Nitrate Utilization by the Diatom Skeletonema costatum

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skektowema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kineffcs, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carriermediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased. Nitrogen appears to be the major nutrient limiting primary production in the oceans of the world (18), as well as in certain freshwater systems. Nitrogen is found in seawater as dissolved N2 (which cannot be fixed by most marine algae) and as inorganic ions: nitrate, nitrite, and ammonium. These ions are present in concentrations ranging from 0.01 to 50 pM for nitrate, from 0.01 to 5 Am for nitrite, and from 0.1 to 5 pM for ammonium. The concentration of organic forms is usually below 10 pm (15). When all three inorganic ions are present, ammonium is preferentially utilized (4). Nitrate and nitrite have to be reduced to ammonium by means of an energy-dependent enzyme system (nitrate reductase and nitrite reductase) prior to their assimilation by the cell (15). As nitrate is the most abundant form ofN in seawater, marine phytoplankton, mostly composed of diatoms and dinoflagellates, utilizes nitrate as the main N source. Its uptake and reduction constitute major functions in these organisms, since N has been identified as the limiting factor controlling their growth. The relevance of the study of these processes arises from the fact that phytoplankton organisms are the major primary producers of the sea. The purpose of this paper is to characterize the kinetics of nitrate uptake in a phytoplankton organism of particular ecological significance because of its wide distribution, such as the diatom Skeletonema costatum. MATERIALS AND METHODS Culture Media and Conditions. Culture media were prepared according to Guillard and Ryther (8), with the modifications 'This research was supported in part by a grant from the Spanish Ministry of Education and Science (PFPI) to J. L. S. 2Present address: Department of Plant Sciences, University of London, King's College, 68 Half Moon Lane, London SE24 9JF, England. 3 Present address: Departamento de Bioquimica, Facultades de Biologia y Veterinaria, Universidad de Oviedo, Le6n, Spain. proposed by the Woods Hole Oceanographic Institution (25). Cells were maintained in the medium h/21. N-deficient cells were obtained by growth on the medium f/21 but with 25 AM nitrite as the sole N source. For the antibiotic treatments, the strain was placed in the medium h/2-lA, to which the corresponding amounts of penicillin G and streptomycin were added (24). All media were prepared with synthetic seawater, containing 307.7 mM NaCl, 20.3 miM MgSO4, 8.0 mM KCI, 2.7 mM CaCl2, and 0.48 mm H3BO3 in distilled H20. The media were adjusted to pH 7.8 with Tris-HCl buffer and autoclaved at 121 C for 30 min. Batch cultures were used in order to minimize bacterial contamination. The cells were grown autotrophically with continuous artificial light, about 3,500 lux, produced by fluorescent tubes (Sylvania cool-white, F 65 T 12/CW), on a horizontal shaker (140 rpm) at 20 ± 2 C. Strain. The diatom used in this study was a strain of S. costatum (Grev.) Cleve, generously provided by the Instituto de Investigaciones Pesqueras, C.S.I.C., Laboratorio de Castell6n, Castell6n, Spain. For maintenance purposes, the strain was grown in 100-ml Erlenmeyer flasks containing 50 ml of culture medium h/21. The cultures were grown under the conditions described above. Fresh medium was inoculated every week with 2 ml of the preceding culture. The purity of the strain was controlled by means of periodic observations under the phase contrast microscope. Kinetics and Assessment of Growth. For the study of growth kinetics, cells were grown in 2-liter Erlenmeyer flasks, containing 800 ml of medium f/2-1, with varying amounts of different inorganic salts as the only N source. The cultures were inoculated with 20 ml of a culture grown for 5 days on ammonium containing (h/21) medium. Aliquots were taken, under sterile conditions, at regular intervals. Growth was measured by a variety oftechniques, turbidimetrically (at 750 nm), hemocytometrically, and by dry weight, with consistent results. Cell density was also measured as Chl a, after acetone extraction of the cell pigments (19). Absorption spectra of the acetone extracts were also used to determine the Margalef index (11) A43o/A^c5, which is the ratio between the absorbancy of the whole of the pigments and the absorbancy of Chl a. This index varies with the different populations and environmental conditions, and has been considered to reflect the physiological state of the cell. Axenic Cultures. Axenic cultures were achieved as proposed by the Woods Hole Oceanographic Institution (24) by repeated inoculation of antibiotic-containing media (h/21A). After three or four steps of antibiotic treatment, the axenic character ofthe strain was checked by inoculating a medium containing synthetic seawater (1 liter), peptone (10.0 g), and yeast extract (1.0 g) (pH 7.4) with 1 ml of the antibiotic-treated culture. This medium was incubated for at least 48 hr in the dark, and bacterial growth was investigated turbidimetrically, and by microscopic observation. Antibiotic treatments were performed when the cultures became contaminated. Nitrogen-deficient Cells. Cells were grown in 2-liter Erlenmeyer flasks containing 800 ml of medium f/2-1, 25 ltM in nitrite, under the conditions described above. The inoculum consisted of 20 ml 987 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. SERRA, LLAMA, AND CADENAS of a 48-hr-old culture, grown on the same medium, except that the initial concentration of nitrite was 50 tiM. The culture was incubated for 120 hr, although no nitrite could be detected in the medium after 62 hr. At the end of the culture, the biomass corresponded to 40 ± 5 ,g Chl all. These cells were considered N-deficient and used for nitrate uptake assays. Nitrate Uptake Assay. Nitrate uptake was assayed by a modification of the method of Eppley and Coatsworth (3). N-deficient cultures were divided into 100-ml aliquots (3.5-4.5 ,ug Chi a) in 250-ml Erlenmeyer flasks, to which different amounts of NaNO3 (0-15 tM) were added. The cells were incubated under the conditions described above. The incubation times were chosen in such a way that at low nitrate concentrations, less than 50%o of the added nitrate was taken up, and, at high (> 10 AM) nitrate concentrations, the excess nitrate was more than 2 liM. After incubation, the cells were harvested by filtration under reduced pressure on glass-fiber GF/C Whatman filters. The filtrates were stabilized with few drops of chloroform and stored at 4 C for nitrate and nitrite analysis.4 vi N03 was calculated as the ratio: Initial nitrate final nitrate + final nitrite Incubation time x amount of Chl a and v. N02was calculated as the ratio: