μM Nitrate Research Articles

Influence of upwelling and river runoff interaction on phytoplankton assemblages in a Middle Galician Ria and Comparison with northern and southern rias (NW Iberian Peninsula)

The first oceanographic research (hydrography, nutrient salts, chlorophyll, primary production and phytoplankton assemblages) in a Middle Galician Ria was carried out in Corme-Laxe during 2001, just a year before the Prestige oil spill, being the only reference to evaluate eventual changes in the phytoplankton community. Due to the small size of this ria (6.5 km 2), oceanographic processes were driven by the continental water supplied by Anllons River during the wet season (20–30 m 3 s −1 in winter), and the strong oceanic influence from the nearby shelf during the dry season. The annual cycle showed a spring bloom with high levels of chlorophyll (up to 14 μg Chl- a L −1) and primary production (3 g C m −2 d −1) and a summer upwelling bloom (up to 8 μg Chl- a L −1 and 10 g C m −2 d −1) where the proximity of the Galician upwelling core (<13.5 °C at sea surface) favors the input of upwelled seawater (up to 9 μM of nitrate and silicate) to the bottom ria layer, even during summer stratification events (primary production around 2 g C m −2 d −1). Thus, phytoplankton assemblages form a “continuum” from spring to autumn with a predominance of diatoms and overlapping species between consecutive periods; only in autumn dinoflagellates and flagellates characterized the phytoplankton community. In the Middle Rias as Corme-Laxe, the nutrient values, Chl -a, primary production and phytoplankton abundance for productive periods were higher than those reported for the Northern (Ria of A Coruña) and Southern Rias (Ria of Arousa) for year 2001; this suggests the importance of the hydrographic events occurring in the zone of maximum upwelling intensity of the Western Iberian Shelf, where a lack of annual cycles studies exists.

N:Si ratios and the ‘balance of organisms’: PROWQM simulations of the northern North Sea

This paper, first presented at the EutWork meeting, includes short reviews of (i) the meaning of balance of organisms in the context of eutrophication and (ii) the simulation of the balance with numerical models. The ratio ψ of flagellate chlorophyll to total chlorophyll is used as a measure of the balance. The PROVESS Water Quality Model (PROWQM) was used to investigate the control of ψ by simulating changes during 1998 in water-column layering and the abundance of diatom-or flagellate-dominated microplankton communities at 59°N, 01°E, in the northern North Sea. This was the site of a study by the PROVESS project in autumn 1998 and near that of the FLEX investigation in spring 1976. Although not considered to be at risk of eutrophication, these waters have a high natural N:Si ratio and provide a good subject for numerical experiments concerning the control of the balance of organisms. PROWQM is a 1-D physical-biological model; its 2 microplankton compartments parameterise the microbial loop and take account of the metabolism of pelagic bacteria and protozoa as well as the photosynthesis, respiration and nutrient uptake of micro-algae. The effect of an initialisation with the long-term mean observed winter concentrations of nutrients in the northern North Sea (10 μM nitrate and 5 μM silicate) was compared with an initialisation with the same amount of nitrate but only 3 μM silicate. Simulations were run with the standard value of the surface drag coefficient and a doubled value. The latter resulted in a more accurate representation of the surface mixed layer, but did not show a midwater maximum of flagellate chlorophyll during summer. The simulation with doubled drag and initial N:Si of 10:5 was in good agreement with the long-term observed seasonal cycle of chlorophyll. Simulations with initial N:Si of 10:3 had substantially more flagellates during summer, as a result of diatom limitation by silica exhaustion. The model results support suggestions that the winter N:Si ratio must be perturbed to greater than 3:1 before the balance of organisms is much changed, if the balance is quantified by peak biomass or annual production. However, annual mean ψ was more influenced by physical conditions than by nutrient initialisation, showing that how data are analysed can have an influence on conclusions about whether the balance is disturbed. The simulated removal of nutrient up to the time of maximum chlorophyll was used to estimate values for diatoms and flagellates of the yield, q, of chlorophyll from nitrate or silicate. The silicate value (0.77 g chl (mol Si) − 1 in the best simulation) is novel. The yields from nitrate were 0.23–0.75 g chl (mol N) − 1, the range including both groups and all simulations. These were less than yields reported for inshore waters. Finally, the need for detailed study of the distributions of pelagic microflora and microfauna in late summer in the northern North Sea is pointed out.

The impact of terrigeneous inputs on the Bay of Ouinné (New Caledonia) phytoplankton communities: A spectrofluorometric and microscopic approach

The impact of terrigeneous inputs on the phytoplankton composition was studied during a 2.5 months daily survey in the Bay of Ouinné, a deep cove on the south-eastern coast of New Caledonia. Surface waters benefited from nutrients originating from nearby land drainage and the Ouinné river outflow during periods of heavy rain. The nutrient composition mirrored the composition of the drained soils, with concentrations reaching 3–4 μM for nitrate and 0.13 μM for phosphate at the river mouth. In addition to nutrient inputs, significant quantities of particulate matter (inorganic and organic compounds) were discharged into the lagoon during heavy rain periods, resulting in transitory decreases of the photic layer depth and enrichments of the water column through remineralization processes. Changes in contributions of the main phytoplankton groups in response to terrigeneous inputs were shown by chlorophyll and phycobiliprotein spectrofluorometric analyses. While dry periods were marked by the dominance of pico- ( Prochlorococcus, and high-phycourobilin (PUB) Synechococcus) and microcyanobacteria ( Trichodesmium spp.), developments of various eukaryote populations resulted from land drainage occurring during the wet periods. This was indicated by the increase of accessory chlorophyll pigments that doubled at a depth of 15 m: chlorophyll b (chlorophytes), chlorophyll c 1+ c 2 (associated with diatoms and dinoflagellates), chlorophyll c 3 (associated with c 1 and/or c 2 in prymnesiophytes, chrysophytes and/or pelagophytes). In addition, Synechococcus with a high phycoerythrobilin (PEB) content also appeared to be stimulated by river outputs. Finally, microscopic observations of the >35 μm net plankton confirmed the greater presence of diatoms and dinoflagellates during periods of rain compared to Trichodesmium, particularly in the surface layer.

Increased reproductive toxicity of landfill leachate after degradation was caused by nitrite

Leachate from the landfill Lindbodarna was suspected to cause reproductive effects on fish in a Swedish lake, called Molnbyggen. The acute toxicity of this landfill leachate is caused by ammonia. In the present study the acute and chronic toxicity of the leachate from the landfill was tested with Ceriodaphnia dubia before and after treatment, either with (inoculated) or without addition of microorganisms from activated sludge, in both 2000 and 2001. On both occasions, the acute toxicity decreased after treatment, more rapidly with inoculum than without, and the cause of the decrease was mainly explained by decreasing concentrations of ammonia. However, the chronic toxicity decreased after treatment with inoculum but increased after treatment without inoculum. Therefore, we performed a series of acute and reproductive tests with ammonia, nitrite and nitrate on C. dubia, and the 24-h EC50s were 1.0, 2.7 and 59 mM, respectively, which are consistent with literature data. However, the chronic toxicity of these compounds gave quite a different picture with 8-day EC50s for reproduction of 3.0 mM for ammonia, 0.016 mM for nitrite and 1.5 mM for nitrate. Thus, the acute–chronic ratios for these compounds were 0.33 for ammonia, 170 for nitrite and 39 for nitrate. These findings show that reproduction is more sensitive than survival for both nitrite and nitrate, and that nitrite is the more hazardous of the two. This implies that the chronic and reproductive toxicity of nitrite and nitrate on zooplankton may in fact increase effects of eutrophication. In this study the toxicity of the fresh leachate was dominated by ammonia, but after treatment the contribution of nitrite increased, and especially the chronic toxicity of the treated landfill leachate was dominated by nitrite toxicity.

Growth, nutrient uptake and proximate composition of Rhodomonas sp. cultured using f/2 medium and agricultural fertilizers

