in the bulk liquid, up to 40 microM, and remained constant thereafter. SND was observed in the aerated activated sludge when O(2) concentration was in the range of 10 to 35 microM.