Steady-state Growth of the Marine Diatom Thalassiosira

Plant Physiol. (1980) 66, 383-3890032-0889/80/66/0383/07/$00.50/0

Steady-state Growth of the Marine Diatom ThalassiosirapseudonanaUNCOUPLED KINETICS OF NITRATE UPTAKE AND NITRITE PRODUCTION'

Received for publication January 2,1980 and in revised form March 17, 1980

ROBERT J. OLSONInstitute ofMarine Resources, A-018, Scripps Institution of Oceanography, La Jolla, California 92093

JANICE BEELER SooHoo AND DALE A. KIEFERDepartment of Biological Sciences, University of Southern California, Los Angeles, California 90007

ABSTRACT

Seasonal studies of the vertical distribution of nitrate, nitrite, andphytoplankton in the oceans and studies using 15N as a tracer of nitratemetabolism indicate that the reduction of nitrate by phytoplankton is asource of nitrite in the upper waters of the ocean. To better understandthis process, the relationship between nitrate uptake and nitrite productionhas been examined with continuous cultures of the small marine diatomThalassiosira pseudonana. In a turbidostat culture, the rates of nitriteproduction by T. pseudonana increase with light intensity. This process isonly loosely coupled to rates of nitrate assimilation since the ratio of netnitrite production to total nitrate assimilation increases with increasedrates of growth. In continuous cultures where steady-state concentrationsof nitrate and nitrite were varied, T. pseudonana produced nitrite at rateswhich increased with increasing concentrations of nitrate. Again, the ratesof nitrite production were uncoupled from rates of nitrate assimilation. Thestudy was used to derive a mathematical description of nitrate and nitritemetabolism by T. pseudonanmThe validity of this model was supported by the results of a study in

which '5N-labeled nitrite was introduced into the continuous culture, andthe model was used to examine patterns in distribution of nitrite in theAntarctic Ocean and the Sargasso Sea.

Because N is often scarce or in a form, such as gaseous N, whichis unavailable for assimilation by many species, the N cycle is ofkey importance to the structure and function of most ecosystems.In aquatic as well as terrestrial systems, the general features of theN cycle are known, but quantitative knowledge of the rates oftransformations, such as assimilation, nitrification, and denitrifi-cation, is poor. Lack of quantitative knowledge about the cycle isdue in part to the simultaneous and competing metabolism ofbacteria and plants and to inadequate quantitative descriptions ofthese processes. An example of confusion resulting from inade-quate knowledge of the factors affecting rates of nitrification andprimary production is the conflicting views on the origins of theprimary nitrite maximum in marine planktonic ecosystems.

In the tropical and temperature open ocean, the distribution of

'This study was supported by the United States Department of Com-merce, National Oceanic and Atmospheric Administration, National En-vironmental Satellite Service Grant 04-7-158-44123, by Department ofEnergy Contract DE-AC03-79EV70020, and by DePaul University GrantC-8 16302.

nitrite within the water column is usually limited to a sharplydefined maximum within the thermocline and slightly above thenitracline. This feature may originate from the process of bacterialnitrification, as suggested by Brandhorst (2), or may originate asa by-product of nitrate assimilation by phytoplankton, as sug-gested by Vaccaro and Ryther (21). Both processes have beensupported by laboratory work (5, 11, 12), and incubation and 5N-tracer experiments have shown that both processes can occur inthe sea (15, 16, 22). Since the two proposed causes of the nitritemaximum represent distinct pathways ofthe N cycle, experimentaland theoretical examination of the proposals may provide insightinto the nature of the cycle. We have begun such an examinationby direct field measurements of rates of nitrification and rates ofnitrate reduction using 15N tracers for ammonia and nitrate as firstdone by Wada and Hattori (23). We have also examined theproblem indirectly by characterizing the kinetics of nitrite pro-duction by phytoplankton grown in the laboratory upon nitrate.Here, we report the results of a quantitative study of nitratemetabolism by the small marine diatom Thalassiosirapseudonana,which has been grown in continuous culture. The study hasyielded information about the relationship between nitrate andnitrite concentrations and rates of nitrate uptake and nitrite pro-duction. It has also yielded information about the relationshipbetween rates of nitrate uptake and nitrite production and levelsof light. The results show that the process of nitrate uptake is onlyloosely coupled to nitrite production. From the study, we havederived a mathematical description of steady-state growth andnitrogen metabolism for T. pseudonana.