We determined the growth, nutrient (PO43–, NO3–, NH4+) uptake and proximate composition of Rhodomonas sp. cultured during seven days in batch culture using f/2 medium and agricultural fertilizers. The growth rate was similar with both media (µ = 0.37 and 0.38 day–1). Protein content in Rhodomonas sp. cultured with f/2 medium was higher than with agricultural fertilizers (10.7 and 8.96 pg cell–1, respectively). Carbohydrate and lipid contents decreased during the exponential growth phase and there were no significant differences (P = 0.027 and 0.08, respectively) between both experiments. Carbohydrate content was higher during the slow growth phase in cultures using f/2 medium, whereas lipid content showed little variation during the slow growth phase, but was higher in cells cultured using agricultural fertilizers. During the first five days, total phosphate uptake by Rhodomonas sp. was 39.9 µM (94.3%) in the f/2 medium and 40 µM (99%) in the agricultural fertilizer medium. A total of 667.3 µM of nitrate (72.2%) was consumed during the first four days in the f/2 medium, whereas in the agricultural fertilizer medium formulated with ammonium nitrate, ammonium uptake (418.1 µM, 78.7%) in the first three days was higher than nitrate uptake (37.7 µM, 7.3%); however, when ammonium concentration decreased to 1.83 µM, the nitrate uptake increased. In conclusion, the cellular quantity and quality of Rhodomonas sp. was similar when both media were used and agricultural fertilizers can be used in marine aquaculture programs.

Uptake of sewage derived nitrogen by Ulva rigida (Chlorophyceae) in Bahía Nueva (Golfo Nuevo, Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahia Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3 μM of ammonium and between 0.77 and 6.21 μM of nitrate + nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: Vmax = 591.2 μmol g−1 h−1, Ks=262.3 μM, nitrate + nitrite: Vmax=12.9 μmol g−1 h−1, Ks=3.5 μM). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.

Seasonal production regimes off south-western Australia: influence of the Capes and Leeuwin Currents on phytoplankton dynamics

Temporal primary production dynamics were investigated off south-western Australia, where the summer upwelling regime of the Capes Current was compared with early winter conditions characterised by strengthened near-shore Leeuwin Current flow. Seasonal upwelling in this region sourced nitrate levels of ≥1 μm from the nutricline at the base of the Leeuwin Current’s mixed layer, with total water column production reaching a maximum of ~950 mg C m−2 day−1 in the Capes Current. Stable isotope signatures of particulate matter indicated that productivity off south-western Australia was heavily reliant on nitrate as a nitrogen source, with mean δ15N ranging from ~4 to 5 ‰ under both upwelling and non-upwelling (winter) conditions. Unexpectedly, significant nutrient enrichment within the Leeuwin Current (up to 3.1 μm nitrate) occurred during winter, likely as a result of the meandering Leeuwin Current flooding the inner shelf north of the study area and entraining relatively high-nutrient shelf waters in its southwards flow. However, early winter production under these nutrient-replete conditions (mean ± s.d. 310 ± 105 mg C m−2 day−1) was significantly lower than in summer (695 ± 140 mg C m−2 day−1) due to light limitation, both as a result of reduced surface irradiance characteristic of the winter months and significantly higher light attenuation within the water column as compared with summer conditions.

Genetic Differences in Nitrate Uptake in Two Clones of the Common Reed, Phragmites australis

Phragmites australis, common reed, could be useful in removing eutrophic substances from river and lake water. In this study, the genetic differences in nitrate uptake ability of the reed were investigated with a view to breeding a reed plant useful for phytoremediation. Two reed clones (W-6 and W-8) isolated from a reed community in the lakeside wetland along Lake Biwa, Japan, were used for a study on the physiological and molecular basis of nitrate uptake. Kms for nitrate uptake were 80.8 and 45.2 μM and Vmaxs for nitrate uptake were 10.62 and 2.37 μmol g−1 root f.w. h−1 in W-6 and W-8, respectively, suggesting that there were critical differences in kinetic parameters for nitrate uptake. To investigate these differences at the molecular level, we isolated a high-affinity nitrate transporter gene (NRT2) from the two reed clones and analyzed the reed NRT2 structure and expression. The deduced amino acid sequence of the reed NRT2 consisted of 523 residues and had a high similarity to NRT2 from other monocots. Reed NRT2 was strongly expressed in roots treated with 200 μM nitrate. There were three amino acid substitutions of the reed NRT2 between W-6 and W-8. Differences in NRT2 transcription were also observed between the two clones. It was not clear whether the difference in kinetic parameters for nitrate was due to the reed NRT2 structure or expression. These results indicate the possibility of selecting genotypes more useful for the removal of nitrate.

