Significance of cellular nitrate content in natural populations of marine phytoplankton growing in shipboard cultures

Phytoplankton intracellular nitrate concentrations have been monitored in a 56-h experiment on a shipboard culture of surface sea water from an upwelling region. These measurements were related to parameters of biomass (particulate nitrogen) and nitrate assimilation using the ¹⁵N isotope technique and the nitrate reducase (NR) assay. The procedure for measuring cellular nitrate concentrations is described. This parameter exhibited diurnal variations, ranging from 3.1 to 20.6 ng-at nitrate per g-at particulate nitrogen, and could be correlated positively with NR activity. Nitrogen budgets show that NR activity represents only 12% of nitrate incorporation in organic phytoplankton material when nitrate is available in the sea water. However, upon depletion of the environmental nitrate (zero uptake), NR activity can fully account for the decrease of internal nitrate. From the results, it seems that internal nitrate content is a better index of nitrate consumption by marine phytoplankton than the external concentration of nitrate-nitrogen.     

Blooms of surf-zone diatoms along the coast of the Olympic Peninsula,Washington. IV. Nitrate reductase activity in natural populations and laboratory cultures of Chaetoceros armatum and Asterionella socialis

Nitrate reductase (NR) activity in the surf-zone diatom Chaetoceros armatum T. West was studied in its natural habitat at Copalis Beach, Olympic Peninsula, Washington (USA) over a period of 18 months. Variations of enzyme activity were found, depending on the season and the time of day. General seasonal trends in NR activity were correlated with nitrate concentrations in the surf-zone. The results also indicated that several factors may limit the activity of the enzyme. This is one of the reasons why NR activity could not be correlated with environmental nitrate concentration on the basis of individual measurements at a given point in time. Enzyme inhibition due to the presence of ammonium occurred on several occasions throughout the study period, and could be detected by the NR assay. This phenomenon for the most part masked any pattern of diel periodicity in NR activity that might have been expected to exist in cells in the natural environment. Cultures of C. armatum and Asterionella socialis Lewin and Norris were also studied in the laboratory. A diel fluctuation in NR activity corresponding to the pattern shown in natural phytoplankton by other authors was exhibited in C. armatum under a light-dark regime of 8 h light – 16 h dark and a light intensity of 400 lux.     

大肠杆菌JM105中硝酸盐还原酶的制备及其性质

近年来,硝酸盐和亚硝酸盐的污染已引起普遍关注。目前,蔬菜和粮食的NO3-含量过高主要由于农药和化肥的使用、工业废水或生活污水灌溉等,食品类主要来自腌制食品和食品添加剂或防腐剂等。另外,向乳制品或食品中参碱、食盐、化肥、脏水、碱性水等也是硝酸盐污染的来源之一。因此,迫切需要快速、简便、可用于现场的硝酸盐检测方法。这就需仰赖生物酶方法,而生物酶方法的关键是酶制剂的制备和检测方法的建立。本研究通过筛选、厌氧和硝酸盐诱导培养、超声波细胞破碎和差速离心提取等方法,从大肠杆菌(Escherichia coli)JM105细胞膜中制备了硝酸盐还原酶并对其性质进行了研究。结果表明:从大肠杆菌JM105中制得的酶制剂活力很高,且在酶过量的情况下可将NO3-完全转化为NO2-,在用磷酸缓冲液清洗并冷冻保藏过夜后不含有亚硝酸盐还原酶。在加入黄素单核苷酸辅酶(FMN)后,该酶的活力可提高64%,比活力达0.42U·mg-1蛋白。该酶十分稳定,在40℃下24h活力无影响,在浓度为1mmol·L-1的Cu2 、Fe3 、Ca2 、Zn2 、Mg2 和Mo6 存在下,其活力亦不改变。因此,该酶可用于测定食品、蔬菜和环境中硝酸盐的含量。     

Hydrobiological parameters during an annual cycle in the Arcachon Basin

Temperature, salinity, nutrients and phytoplankton biomass were monitored on a weekly to bimonthly frequency at six stations in the Bay of Arcachon from July 1984 to July 1985. This particular period appears to have differed from the last ten years in displaying higher amplitudes of both temperature and salinity (1.5° to 24.5°C and 20 to 34.5) at high tide. However, although in spring both temperature and salinity were normal (14°C, 28), an important phytoplankton spring bloom occurred, with maximum chlorophyll levels reaching 15 g l^-1 in April. Utilization of nutrients was high, particularly for nitrate and silicate, the concentrations of which decreased, respectively, from 10–15 to 0.1–2 M and 10–20 to 0.25–3 M from February to May. Exhaustion of nitrate was observed in May, except in areas subjected to river input. In contrast, silicate increased throughout the study area from May to July.     

Iron-mediated effects on nitrate reductase in marine phytoplankton

The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania huxleyi, Isochrysis galbana and Tetraselmis sp. Cells from iron-depleted cultures had 15 to 50 percent lower enzyme activity than those from iron-replete cultures. Upon addition of iron, nitrate reductase activity was enhanced in depleted cells up to levels comparable to those of the replete cells. Bioassays in the northern North Sea conducted in 1993, under low iron conditions, demonstrated similar results. Upon addition of 2.5 nM iron, a distinct enhancement, to a maximum of three times, of nitrate reductase activity was observed within 32 h after addition. Therefore, iron can stimulate nitrate reductase activity. In spite of the clean techniques used, some nitrate reductase activity was always observed. Iron deficiency was shown to impair nitrate reductase activity, but it is unlikely that nitrate reduction would cease completely.     

Nitrate storage by phytoplankton in a coastal upwelling environment

The storage of nitrate by phytoplankton cells during the early phases of upwelling was studied in coastal stations off northern Spain (southern Bay of Biscay) between 1990 and 1994. In this region, a persistent upwelling during summer is characterised by intermittent pulses of variable intensity, and increased nutrient concentrations in the surface layer. The main effect of an upwelling pulse on phytoplankton distribution is the shifting of the chlorophyll a and primary production maxima to near the surface. When the upwelling relaxes, thermal stratification of the water column occurs, and a distinct subsurface chlorophyll maximum develops below the production maximum. An accumulation of intracellular nitrate characterized the early phases of upwelling (mean = 2.73 μmol N m⁻³), maximum concentrations being attained at depths where biomass and production values were moderate. In contrast, phytoplankton cells from non-upwelling situations contained significantly lower concentrations of intracellular nitrate (mean = 0.17 μmol N m⁻³). The variations in the intracellular pool of nitrate may result from the differential allocation of resources within the cell as a result of variations in the energy available, since the uptake and assimilation of nitrate is a relatively expensive process involving several enzymatic systems. We hypothesize that nitrate storage by phytoplankton cells is characteristic of early phases of upwelling and is linked to patterns of carbon fixation. Average nitrogen budgets for upwelling and non-upwelling situations indicate that intracellular nitrate reserves are not responsible for maintaining high phytoplankton growth rates, since they only account for <2% of daily primary production during upwelling events. Received: 28 August 1996 / Accepted 3 December 1996     

Light regulation of nitrate reductase in Ulya fenestrata (Chlorophyceae)

Observations were made on the behavior of nitrate reductase activity in the green alga Ulva fenestrata under controlled light:dark regimes. The activity of nitrate reductase (NR) was examined in response to normal seasonal photoperiods as well as in response to shortened or extended periods of darkness. NR activity exhibits a light-dependent diurnal rhythm under both normal summer and winter photoperiods, with a maximum in the early morning (2 to 2.5 h after the start of illumination). This peak of activity is followed by a lower steady-state level of activity which is sustained throughout the light period. There is a sustained minimal level of activity in darkness. The morning peak in activity is always observed as long as tissue is illuminated, irrespective of the previous light or dark treatments. As such, it appears that nitrate reductase activity in U. fenestrata is under circadian control. There is no major difference in the NR activity pattern between summer and winter plants, except that the peak activity values in winter plants are consistently much higher (5 times) than in summer plants. The study also suggests that illumination prior to the normal start of photoperiod triggers a different set of regulatory mechanisms, indicating that the physiological state of plants is important in dictating the NR activity response to illumination.Please address all correpondence and requests for reprints to G. J. Smith at his present address (Hopkins Marine Station)     

