μM NH Research Articles

The effect of inhibitors of plasma membrane H+ - ATPase and oxidoreductases on NH4+ uptake by Pisum arvense roots

The effect of inhibitors of plasma membrane oxidoreductases (quinacrine and dicumarol) and H<sup>+</sup>-ATPase (dicyclohexylcarbodiimide and orthovanadate) on ammonium uptake by <i>Pisum arvense</i> seedlings and the activities of H<sup>+</sup>-ATPase and NADH-ferricyanide oxidoreductase was investigated. The uptake solution contained 50 µM NH<sub>4</sub><sup>+</sup>. In I h experiments, quinacrine and dicumarol depressed strongly and irreversibly the rate of NH<sub>4</sub><sup>+</sup> uptake and markedly inhibited the activity of NADH-ferri-cyanide oxidoreductase in the plasma membrane vesicles prepared from root cells. Simultaneously, sodium orthovanadate inhibited the activity of plasma membrane H<sup>+</sup>-ATPase increased the rate of NH<sub>4</sub><sup>+</sup> uptake. Dicyclohexylcarbodiimide inhibited H<sup>+</sup>-ATPase activity and increased efflux of NH<sub>4</sub><sup>+</sup> from roots to ambient solution. The results indicate on the lack of direct connection between uptake rate of 50 µM NH<sub>4</sub><sup>+</sup> and H<sup>+</sup>-ATPase activity, and suggest that membrane redox systems play a predominant role in this process.

Morphology and bloom dynamics of Cochlodinium geminatum (Schütt) Schütt in the Pearl River Estuary, South China Sea

An unarmored dinoflagellate bloom of Cochlodinium g eminatum (Schütt) Schütt has been identified in the Pearl River Estuary, South China Sea during the severe dry season, from late October to early November, 2009, when temperature and salinity ranged between 20.0–27.2 °C and 10.6–33.4, respectively. Light and scanning electron microscopy were used to identify the characteristics of C. g eminatum and provided the clear morphological structure for this species. The organism was primarily found in chains of two cells or single cell, and no longer chains were observed. Cells were irregularly spherical or slightly dorso-ventrally, with size ranged between 28 and 36 μm and longer than wide. A large nucleus in the center with numerous golden chloroplasts was present, and the cingulum made 1.5 turns around the cell. The concentration of C. g eminatum ranged from 10 2 to greater than 10 7 cells l −1 during the bloom period. Nutrient concentration ranges during the bloom were 1.29–81.00 μM NO 3, 0.14–12.14 μM NO 2, 0.21–6.29 μM NH 4, 0.23–6.26 μM PO 4 and 3.29–171.43 μM SiO 3, respectively. Total biomass expressed in terms of chlorophyll a ranged from 2.44 to 135.45 μg l −1, with an average 19.9 μg l −1 in surface water throughout the PRE. Two main clusters corresponding to the water sectors were defined with multivariate analysis (cluster and nMDS). Based on the composition and abundance of phytoplankton, spatial variations were observed at a significant level (ANOSIM, R = 0.44, P < 0.01). Although the pairwise correlation analysis detected no significant effect of any single environmental variable on the abundance of C. g eminatum, the multivariate analysis (BIO-ENV) between biotic and abiotic variables resulted in the best variables combination with all measured factors involved (temperature, salinity, turbidity, NO 3, NO 2, NH 4, PO 4 and SiO 3) which showed a combined effect during the bloom of C. g eminatum in the Pearl River Estuary ( ρ w = 0.477).

Sedimentary nitrogen uptake and assimilation in the temperate zooxanthellate sea anemone Anthopleura aureoradiata