Relationship between land-use in the agro-forestry system of les Landes, nitrogen loading to and risk of macro-algal blooming in the Bassin d'Arcachon coastal lagoon (SW France)

Nitrogen loading to the Bassin d'Arcachon coastal lagoon (SW France) was evaluated by studying land-use and nitrogen output in its 3001 km 2 catchment. At present, the catchment is dominated by forestry (79%), while intensive agriculture occupies 9% of the surface. The N-output of two hydrological subunits, i.e. the Tagon subunit dominated by pine forestry and the Arriou II subunit comprising both forestry and intensive agriculture, were monitored for a seven year period (1996–2002). From these observations it was calculated that forestry contributes on average 1.6 kg total N ha −1 yr −1, which is dominated by organic nitrogen (DON + PON are 70% of N). On an areal basis, intensive agriculture contributes 26 times more than forestry, i.e. 41.6 kg total N ha −1 yr −1, which is mainly in the form of nitrate (65% of N). These data were upscaled to the catchment and the upscaling was validated by comparison to gauged nitrogen throughputs for the catchment of the Leyre river that is the major tributary to the system. Taking into account the other known N sources and the interannual variability in the catchment it was estimated that nitrogen loading to the lagoon was on average 90 kg ha −1 yr −1 (range from 54 to 126 kg ha −1 yr −1). The sandy soils of the catchment have a clear potential for denitrification, but anoxic conditions (waterlogged) and input of organic matter to fuel this process are required. Currently, agricultural practices and spatial planning do not make use of this potential. Nitrogen loading in the Bassin d'Arcachon is reflected by 10–40 μM nitrate concentrations in winter, which became depleted during spring as a result of uptake by vegetation. Short-term uptake experiments showed that the macroalga Monostroma obscurum is well adapted to temperatures between 10 to 20 °C and competitive with respect to the seagrass Zostera noltii when the nitrate concentrations are above 10 μM. Spring conditions with high nitrate and high insolation are therefore favourable for M. obscurum and this species presents a high risk for algal blooming. In contrast, the macroalga Enteromorpha clathrata well adapted to summertime temperatures around 25 °C, forms occasionally blooms in the lagoon. This phenomenon is limited due to the low DIN concentrations in summer.

Simultaneous determination of nitrate and nitrite in biological fluids by capillary electrophoresis and preliminary study on their determination by microchip capillary electrophoresis

In order to develop a highly sensitive and high-throughput screening method for nitrogen monoxide metabolites in biological fluids, we have investigated the simultaneous determination of nitrite and nitrate, using capillary electrophoresis and microchip capillary electrophoresis. In capillary zone electrophoresis, a running buffer based on human serum components with high ionic strength has been developed for the determination of nitrite and nitrate in human serum and human saliva. We obtained successful separation of nitrite and nitrate in the serum and the saliva within 7 min under optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and area were obtained by a standard addition method. The limits of detection obtained at a signal-to-noise ratio ( S/ N) of 3 for nitrite and nitrate in the serum were 2.6 and 1.5 μM, respectively. The values of the relative standard deviation of peak height for the serum with 9.2 μM nitrite and 20.9 μM nitrate were 5.7 and 4.1%, respectively. For on-site analysis with high-throughput screening, a microchip capillary electrophoresis method using a microchip made of quartz with a UV detector was developed. In this high-throughput format, using a running buffer with an electroosmotic flow modifier, the peaks of nitrite and nitrate in an artificial serum sample were obtained within 8 s. In high-resolution mode, using the buffer without electroosmotic flow modifier, the separation of nitrite and nitrate was obtained within 15 s. In high-resolution mode, using an artificial serum sample with 50 μM NO 2 − and 50 μM NO 3 −, the limits of detection ( S/ N = 3) of 41 μM for NO 2 − and 26 μM for NO 3 − were obtained. The method was applied to human serum and saliva. We obtained peaks due to nitrite and nitrate in 10-fold diluted saliva.

Semicontinuous PM2.5 Sulfate and Nitrate Measurements at an Urban and a Rural Location in New York: PMTACS-NY Summer 2001 and 2002 Campaigns