MATERIALS AND METHODS

Turbidostat. Rates of growth, nitrate uptake, and nitrite pro-duction by T. pseudonana (clone 66-A, isolated from the centralNorth Pacific gyre; Food Chain Research Group culture collec-tion, SIO) were most often measured in a turbidostat, a devicewhich continuously monitors the turbidity of the cell suspension.The turbidostat consisted of a dual optical system: one photocelldetected the diffuse light emitted from an IR light source andpassed through the reaction vessel and a second photocell detectedthe diffuse light from the same light source which passed directlythrough a fiber optics probe. The ratio of the voltages from thereference and sample photocells was then the final signal ofturbidity changes (13). When the ratio exceeded a preset value,the electrical circuit activated a solenoid which allowed media toenter the reaction vessel until a balancing drop in turbidity wasachieved. This turbidostat was used to determine steady-state ratesof nitrate metabolism as functions of both light levels and concen-trations of nitrate and nitrite within the reaction vessel. Additional

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OLSON, SOOHOO, AND KIEFER

experiments were undertaken with a spin filter chemostat, whichproved to be a useful culturing method when cells were growingat lower concentrations of nitrate and nitrite. The spin filter(VirTis, Inc.) allows independent control of cell growth rate andrates of medium flow-through, analogous to dialysis culture tech-niques. The turbidostat or spin-fiter chemostat sat in an aquariumin which temperature was maintained constant at 18 C by circu-lating water. The light field, which was oriented at right angles tothe measuring beam, was of continuous duration and of intensityselected through the use of neutral density screening. Cool-whitefluorescent lights were used for all but the highest light intensityexperiments; the light source for these experiments was a 500-wQuartzlite lamp (Appleton Electric Co., Chicago) whose outputpassed through 40 cm water before reaching the culture vessel.Measurements of the diffuse light field were obtained with a

quantum scalar irradiance meter (1).Medium. The culture medium was IMR/2 (9) in which nitrate

was the source of inorganic N. Surface seawater from the SIO pierwas depleted of nitrate and nitrite by placing it in front of a bankof 40-w fluorescent lights for 3 to 5 days before filtering it for use

as the medium base. The concentrations of nitrate in the influentwere varied according to experimental design, but its concentra-tions were always such that no other nutrient would limit cellgrowth before nitrate. In one set of experiments nitrite was alsoadded to the medium as 15N.

Monitoring of the Continuous Cultures. After sterile inoculationof the culture, growth of T. pseudonana within the reaction vesselreached a steady-state in which nitrate, nitrite, and cell numberremained constant. During this time, samples were taken fordeterminations of rates of dilution and concentrations of cells,nitrate, and nitrite. For selected studies, cellular nitrogen and 15N

incorporation into cellular nitrogen were also measured. Cellswere counted with a hemocytometer and nitrate and nitrite weremeasured colorimetrically following the procedures outlined inStrickland and Parsons (20). Cellular N was measured by collect-ing cells on a glass fiber filter (GF/C) and combusting the filterin a Hewlett-Packard 185-B CHN analyzer. Enrichment of 15N

was measured with a mass spectrometer (24) and rates of assimi-lation were calculated according to the procedures of Dugdale andGoering (8).