The sea anemone Anthopleura aureoradiata (Carlgren), which harbours symbiotic dinoflagellates (zooxanthellae), is abundant on mudflats and rocky shores around New Zealand. We measured the potential for particulate nitrogen uptake from sediment by A. aureoradiata and the subsequent consequences of this uptake on the nitrogen status of its zooxanthellae. Sediment was rinsed, labelled with ( 15NH 4) 2SO 4, and provided to anemones at low (0.23 g ml − 1 ) and high (1.33 g ml − 1 ) sediment loads for 6 h. Both anemone tissues and zooxanthellae became enriched with 15N. Enrichment of anemone tissues was similar at both high and low sediment loads, but the zooxanthellae became more enriched at the lower load. This was presumably because the uptake of ammonium, arising from host catabolism, by zooxanthellae is light driven and because the anemones at the lower load were able to extend their tentacles into the light while those at the higher load were not. The influence of sediment uptake on the nitrogen status of the zooxanthellae was determined by measuring the extent to which 20 μM NH 4 + enhanced the rate of zooxanthellar dark carbon fixation above that seen in filtered seawater (FSW) alone; the ammonium enhancement ratio (AER) was expressed as [dark NH 4 + rate/dark FSW rate], where ‘rate’ refers to C fixation and a ratio of 1.0 or less indicates nitrogen sufficiency. When anemones were starved with and without rinsed sediment in nitrogen-free artificial seawater for 8 weeks, zooxanthellar nitrogen deficiency became apparent at 2–4 weeks and reached similar levels in both treatments (AER = ~ 2). In contrast, anemones fed 5 times per week for 8 weeks with Artemia nauplii were nitrogen sufficient (AER = 1.03). In the field, zooxanthellae from mudflat anemones were largely nitrogen sufficient (AER = 1.26), while nitrogen deficient zooxanthellae were present in anemones from a rocky intertidal site (AER = 2.93). These results suggest that, while there was evidence for particulate nitrogen uptake, dissolved inorganic nitrogen (especially ammonium) in interstitial pore water may be a more important source of nitrogen for the zooxanthellae in mudflat anemones, and may explain the marked difference in nitrogen status between the mudflat and rocky shore populations.

The source of 3-μm absorption in Jupiter’s clouds: Reanalysis of ISO observations using new NH 3 absorption models

A prominent characteristic of jovian near-IR spectra is the widely distributed presence of a strong absorption at wavelengths from about 2.9 μm to 3.1 μm, first noticed in a 3-μm spectrum obtained by the Infrared Space Observatory (ISO) in 1996. While Brooke et al. (Brooke, T.Y., Knacke, R.F., Encrenaz, T., Drossart, P., Crisp, D., Feuchtgruber, H. [1998]. Icarus 136, 1–13) were able to fit the ISO spectrum very well using ammonia ice as the sole source of particulate absorption, Irwin et al. (Irwin, P.G.J., Weir, A.L., Taylor, F.W., Calcutt, S.B., Carlson, R.W. [2001]. Icarus 149, 397–415) noted that their best-fit cloud model implied a strong absorption at 2 μm that was not observed in Galileo NIMS spectra, raising questions about the source of the absorption. Subsequent significant revisions in ammonia gas absorption models (Bowles, N., Calcutt, S., Irwin, P., Temple, J. [2008]. Icarus 196, 612–614) also raised questions about these conclusions because ammonia gas absorption overlaps regions of ammonia ice absorption. Our reanalysis, based on improved ammonia absorption models, finds that the ISO spectrum can be well fit by models that include both NH 3 ice and solid NH 4SH, with the latter substance providing most of the absorption. The component due to NH 3 is very possibly due to NH 3 present as a coating on either large ( r ∼ 15 μm) NH 4SH particles in a deeper layer at ∼550 mb or on small ( r ∼ 0.3 μm) photochemical haze particles in a lower pressure layer at ∼370 mb. Neither option creates conflict with the lack of significant NH 3 absorption features at thermal wavelengths.

Removal of pathogenic bacteria and nitrogens by Lactobacillus spp. JK-8 and JK-11

The feasibility of using Lactobacillus strains for the simultaneous removal of pathogenic bacteria and nitrogens, with the ultimate aim of application for the improvement of water quality in shrimp farms, was explored. Initially, strains of Lactobacillus spp. JK-8 and JK-11 in shrimp ponds were screened for their ability to remove pathogenic bacteria and nitrogen compounds. At the end of the incubation period in cultures of JK-8 and JK-11, JK-8 had produced 192.8 mM lactic acid and 43.6 mM acetic acid, and the pH of the culture had decreased from 7.0 to 3.8. However, the JK-11 strain produced only small amounts of organic acids, and the pH of the culture decreased from 7.0 to pH 5.4. Pathogen survival following exposure to concentrated cell-free supernatants derived from JK-8 and JK-11 cultures was also investigated. Pathogens were completely removed within 4 min of exposure to either culture supernatant. Cell-free supernatants concentrated from pH-nonadjusted JK-8 cultures and either pH-adjusted or pH-nonadjusted JK-11 cultures demonstrated remarkable antimicrobial activities against target bacteria (e.g., Vibrio parahemolyticus, V. harveyi, Edwardsiella tarda). Scanning electron microscopy analyses revealed perforations in bacterial cells and irregular rod shapes with wrinkled surfaces following incubation with the concentrated supernatants. Both JK-8 and JK-11 cultures facilitated the removal of up to 400 µM NH 4 +, NO 2 −, and NO 3 −. The results of this work are applicable for the simultaneous removal of pathogens and nitrogens from contaminated shrimp farms.

Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates ( Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte ( Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO 4 3− (median = 0.3 μM), 0.7–30.2 μM NO x (median = 12.9 μM), and 0–19.4 μM NH 4 + (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.

Ammonium uptake kinetics in root and leaf cells of Zostera marina L.

Ammonium uptake rates and the mechanism for ammonium transport into the cells have been analysed in Zostera marina L. In the cells of this species, a proton pump is present in the plasmalemma, which maintains the membrane potential. However, this seagrass shows a high-affinity transport mechanism both for nitrate and phosphate which is dependent on sodium and is unique among angiosperms. We have then analysed if the transport of another N form, ammonium, is also dependent of sodium. First, we have studied ammonium transport at the cellular level by measurements of membrane potentials, both in epidermal root cells and mesophyll cells. And second, we have monitored uptake rates in whole leaves and roots by depletion experiments. The results showed that ammonium is taken up by a high-affinity transport system both in root and leaf cells, although two different of kinetics could be discerned in mesophyll cells (with affinity constants of 2.2 ± 1.1 μM NH 4 +, in the range 0.01–10 μM NH 4 +, and 23.2 ± 7.1 μM NH 4 +, at concentrations between 10 and 500 μM NH 4 +). However, only one kinetic could be observed in epidermal root cells, which showed a K m = 11.2 ± 1.0 μM NH 4 +, considering the whole ammonium concentration range assayed (0.01–500 μM NH 4 +). The higher affinity of leaf cells for ammonium was consistent with the higher uptake rates observed in leaves, with respect to roots, in depletion experiments at 10 μM NH 4 + initial concentration. However, when an initial concentration of 100 μM was assayed, the difference between uptake rates was reduced, but still being higher in leaves. Variations in proton or sodium-electrochemical gradient did not affect ammonium uptake, suggesting that the transport of this nutrient is not driven by these ions and that the ammonium transport mechanism could be different to the transport of nitrate and phosphate in this species.

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH 4 +-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH 4 + and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH 4 +-N, and the biomass production was reduced by 47% at 3700 μM NH 4 +-N compared to NO 3 −-N. Similar responses were not found in Phragmites. The amounts (mg g −1 dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH 4 + (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO 3 − treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g −1 dry wt) of soluble sugars, TNC, K +, and Mg 2+ in roots under NH 4 + (5.6; 14.3; 20.6; 1.9) compared to NO 3 − nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K +, and Mg 2+, respectively), while root internal levels of NH 4 + and Ca 2+ (0.29; 4.6 mg g −1 dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca 2+, K +, and Mg 2+ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g −1 dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH 4 + supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH 4 +-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH 4 + is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH 4 + levels when P availability is high and NO 3 − is also provided.

Effects of host nutritional status and seasonality on the nitrogen status of zooxanthellae in the temperate coral Plesiastrea versipora (Lamarck)

