Biosensor Determination of the Microscale Distribution of Nitrate, Nitrate Assimilation, Nitrification, and Denitrification in a Diatom-Inhabited Freshwater Sediment

High-resolution NO₃⁻ profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO₃⁻ biosensor characterized by absence of interference from chemical species other than NO₂⁻ and N₂O. Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO₃⁻ assimilation during illumination. Nitrification during darkness could be induced by purging the bulk water with O₂ gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O₂. NH₄⁺ addition did not stimulate nitrification during darkness when O₂ was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment.     

Estimation of Nitrification and Denitrification from Microprofiles of Oxygen and Nitrate in Model Sediment Systems

The coupling between nitrification and denitrification and the regulation of these processes by oxygen were studied in freshwater sediment microcosms with O₂ and NO₃^- microsensors. Depth profiles of nitrification (indicated as NO₃^- production), denitrification (indicated as NO₃^- consumption), and O₂ consumption activities within the sediment were calculated from the measured concentration profiles. From the concentration profiles, it was furthermore possible to distinguish between the rate of denitrification based on the diffusional supply of NO₃^- from the overlying water and the rate based on NO₃^- supplied by benthic nitrification (D_w and D_n, respectively). An increase in O₂ concentration caused a deeper O₂ penetration while a decrease in D_w and an increase in D_n were observed. The relative importance for total denitrification of NO₃^- produced by nitrification thus increased compared with NO₃^- supplied from the water phase. The decrease in D_w at high oxygen was due to an increase in diffusion path for NO₃^- from the overlying water to the denitrifying layers in the anoxic sediment. At high O₂ concentrations, nitrifying activity was restricted to the lower part of the oxic zone where there was a continuous diffusional supply of NH₄⁺ from deeper mineralization processes, and the long diffusion path from the nitrification zone to the overlying water compared with the path to the denitrifying layers led to a stimulation in D_n.     

Sediment Nitrification, Denitrification, and Nitrous Oxide Production in a Deep Arctic Lake

We used a combination of ¹⁵N tracer methods and a C₂H₂ blockage technique to determine the role of sediment nitrification and denitrification in a deep oligotrophic arctic lake. Inorganic nitrogen concentrations ranged between 40 and 600 nmol · cm⁻³, increasing with depth below the sediment-water interface. Nitrate concentrations were at least 10 times lower, and nitrate was only detectable within the top 0 to 6 cm of sediment. Eh and pH profiles showed an oxidized surface zone underlain by more reduced conditions. The lake water never became anoxic. Sediment Eh values ranged from −7 to 484 mV, decreasing with depth, whereas pH ranged from 6.0 to 7.3, usually increasing with depth. The average nitrification rate (49 ng of N · cm⁻³ · day⁻¹) was similar to the average denitrification rate (44 ng of N · cm⁻³ · day⁻¹). In situ N₂O production from nitrification and denitrification ranged from 0 to 25 ng of N · cm⁻³ · day⁻¹. Denitrification appears to depend on the supply of nitrate by nitrification, such that the two processes are coupled functionally in this sediment system. However, the low rates result in only a small nitrogen loss.     

Differential Inhibition by Allylsulfide of Nitrification and Methane Oxidation in Freshwater Sediment

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.     

Effects of Methane Metabolism on Nitrification and Nitrous Oxide Production in Polluted Freshwater Sediment

We report the effect of CH₄ and of CH₄ oxidation on nitrification in freshwater sediment from Hamilton Harbour, Ontario, Canada, a highly polluted ecosystem. Aerobic slurry experiments showed a high potential for aerobic N₂O production in some sites. It was suppressed by C₂H₂, correlated to NO₃^- production, and stimulated by NH₄⁺ concentration, supporting the hypothesis of a nitrification-dependent source for this N₂O production. Diluted sediment slurries supplemented with CH₄ (1 to 24 μM) showed earlier and enhanced nitrification and N₂O production compared with unsupplemented slurries (≤1 μM CH₄). This suggests that nitrification by methanotrophs may be significant in freshwater sediment under certain conditions. Suppression of nitrification was observed at CH₄ concentrations of 84 μM and greater, possibly through competition for O₂ between methanotrophs and NH₄⁺ -oxidizing bacteria and/or competition for mineral N between these two groups of organisms. In Hamilton Harbour sediment, the very high CH₄ concentrations (1.02 to 6.83 mM) which exist would probably suppress nitrification and favor NH₄⁺ accumulation in the pore water. Indeed, NH₄⁺ concentrations in Hamilton Harbour sediment are higher than those found in other lakes. We conclude that the impact of CH₄ metabolism on N cycling processes in freshwater ecosystems should be given more attention.     