Rates of Metabolism. From measurements of nitrate [NO3Jand nitrite [NO2 Ji concentrations in the influent and concentra-tions within the reaction vessel or the effluent [NO3j1O, [NO2 lO,one may calculate basic rates of nitrate metabolism. According toFigure 1, the three major species of N within the reaction vesselare nitrate, nitrite, and cellular N, ammonia concentrations beingquite low. These three compartments are related by three fluxes:

nitrate assimilation into cellular N (jNo3), gross nitrite productionfrom nitrate (jNo2), and nitrite assimilation into cellular N (jNO2).Measurements of dilution rates and nutrient concentrations were

used to calculate rates of net nitrite production (jNO2 = jPNo-jO2) and rates of nitrate uptake (jNO3 = jNo3 + jNo2). The intro-

AMBIENT iNO3 CELL|NO3 | NITRATE ASSIMILATION NITROGEN

<eXF AMBIENT f

FIr. 1. Nitrogzen compartments and fluxes in the turbidostat culturesupplied with nitrate as N source.

duction of '5N-labeled nitrite into the continuous culture was usedto calculate each of the three fluxes shown in Figure 1. All fluxesare expressed as N-specific rates, i.e. (nutrient-N) (cellular-N)-'time-', or simply time-'.

Short Term Competitive Uptake of Nitrate and Nitrite. Severalexperiments were designed to measure the short term kinetics ofnitrate and nitrite uptake by cells from a nitrate-limited continuousculture. Aliquots taken from a chemostat culture with a doublingtime of 24 h were diluted with filtered chemostat overflow toprovide sufficient volume to measure uptake oflow concentrationsof substrate without causing substrate depletion. The dilutedaliquots were added to flasks containing the appropriate amountsof nitrate and nitrite, one of the species being labeled with 15N.These were incubated for 10 min in light of the same intensity asthat of the growth chamber (1.3 x 1016 quanta cm-2 s-1), the cellsthen were collected by filtration and dried, and the 15N enrichmentwas determined. Values for Vm. and apparent half-saturationconstants were determined from Lineweaver-Burk transforma-tions of the data fitted by the weighted linear regression methodof Wilkinson (25).

Field Studies. In our discussion of the laboratory study we willcompare the results of the culture work with observations ofnitrate uptake and nitrite production in the ocean. These obser-vations required sampling of seawater at selected depths, inocula-tion of the sample with '5N-labeled nitrate to a final enrichmentof from 20 to 30% "5N, and incubation of the sample either in situor simulated in situ on board ship. The particulate N was collectedupon glass fiber filters and the filtrate was saved. The rates ofnitrate assimilation were calculated from the three measurementsof nitrate concentration within the sample, the concentration ofparticulate N, and 15N enrichment of the particulate N. Rates ofnitrite production likewise were calculated from concentrations ofnitrate and nitrite in the samples and 15N enrichment of the nitritepresent. The latter measurement was obtained by a proceduresimilar to that described by Schell (17). In this method, anilineand 8-naphthol are added to the filtrate, leading to the incorpo-ration of the nitrite into an azo dye, which is extracted in chloro-form. The extract then is evaporated onto a glass fiber filter andsubjected to MS.

RESULTS

Effects of Nitrate Concentration on Nitrite Production. A largenumber of measurements were made of continuous nitrate uptakeand net nitrite production under conditions where the influentcontained nitrate and not nitrite, and light intensities were mod-erate and constant (6.3 x 1015 quanta cm-2 s-'). In these cultures,the concentrations of nitrate and nitrite within the reaction vesselwere affected either by changing the nitrate concentration in theinfluent medium or by adjusting the levels of turbidity of the cellsuspension (or rates of dilution in the chemostat); the steady-stateconcentrations of nitrate ranged from 0.3 to 44 ,UM (Fig. 2). Overthis range of nitrate concentrations, the uptake rate of nitrateremained relatively constant, whereas the rate of nitrite productiondecreased monotonically below nitrate concentrations of about 20ELM. In Figure 3, we plot the same data in terms of the ratio of netnitrite production to nitrate uptake (jNO2/jNo3). This ratio variesfrom 0.001 to 0.05, indicating that nitrite production is not wellcoupled to nitrate uptake.