Nitrate reductase and glutamine synthetase activity,internal N pools,and growth of<Emphasis Type="Italic"> Ulva lactuca</Emphasis>: responses to long and short-term N supply

Fast-growing macroalgae, including Ulva lactuca Linnaeus, respond rapidly to changes in nutrient conditions, particularly to short-term N supply. This ability to rapidly take up and assimilate N contributes to the increasing occurrence of macroalgal blooms in heavily N loaded coastal ecosystems. To determine whether long-term nutrient histories affect short-term responses in activity of N-assimilating enzymes, including nitrate reductase (NRA) and glutamine synthetase activity (GSA), internal N storage, and macroalgal growth, we conducted an in situ nitrate fertilization experiment between 7 and 22 July 2004, with fronds of U. lactuca collected from estuaries with high and low N loads in Waquoit Bay, Cape Cod, Massachusetts, USA (N 41° and W 70°). Initial NRA, GSA, % N, δ¹⁵N, and growth of U. lactuca fronds were higher in the site where nitrate was in high supply. Differences in NRA persisted even after short-term experimental enrichment. Differences in internal N pools, δ¹⁵N, and growth, in contrast, mirrored the changes of nutrient supply. The rate of turnover of the internal N content of U. lactuca was quite short (<2 d), and turnover of enzyme activity may have been even shorter. N isotopic fractionation by U. lactuca appeared to be of small magnitude, unlike the case of phytoplankton, and similar to that of vascular plants. δ¹⁵N was a better indicator of short-term response to external and internal nutrient supplies in U. lactuca than enzyme activity or N content, and may reliably detect rapid changes in N availability, source, and uptake and assimilation processes.     

Nitrate reductase activity in Zostera marina

Eelgrass (Zostera marina L.) has access to nutrient pools in both the water column and sediments. We investigated the potential for eelgrass to utilize nitrate nitrogen by measuring nitrate reductase (NR) activity with an in vivo tissue assay. Optimal incubation media contained 60 mM nitrate, 100 mM phosphate, and 0.5% 1-propanol at pH 7.0. Leaves had significantly higher NR activity than roots (350 vs 50 nmoles NO ₂ ^– produced g FW^–1 h^–1). The effects of growing depth (0.8 m MLW, 1.2 m, 3.0 m, 5.0 m) and location within the eelgrass meadow (patch edge vs middle) on NR activity were examined using plants collected from three locations in the Woods Hole area, Massachusetts, USA, in July 1987. Neither depth nor position within the meadow appear to affect NR activity. Nitrate enrichment experiments (200 M NO ₃ ^– for 6 d) were conducted in the laboratory to determine if NR activity could be induced. Certain plants from shallow depth (1.2 m) showed a significant response to enrichment, with NR activity increasing from >100 up to 950 nmoles NO ₂ ^– g FW^–1 h^–1 over 6 d. It appears that Z. marina growing in very shallow water (0.8 m) near a shoreline may be affected by ground water or surface run-off enrichments, since plants from this area exhibited rates up to 1 600 nmol NO ₂ ^– g FW^–1 h^–1. Water samples from this location consistently had slightly higher NO ₃ ^– concentrations (1.4 M) than all other collection sites (0.7 M). Thus, it is possible that chronic run-off or localized groundwater inputs can create sufficient NO ₃ ^– enrichment in the water column to induce nitrate reductase activity in Zostera leaves.     

Continuous plankton records: Changes in the composition and abundance of the phytoplankton of the north-eastern Atlantic ocean and North Sea, 1958–1974

In most areas of the north-eastern Atlantic Ocean, diatoms have declined drastically in abundance in the last decade. Additionally, in areas to the north of 59°N Ceratium species and an index of total phytoplankton have also declined. South of 59°N the phytoplankton index has increased, diatoms have declined and Ceratium species have remained at a constant level of abundance. A possible explanation of the increase in the phytoplankton index at a time when the diatoms were declinig south of 59°N is the development of unidentified phytoplankton organisms such as microflagellates. As many of the variables influencing phytoplankton standing crop are governed in turn by the prevailing weather, the phytoplankton changes may well be a consequence of the general deterioration, since 1940, of North Atlantic weather. Changes in phytoplankton which may be attributed to an amelioration of climate since 1971 are evident as yet only in the southern North Sea.     