Several collocated semicontinuous instruments measuring particulate matter with particle sizes ≤2.5 μm (PM2.5) sulfate (SO4 22−) and nitrate (NO3 −) were intercompared during two intensive field campaigns as part of the PM2.5 Technology Assessment and Characterization Study. The summer 2001 urban campaign in Queens, NY, and the summer 2002 rural campaign in upstate New York (Whiteface Mountain) hosted an operation of an Aerosol Mass Spectrometer, Ambient Particulate Sulfate and Nitrate Monitors, a Continuous Ambient Sulfate Monitor, and a Particle-Into-Liquid Sampler with Ion Chromato-graphs (PILS-IC). These instruments provided near realtime particulate SO4 2− and NO3 − mass concentration data, allowing the study of particulate SO4 2−/NO3 − diurnal patterns and detection of short-term events. Typical particulate SO4 2− concentrations were comparable at both sites (ranging from 0 to 20 μg/m3), while ambient urban particulate NO3 − concentrations ranged from 0 to 11 μg/m3 and rural NO3 − concentration was typically less than 1 μg/m3. Results of the intercomparisons of the semicontinu-ous measurements are presented, as are results of the comparisons between the semicontinuous and time-integrated filter-based measurements. The comparisons at both sites, in most cases, indicated similar performance characteristics. In addition, charge balance calculations, based on major soluble ionic components of atmospheric aerosol from the PILS-IC and the filter measurements, indicated slightly acidic aerosol at both locations.

EFFECT OF 4-AMINO TETRAHYDROBIOPTERIN ON SURFACE MARKER EXPRESSION AND T-CELL STIMULATORY CAPACITY OF MURINE BONE MARROW DERIVED DENDRITIC CELLS.

P1024 Aims: The purpose of the present study was to investigate effects of 4-amino tetrahydrobiopterin on dendritic cells in order to identify a potential immunosuppressive mechanism, with particular emphasis on a role of inducible nitric oxide (NO) synthase (iNOS). 4-amino tetrahydrobiopterin is a tetrahydrobiopterin-competitive inhibitor of NO-synthases, which in a murine cardiac allograft model prolongued allograft survival comparable to cyclosporin-A treatment. Methods: We used bone-marrow derived dendritic cells of wild-type and iNOS(−/−) C57/BL10 mice. Surface antigen expression was determinend by FACS analysis. To assess T-cell stimulatory capacity, dendritic cells were incubated for 48 h with and without LPS (10 ng/ml) and with and without pteridines (50 μM - 500 μM) or the iNOS inhibitor N-iminoethyl-L-lysine (NIL, 500μM). After washing, varying amounts of dendritic cells were cocultivated with periodate-treated T-cells of the same strain for another 48 h. T-cell proliferation was then assessed by 3H-thymidine incorporation. In a separate set of experiments, LPS and pteridines were added simultaneously with periodate-treated T-cells to the dendritic cells, and proliferation was determined as described above. Apopotosis was measured by annexin V and propidium iodide staining. Nitrite and nitrate released into the supernatants was assessed by reversed phase HPLC, with cadmium reduction of nitrate and post-column mixing with the Griess reagent. Results: Dendritic cells produced up to 100 μM nitrite and nitrate upon LPS treatment, which was inhibited to baseline levels by NIL. LPS also induced apoptosis in wild type dendritic cells, which was blocked by NIL. In iNOS(−/−) dendritic cells, however, apoptosis was independent of LPS and NIL treatment. Pteridines had little effects on NO formation and apoptosis of dendritic cells. Among 6 surface markers, only 4-amino tetrahydrobiopterin, but not the other agents, downregulated MHC II and attenuated LPS-induced decrease of ICOS-ligand surface expression in both wild-type and iNOS(−/−) dendritic cells and led to a significantly decreased T-cell stimulatory capacity. When added together with dendritic cells to T-cells, 4-amino tetrahydrobiopterin and tetrahydrobiopterin caused apoptosis of T-cells. Catalase inhibited the apoptotic action of tetrahydrobiopterin, but not of 4-amino tetrahydrobiopterin. Conclusions: These results suggest the following immunosuppressive mechanism of 4-amino tetrahydrobiopterin: When present during dendritic cell maturation, it suppresses MHC-II expression and attenuates downregulation of ICOS-ligand. This results in lower T-cell stimulatory capacity and thus in an attenuated immune response. In addition, 4-amino tetrahydrobiopterin causes apoptosis of T-cells, which is independent of hydrogen peroxide formation during autooxidation of tetrahydropteridines. Our results with the iNOS inhibitor NIL and with cells from iNOS(−/−) mice clearly indicate that these mechanisms are independent of iNOS and hence describe a novel immunosuppressive mechanism of tetrahydropteridines.