The nitrogen status of endosymbiotic dinoflagellates (zooxanthellae) in the temperate coral Plesiastrea versipora (Lamarck) was determined by measuring the extent to which ammonium (40 μM NH 4 +) enhanced the rate of zooxanthellar dark carbon fixation above that seen in filtered seawater (FSW) alone; the enhancement ratio was expressed as [dark NH 4 + rate/dark FSW rate]. V D′/ V L, a further index of nitrogen status, was also calculated where V D′ = [dark NH 4 + rate − dark FSW rate] and V L = rate of carbon fixation in the light. When corals were starved for 2–8 weeks, zooxanthellar nitrogen deficiency became apparent at ≥ 4 weeks, with NH 4 +/FSW and V D′/ V L averaging up to 2.08 and 0.0061, respectively. A decrease in light-saturated photosynthesis per zooxanthella also occurred, with the photosynthetic rate after 4–6 weeks being just 81% of that seen prior to starvation. In comparison, when corals were fed 5 times per week for 8 weeks the addition of ammonium had little effect, indicating nitrogen sufficiency; NH 4 +/FSW and V D′/ V L were 1.03 and 0.0003, respectively. Photosynthetic rates of zooxanthellae from well-fed corals were up to 1.7 times greater than those of zooxanthellae from starved corals. The nitrogen status of zooxanthellae from corals in the field exhibited seasonal differences. Autumn samples were nitrogen sufficient, with NH 4 +/FSW = 1.003 and V D′/ V L = 0.0001. In contrast, a small degree of nitrogen deficiency was seen in winter and spring, when NH 4 +/FSW averaged 1.075 and 1.249, and V D′/ V L averaged 0.0013 and 0.0014, respectively. The greatest degree of nitrogen deficiency was observed in summer, when NH 4 +/FSW averaged 1.318 and V D′/ V L averaged 0.0036. Given the clear links between food supply and nitrogen status seen under experimental conditions, and the likelihood that the zooxanthellae are also able to take up nutrients directly from the seawater, the fluctuations in nitrogen status may reflect temporal fluctuations in seawater nutrient concentrations and plankton abundance. The nutrient status of these temperate zooxanthellae in the field is in contrast to the marked nitrogen deficiency seen in zooxanthellae from nutrient-poor coral reef waters, and raises the possibility that temperate zooxanthellae can store nitrogen for use when exogenous nutrients and food are less readily available. This, in turn, may contribute to the considerable stability of temperate zooxanthellar populations under highly variable environmental conditions.

Monochloramine potently inhibits arachidonic acid metabolism in rat platelets

In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH 2Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB 2, and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4 × 10 8/ml) were preincubated with drugs for 5 min at 37 °C prior to the incubation with AA (40 μM) for 2 min at 37 °C. HOCl (50–250 μM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5–67% inhibition) and COX metabolites (TXB 2, 33–73% inhibition; 12-HHT, 27–74% inhibition). Although Tau-Cl and Glu-Cl up to 100 μM were without effect on the formation of 12-HETE, TXB 2 and 12-HTT, NH 2Cl showed a strong inhibition on the formation of all three metabolites (10–100 μM NH 2Cl, 12-HETE, 21–92% inhibition; TXB 2, 58–94% inhibition; 12-HHT, 36–92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH 2Cl, and taurine restoring that induced by both NH 2Cl and HOCl. These results suggest that NH 2Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH 2Cl-induced alterations in the COX and LOX pathways in vivo.

Assessing the improvement of the Bilbao estuary water quality in response to pollution abatement measures

To evaluate the success of almost 20 years of pollution abatement in the Bilbao estuary watershed in northern Spain, we analyzed temporal trends in pollution discharges and water quality from 1993 to 2003. Over that period a great portion of the raw wastewater discharge was intercepted and treated, leading to a significant reduction in the pollution load to the estuary (51.8% in biochemical oxygen demand, 70.9% in ammonia nitrogen and 81.9% in faecal coliforms). Temporal trends of mean annual levels of water quality variables showed statistically significant increases in dissolved oxygen saturation (between 2.04 and 4.11%/year) and decreases in ammonia nitrogen (between −4.15 and −175.75 μM NH 3/year) and faecal coliforms concentrations (from 2.55×10 5 to 2.13×10 4 CFU/100 ml). The improvement of the Bilbao estuary water quality reported in this paper is primarily attributed to the pollution abatement measures accomplished by the local water authority. Finally, as a result of these pollution control efforts, European bathing water quality standards were met at local beaches.