Effect of temperature and light on the growth of micro- nano- and pico-plankton: impact on algal succession

The influence of light and temperature on the phytoplankton succession in a temperate sea area was investigated in laboratory experiments with natural assemblages of micro-, nano-, and picoplankton collected from the northern Baltic Sea during 1989 and 1990. Respiration increased from 0 to 30°C in all groups of phytoplankton, while gross photosynthesis stabilised at 10 to 15°C. Light saturation occurred at 25 to 75 mol quanta m^-2 s^-1, indicating low light adaptation of the algae. Picoplankton showed the strongest temperature response, and at temperatures above 10°C picoplankton obtained a higher biomass specific photosynthesis than that of the other groups. Different light treatments had no effect on the species composition in experiments with natural algal assemblages, while different temperature conditions had a marked effect. With a temperature increase from 0 to 10°C, the algal community changed from a typical spring community, with diatoms and dinoflagellates, to a summer community, dominated by mixotrophic nanoflagellates. The small, or often non-existing, autumn bloom in the sea area studied, can be explained by short day lengths combined with relatively high temperatures, causing high community respiration rates and low gross photosynthesis, resulting in a negative energy balance. The net energy gain depends on a differential temperature effect on gross photosynthesis and endogenous respiration in various plankton groups. This gives the phytoplankton groups diverse competitive advantages during different seasons and thus may be an important factor in controlling algal succession.     

Seasonal variations in the utilization of ammonium and nitrate by photoplankton in Vineyard Sound,Massachusetts, USA

The nitrogenous nutrition of the phytoplankton in Vineyeard Sound, Massachusetts, USA was investigated over a 15-month period. Highest rates of ammonium uptake were observed immediately prior to, or during, the diatom bloom periods, and with one exception were found in the <10 m size class. The saturating rate of ammonium uptake correlated well with temperature and gave Q₁₀ values of 2.6–3.2; correlations with ambient solar irradiation were not nearly so clear. Uptake rates of ammonium exceeded those of nitrate except during the winter bloom of the diatom Rhizosolenia delicatula; yet calculation of the f ratio revealed that nitrate was relatively important in the nitrogenous nutrition of the phytoplankton throughout the year.Contribution no. 5096 from the Woods Hole Oceanographic Institution     

Patterns and UV sensitivity of carbon anhydrase and nitrate reductase activities in south Pacific macroalgae

This study describes the activities of the key enzymes involved in carbon incorporation (carbonic anhydrase, CA) and inorganic nitrogen reduction (nitrate reductase, NR) in 25 intertidal macroalgae of southern Chile (39°S). UV radiation as a factor affecting the nutrient metabolism of algae was also examined. The results of the enzyme activities and the UV sensitivity were related to the position of the algae on the shore, species/taxonomic groups and morpho-functional patterns. The CA activity in the studied algae ranged from 42 to 165 REA g⁻¹ FW, and was neither related to growing depth nor to taxonomic or morpho-functional groups. The NR activities ranged from 0.1 to 8.9 μmol NO₂⁻ g⁻¹ FW min⁻¹, with the highest levels observed in red algae. In contrast to CA, the NR activities showed a decreasing tendency from supra/midlittoral to infra/sublittoral. Also, differences between morpho-functional groups were seen. The impact of artificial UV radiation on CA and NR activities was variable as in some species it provoked an increase while in other species a decrease was observed, suggesting species-specific responses and UV sensitivity. The CA activity was the most UV sensitive in the taxonomic group Chlorophyta and in the supralittoral algae. The UV sensitivity of NR activity could not be related to any patterns related to morpho-functional or taxonomic groups and habitat depth.     

