Nitrate reductase activity in macroalgae and its vertical distribution in macroalgal epiphytes of seagrasses.

Macroalgal epiphytes within seagrass meadows make a significant contribution to total primary production by assimilating water column N and transferring organic N to sediments. Assimilation of NO3 – requires nitrate reductase (NR, EC 1.6.6.1); NR activity represents the capacity for NO3 – assimilation. An optimised in vitro assay for determining NR activity in algal extracts was applied to a wide range of macroalgae and detected NR activity in all 22 species tested with activity 2 to 290 nmolNO3 – min–1 g–1 frozen thallus. With liquid-N2 freezing immediately after sample collection, this method was practical for estimating NR activity in field samples. Vertical distribution of NR activity in macroalgal epiphytes was compared in contrasting Posidonia sinuosa and Amphibolis antarctica seagrass meadows. Epiphytes on P. sinuosa had higher mass-specific NR activity than those on A. antarctica. In P. sinuosa canopies, NR activity increased with distance from the sediment surface and was negatively correlated with [NH4 +] in the water but uncorrelated with [NO3 –]. This supported the hypothesis that NH4 + released from the sediment suppresses NR in epiphytic algae. In contrast, the vertical variation in NR activity in macroalgae on A. antarctica was not statistically significant although there was a weak correlation with [NO3 –], which increased with distance from the sediment. Estimated capacities for NO3 – assimilation in macroalgae epiphytic on seagrasses during summer (24 and 46 mmolN m–2 d–1 for P. sinuosa and A. antarctica, respectively) were more than twice the estimated N assimilation rates in similar seagrasses. When the estimates were based on annual average epiphyte loads for seagrass meadows in other locations, they were comparable to those of seagrasses. We conclude that epiphytic algae represent a potentially important sink for water-column nitrate within seagrass meadows.

Nitrate and phosphate uptake characteristics of three species of brown algae cultured at low salinity.

Nitrate and phosphate uptake mechanisms have been characterised under conditions of 100 and 50% seawater in 3 common brown algae of NW Europe: Fucus vesiculosus, F. serratus and Laminaria digitata. Under low salinity, the growth rate and internal nitrate accumulation of F. serratus significantly increased (20 and 48%, respectively), but no significant changes were observed for F. vesiculosus and L. digitata. However, nitrate uptake rates were reduced in L. digitata, so that this species was less adaptable to low salinity than the Fucus species. Both F. vesiculosus and F. serratus reached a steady-state uptake rate after acclimation regardless of the salinity treatment. All 3 species had a high capacity for storing inorganic N and P intracellularly. The results for F. serratus pointed to a dual mechanism of adaptation to the special characteristics of the intertidal environment where it grows. Non-saturating (low affinity) nitrate uptake and biphasic (double Michaelis-Menten curve) phosphate uptake are adaptations to high nutrient concentrations. Temporal partition of cellular energy for carbon metabolism and nutrient uptake is also suggested as an adaptation to the transient nutrient inputs occurring in these environments.

Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations.

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO3- storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO3-, with highest values in winter, when ambient NO3- was maximum, and declined with NO3- during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 ± 33 nmol NO3- min1 g1 in F. vesiculosus versus 55 ± 17 nmol NO3- min1 g1 in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO3- assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7–50.0, yet seaweeds stored significant NO3- (up to 40–86 µmol g1). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.

Nitrogen-15 and Oxygen-18 Natural Abundance of Potassium Chloride Extractable Soil Nitrate Using the Denitrifier Method

In agroecosystems, most isotopic investigations of NO3- involve the use of tracers that are artificially enriched in 15N. Although the dual isotope composition of NO3-— d15N and d18O is especially beneficial for understanding the origin and fate of NO3-, its use for KCl-extractable soil NO3- has been hampered by the lack of a suitable analytical technique. Our objective was to test whether the denitrifier method, whereby NO3- is reduced to N2O before mass spectrometric analysis, can be used to determine the N and O isotopic composition of NO3- from 2 M KCl soil extracts. Several internationally accepted NO3- standards were dissolved in 2 M KCl, the conventional extractant for soil inorganic N, and inoculated with the bacterial strain Pseudomonas aureofaciens (ATCC no. 13985). The standard deviation of the NO3- standards analyzed did not exceed 0.2‰ for d15N and 0.3‰ for d18O values. After appropriate corrections, differences between our measured and consensus d15N and d18O values of standard NO3- generally were within the standard deviations given for the consensus values. Both d15N and d18O values were reproducible among separate analytical runs. The method was also tested on genuine 2 M KCl extracts from unfertilized and fertilized soils. Depending on N fertilization, the soils had distinct d15N and d18O values, which were attributed to amendment with NH4NO3 fertilizer. Hence, our data indicate that the denitrifier method provides a fast, reliable, precise, and accurate way of simultaneously analyzing the natural abundances of 15N and 18O in KCl-extractable soil NO3-.

Tracing nitrate and sulfate in river basins using isotope techniques

The objective of this paper is to outline how stable isotope techniques can contribute to the elucidation of the sources and the fate of riverine nitrate and sulphate in watershed studies. The example used is the Oldman River Basin (OMRB), located in southern Alberta (Canada). Increasing sulphate concentrations and decreasing d34S values along the flowpath of the Oldman River indicate that oxidation of pyrite in tills is a major source of riverine sulphate in the agriculturally used portion of the OMRB. Chemical and isotopic data showed that manure-derived nitrogen contributes significantly to the increase in nitrate concentrations in the Oldman River and its tributaries draining agricultural land. It is suggested that hydrological conditions control agricultural return flows to the surface water bodies in southern Alberta and impart significant seasonal variations on concentrations and isotopic compositions of riverine nitrate. Combining isotopic, chemical, and hydrometric data permitted us to estimate the relative contribution of major sources to the total solute fluxes. Hence, we submit that isotopic measurements can make an important contribution to the identification of nutrient and pollutant sources and to river basin management.