Effects of Light Levels on Nitrite Production. A second seriesof culture studies were designed to examine the relationshipbetween light levels and rates of net nitrite production and nitrateuptake. In these cultures, nitrate was the sole N source and theturbidity of the cell suspension was adjusted so that nitrate con-

centrations within the reaction vessel were high (>20 LM) and not

limiting to nitrite production. By varying the number of fluores-cent bulbs in the light bank and by using neutral density screening,we established a range of light-limited growth rates. In Figures 4

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Plant Physiol. Vol. 66, 1980 STEADY-STATE NITRITE PRODUCTION

[NOj] ,M

FIG. 2. Steady-state rates of net nitrite production (jNo2) and net nitratedisappearance (jNo,) as functions of nitrate concentration. E = 6.3 x 10i5quanta cm-' s-'.

0.08

0.07

0.06

_N2jNO3

0.05

0.04

0.03

0.02

0.01

10 20 30 40

[NO3],,MFIG. 3. Ratio of net nitrite production to net nitrate disappearance as

a function of nitrate concentration in the turbidostat culture. E = 6.3 x

1O05 quanta cm-2 s-'.

and 5, we present the rates of nitrate assimilation and nitrateproduction as functions ofquantum irradiances. As levels of scalarirradiance increased from 5 x 1014 to 3 x 1016 quanta cm-2 s-1,rates of both nitrate uptake and nitrite production increased asshown in Figure 4, but rates of nitrite production increased at afaster rate than nitrate uptake. The changing stoichiometry ofnitrate uptake and nitrite production under conditions of light-limited continuous growth is evident from the curve of Figure 5.

Short-term Uptake of Nitrate and Nitrite. The steady-statecultures described above yielded information about the depend-ence of nitrite production upon ambient concentrations of nitrateand nitrite and upon light intensity; however, the data yielded noinformation about the dependence of nitrate and nitrite assimila-tion (jNo,, j&o2) upon ambient concentrations of nitrate and nitrite.Much lower concentrations than those measured in our continuouscultures affect both growth and rates of assimilation (3, 4). Inaddition, these cultures yielded little information about the com-petitive nature of nitrate and nitrite uptake. The short-term uptakeexperiments summarized in Figures 6 to 8 help supply someinsight in these two areas.

Lineweaver-Burk plots of the short term uptake experiments(Figs. 6 and 7) appear to exhibit classical competitive inhibitionkinetics: increasing slopes with increasing competitor concentra-tions and convergence of the lines to a common V.. value. The

j NO2 j N03( d- ) ( d-')

0.05 - 0.5

0.5 2 3x1O0

E(quanta s'I cm-2)

FIG. 4. Steady-state rates of net nitrite production (jNO2) and net nitratedisappearance (jNo2) and growth rate constant (,i) as functions of irradi-ance in the turbidostat culture.

0.08

0.07

0.06

0.05

jNO2

jNO3 0.04

0.03

0.02

0.01

0

0 1 2 5 10 20

E (quanto * s7l-ccmh2)30x 1015

FIG. 5. Ratio of net nitrite production to net nitrate disappearance as

a function of irradiance in the turbidostat culture.

inhibitory effect of nitrate on '5N-labeled nitrite uptake is clearlygreater than the reverse situation, indicating greater affimity of theuptake system for nitrate. It should also be noted that the VK. fornitrate is significantly greater than that for nitrite (3.37 + 0.50 and2.19 + 0.25 atom %/10 min, respectively; 95% confidence limits).The mean values for inhibitor constants (KI), as determined

graphically by the method of Dixon (6), are 0.10 ,UtM for nitrateand 0.20 ,UM for nitrite. Half-saturation constants for uptake (K.)of nitrate aind nitrite were determined on the same plot by usingthe relationship [I] = -K, ([S]/K8 + 1) at the intersection of eachline with the base line (6). The values obtained were 0.06 ,tM fornitrate and 0.14 ,iM for nitrite; these agree fairly well with the K,

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I/V

(%0 lOmin'8)'

I/S (,FM)-

FIG. 6. Lineweaver-Burk plot of short-term '5N-labeled nitrite uptakein the presence of varying nitrate concentrations.

