Intracellular Nitrate Concentration Research Articles

Changes in isotope fractionation during nitrate assimilation by marine eukaryotic and prokaryotic algae under different pH and CO2 conditions

AbstractThe impact of environmental factors on nitrogen (N) and oxygen (O) isotope effects during algal nitrate assimilation causes uncertainty in the field application of sedimentary N isotope records and nitrate isotopes to understand the marine nitrogen cycle. Ocean acidification is predicted to change nitrogen cycling including nitrate assimilation, but how N and O isotope effects during algal nitrate assimilation vary in response to changes in seawater pH and partial pressure CO2 (pCO2) remains unknown. We measured N and O isotope effects during nitrate assimilation and physiological states of the marine diatom Thalassiosira weissflogii and Synechococcus under different pH (8.1 or 7.8) and pCO2 (400 or 800 μatm) conditions. Low pH and/or high pCO2 equally decreased N and O isotope effects during nitrate assimilation by diatoms possibly due to reducing cellular nitrate efflux/uptake ratio and decreased isotope effects for nitrate uptake, whereas they did not affect those by Synechococcus with low intracellular nitrate concentration and limited nitrate efflux. Our results provide compelling experimental evidence showing different changes in N and O isotope effects during nitrate assimilation by marine eukaryotic and prokaryotic phytoplankton at low pH and/or high pCO2. These findings suggest new insight into environmental controls on variability in the isotope effect during algal nitrate assimilation, and have implications for improving a predictive understanding of N and O isotope tools in acidified oceans.

Benthic foraminifera and gromiids from oxygen-depleted environments – survival strategies, biogeochemistry and trophic interactions

Abstract. The oceans are losing oxygen (O2), and oxygen minimum zones are expanding due to climate warming (lower O2 solubility) and eutrophication related to agriculture. This trend is challenging for most marine taxa that are not well adapted to O2 depletion. For other taxa this trend might be advantageous because they can withstand low O2 concentrations or thrive under O2-depleted or even anoxic conditions. Benthic foraminifera are a group of protists that include taxa with adaptations to partly extreme environmental conditions. Several species possess adaptations to O2 depletion that are rare amongst eukaryotes, and these species might benefit from ongoing ocean deoxygenation. In addition, since some foraminifera can calcify even under anoxic conditions, they are important archives for paleoceanographic reconstruction in O2-depleted environments. This paper reviews the current state of knowledge about foraminifera from low-O2 environments. Recent advances in our understanding of specific survival strategies of foraminifera to withstand O2 depletion are summarized and discussed. These adaptations include an anaerobic metabolism, heterotrophic denitrification, symbiosis with bacteria, kleptoplasty and dormancy and have a strong impact on their preferred microhabitat in the sediments, especially the ability of some benthic foraminiferal species to denitrify. Benthic foraminifera also differ regarding their trophic strategies, which has an additional impact on the selection of their microhabitat. For example, some species are strict herbivores that feed exclusively on fresh phytodetritus and live close to the sediment surface, while some species are non-selective detrivores that occupy intermediate to deep infaunal habitats. There is evidence that foraminifers have the capacity to undergo phagocytosis, even under anoxia, and some foraminiferal species which can withstand low-O2 conditions seem to prey on meiofauna. Also, due to their high abundances in O2-depleted environments and their metabolic adaptations, benthic foraminifera are key players in marine nutrient cycling, especially within the marine N and P cycles. This review summarizes the denitrification rates for the species that are known to denitrify and the intracellular nitrate concentrations of the species that are known to intracellularly store nitrate. Finally, equations are provided that can be used to estimate the intracellular nutrient storage and denitrification rates of foraminifera and might be integrated into biogeochemical models.

