Impact of bacterial NO3(-) transport on sediment biogeochemistry

Experiments demonstrated that Beggiatoa could induce a H2S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO3(-) reduction from denitrification to dissimilatory NO3(-) reduction to ammonium. pH, O2, and H2S profiles indicated that the bacteria oxidized H2S with NO3(-) and transported S0 to the sediment surface for aerobic oxidation.     

Denitrification, dissimilatory reduction of nitrate to ammonium, and nitrification in a bioturbated estuarine sediment as measured with N and microsensor techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of N isotope (NO(3)), specific inhibitor (C(2)H(2)), and microsensor (N(2)O and O(2)) techniques in a continuous-flow core system. The estuarine water was NO(3) rich (125 to 600 muM), and NO(3) was consistently taken up by the sediment on the four occasions studied. Total NO(3) uptake (3.6 to 34.0 mmol of N m day) corresponded closely to N(2) production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO(3) reduction to NH(4) was insignificant. When C(2)H(2) was applied in the flow system, denitrification measured as N(2)O production was often less (58 to 100%) than the NO(3) uptake because of incomplete inhibition of N(2)O reduction. The NO(3) formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from NO(3) dilution and from changes in NO(3) uptake before and after C(2)H(2) addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m day on the four occasions. Attempts to measure total nitrification activity by the difference between NH(4) fluxes before and after C(2)H(2) addition failed because of non-steady-state NH(4) fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N(2)O and O(2) microelectrodes. The N(2)O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N(2)O after C(2)H(2) was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O(2) uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Community-specific pH response of denitrification: experiments with cells extracted from organic soils

Denitrifying prokaryotes are phylogenetically and functionally diverse. Little is known about the relationship between soil denitrifier community composition and functional traits. We extracted bacterial cells from three cultivated peat soils with contrasting native pH by density gradient centrifugation and investigated their kinetics of oxygen depletion and NO2 -, NO, N(2) O and N(2) accumulation during initially hypoxic batch incubations (0.5-1 μM O(2)) in minimal medium buffered at either pH 5.4 or 7.1 (2 mM glutamate, 2 mM NO3 -). The three communities differed strikingly in NO2 - accumulation and transient N(2) O accumulation at the two pH levels, whereas NO peak concentrations (24-53 nM) were similar across all communities and pH treatments. The results confirm that the communities represent different denitrification regulatory phenotypes, as indicated by previous denitrification bioassays with nonbuffered slurries of the same three soils. The composition of the extracted cells resembled that of the parent soils (PCR-TRFLP analyses of 16S rRNA genes, nirK, nirS and nosZ), which were found to differ profoundly in their genetic composition (Braker et al., ). Together, this suggests that direct pH response of denitrification depends on denitrifier community composition, with implications for the propensity of soils to emit N(2) O to the atmosphere.     

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2(-) and O2 concentrations.

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at 相似文献   

Vertical distribution of denitrification in an estuarine sediment: integrating sediment flowthrough reactor experiments and microprofiling via reactive transport modeling

Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N(2)O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N(2)O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm(-3) h(-1). The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (+/- 10) nmol cm(-2) h(-1), which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.     

Small-scale distribution of interstitial nitrite in freshwater sediment microcosms: the role of nitrate and oxygen availability,and sediment permeability

The spatial distribution of interstitial NO2(-) concentrations was studied in NO3(-)-exposed freshwater sediment microcosms, using pore water extractions as well as ion-selective microsensors. Porewater extractions revealed ecotoxicologically critical NO2(-) concentrations in hypoxic and anoxic sediment layers in which significant NO3(-) consumption took place. In contrast, the use of ion-selective microsensors demonstrated the high capacity of the thin oxic surface layer of the sediments to consume NO2(-) and to produce NO3(-). Two modes of NO3(-) supply to the sediments were compared: In treatments with NO3(-) supply to the overlying water, a subsurface maximum of NO2(-) concentration was observed, coinciding with the site of maximum NO3(-) consumption. When NO3(-) was perfused up through the sediment cores, however, NO2(-) accumulated throughout the entire sediment column. Such spatially extensive NO2(-) accumulations were only observed in sediments poor in organic matter with a relatively high permeability. By manipulating the O2 content of the overlying water, the release of NO2(-) from the sediments could be influenced: In treatments with air-saturated overlying water, the sediments did not release detectable amounts of NO2(-) into the water phase. When kept hypoxic (25% air saturation) instead, significant NO2(-) accumulations were recorded in the overlying water. These findings suggest that in treatments with air-saturated overlying water, NO2(-) that was produced in deeper sediment layers (denitrifying conditions) was completely consumed at the oxic sediment surface (nitrifying conditions) before it could reach the overlying water.     