('5NO- UPTAKE)

I/V

(15N atom% XS/IO min )'4/iM t

/S ( FM)I

FIG. 7. Lineweaver-Burk plot of short-term "5N-labeled nitrate uptakein the presence of varying nitrite concentrations.

values, as expected. When the data are plotted for this determi-nation, as 1/ V versus [I] for constant [SI, curvature of the linesbecomes apparent at high concentrations of nitrite (but not ofnitrate). This curvature was even more pronounced in anotherexperiment in which nitrite levels were extended to 10 ,uM (Fig. 8).The saturation of inhibitory effectiveness of nitrite indicated bythe curvature suggests that partially competitive inhibition maybe occurring (7). This effect indicates that the regulation of nitrateand nitrite uptake may be more complicated than that of classicalcompetitive kinetics. Eppley (10) also noted a quasicompetitiverelationship between nitrate and nitrite uptake by the diatomDitylum brightwellii. The concentrations of nitrate and nitritewhich cause diminished rates of uptake are more than 1 order ofmagnitude lower than the concentrations causing decreases innitrite production.

Effects of Nitrite Concentration on Nitrite Production. In fourcultures the influent contained both nitrate and nitrite, the nitritebeing labeled with 15N. These cultures were examined in order todetermine both the effects of nitrite concentration on nitriteproduction and to determine all three rates of steady-state metab-olism, jNo2, jNo3, and jio2 (Table I). In examining the first twocultures, which were grown at the same light intensities and withhigh nitrate concentrations, we see that an increased concentrationof extracellular nitrite in the second culture caused an increasedrate of nitrite uptake and a decreased rate of net nitrite production(jNO2) (in fact, negative); the gross rate of nitrite production (jNo2)

[ i]=

FIG. 8. Dixon plot of short-term "5N-labeled nitrate uptake in thepresence of varying nitrite concentrations.

was unaffected. In the fourth experiment, at a much higher lightintensity, the ratio jPO2/jaNO was higher and there was again netnitrite production, in spite of the high extracellular nitrite concen-tration. The results also show that nitrate is the favored substratesince isotopic enrichment of the cellular N in each case wasroughly half that of the inorganic N supplied in the medium. Itappears likely that, for given values of nitrate and light intensity,there exists a concentration of nitrite at which there is no netproduction: lower concentrations of nitrite lead to net productionand higher concentrations lead to net assimilation. Table I alsocontains (in parentheses) predicted fluxes based upon our mathe-matical description of all the experiments.

DISCUSSION

The study aimed to provide a general description of continuousnitrite production by T. pseudonana and did not examine cellularregulation of the steps of nitrate assimilation. Neither the sizes ofinternal pools of nitrate, nitrite, and ammonia nor activities ofpermeases and reductases were measured. We do feel that anymodel of the mechanism of nitrate uptake for diatoms, such asthat recently described by Serra et al. (18, 19), should be consistentwith the metabolism described for continuous culture. In partic-ular, understanding of the large variations in the ratio jNO2/jNO,affected by varying nutrient concentrations and light levels asshown in Figures 3 and 5 may provide insight into the regulationof the steps of nitrate assimilation. Likewise, the "quasicompeti-tive" nature of nitrate and nitrite uptake in both continuousculture (Table I) and short-term experiments is of interest tonutrient biochemists.

Analytical Expressions. To summarize the characteristics ofnitrate metabolism by T. pseudonana presented here, we havederived several empirical functions which attempt to describe thethree metabolic fluxes shown in Figure 1. These functions haverecently been introduced into a model of the seasonal productionof nitrite by phytoplankton in the temperate ocean (D. Kiefer andJ. Kremer, manuscript in preparation) and in our discussion herewill be applied to selected field observations of nitrite productionand distributions of nitrite. The three functions of interest are

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Table I. Steady-state Culture Parameters and Fluxes of Nitrate and Nitrite by T. pseudonana in Continuous CultureSubscripts i and o refer to inflowing and outflowing medium, respectively. '5N was added as '5NO2- and fluxes were calculated using the measured

cellular '5N, nutrient concentrations, and flow rate. Values in parentheses are fluxes predicted by the model.