乙炔抑制法在硝化与反硝化过程中的应用

硝化和反硝化作用在土壤氮素循环中扮演重要作用,由于硝化和反硝化作用一方面能够导致土壤中氮素的损失,另一方面能够产生温室气体-N2O,所以硝化和反硝化作用的研究备受关注.乙炔抑制法能同时测定硝化和反硝化作用,在硝化和反硝化作用中有着重要的应用.该文主要论述了乙炔抑制法的研究进展;以及对应用乙炔气体时存在的一些问题进行了综述.     

Free Cyanide Sorption on Freshwater Sediment and Model Components

The sorption of free cyanide (HCN) on mineral components of sediment, activated carbon, and a freshwater sediment was studied via batch experiments in synthetic freshwater at pH 6.4–7.6. It was found that free cyanide did not sorb to any significant extent on sediment mineral components, but did sorb strongly to activated carbon and moderately to a freshwater sediment. Results of experiments with 100 and 150 μ g/L free cyanide spike amounts resulted in no observed sorption to kaolin clay, Ottawa sand, or alumina in the synthetic freshwater. Extensive removal of free cyanide from the aqueous phase was observed in the partitioning experiments with the powdered activated carbon. Results with whole sediment indicated up to 46% removal of free cyanide from the aqueous phase in experiments with 20 and 50 μ g/L free cyanide. The organic-carbon-normalized distribution coefficient K_oc (= C_s/C_wf_oc) for free cyanide sorption on the activated carbon (f_oc = 1.0), 4.2 L/g_s, was similar to the K_oc value for free cyanide sorption on the freshwater sediment (f_oc = 0.0031), approximately 12.9 L/g_s. The results indicate that free cyanide can sorb to sediments with organic carbon content under freshwater conditions, primarily through interaction of HCN with organic carbon in the sediment.     

Weak Coupling between Heterotrophic Nanoflagellates and Bacteria in a Eutrophic Freshwater Environment

In a study on the dynamics and trophic role of the heterotrophic nanoflagellate (HNAN) assemblage in the microbial food web of a eutrophic oxbow lake abundances, biomass, and production rates of HNAN and their potential prey organisms, namely heterotrophic bacteria and autotrophic picoplankton, were monitored for a period of 2 years. No coupling between HNAN abundance and biomass and the abundance and biomass of their picoplanktonic prey was observed for the investigation period. The ratio of heterotrophic bacterial to HNAN abundance ranged from 2.2 x 103 to 8.6 x 103 (mean: 4.2 x 103 +/- 1.8 x 103). HNAN carbon consumption could account for only 10% to 40% of bacterial secondary production. The lack of coupling between HNAN and their potential prey and the low HNAN abundance relative to bacterial abundance suggested (a) that HNAN grazing was an insignificant factor in the regulation of bacterial abundance and (b) that HNAN abundance was regulated by predation rather than by prey abundance. This hypothesis was supported by the fact that HNAN growth rates were high (in the range of 0.45 d-1 to 1.00 d-1 during spring and summer, yearly mean: 0.52 d-1), and only weakly correlated with prey abundance and biomass. The results indicated strong top-down control of HNAN and consequently a weak coupling of HNAN and picoplankton in the investigated eutrophic freshwater environment.     

Distinguishing between Nitrification and Denitrification as Sources of Gaseous Nitrogen Production in Soil

The source of N₂O produced in soil is often uncertain because denitrification and nitrification can occur simultaneously in the same soil aggregate. A technique which exploits the differential sensitivity of these processes to C₂H₂ inhibition is proposed for distinguishing among gaseous N losses from soils. Denitrification N₂O was estimated from 24-h laboratory incubations in which nitrification was inhibited by 10-Pa C₂H₂. Nitrification N₂O was estimated from the difference between N₂O production under no C₂H₂ and that determined for denitrification. Denitrification N₂ was estimated from the difference between N₂O production under 10-kPa C₂H₂ and that under 10 Pa. Laboratory estimates of N₂O production were significantly correlated with in situ N₂O diffusion measurements made during a 10-month period in two forested watersheds. Nitrous oxide production from nitrification was most important on well-drained sites of a disturbed watershed where ambient NO₃⁻ was high. In contrast, denitrification N₂O was most important on poorly drained sites near the stream of the same watershed. Distinction between N₂O production from nitrification and denitrification was corroborated by correlations between denitrification N₂O and water-filled pore space and between nitrification N₂O and ambient NO₃⁻. This technique permits qualitative study of environmental parameters that regulate gaseous N losses via denitrification and nitrification.     