Nitrous oxide emissions from denitrifying activated sludge of urban wastewater treatment plants, under anoxia and low oxygenation

Batch experiments were made to better understand the mechanisms of N2O emissions from activated sludge in denitrifying conditions found in urban WWTPs, i.e. under anoxic and low oxygenation conditions. The results showed that in completely anoxic conditions, denitrification, related to a periplasmic nitrate reductase activity, is the major producer of N2O (100% of the N2O production), whereas the nitrate ammonifying activity is not significant. In a gradient of low oxygenation, the highest N2O emissions (49.7+/-3.8 microg N2O-N/g SS/h on average) occurred at a dissolved-oxygen concentration of around 0.3mg O2/L. Below 0.3mg O2/L, heterotrophic denitrification appeared to be the major process responsible for the N2O emission (100% at zero oxygenation). From 0.4 to 1.1mg O2/L, N2O emissions were due to two processes: (i) heterotrophic denitrification that represented about 40% of the N2O production, and (ii) autotrophic nitrifier denitrification that accounted for about 60%. The N2O emissions from activated sludge represented on average 0.4% of reduced NO3(-) in anoxic conditions. The N2O emissions associated with denitrification of entire nitrogen load would amount to 155 T N2O-N/year, if all the Paris wastewater was treated by a process using activated sludge.     

Nitrous oxide as end product of denitrification by strains of fluorescent pseudomonads.

Growing cultures of several strains of Pseudomonas fluorescens and Pseudomonas chlororaphis produced N2O as the only detectable gaseous product of denitrification, and other strains produced N2 as the gaseous end product of denitrification. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2O-producing strains P. fluorescens PJ 185 and P. chlororaphis B-560 was recovered as N2O. All of the nitrogen in NO3- or NO2- added to cell suspensions of the N2-producing strain P. fluorescens PJ70 was converted to N2. Cell extracts of P. fluorescens PJ 70, PJ 185, and P. chlororaphis B-560 exhibited NO3- reductase activity when sodium succinate was the electron donor. Reduced nicotinamide adenine dinucleotide and flavine adenine dinucleotide were required to demonstrate NO2- reductase activity in cell extracts.     

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.     

Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus

We screened actinomycete strains for dinitrogen (N(2))-producing activity and discovered that Streptomyces antibioticus B-546 evolves N(2) and some nitrous oxide (N(2)O) from nitrate (NO(3)(-)). Most of the N(2) that evolved from the heavy isotope ([(15)N]NO(3)(-)) was (15)N(14)N, indicating that this nitrogen species consists of two atoms, one arising from NO(3)(-) and the other from different sources. This phenomenon is similar to codenitrification in fungi. The strain also evolved less, but significant, amounts of (15)N(15)N from [(15)N]NO(3)(-) in addition to (15)N(15)NO with concomitant cell growth. Prior to the production of N(2) and N(2)O, NO(3)(-) was rapidly reduced to nitrite (NO(2)(-)) accompanied by distinct cell growth, showing that the actinomycete strain is a facultative anaerobe that depends on denitrification and nitrate respiration for anoxic growth. The cell-free activities of denitrifying enzymes could be reconstituted, supporting the notion that the (15)N(15)N and (15)N(15)NO species are produced by denitrification from NO(3)(-) via NO(2)(-). We therefore demonstrated a unique system in an actinomycete that produces gaseous nitrogen (N(2) and N(2)O) through both denitrification and codenitrification. The predominance of codenitrification over denitrification along with oxygen tolerance is the key feature of nitrate metabolism in this actinomycete.     

The impact of desiccation of a freshwater marsh (Garcines Nord,Camargue, France) on sediment-water-vegetation interactions