Inorganic nitrogen transformations at high loading rates in an oligohaline estuary

A well-defined nitrogen retention and turnover budget was estimated for a shallow oligohaline lake (Lake Pontchartrain, Louisiana, USA). In 1997 a month-long diversion of the Mississippi River filled the Lake with highly concentrated river water (80 µM nitrate) and lowered the salinity to 0 psu within 2 weeks. After the spillway was closed the Lake mixed with estuarine tidal waters and came to equilibrium over 4 months with the riverine, atmospheric and offshore water nitrogen sources. A flushing rate of 1.78% d−1 was estimated by analyzing a plot of ln salinity versus time for the first 120 days after the diversion ceased. This flushing rate was similar to the loss rate for total nitrogen (1.75% d−1), implying no significant net nitrogen losses or gains were occurring inside the Lake. The percent loss of dissolved inorganic nitrogen was higher than that for TN (4.11% d−1), whereas the loss of organic nitrogen was lower (0.94% d−1), which suggests a net transfer from inorganic to organic nitrogen. These changes occurred steadily as chlorophyll a concentration ranged from 5 to 200 µg l−1. The results demonstrate the potential significance of the organic nitrogen and interconversion of nitrogen forms when calculating estuarine nitrogen retention budgets and the necessity of measuring all nitrogen forms when performing mass balance estimates. The significance of denitrification in nitrogen removal is minimal at the high loading rates observed during this study. An implication to estuarine water quality management is that the relationships between nitrogen loading and retention are not linear and are controlled by factors other than water residence time.

Physiological responses of phytoplankton communities in the Irish Sea to simulated upwelling

Understanding the dynamics of upwelling systems, especially the interactions between nutrients and light, has benefited from the application of models of varying complexity. Validation of such models using unialgal cultures or field observations has often proven difficult, but short-term incubations of contained natural assemblages and use of instantaneous physiological indicators offer an alternative approach. In May and June 1996, phytoplankton communities deep in the euphotic zone were sampled from nearly identical physical environments. Replicate samples (20 l volume) were incubated on deck at 50% surface irradiance with either no nutrient additions (Controls) or additions of 20 µM nitrate (Enrichments). Over 24 h, variable fluorescence (Fv:Fm), nitrate reductase activity (NR), nutrients, chlorophyll a and particulate C and N were monitored. Initial chlorophyll a (∼3 µ gl −1 ), phosphate (∼0.2 µM), nitrate (∼1.5 µM) and silicate (∼3 µM) were similar in both months. Changes in NR and Fv:Fm indicated clear physiological responses to changes in irradiance and added nitrate that differed between months. In May, Controls and Enrichments responded in the same way. Fv:Fm stayed constant (0.5), chlorophyll a increased slightly, and NR activity increased markedly in all samples. In contrast, in June, treatments responded quite differently. Fv:Fm was near the theoretical maximum (0.7–0.8) initially and remained constant in Enrichments, but fell sharply in Controls. Declines in controls were also seen for chlorophyll a, and NR activity. Thus, the addition of 20 µM nitrate had a significant effect even though ambient levels of nitrate (>1 µM) should not have been limiting. Small (<20 µm) flagellates predominated in the May samples, but in June large and chain-forming centric diatoms constituted a significant proportion of the phytoplankton community. We conclude that the response of a phytoplankton community to environmental changes can depend on factors that are poorly represented by bulk measurements of chlorophyll, nutrients and particulate elements.

Intraperitoneal Nitric Oxide Production in Patients Treated by Continuous Ambulatory Peritoneal Dialysis