Coupling between Marine Plankton and Freshwater Flow in the Plumes off a Small Estuary

Freshwater discharge from rivers is a powerful forcing agent in coastal ecosystems. It not only generates strong ecological effects in estuaries, but also drives the dynamics of nearshore marine waters where prominent river plumes form biogeochemical hot spots in coastal seas worldwide. Large plumes from major rivers exert important controls on pelagic processes. The majority of estuaries are smaller, however, and the importance of the smaller plumes they generate is unknown. We measured the degree of coupling between freshwater flow and inshore zooplankton in such a plume from a subtropical estuary on the east coast of Australia. Flow regimes encompassed long periods of low freshwater input, punctuated by pulsed freshets that initiated the formation of buoyant, lower‐salinity plumes in the nearshore marine zone. Plumes stimulated phytoplankton biomass in the receiving waters, and ultimately changes in zooplankton assemblages. Zooplankton responded strongly to river discharge: (1) in the absence of substantial freshwater flows and plumes, zooplankton was broadly similar in density and biomass across the estuarine‐marine gradient; (2) freshets that generated significant plumes strongly modified hydrological conditions and lowered zooplankton in the estuarine and nearshore waters, and (3) after the initial freshet, zooplankton in the residual plume was at a higher density in nearshore than shelf waters. We demonstrate that coupling between riverine and coastal pelagic systems operates in small plumes, but that there is substantial temporal variance linked to fluctuations in freshwater delivery. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

异养硝化细菌脱氮特性及研究进展

异养硝化细菌能够在利用有机碳源生长的同时将含氮化合物硝化生成羟胺、亚硝酸盐、硝酸盐等产物, 多数还能同时进行好氧反硝化作用, 直接将硝化产物转化为含氮气体。因此, 这类细菌已成为废水处理中生物脱氮新工艺的重要研究对象。本文综述了目前所分离出的一些异养硝化菌的脱氮特性, 分析了各种环境条件如温度、pH、溶解氧、碳源类型、C/N以及抑制剂等对异养硝化菌的影响, 并介绍了异养硝化菌的应用现状及前景。     

Estimates of Denitrification and Nitrification in Coastal and Estuarine Sediments

Denitrification and nitrification in sediments of Tama Estuary and Odawa Bay, Japan, were investigated by the combined use of a continuous-flow sediment-water system and a ¹⁵N tracer technique. At Odawa Bay, the nitrification rate was comparable to the nitrate reduction rate, and 70% of the N₂ evolved originated from nitrogenous oxides (nitrate and nitrite) which were produced by the action of nitrifying bacteria in the sediments. At Tama Estuary, the nitrate reduction rate was 11 to 17 times higher than the nitrification rate, and nitrogenous oxides derived from ammonium accounted for only 6 to 9% of the N₂ evolution by denitrification.     

Denitrification, Dissimilatory Reduction of Nitrate to Ammonium, and Nitrification in a Bioturbated Estuarine Sediment as Measured with 15N 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 ¹⁵N isotope (NO₃^-), specific inhibitor (C₂H₂), and microsensor (N₂O and O₂) techniques in a continuous-flow core system. The estuarine water was NO₃^- rich (125 to 600 μM), and NO₃^- was consistently taken up by the sediment on the four occasions studied. Total NO₃^- uptake (3.6 to 34.0 mmol of N m^-2 day^-1) corresponded closely to N₂ production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO₃^- reduction to NH₄⁺ was insignificant. When C₂H₂ was applied in the flow system, denitrification measured as N₂O production was often less (58 to 100%) than the NO₃^- uptake because of incomplete inhibition of N₂O reduction. The NO₃^- formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from ¹⁵NO₃^- dilution and from changes in NO₃^- uptake before and after C₂H₂ addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m^-2 day^-1 on the four occasions. Attempts to measure total nitrification activity by the difference between NH₄⁺ fluxes before and after C₂H₂ addition failed because of non-steady-state NH₄⁺ fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N₂O and O₂ microelectrodes. The N₂O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N₂O after C₂H₂ 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 O₂ uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min⁻¹ mg of protein⁻¹. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a “bottleneck” in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be “poor” nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.     

Isolation and Characterization of Acetate-Utilizing Anaerobes from a Freshwater Sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5–1 μm × 3–4 μm long). Doubling times with acetate at 30–35°C were 5.6–8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related toMethanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5–0.7 μm × 1.5–3.5 μm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30°C the doubling times with acetate were 3.9–5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H₂, CO₂, and sulfate. The closest relative of strain ASRB-Zg isDesulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5–0.7 μm × 0.7–1 μm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20–25°C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H₂, CO₂, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg isVariovorax paradoxus.     