Comparison of the modified Monod and Droop function combined with Logistic function for describing algae growth

Microcystis aeruginosa (M. aeruginosa) are algae found in common freshwater blooms in China, and Dunaliella tertiolecta (D. tertiolecta) are economically important marine algae. Understanding of the microbial growth kinetics plays a significant role in the management of M. aeruginosa’s blooms and biodiesel production by D. tertiolecta. This study has shown that the combination of mechanistic models (Logistic and Monod) proved to be efficient in describing relationship between M. aeruginosa growth rates and specific concentrations of total dissolved phosphorus (TDP), orthophosphate (PO4 3--P), total dissolved nitrogen (TDN) and ammonia (NH4 +-N) reasonably with R2=0.28-0.93. Meanwhile, results also show that both PO43--P and NH4 +-N are important forms of TDP and TDN in influencing M. aeruginosa growth. It was also noted that the combination of modified Monod and Logistic functions is suitable for describing specific growth rates of D. tertiolecta versus extracellular nitrate concentrations (R2=0.24-0.72). In terms of the combination of Droop and Logistic functions, it was analysed to better explain the relationships between M. aeruginosa specific growth rates and cellular P and N concentrations (R2=0.41-0.86) as compared to the application of Droop function alone. It is also observed that the relationship between D. tertiolecta specific growth rates and intracellular nitrate concentrations also can be well described by the combination of Logistic and Droop functions. In addition, M. aeruginosa growth was affected by less intracellular P concentrations than intracellular N concentrations. In sum, the combination of modified Monod and Logistic functions and the combination of Droop and Logistic functions all can predict algae growth reasonably well, while the combination of Droop and Logistic functions is slightly better. Meanwhile, it is through these two combinations that two sets of better parameters in modified Monod and Droop functions can be respectively obtained to characterize algal population kinetics with changing nutrient concentrations.

Sinks and Sources of Intracellular Nitrate in Gromiids.

A substantial nitrate pool is stored within living cells in various benthic marine environments. The fate of this bioavailable nitrogen differs according to the organisms managing the intracellular nitrate (ICN). While some light has been shed on the nitrate carried by diatoms and foraminiferans, no study has so far followed the nitrate kept by gromiids. Gromiids are large protists and their ICN concentration can exceed 1000x the ambient nitrate concentration. In the present study we investigated gromiids from diverse habitats and showed that they contained nitrate at concentrations ranging from 1 to 370 mM. We used 15N tracer techniques to investigate the source of this ICN, and found that it originated both from active nitrate uptake from the environment and from intracellular production, most likely through bacterial nitrification. Microsensor measurements showed that part of the ICN was denitrified to N2 when gromiids were exposed to anoxia. Denitrification seemed to be mediated by endobiotic bacteria because antibiotics inhibited denitrification activity. The active uptake of nitrate suggests that ICN plays a role in gromiid physiology and is not merely a consequence of the gromiid hosting a diverse bacterial community. Measurements of aerobic respiration rates and modeling of oxygen consumption by individual gromiid cells suggested that gromiids may occasionally turn anoxic by their own respiration activity and thus need strategies for coping with this self-inflicted anoxia.

Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates.

Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations >60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2–3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

The contributions of nitrate uptake and efflux to isotope fractionation during algal nitrate assimilation