The role of ammonium in photoprotection against high irradiance in the red alga Grateloupia lanceola

Combined and/or interactive effects of inorganic nitrogen (as ammonium) and irradiance on the accumulation of nitrogenous compounds, like UV-absorbing mycosporine-like amino acids (MAAs), chlorophyll a and phycobiliproteins, were examined in the red alga Grateloupia lanceola (J. Agardh) J. Agardh in a high irradiance laboratory exposure and a subsequent recovery period under low light. Also, photosynthetic activity as in vivo chlorophyll fluorescence of photosystem II, i.e. optimum quantum yield ( F v/ F m), electron transport rate (ETR) and quantum efficiency, were examined. Photosynthetic activity, phycobiliproteins and internal nitrogen content declined during the 3-day PAR (photosynthetically active radiation; 600 μmol s −1 m −2) and PAR + UVR (ultraviolet radiation; UVB 280–315 nm 0.8 W m −2, UVA 315–400 nm 16 W m −2) exposure. Ammonium supplied in the culture medium (0, 100 and 300 μM NH 4Cl) modified the responses of the alga to high irradiance exposures in a concentration dependent manner, mainly with respect to recovery, as the highest recovery during a 10-day low light period was produced under elevated concentration of ammonium (300 μM). The recovery of photosynthetic activity and phycobiliproteins was enhanced in the algae previously incubated under PAR + UVR as compared to exposure to only PAR, suggesting a beneficial effect of UVR on recovery or photoprotective processes under enriched nitrogen conditions. However, the content of MAAs did not follow the same pattern and thus it could not be concluded as the cause of observed enhanced recovery.

GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices

We report the cloning and characterization of the first NH 4 + transporter gene ( GintAMT1) in an arbuscular mycorrhizal fungus. GintAMT1 encodes a polypeptide of 479 amino acids sharing high sequence similarity with previously characterized NH 4 + transporters from other fungi. Heterologous expression of GintAMT1 in the yeast triple mep mutant complemented the defect of this strain to grow in the presence of less than 1 mM NH 4 +. As revealed by [ 14C]methylammonium uptake experiments carried out in yeast, GintAMT1 encodes a high-affinity NH 4 + transporter. In mycelia developed in the presence of 0.9 mM NO 3 −, GintAMT1 transcription was increased after the addition of 30 μM NH 4 + but decreased after the addition of 3 mM NH 4 +. However, in mycelia grown in the presence of higher N concentrations, GintAMT1 transcripts decreased after the addition of NH 4 +, irrespective of the concentration used. These data suggest that GintAMT1 is involved in NH 4 + uptake by the extraradical mycelia from the surrounding media when it is present at micromolar concentrations.

Effects of sediment and nutrient enrichment on water quality in the Archipelago Sea, northern Baltic: An enclosure experiment in shallow water

The effects of bottom sediment and nutrient enrichment on water quality were studied in an enclosure experiment in the Archipelago Sea, northern Baltic. The three-week experiment was conducted in a small and shallow bay, where the organic content of the sediment is low. The enclosures were large (diameter 3.6 m, depth 3.5 m), and reached from the surface to the bottom. Some of the enclosures included the natural sediment, some had a plastic bottom without contact with the sediment. Concentrations of nutrients and chlorophyll a and physical variables in the water column, concentrations of inorganic nutrients in the pore water of the sediment, and numbers and biomasses of the benthic macrofauna were measured. Both the presence of sediment and nutrient enrichment significantly increased the concentrations of total nitrogen, total phosphorus and chlorophyll a in the water column. The concentration of chlorophyll a doubled in the sediment-bottomed enclosures without nutrient enrichment; the increase was similar to that in the plastic-bottomed enclosures with nutrient enrichment (7.2 μM NH 4 + and 0.46 μM PO 4 3− during the three-week study). The concentration of silica doubled or tripled in the sediment-bottomed enclosures. No shortage of oxygen was found in the water column during the experiment. The results show that sediment with a low content of organic matter may serve as an important source of nutrients in shallow and littoral oxic waters and may be important in sustaining their eutrophic state during the productive season. It is suggested that an important part of the nutrients released from this erosion bottom had originated from the surface layer of the sediment, which had been sedimented and transported to the area quite recently. The results indicate that it is important to include sediment in mesocosm studies dealing with nutrient dynamics, especially in shallow waters.