Background: Nitric oxide (NO) generation within the peritoneum could potentially affect peritoneal transport by increasing capillary vasodilatation, and increase peritoneal permeability during episodes of bacterial peritonitis. As peritoneal mesothelial cells have a common embryological derivation with endothelial cells, then mesothelial cells could potentially be a major source of locally produced NO. Methods: NO was measured using the Griess reaction in fresh and spent dialysate effluent (SPDE) from uninfected CAPD patients, and from those during episodes of bacterial peritonitis. Human peritoneal mesothelial cells (HPMC) were cultured and NO production determined in the presence of SPDE and the effect of a potential NO substrate, L-arginine, and NO synthase inhibitor, L-NMMA. NO production by peritoneal macrophages (MØ), obtained from SPDE and the effect of staphylococci was also determined. RNA for inducible nitric oxide synthase (iNOS) was sought using Northern blotting technique following combination stimulation with lipopolysaccharide and cytokines (IL-1β, TNF-α and γ-INF, and/or spent dialysate from patients with bacterial peritonitis). Results: Whereas fresh CAPD dialysate was nitrite-free, SPDE from the day time exchange contained 41 ± 3 µM (nitrite and nitrate), and that from the overnight dwell 91 ± 8 µM. During CAPD peritonitis, dialysate nitrite and nitrate increased from 9.3 ±0.8 to 17.5 ± 2.4 µM/l·h, for the first CAPD bag at presentation, and 15.2 ± 1.8 for the second and 16.2 ± 2.4 for the third exchange (p < 0.01 compared to non-infected control). By the second day, levels had returned to baseline, 7.3 ± 0.9 µM/l·h. HPMC produced 261 nmol nitrate and nitrite/mg cell protein, and this increased in a dose-dependent manner with the addition of spent uninfected CAPD dialysate, to 365 nmol/mg with 1:10 dilution and 655 nmol/mg with 1:2 dilution, p < 0.001. The addition of the substrate, L-arginine, resulted in a 10% increase in nitrite and nitrate production, whereas the addition of L-NMMA produced a 10% reduction. Peritoneal MØ obtained from SPDE produced similar quantities of nitrite and nitrate to peritoneal mesothelial cells, and cultures of Staphylococcus aureus resulted in a reduction in nitrite and nitrate levels, as they were used as a growth requirement. However, we could not demonstrate RNA production for iNOS by HPMC following cytokine or SPDE stimulation. Conclusions: This suggests that HPMC may be an important source of locally generated NO within the peritoneal cavity under basal conditions, but as they do not contain iNOS, the increased NO produced during episodes of acute bacterial peritonitis is more likely due to a combination of increased NO production by peritoneal endothelial cells and transmigrating macrophages.

Growth and uptake kinetics for nitrate, ammonium and phosphate by the toxic dinoflagellate Gymnodinium catenatum isolated from Hiroshima Bay, Japan

: Growth and uptake kinetics for nitrate, ammonium and phosphate by Gymnodinium catenatum isolated from Hiroshima Bay were experimentally investigated. Maximum uptake rate (ρmax) and half saturation constant (Ks), which were obtained by short-term uptake experiments, were 6.48 pmol/cell per h and 7.60 µM for nitrate, 3.37 pmol/cell per h and 33.6 µM for ammonium, and 1.42 pmol/cell per h and 3.40 µM for phosphate, respectively. Specific maximum growth rate (µ′m) and minimum cell quota (Q0), which were obtained by semicontinuous growth experiments, were 0.36/day and 31.3 pmol/cell for the N-limited conditions, respectively. The µ′m and QP0 for P-limited conditions were 0.37/day and 1.83 pmol/cell, respectively. These results indicate that G. catenatum is a poor competitor in terms of utilization of inorganic nutrients and is unlikely to form blooms in Hiroshima Bay where phosphate is chronically depleted unless the growth is supported by organic species (i.e. dissolved organic phosphorus) of the nutrients.

171 Light‐Induced Growth of Winter Phytoplankton Collected from the Benthic Boundary Layer in Coastal Oregon Waters

It has been speculated that overwintering phytoplankton in the benthic boundary layer (BBL) are seed stocks for spring blooms in coastal upwelling systems. Experiments were conducted in Jan/Feb 2003 to test the hypothesis that phytoplankton in the BBL are light limited. Water collected from the BBL at four sites across the northern Oregon shelf was incubated at in situ temperature and nutrient concentrations (∼1 μM phosphate, 10 μM nitrate, 15 μM silicate) and exposed to two different daylengths and four light levels. The daylengths were 9 h (winter) and 12 h (early spring), and the light levels (in μmol photons·m−1 ·s−1) were 0 (Control), 20 (L), 70 (M), and 160 (H). Light‐induced growth was observed in all four incubations. Exponential growth was reached in the 12 H, 12 M, and 9 H treatments after 4 to 6 days, and stationary growth was reached after 5 to 10 days. Maximum in vivo fluoresence was obtained in the 12 H, 12 M, and 9 H treatments, while maximum particulate organic carbon (POC) was obtained in the 12 H treatment. In vivo fluorescence increased in the L light treatments relative to the control, but POC did not. Blooms were terminated by nitrate or silicate depletion. These results demonstrate that growth of winter phytoplankton off Oregon is primarily light‐limited, and that input of light of sufficient intensity and constancy (i.e. water column stabilization) can lead to blooms on time scales typical of upwelling blooms (&lt;1 week).