一株好氧反硝化-异养硝化菌的筛选及脱氮特性研究

针对渗滤液中硝酸盐氮和亚硝酸盐氮难以转化的问题,从好氧生物反应器填埋场中筛选出一株好氧反硝化菌-异养硝化菌HN,初步鉴定该菌为假单胞菌(Pseudomonas)。通过对不同氮源的生物转化作用研究了HN的脱氮特性,并对其脱氮条件进行了优化。结果表明,在有氧条件下,以硝酸钾为唯一氮源,HN在96 h时对硝酸盐氮的去除率达到95.44%;以硫酸铵为唯一氮源时,HN在60 h时对氨氮的去除率达到85.14%;当碳源为乙醇,碳氮比为7∶1,初始p H为7.5,温度为35℃,接种量为10%时,菌株HN脱氮效率最高。     

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.     

Annual Pattern of Denitrification and Nitrate Ammonification in Estuarine Sediment

The seasonal variation and depth distribution of the capacity for denitrification and dissimilatory NO₃⁻ reduction to NH₄⁺ (NO₃⁻ ammonification) were studied in the upper 4 cm of the sediment of Norsminde Fjord estuary, Denmark. A combination of C₂H₂ inhibition and ¹⁵N isotope techniques was used in intact sediment cores in short-term incubations (maximum, 4 h). The denitrification capacity exhibited two maxima, one in the spring and one in the fall, whereas the capacity for NO₃⁻ ammonification was maximal in the late summer, when sediments were progressively reduced. The denitrification capacity was always highest in the uppermost 1 cm of the sediment and declined with depth. The NO₃⁻ ammonification was usually higher with depth, but the maximum activity in late summer was observed within the upper 1 cm. The capacity for NO₃⁻ incorporation into organic material was investigated on two occasions in intact sediment cores and accounted for less than 5% of the total NO₃⁻ reduction. Denitrification accounted for between 13 and 51% of the total NO₃⁻ reduction, and NH₄⁺ production accounted for between 4 and 21%, depending on initial rates during the time courses. Changes of the rates during the incubation were observed in the late summer, which reflected synthesis of denitrifying enzymes. This time lag was eliminated in experiments with mixed sediment because of preincubation with NO₃⁻ and alterations of the near-environmental conditions. The initial rates obtained in intact sediment cores therefore reflect the preexisting enzyme content of the sediment.     

Assessing Nitrification and Denitrification in the Seine River and Estuary Using Chemical and Isotopic Techniques

Downstream from metropolitan Paris (France), a large amount of ammonium is discharged into the Seine River by the effluents of the wastewater treatment plant at Achères. To assess the extent of nitrification and denitrification in the water column, concentrations and isotopic compositions of ammonium (δ¹⁵N–NH₄⁺) and nitrate (δ¹⁵N–NO₃⁻, δ¹⁸O–NO₃⁻) were measured during summer low-flow conditions along the lower Seine and its estuary. The results indicated that most of the ammonium released from the wastewater treatment plant is nitrified in the lower Seine River and its upper estuary, but there was no evidence for water-column denitrification. In the lower part of the estuary, however, concentration and isotopic data for nitrate were not consistent with simple mixing between riverine and marine nitrate. A significant departure of the nitrate isotopic composition from what would be expected from simple mixing of freshwater and marine nitrates suggested coupled nitrification and denitrification in the water, in spite of the apparent conservative behavior of nitrate. Denitrification rates of approximately 0.02 mg N/L/h were estimated for this part of the estuary.     

Denitrification Potential in Lake Sediment Increases Across a Gradient of Catchment Agriculture

Intensification of catchment agriculture has increased nutrient loads and accelerated eutrophication in some lakes, often resulting in episodic harmful algal blooms or prolonged periods of anoxia. The influence of catchment agriculture on lake sediment denitrification capacity as a nitrogen (N) removal mechanism in lakes is largely unknown, particularly in contrast to research on denitrification in agricultural streams and rivers. We measured denitrification enzyme activity (DEA) to assess sediment denitrification potential in seven monomictic and three polymictic lakes that range in the proportion of agriculture in the catchment from 3 to 96% to determine if there is a link between agricultural land use in the lake catchment and sediment denitrification potential. We collected sediment cores for DEA measurements over 3 weeks in austral spring 2008 (October–November). Lake Okaro, with 96% catchment agriculture, had approximately 15 times higher DEA than Lake Tikitapu, with 3% catchment agriculture (232.2 ± 55.9 vs. 15.9 ± 4.5 μg N gAFDM⁻¹ h⁻¹, respectively). Additionally, sediment denitrification potential increased with the proportion of catchment in agriculture (R ² = 0.85, P < 0.001). Our data suggest that lakes retain a high capacity to remove excess N via denitrification under increasing N loads from higher proportions of catchment agriculture. However, evidence from the literature suggests that despite a high capacity for denitrification and longer water residence times, lakes with high N loads will still remove a smaller proportion of their N load. Lakes have a denitrification potential that reflects the condition of the lake catchment, but more measurements of in situ denitrification rates across lake catchments is necessary to determine if this capacity translates to high N removal rates.