Concentration of Parameters ja /jaO

E y [NO3-1i [NO3-1o [NO2-]i [NO2-1. 2AO.J'NON jNO, [NO 10/ar ~~~~~~~~~~~~~[NO:31.Jquanta/s. day' ,UM atom % day' ratio

cm2 excess6 x IO' 1.61 81.0 38.0 3.85 4.94 2.00 0032 0.021 0.075 0.022 1.53 0.26

0.053(0.044) (0.078) (0.034) (1.57)

6 x 10'1 1.65 59.6 28.7 10.07 9.37 5.65 0093 0.012 0.069 -0.37 1.54 0.21

0.105(0.108) (0.077) (-0.031) (1.54)

6 x 1015 0.98 49.5 2.47 0.64 0.69 1.14 0011 0.061 0.073 0.001 0.91 0.28

0.072(0.055) (0.057) (0.002) (0.92)

3 x 10'6 1.30 76.3 44.0 10.20 10.90 3.84 0050 0.011 0.089 0.029 1.24 0.20

0.061(0.066) (0.125) (0.059) (1.23)

cellular rates of nitrate assimilation (j o3), cellular rates of grossnitrite production (j' o2), and cellular rates of nitrite assimilation(j'No2) (Fig. 1). As shown under "Results," each of these fluxesmay be affected by light intensity (which may act by affectinggrowth rates) and concentrations of nitrate and nitrite. Tempera-ture effects upon the three fluxes were not examined and will beignored.The model of steady-state N metabolism by T. pseudonana will

be based upon the assumption that the rates of N metabolism aregoverned by Liebig's principle of the most limiting factor. In thecase of rates of N assimilation or the specific growth rate of thecells, we will assume that these rates are determined by eitherlimiting ambient concentrations of nitrate and nitrite or limitinglevels of light intensity. Thus, the growth rate of the cell is afunction of only one of the two factors and there is no interactionbetween these factors. Likewise, we will assume that the rate ofgross nitrite production is limited by either light intensity or nitrateconcentration. To verify this assumption the experiments de-scribed here must be greatly expanded to include various combi-nations of light intensity and nutrient concentrations.We will also assume that the assimilation of nitrate and nitrite

obeys the kinetics of competitive uptake. As mentioned earlier(Figs. 4 and 5), such an assumption is only an approximation todescribing the steady-state growth of T. pseudonana. As waspointed out by Serra et al. (19) most recently, rates of assimilationare most likely regulated (and complicated) by the negative feed-back inhibition of ammonia or another intermediate of the path-way ofN assimilation. Where N concentrations are more limitingto the growth rate of cells than light levels, we describe suchgrowth according to competitive uptake of nitrate and nitrite:

jVKNO2NO3 + janKNoxNO2= KNo2KNO3 + KNo2NO3 + KNOjNO2 (1)

IL is the specific growth rate of the cells in units of/M N (AM cellN)-' day-, j'Nn3x and j'N-x are constants for maximum specificrates of assimilation for the two species ofN in the same units as

A, KNO2 and KNO3 are half-saturation constants for nitrate andnitrite uptake, respectively, in units of tmM, and NO2 and NO3 are

variables, concentrations of nitrate and nitrite in units of iM. From

an examination of the short-term uptake experiments (Figs. 6 and7) and the isotopic study with the turbidostat, we have estimatedthe four constants in this equation to be: (jo-ax = 1.6 day-', j'-"x= 0.8 day-', KNO:, = 0.06 isM, and KNO2 = 0.14 .LM. According tothese constants, the maximum doubling time for T. pseudonanagrowing on nitrate (18 C) is about 10 h and that for nitrite is abouttwice as long; also, the cellular affinity for nitrate is twice that ofnitrite. The two pairs of constants indicate a 4-fold increase inpreference for nitrate over nitrite. KNO3 and j"Y' values agree wellwith studies of Caperon and Meyer (3, 4).When light levels are more limiting to growth rate than nutri-

ents, ,u is calculated from a second function:

mI E+E -RA = max KE'

(2)