The impact of desiccation on a marsh sediment was studied both in the laboratory and in the field. Changes in the sediment chemistry of a homogenized sediment suspension during desiccation were studied in the laboratory. FeS was oxidized completely. A considerable mineralization of organic phosphate took place, from both the acid soluble organic phosphate fraction and from the residual organic phosphate fraction, but no significant mineralization of organic matter was observed. The o-P formed during the mineralization was recovered partly in the Fe(OOH) ≈P fraction and partly in the CaC₃≈P fraction. An upward flux was found. During spring and summer 1990 the water inlet to a shallow permanent freshwater marsh with a surface of about 1.5 hectares was blocked, in order to desiccate the marsh by evaporation. The sediments initially consisted of a black anoxic organic top layer and a less organic anoxic gray layer. During the desiccation of the sediment a brown oxic surface layer was formed from the black layer and an increase of pH and Eh occurred. Subsequent rainfall made the Eh increase further but caused a decrease in pH indicating an increase in bacterial activity. A progressive oxidation of FeS was observed. An increase in Tot-P in the surface layer and a decrease in the gray and the black layer of the sediment occurred, probably due to a capillary upward flux. A mineralization of organic matter was observed in the two deeper layers. In the upper brown layer this mineralization was less evident, probably because it has been masked by the capillary movement. A net C loss of 40% was calculated to have occurred in the layer 0–40 cm. In the deeper layers a decrease in Tot-N was observed, whereas no important increase occurred in the top layer. Over a sediment layer of 40 cm a N loss of 50% was calculated. C- and N losses occurred simultaneously, suggesting the importance of mineralization as a source of inorg-N for denitrification. The chemical and physical changes in the sediment during desiccation affected layers down to 40 cm. This means that not only the top layer of a sediment but also deeper layers are active in systems of which part of the sediment dries occasionally. Fractionation of the surface sediment phosphate showed an increase of Fe(OOH) ≈ P in the top layer due to the oxidation of FeS to Fe(OOH), enlarging the P-adsorption capacity of the sediment. A mineralization of about 50% of acid soluble organic phosphate occurred. After rainfall, a net increase in residual organic phosphate occured presumably due to an increase of bacterial activity. Drying may therefore be utilized as a tool, in wetland management, to eliminate organic nitrogen and carbon from the sediment. In rice culture, it may be used to make part of the organic nitrogen available to the rice.     

Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity

Nitrous oxide production by Escherichia coli seems to result from the reduction of NO2- by NO3- reductase. This hypothesis is consistent with previous observations and with the observation that molybdenum was required for both NO3- reduction and N2O production. Several E. coli NO3- reductase mutants were assayed for both N2O-producing and NO3--reducing activity. The hypothesized role of NO3- reductase is supported by the correlation of these two activities. Nitrate reduction to NH4+ enhanced growth, but NO2- reduction to N2O apparently did not. Therefore, this process differs significantly from respiratory denitrification.     

Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

Denitrifying prokaryotes use NO(x) as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N(2)O and N(2), depending on the relative activity of the enzymes catalysing the stepwise reduction of NO(3)(-) to N(2)O and finally to N(2). Cultured denitrifying prokaryotes show characteristic transient accumulation of NO(2)(-), NO and N(2)O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N(2)O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N(2)O produced (N(2)O/(N(2)+N(2)O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N(2)O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N(2)O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level.     

Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity.

Nitrous oxide production by Escherichia coli seems to result from the reduction of NO2- by NO3- reductase. This hypothesis is consistent with previous observations and with the observation that molybdenum was required for both NO3- reduction and N2O production. Several E. coli NO3- reductase mutants were assayed for both N2O-producing and NO3--reducing activity. The hypothesized role of NO3- reductase is supported by the correlation of these two activities. Nitrate reduction to NH4+ enhanced growth, but NO2- reduction to N2O apparently did not. Therefore, this process differs significantly from respiratory denitrification.     

Nitrate and nitrite microgradients in barley rhizosphere as detected by a highly sensitive denitrification bioassay.

A highly sensitive denitrification bioassay was developed for detection of NO3- and NO2- in rhizosphere soil samples. Denitrifying Pseudomonas aeruginosa ON12 was grown anaerobically in citrate (30 mM) minimal medium with KClO3 (10 mM) and NaNO2 (3 mM), which gave cells capable of NO2- reduction to N2O but incapable of NO3- reduction to NO2-. Growth on citrate minimal medium further resulted in the absence of N2O reduction. When added to small soil samples in O2-free vials, such cells could be used to convert the indigenous NO2- pool to N2O, which was subsequently quantified by gas chromatography. Cells grown in KClO3-free citrate medium with 10 mM NaNO3 as the electron acceptor were capable of reducing both NO3- and NO2-, and these cells could subsequently be added to the sample to convert the indigenous NO3- pool to N2O. Concentrations of both NO3- and NO2- were thus determined as N2O, with a detection limit of approximately 10 pmol of N. The bioassay could be used to determine NO3- and NO2- pools in 10-mg soil samples taken along a microgradient in the rhizosphere of field-grown barley plants. At both low (10%, wt/wt) and high (18%, wt/wt) water content, relatively high levels of NO2- were found in the rhizosphere compared with bulk soil. Under dry conditions, NO3- was also more abundant in the rhizosphere than in the bulk soil, whereas such a difference was not observed at the high water content. The roles of plant metabolism and bacterial nitrification and denitrification processes for NO3- and NO2- availability in the rhizosphere are discussed.     

Nitrate and nitrite microgradients in barley rhizosphere as detected by a highly sensitive denitrification bioassay.