In order to strengthen environmental application of nitrate N and O isotopes, we measured the N and O isotopic fractionation associated with cellular nitrate uptake and efflux in the nitrate-assimilating marine diatom Thalassiosira weissflogii. We isolated nitrate uptake and efflux from nitrate reduction by growing the cells in the presence of tungsten, which substitutes for molybdenum in assimilatory nitrate reductase, yielding an inactive enzyme. After growth on ammonium and then N starvation, cells were exposed to nitrate. Numerical models fit to the evolution of intracellular nitrate concentration and N and O isotopic composition yielded distinct N isotope effects (15ε) for nitrate uptake and nitrate efflux (2.0±0.3‰ and 1.2±0.4‰, respectively). The O isotope effects (18ε) for nitrate uptake and nitrate efflux were indistinguishable (2.8±0.6‰), yielding a ratio of O to N isotopic fractionation for uptake of 1.4±0.4 and for efflux of 2.3±0.9. The 15ε for nitrate uptake can account for at most 40% of the organism-level N isotope effect (15εorg) measured in laboratory studies of T. weissflogii and in the open ocean (typically 5‰ or greater). This observation supports previous evidence that most isotope fractionation during nitrate assimilation is due to intracellular nitrate reduction, with nitrate efflux allowing the signal to be communicated to the environment. An O to N fractionation ratio (18εorg:15εorg) of ∼1 has been measured for nitrate assimilation in algal cultures and linked to the N and O isotope effects of nitrate reductase. Our results suggest that the ratios of O to N fractionation for both nitrate uptake and efflux may be distinct from a ratio of 1, to a degree that could cause the net 18εorg:15εorg to rise appreciably above 1 when 15εorg is low (e.g., yielding a ratio of 1.1 when 15εorg is 5‰). However, field and culture studies have consistently measured nearly equivalent fractionation of N and O isotopes in association with low isotope effects, calling for isotopic studies of nitrate transport by other phytoplankton strains.

Potential importance of physiologically diverse benthic foraminifera in sedimentary nitrate storage and respiration

Until recently, the process of denitrification (conversion of nitrate or nitrite to gaseous products) was thought to be performed exclusively by prokaryotes and fungi. The finding that foraminifera perform complete denitrification could impact our understanding of nitrate removal in sediments as well as our understanding of eukaryotic respiration, especially if it is widespread. However, details of this process and the subcellular location of these reactions in foraminifera remain uncertain. For example, prokaryotic endobionts, rather than the foraminifer proper, could perform denitrification, as has been shown recently in an allogromiid foraminifer. Here, intracellular nitrate concentrations and isotope ratios (δ15NNO3 and δ18ONO3) were measured to assess the nitrate dynamics in four benthic foraminiferal species (Bolivina argentea, Buliminella tenuata, Fursenkoina cornuta, Nonionella stella) with differing cellular architecture and associations with microbial endobionts, recovered from Santa Barbara Basin, California. Cellular nitrate concentrations were high (12–217 mM) in each species, and intracellular nitrate often had elevated δ15NNO3 and δ18ONO3 values. Experiments including suboxic and anoxic incubations of B. argentea revealed a decrease in intracellular nitrate concentration and an increase in δ15NNO3 and δ18ONO3over time, indicating nitrate respiration and/or denitrification within the foraminifera. Results illustrate that nitrate reduction occurs in a range of foraminiferal species, including some possessing endobionts (including a chloroplast‐sequestering species) and others lacking endobionts, implying that microbial associates may not solely be responsible for this process in foraminifera. Furthermore, we show that benthic foraminifera may represent important reservoirs of nitrate storage in sediments, as well as mediators of its removal.

Nitrate Control of Root Hydraulic Properties in Plants: Translating Local Information to Whole Plant Response

The sessile lifestyle of plants constrains their ability to acquire mobile nutrients such as nitrate. Whereas proliferation of roots might help in the longer term, nitrate-rich patches can shift rapidly with mass flow of water in the soil. A mechanism that allows roots to follow and capture this source of mobile nitrogen would be highly desirable. Here, we report that variation in nitrate concentration around roots induces an immediate alteration of root hydraulic properties such that water is preferentially absorbed from the nitrate-rich patch. Further, we show that this coupling between nitrate availability and water acquisition results from changes in cell membrane hydraulic properties and is directly related to intracellular nitrate concentrations. Split-root experiments in which nitrate was applied to a portion of the root system showed that the response is both localized and reversible, resulting in rapid changes in water uptake to the portions of the roots exposed to the nitrate-rich patch. At the same time, water uptake by roots not supplied with nitrate was reduced. We believe that the increase in root hydraulic conductance in one part causes a decline of water uptake in the other part due to a collapse in the water potential gradient driving uptake. The translation of local information, in this case nitrate concentration, into a hydraulic signal that can be transmitted rapidly throughout the plant and thus coordinate responses at the whole plant level, represents an unexpected, higher level physiological interaction that precedes the level of gene expression.