Effect of salinity and inorganic nitrogen concentrations on nitrification and denitrification rates in intertidal sediments and rocky biofilms of the Douro River estuary, Portugal

The regulatory effects of salinity and inorganic nitrogen compounds on nitrification and denitrification were studied in intertidal sandy sediments and rocky biofilms in the Douro River estuary, Portugal, over a 12-month period. Nitrification and denitrification rates were measured in slurries of field samples and enrichment experiments using the difluoromethane and the acetylene inhibition techniques, respectively. Salinity did not regulate denitrification in either environment, suggesting that halotolerant bacteria dominated the denitrifier communities. However, nitrification rates were stimulated when salinity increased from 0 to 15 practical salinity units. NO 3 − addition experiments revealed that NO 3 − availability stimulates denitrification rates in sandy sediments, but not in rocky biofilms; however, in rocky biofilms a positive and linear relationship was observed between denitrification rates and water column NO 3 − concentrations ( r = 0.92 ) during the monthly surveys. The N 2O:N 2 ratios increased rapidly when NO 3 − increased from 63 to 363 μM; however, results from monthly surveys showed that environmental parameters other than NO 3 − availability may be important in controlling the variation in N 2O production via denitrification. Ammonium additions to sandy sediments stimulated nitrification rates by 35% for the 20 μM NH 4 + addition, but NH 4 + appeared to inhibit nitrification at high concentration addition (200 μM NH 4 +). In contrast, rocky biofilm nitrification was stimulated by 65% when 200 μM NH 4 + was added.

Characterization of monochloramine toxicity on PC12 cells and protective effect of tocopherol via antioxidative function

Monochloramine (NH 2Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH 2Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 μM NH 2Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH 2Cl cytotoxicity, we examined the effect of various kinds of antioxidants including α-tocopherol (α-Toc) and β-tocopherol (β-Toc). These antioxidants exerted a protective effect against NH 2Cl-induced cell death, and α-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 μM NH 2Cl. The formation of reactive oxygen species (ROS) was also measured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an antioxidant, such as α-Toc, suppressed the increase in ROS. Interestingly, β-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH 2Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function.

An NH 4+ biosensor based on ammonia-oxidizing bacteria for use under anoxic conditions

An ammonium biosensor based on bacterial oxidation of ammonia with oxygen was constructed. The biosensor contained an internal oxygen reservoir from which oxygen was supplied to the biomass of ammonia oxidizing bacteria (AOB). The biosensor was thus designed to have best performance under ambient anoxic conditions. An oxygen microsensor was used to monitor the internal oxygen gradient. Due to the ammonia-oxidizing and respiratory activity of the AOB the signal from this oxygen microsensor was proportional to the ambient ammonium concentration in a range of 0–200 μM. Oxygen in the environment interfered with the reading, but it was in principle possible to compensate for a constant oxygen concentration in the analyzed medium. An increase in pH from 6 to 8.5 increased the sensitivity, while a range of chemicals affected the signal in various ways. A total of four biosensors were constructed, and one exhibited a lifetime of >2 months. Sensors containing bacteria that were starved for ammonium could be revived within 2 days by placing them in ammonium-containing medium. The 90% response times of the sensors were about 2 min, and the difference in signal between vigorously stirred and stagnant medium corresponded to about 10 μM NH 4 + within the linear range. A sensor was tested in a wastewater treatment plant. The sensor signal during the anoxic periods corresponded well to the concentration of NH 4 + analyzed by colorimetry.

Effect of ammonium and oxygen on methane and nitrous oxide fluxes across sediment–water interface in a eutrophic lake

Eutrophication has decreased the O 2 content and increased the NH 4 + availability in freshwaters. These changes may affect carbon and nitrogen transformation processes and the production of CH 4 and N 2O, which are important greenhouse gases. We studied release of CH 4 and N 2O from a eutrophic lake sediment under varying O 2 and NH 4 + conditions. Intact sediment cores were incubated in a laboratory microcosm with a continuous anoxic or oxic water flows containing 0, 50, 500, 5000, or 15 000 μM NH 4 +. With the anoxic flow, the sediment released CH 4, up to 7.9 mmol m −2 d −1. With the oxic flow, the CH 4 emissions were small indicating limited CH 4 production and/or effective CH 4 oxidation. Addition of NH 4 + did not affect sediment CH 4 release, evidence that the CH 4 oxidizing bacteria were not disturbed by the extra NH 4 +. The release of N 2O from the sediment was highest, up to 7.6 μmol m −2 d −1, with the oxic flow without NH 4 + addition. Oxygen was the key factor regulating the production of NO 3 −, which enabled denitrification and production of N 2O. However, the highest NH 4 + addition increased nitrification and associated O 2 consumption causing a decrease in sediment O 2 content and in accumulation of NO 3 − and N 2O, which were effectively reduced to N 2 in denitrification. In summary, sediment CH 4 and N 2O dynamics are regulated more by the availability of O 2 than extra NH 4 +. Anoxia in eutrophic lakes favouring the CH 4 production, is the major contributor to the atmospheric consequences of water eutrophication.