Egg production and feeding by copepods prior to the spring bloom of phytoplankton in Fram Strait,Greenland Sea

Reproductively activeCalanus hyperboreus (Krøyer) andC. glacialis Jaschnov were captured in the upper 100 m of Fram Strait (77° to 79°N) in late winter 1987. There was no evidence of a phytoplankton bloom; chlorophylla concentrations were uniformly low (<0.1 mg m^–3), and nitrate concentrations were uniformly high (>11.3 mg-at m^–3). Gut-fullness measurements indicated that females were ingesting very little. The maturation state of gonads of bothC. hyperboreus andC. glacialis indicated that 75% of females were in a ripe condition consistent with observed egg laying. The lipid content of females laying eggs was reduced in both species compared to that of females not laying eggs. InC. hyperboreus the reduction was 39% and inC. glacialis it was 44%. All the evidence suggests that bothC. hyperboreus andC. glacialis were laying eggs in late winter by using lipids stored previously; they were not relying on ambient concentrations of phytoplankton. The daily rate of egg laying byC. glacialis using lipids in late winter exceeded the rate reported for summer when ambient food supplies have been shown to be necessary. It is suggested that individuals, spawned well in advance of the spring bloom of phytoplankton, may comprise a major portion of the annual recruitment to the entire population ofC. glacialis in this area, and that their life cycle can be completed within 1 yr. NeitherMetridia longa (Lubbock) norC. finmarchicus (Gunnerus) laid eggs during this study.     

Aspects of the biology of resting spores of Thalassiosira nordenskioeldii and Detonula confervacea

Large numbers of resting spores of Thalassiosira nordenskioeldii Cleve and Detonula confervacea (Cleve) Gran were produced when these species were grown at low levels of ammonia and nitrate. The production of resting spores by T. nordenskioeldii was inversely related to temperature. At 0° and 5°C between 68 and 96% of the total cells were resting spores, while at 15°C resting spores were not produced. Resting spores of both species would not survive 7 days in the dark at 20°C. At 0°, 5°, 10° and 15°C, the length of time that the resting spores of both T. nordenskioeldii and D. confervacea remained viable was inversely related to temperature. At 0°C, T. nordenskioeldii remained viable for 576 days. The data suggest that the production of resting spores by these two species does not aid them in the survival of unfavorably high temperatures such as are found in temperate estuaries during the summer. Rather, they appear to be an adaptation for the survival of long periods of darkness in polar regions.     

Enzymes from marine phycophages that degrade cell walls of seaweeds

Agarase, cellulase and alginate lyase activities from crude extracts of Aplysia dactylomela Rang, Haliotis coccinea canariensis Nordsieck, Littorina striata King et Broderip and Diadema antillarum Phillipi were measured in vitro to compare digestive efficiencies against several components of complex seaweed cell walls. Commercial abalone acetone powder (AAP, an extract from Haliotis sp.; Sigma, Ref. A-7514) and purified (Sigma, Ref. A-6306) and non-purified agarases from Pseudomonas atlantica were used with the same objective. Optimum conditions for agarase and cellulase activities were 40°C and pH 6.0. For alginate lyase, optimum temperature and pH were species-dependent. Highest reducing sugar release was shown by crude extracts from A. dactylomela. These crude extracts displayed high agarase activity compared with bacterial agarases at 40°C, and were significantly higher at 25°C. AAP and crude extracts from L. striata and D. antillarum exhibited high specific activities on all the substrates. Cold extract from Gracilaria spp. was the best substrate with which to measure agarase activity.     

Nitrogen regeneration by the surf zone penaeid prawn Macropetasma africanus

Nitrogen excretion of individual Macropetasma africanus (Balss) from an exposed beach/surf zone in Algoa Bay, South Africa was monitored under laboratory and field conditions in relation to body mass, temperature and feeding during 1985. Excretion rate experiments were performed on starved prawns at 15°, 18°, 20° and 25°C, as well as on individuals fed on four different diets (mussel, fish, shrimp and natural diet) at 15° and 20°C. The ratios of the excreted compounds to total nitrogen excreted were similar for the four diets despite differences in their nitrogen content and in the amount of food consumed. At 15° and 20°C, ammonia excretion rates of fed individuals were four to seven times higher than in starved prawns. the excretion rates were not correlated with nitrogen content of diets. M. africanus recycles 1 557 g NH₄–N per metre strip per year or 1 832 g total nitrogen m^-1 yr^-1, which constitute 12 and 14%, respectively, of total phytoplankton requirements of the surf zone. This study indicates that large motile crustaceans, when abundant, can play an important role in nutrient recycling in turbulent marine environments.     