Vertical migration of the toxic dinoflagellate Gymnodinium catenatum under different concentrations of nutrients and humic substances in culture

Vertical migration behaviour by the chainforming dinoflagellate Gymnodinium catenatum Graham was investigated using vertically-stratified laboratory columns. Under surface nutrient-deplete conditions, with nutrients added only at depth, 100% of cells underwent vertical migration (VM), starting downwards migration 3 h before the end of the light period and beginning upwards migration 3 h before the start of the light period. Cells in nutrient-replete columns showed no VM, but they were more dispersed in the upper layer during the dark compared to the light period. When surface layers (S) were nitrate-deplete (−N) and enriched with humic substances (H) contained in Huon River water and bottom waters (B) were nutrient replete (R) (SH −NB R), the pattern of VM was altered—50% of cells underwent migration and 50% remained at the pycnocline. In columns with nitrate-replete and humic-enriched surface layers (SH RB R), most cells underwent VM, while 30% remained at the surface. Cells in SH −NB R columns showed increased N quotas and intra-cellular nitrate concentrations after 4 days, indicating nitrate uptake by G. catenatum in bottom layers. The concomitant increase in particulate organic nitrogen (PON) with the decrease in external nitrate concentrations in bottom layers provide convincing evidence that VM by G. catenatum facilitates nutrient retrieval at depth. However, addition of humic substances (a potential source of organic nitrogen) to surface layers did not ameliorate G. catenatum N depletion sufficiently to preclude the need for NO 3 − uptake at depth. Furthermore, there was no detectable pattern of increasing carbon (C) quota during the day (photosynthate accumulation) or increasing N quota during the night (nitrate assimilation). Toxic dinoflagellate G. catenatum blooms are commonly associated with nitrate depletion in surface waters in south-east Tasmanian waters (Australia). Therefore, vertical migration, facilitating N uptake at depth, could play an important role in this organism's ecological strategy, enabling it to exploit environments where light and nutrients are vertically separated.

Simultaneous production of ginseng saponin and polysaccharide by suspension cultures of Panax ginseng: Nitrogen effects

The effects of the nitrogen ( n) source on the kinetics of cell growth, major nutrient consumption, and production of ginseng saponin and polysaccharide in suspension cultures of Panax ginseng cells were studied. The ratio of NO 3 − NH 4 + and initial total medium n were altered. Cell growth was better at 60 m m n and higher ratios of NO 3 − NH 4 + . With nitrate alone or a mixture of nitrate and ammonium (at a ratio of 2:1) provided as n source, 10 m m n was found to be a crucial point for the cell mass accumulation. Nitrate was a favorable n source for ginseng cell growth. A cell growth rate of 0.11 day −1 and a dry cell density of 13 g l −1 were obtained at 10 m m nitrate (sole n source). At an initial ammonium concentration above 20 m m, the ginseng cell growth was inhibited, and a higher intracellular nitrate concentration and lower soluble sugar content in cells were observed. The results also indicate that the specific production ( i.e., content) of ginseng polysaccharide was not significantly affected by the alteration of n source while the maximum production of polysaccharide of 1.19 g l −1 was achieved at NO 3 − NH 4 + of 60:0 under 60 m m of total n ( i.e., sole nitrate). The saponin production was relatively higher within initial n concentrations of 5–20 m m (with nitrate alone or a NO 3 − NH 4 + ratio of 2:1). The highest content (5.2%) and total production (0.6 g l −1) of ginseng saponin were obtained at an initial nitrate of 5 m m.

Oxygen increases the steady-state level of nitrate in denitrifying cells of, Paracoccus denitrificans

The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [ 14C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.

Oxygen increases the steady-state level of nitrate in denitrifying cells of Paracoccus denitrificans.