Seasonal changes of dissolved nutrients within and around Port Foster Deception Island, Antarctica

Abstract Temporal and spatial distribution of dissolved macronutrients (ammonia, nitrate, phosphate and silica) and productivity were investigated within and around Port Foster, the flooded 160-m-deep caldera of Deception Island, Antarctica. This study was part of the Erupt Project, which included five seasonal cruises over a complete annual cycle during 1999–2000. Seawater samples were collected and physical properties were monitored from seven stations within Port Foster and 12 stations in the adjacent Bransfield Strait. In addition, shallow-water and beach interstitial-water samples were collected along the shorelines of the peripheral coves. Port Foster macronutrient/depth profiles were typical for a normal shallow seawater column in a polar region. The water column in early austral spring was well mixed and changed to a stratified water column with a weak thermocline during the summer. By early winter, the thickness of the well-mixed surface layer increased until the entire water column returned to well-mixed conditions. This early winter transition from stratified conditions to well-mixed conditions occurred in June and appeared to be abrupt. During the seasons of light limitation and low-primary productivity, local currents were effective at redistributing dissolved biochemical components throughout the bay. During the summer season, the dissolved nutrient and oxygen distributions reflected consumption of nutrients by primary producers. The mid-depth maximum observed in the ammonia profiles implies the excretion of metabolites from resident animal populations. Residence time of dissolved ammonia must have been shorter than the circulation time within Port Foster because ammonia is not as uniformly distributed during the summer months as it was during the winter and spring. Dissolved nitrate concentrations in the Bransfield Strait during this study were similar to those measured in previous studies. The mean concentrations of phosphate, nitrate, and silica in the beach interstitial samples were significantly higher (2.8–9.5 times) than in the shore, offshore and CTD samples. Possible sources for the high phosphate, nitrate, and ammonia concentrations in the beach interstitial and shore waters include decaying organic matter and bird and mammal excrement. Elevated silica concentrations appear to be associated with hydrothermal heating of beach and near-shore waters. However, the elevated macronutrient concentrations measured in the beach interstitial water were not traceable beyond 5 m of the shoreline. Phytoplankton biomass in Port Foster exhibited temporal variability similar to other coastal and continental shelf zones (CCZ) of the Antarctic Peninsula. Blooms in February and November 2000 were dominated by the centric diatoms Thalassiosira spp. and Rhizosolenia spp. Chlorophyll a (chl a ) values of 15 and 19 mg m −3 in Port Foster during these blooms were comparable to maximum-recorded levels in western Antarctic Peninsula CCZ (30–40 mg chl a m −3 ), while chl a values from November 1999 and June 2000 (non-bloom conditions) corresponded to historical monthly chl a averages of western Antarctic Peninsula CCZ ( −3 ). During blooms, phytoplankton standing stock could account for about 15 μM nitrate, which corresponds to the observed surface nitrate depletion.

Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism.

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 microm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.

A shipboard natural community continuous culture system for ecologically relevant low‐level nutrient enrichment experiments

Inputs of low concentrations of new and regenerated forms of nitrogen (≤1 µM) have large impacts on phytoplankton community structure and ocean biogeochemistry in the oligotrophic central gyres. However, current manipulative experimental methods cannot effectively simulate low‐level, continuous supplies of nitrate, such as those from upwelling or eddy events, or steady‐state inputs of regenerated ammonium and dissolved organic nitrogen from zooplankton grazing. Using a new shipboard continuous culture system based on laboratory chemostat methodology, we compared the effects of a continuous supply of 1 µM nitrate, ammonium, and urea‐N at a dilution rate of 0.5 d−1 on algal community composition in the North Atlantic. In the Gulf Stream, continuous inputs of 1 µM nitrate dramatically changed phytoplankton community structure from dominance by cyanobacteria to dominance by diatoms. Equimolar continuous supplies of ammonium resulted in much smaller increases in total phytoplankton biomass, and favored a community co‐dominated by diatoms and cyanobacteria, and promoted the growth of pelagophytes. In the Sargasso Sea, continuous 1 µM urea‐N inputs greatly increased the biomass and dominance of Synechococcus relative to the initial community and compared with control and 1 µM nitrate additions. The shipboard natural community continuous culture system is uniquely suited for realistically simulating inputs of low levels of limiting nutrients, allowing new types of prognostic enrichment experiments that give novel insights into the processes that control phytoplankton community structure in the ocean.