Light-saturated photosynthesis by phytoplankton size fractions in the New York bight,USA

The relationships between netplankton and nanoplankton assimilation numbers, temperature, and major nutrient concentrations were studied and evaluated in the context of seasonal patterns in the biomass of these phytoplankton size fractions. Netplankton and nanoplankton blooms typically occur during late winter (2° to 8°C) and summer (18° to 24°C), respectively. Variations in nanoplankton and netplankton assimilation numbers were not statistically related to the development or collapse of specific blooms based on weekly sampling, but assimilation numbers were higher during the bloom periods than during transition periods of rapid temperature change (8° to 18°C). Differences in the assimilation numbers between size fractions could account for the dominance of the nanoplankton fraction during the summer bloom period but not for the dominance of netplankton during the winter bloom period. Nanoplankton and netplankton assimilation numbers were exponential functions of temperature between 8° and 24°C and 8° and 20°C, respectively. Below 8°C the assimilation numbers of both fractions were higher than expected on the basis of temperature. Above 20°C netplankton assimilation numbers declined with temperature. Netplankton and nanoplankton assimilation numbers were occasionally correlated with dissolved inorganic nitrogen concentrations from less than 1.0 to more than 15 g-at l^-1. Under these conditions, nanoplankton growth rates (calculated from assimilation number and carbon:chlorophyll) were higher and increased more rapidly with dissolved inorganic nitrogen than netplankton growth rates.     

The effects of varied dissolved oxygen concentrations and temperature on the wood-boring isopod genus Limnoria

Three species of the marine wood-boring genus Limnoria were subjected to low dissolved oxygen concentrations at different temperatures under laboratory conditions. 28-day median tolerance limits (TL_m) were 1.0 mg/l of dissolved oxygen at 15° to 16°C and 19° to 20°C for L. lignorum, 0.75 and 0.60 mg/l at 15° to 16°C and 22° to 25°C, respectively, for L. quadripunctata, and 1.0 and 1.18 mg/l at 15° to 16°C and 22° to 25°C, respectively, for L. tripunctata. The amount of burrowing activity, as measured by the egestion rate, was directly related to the amount of dissolved oxygen. A daily egestion rate of 0.116 mg per day in L. tripunctata at 22° to 25°C was the highest figure measured. The daily egestion rate was sharply reduced at dissolved oxygen concentrations below 3.0 mg/l.     

Bacterial activity related to sedimenting particulate matter

The rates of sedimentation of particulate matter were measured at a station in the Eckernförde Bight, Baltic Sea, using sediment traps. Different parameters (C, N, chlorophyll a, phaeopigments, adenosine triphosphate (ATP), heterotrophic activity (glucose maximum uptake velocity, v_max), biological oxygen demand (BOD), and electron-transport-system (ETS) activity) were measured during a 1-year period to evalnate the composition, vertical distribution, and activity of microorganisms associated with sedimenting particulate matter. Measurements indicated that the organic fractions of particulate sedimenting matter were transformed considerably during sedimentation. The seasonal fluctuations showed higher heterotrophic activity durang summer and lower activity during winter. Laboratory experiments also indicated that an important part of planktonic particulate material was susceptible to mineralization within a short period of time: phytoplankton was mineralized by about 35% and zooplankton by about 18% per day, at 20°C; at 5°C mineralization was considerably lower (about 3 and 8%, respectively).Please address all correspondence to: Dr. R. Zimmermann, Institut für Meereskunde an der Universität Kiel, Abteilung Marine Mikrobiologie, Düsternbrooker Weg 20, 2300 Kiel 1, Germany (FRG)