The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [14C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.

Two‐stage batch process for the production of ajmalicine by Catharanthus roseus: The link between growth and production stage

The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. (c) 1995 John Wiley & Sons, Inc.

Nitrate uptake kinetics by two marine diatoms using the radioactive tracer 13N

The response of the nitrate uptake system to nitrate concentrations in the range 1 to 500 μM was examined with the radioactive isotope 13N for two marine diatoms ( Skeletonema costatum and Thalassiosira weissflogii) under various states of nitrgenous nutrition. Classical saturation kinetics were observed in the lower range of concentrations (1–40 μM), but a further increase in uptake was observed above 60 μM. Intracellular nitrate concentrations between 5 and 17 mM indicate that it is an active uptake system rather than a diffusion phenomenon which is responsible for uptake at those high external nitrate levels.

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling

Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m(-3) nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m(-3) (n=19) and 41.8 ± 2.6 mol · m(-3) (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m(-3) (n = 4) and 72.8 ± 8.4 mol · m(-3) (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m(-3) (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m(-3) (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m(-3), while the vacuole accumulated nitrate to 30-50 mol · m(-3). The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.

Measurement of intracellular nitrate concentrations in Chara using nitrate-selective microelectrodes

Nitrate-selective microelectrodes have been made using a quaternary ammonium sensor, methyl-tridodecylammonium nitrate, in a Polyvinylchloride matrix. These electrodes showed a log-linear response from 0.1 to 100 mol · m(-3) nitrate with a typical slope of 55.6 mV per decade change in nitrate concentration. The only physiologically significant interfering anion was chloride but the lower limit of nitrate detection was 0.5 mol · m(-3) in the presence of 100 mol · m(-3) chloride which means this interference will not be important in most physiological situations. These microelectrodes were used to measure nitrate concentrations in internodal cells of Chara corallina cultured under low nitrate and nitrate-replete conditions for 6 to 30 weeks. Cells maintained in low nitrate only showed measurements which were less than the detection limit of the electrodes, while cells grown under nitrate-replete conditions showed two populations of measurements having means of 1.6 and 6.2 mol · m(-3). Chemical analysis of the high-nitrate cells indicated that they contained a mean nitrate concentration of 5.9 mol · m(-3). As vacuolar nitrate concentration would dominate this whole-cell measurement, it is concluded that the higher concentration measured with the electrodes represents vacuolar nitrate concentration and the lower value represents the cytoplasmic concentration. This intracellular distribution of nitrate could only be achieved passively if the electrical potential difference across the tonoplast is between +25 and + 35 mV.

Manipulation, by nutrient limitation, of the biosynthetic activity of immobilized cells of Capsicum frutescens Mill. cv. annuum

The relationship between the synthesis and accumulation of protein and capsaicin was investigated in cultured cells of Capsicum frutescens Mill. cv. annuum immobilized in reticulate polyurethane. Cells were cultured in media containing reduced concentrations of essential nutrients, in an attempt to manipulate the rates of protein synthesis. Cells cultured in the absence of orthophosphate for 7 d demonstrated no reduction in the incorporation of L-[U-(14)C]phenylalanine into soluble protein or an increase in incorporation into capsaicin, compared with controls supplied with orthophosphate. By day 15 of culture, however, a differential incorporation of label was observed. Over a 21-d culture period the intracellular phosphate did not completely disappear. Cells cultured in the absence of nitrate and phosphate combined, however, exhibited some reduction in incorporation of [(14)C]phenylalanine into protein and an increased incorporation into capsaicin after 7 d of culture, but the differences were greater at day 15, when increases in the total capsaicin content of the cultures were apparent. There was observed a relationship between the intracellular nitrate concentration, the culture growth index, and the incorporation of [(14)C]phenylalanine into soluble protein - each of these factors was inversely related to the incorporation of label into capsaicin and the total capsaicin content of the cultures.

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 μg-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.