Effects of mesozooplankton removal and ammonium addition on planktonic trophic structure during a bloom of the Texas 'brown tide': a mesocosm study

A bloom of the alga Aureoumbra lagunensis, known as the Texas 'brown tide', persisted in the Laguna Madre of Texas for most of the 1990s. The dominant mesozooplankter in Laguna Madre, Acartia tonsa, does not feed on A. lagunensis, and during blooms there are few other suitably sized phytoplankton cells available to feed on. We hypothesized that these copepods increased their feeding on microzooplankton, thereby reducing grazing pressure by microzooplankton on A. lagunensis and contributing to the persistence of this bloom. A mesocosm experiment was carried out to test this hypothesis during the summer of 1999. Twelve fiberglass corral-type mesocosms were deployed in the field for 16 days, each enclosing ∼1.2 m 3 of Laguna Madre water and 1.1 m 2 of natural benthos. Mesozooplankton were removed from six mesocosms with a 153 μm mesh dip net every 4 days; the other six mesocosms were treated in the same way, except that the contents of the net were returned to the mesocosm. For each zooplankton treatment, half of the mesocosms were dosed with ∼40 μM NH 4 at 4 day intervals, and half received no additions. Phytoplankton populations in these mesocosms at the start of the experiment were dominated by A. lagunensis and the cyanobacterium Synechococcus spp. Growth rates of A. lagunensis were higher in mesocosms without ammonium additions, providing no evidence for nitrogen limitation. Acartia tonsa populations were reduced by ∼50% in the zooplankton removal mesocosms, and ciliate populations were significantly higher. The increase in ciliate population had no significant impact on A. lagunensis population dynamics, however, providing no evidence to support the hypothesis that a trophic cascade reducing microzooplankton populations contributed to the persistence of the brown-tide bloom. In contrast, populations of Synechococcus sp. showed evidence of both 'top-down' and 'bottom-up' control; they grew faster in nutrient addition mesocosms and had lower populations in mesocosms with increased densities of ciliate graders.

Surge ammonium uptake of the cultured seaweed, Kappaphycus alvarezii (Doty) Doty (Rhodophyta: Gigartinales)

The possibility that the cultured seaweed Kappaphycus alvarezii may demonstrate surge ammonium uptake was studied using the perturbation method. Cultivars of the green morphotype of K. alvarezii were incubated at three different initial NH 4 + concentrations (i.e., 10, 20, 30 μM) at the same light intensity. Additional incubations were conducted at 30 μM initial NH 4 + concentration in the dark. K. alvarezii were also placed in a 30 μM NH 4 +-enriched seawater medium for 1 h before incubation at 30 μM initial NH 4 + concentration. Levels of enrichment and time had significant effects on ammonium uptake rates. Surge ammonium uptake tended to be higher with higher initial ammonium concentration. Mean uptake rates at 10 μM NH 4 + initial concentration were significantly lower compared to other treatments. Higher variability in uptake was noted during the first hour of incubation. Ammonium uptake in the dark was not significantly different from that in light (at 30 μM NH 4 + initial concentration). Surge uptake was higher for incubations without additional enrichment than when the alga was additionally exposed to enriched medium before the experiment. On average, surge uptake ( V S) occurred within the first 30 min and ranged from 15 to 35 μmol NH 4 + g −1 DW h −1. Internally controlled uptake ( V I) ranged from 5 to 15 μmol NH 4 + g −1 DW h −1 and occurred from 30 min to 6 h after exposure to the nutrient. Externally controlled uptake ( V E) was less than 5 μmol NH 4 + g −1 DW h −1 and occurred from 6 to 8 h after exposure when the medium was significantly depleted of ammonium (as in 10 and 20 μM NH 4 + initial concentrations). K. alvarezii from Danajon Reef, central Philippines thus exhibits surge ammonium uptake and some form of nitrogen limitation. Such a strategy is needed for surviving in environments with low or erratic nutrient supplies.