A highly sensitive denitrification bioassay was developed for detection of NO3- and NO2- in rhizosphere soil samples. Denitrifying Pseudomonas aeruginosa ON12 was grown anaerobically in citrate (30 mM) minimal medium with KClO3 (10 mM) and NaNO2 (3 mM), which gave cells capable of NO2- reduction to N2O but incapable of NO3- reduction to NO2-. Growth on citrate minimal medium further resulted in the absence of N2O reduction. When added to small soil samples in O2-free vials, such cells could be used to convert the indigenous NO2- pool to N2O, which was subsequently quantified by gas chromatography. Cells grown in KClO3-free citrate medium with 10 mM NaNO3 as the electron acceptor were capable of reducing both NO3- and NO2-, and these cells could subsequently be added to the sample to convert the indigenous NO3- pool to N2O. Concentrations of both NO3- and NO2- were thus determined as N2O, with a detection limit of approximately 10 pmol of N. The bioassay could be used to determine NO3- and NO2- pools in 10-mg soil samples taken along a microgradient in the rhizosphere of field-grown barley plants. At both low (10%, wt/wt) and high (18%, wt/wt) water content, relatively high levels of NO2- were found in the rhizosphere compared with bulk soil. Under dry conditions, NO3- was also more abundant in the rhizosphere than in the bulk soil, whereas such a difference was not observed at the high water content. The roles of plant metabolism and bacterial nitrification and denitrification processes for NO3- and NO2- availability in the rhizosphere are discussed.     

Denitrification Rates in a Marine Sediment as Measured by the Acetylene Inhibition Technique

A method has been developed for measurement of denitrification activity in sediments by application of the acetylene inhibition technique. Acetylene-saturated water was injected, at close intervals, into sediment cores which were then incubated for a few hours at the in situ temperature. Frozen segments of the cores were assayed for accumulation of N₂O by a combined gas extraction and detection system. The segments were thawed under a stream of helium from which N₂O (and other gases) was collected in a liquid N₂ trap, and the quantity of N₂O was measured by gas chromatography. The maximum rate of denitrification in a coastal marine sediment was 35 nmol of N per cm³ of sediment per day at 2.5°C, and the rate of denitrification for the total sediment was 0.99 nmol of N per m² per day.     

Ecology of Thioploca spp.: nitrate and sulfur storage in relation to chemical microgradients and influence of Thioploca spp. on the sedimentary nitrogen cycle.

Microsensors, including a recently developed NO3(-) biosensor, were applied to measure O(2) and NO3(-) profiles in marine sediments from the upwelling area off central Chile and to investigate the influence of Thioploca spp. on the sedimentary nitrogen metabolism. The studies were performed in undisturbed sediment cores incubated in a small laboratory flume to simulate the environmental conditions of low O(2), high NO3(-), and bottom water current. On addition of NO3(-) and NO2(-), Thioploca spp. exhibited positive chemotaxis and stretched out of the sediment into the flume water. In a core densely populated with Thioploca, the penetration depth of NO3(-) was only 0.5 mm and a sharp maximum of NO3(-) uptake was observed 0.5 mm above the sediment surface. In sediments with only few Thioploca spp., NO3(-) was detectable down to a depth of 2 mm and the maximum consumption rates were observed within the sediment. No chemotaxis toward nitrous oxide (N2O) was observed, which is consistent with the observation that Thioploca does not denitrify but reduces intracellular NO3(-) to NH(4)(+). Measurements of the intracellular NO3(-) and S(0) pools in Thioploca filaments from various depths in the sediment gave insights into possible differences in the migration behavior between the different species. Living filaments containing significant amounts of intracellular NO3(-) were found to a depth of at least 13 cm, providing final proof for the vertical shuttling of Thioploca spp. and nitrate transport into the sediment.     

Temporal Variation of Denitrification Activity in Plant-Covered, Littoral Sediment from Lake Hampen, Denmark

Diel and seasonal variations in denitrification were determined in a littoral lake sediment colonized by the perennial macrophyte Littorella uniflora (L.) Aschers. In the winter, the activity was low (5 μmol of N m⁻² h⁻¹) and was restricted to the uppermost debris layer at a depth of 0 to 1 cm. By midsummer, the activity increased to 50 μmol of N m⁻² h⁻¹ and was found throughout the root zone to a depth of 10 cm. The root zone accounted for as much as 50 to 70% of the annual denitrification. A significant release of organic substrates from the roots seemed to determine the high activities of root zone denitrification in the summer. The denitrification in the surface layer and in the root zone formed two distinct activity zones in the summer, when the root zone also contained nitrification activity, as indicated from the accumulations of NO₃⁻. Light conditions inhibited denitrification in both the surface layer and the upper part of the root zone, suggesting that a release of O₂ by benthic algae and from the roots of L. uniflora controlled a diel variation of denitrification. In midsummer, the rate of denitrification in both the surface layer and the upper part of the root zone was limited by NO₃⁻. In the growth season, there was evidence for a significant population of denitrifiers closely associated with the root surface.