E is the scalar irradiance in units of quanta cm-2 s-' to which thecells are exposed and tlmax, KE, and R are empirically derivedconstants. A best fit of the growth response of T. pseudonana toirradiances of 1 x 1016 quanta cm-2 s-' or less yielded values forKE ttmax, and R of 1.5 x 1015 quanta cm-2 s-', 1.67 day-' and 0.16day-', respectively. For values of E greater than 3 x 1016 quantacm-2 s-', ,[ is equal to maximal growth rates of 1.67 day-'.Once the steady-state growth rates for T. pseudonana are deter-

mined, one can calculate the two fluxes, jNo2 and jao3, on the basisof competitive kinetics:

NO2 = I' a.jN0KNO3NOJN03KNO NO3 + jOKNO.,NO3ja= K jNO2KNO2NO3

JN KNo2NO3 + jaxKNO3NO2

(3)

(4)

The functions describing gross nitrite production (jNo), foreither nutrient limitation or light limitation were derived from theresults shown in Figures 2 and 4. In the case when nutrientconcentrations are more limiting to production, jno2 appears to bewell described by a rectangular hyperbola:

.p°2 = jtNO3JN02 = K + NO3 (5)

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jNO2 is a constant, the maximum specific rate of gross nitriteproduction in units of N02- (riM cell N)-1 day-l, and K is ahalf-saturation constant. We have applied a best fit analysis to theresults shown in Table I using equations 2 and 5 and obtainedvalues of 0.08 day-' and 1.0 ,UM for jNoa and K. The differencebetween values for KNO3 (0.06 ,uM) and K (1.0 uM) are consistentwith the loose stoichiometry of nitrite production and nitrateuptake. In the case where nitrite production is more limited bylight than nutrient concentration (Fig. 4), gross nitrite productionmay be described by an equation like that introduced earlier fordeterminations of growth rate.

(6)E

KE + E

A best fit analysis to the results shown in Figure 4 using equations2 and 6 yielded values for KE, N2j, and R' of 6.5 x 10'5 quantacm-2 s- , 0.13 dayf1, and 0.085 day-', respectively.

Testing of Nutrient Model. Equations 1 through 6 represent amodel of steady-state nitrate metabolism by T. pseudonana, inwhich rates of nitrate assimilation, nitrite assimilation, and nitriteproduction may be calculated for cells growing at moderate tem-peratures exposed to differing light intensities and differing con-centrations of nitrate and nitrite. We have tested the validity ofthis model by comparing predicted fluxes with fluxes measuredwith continuous culture in which fluxes were observed by thecontinuous addition of '5N-labeled nitrite. Table I contains pre-dicted fluxes for the three steady states (in parentheses) as well asthe measured fluxes. A comparison of the predicted and observedfluxes indicates good agreement for the first two steady states,which had similar light intensities and nitrate present at highconcentrations. The model predicted the net production of nitritein the first culture and the net assimilation of nitrite in the secondculture; the relative rates of nitrate and nitrite assimilation werealso predicted well. The fluxes for the third culture, which wasgrowing at a low nitrate concentration for which net nitriteproduction was very small, were also satisfactorily described.The last steady state was achieved at a high light intensity and

the fluxes were adequately described by the model after raisingthe estimates ofjNOandK (to 0.13 day-i and 1.7 pM) to correspondto the observed increase in nitrite production at this light level(Fig. 5).

Field Studies. We conclude our discussion with selected exam-ples of conditions in the ocean where the production of nitrite byphytoplankton appears to be most likely. Figure 9 summarizesstudies of nitrate metabolism made in the Antarctic Ocean duringthe early spring of 1978. Water sampled from the surface or the10%1o light level was inoculated with '5N-labeled nitrate and fluxesinto particulate N and ambient nitrite were determined. Concen-trations of nitrate and nitrite in these waters were 20 to 30 AM and0.1 to 0.3 ,UM in the upper mixed layer, and the flora consistedprimarily of diatoms. Both nitrate uptake and nitrite productionfrom nitrate ceased in the dark, an indication of mediation byphytoplankton. Figure 9 shows rates of nitrite production fromnitrate as a function of rates of nitrate assimilation. The rates ofnitrite production increase with rates of nitrate assimilation asexpected for light-limited growth and, most interestingly, the ratio; N02/j NO:, values are similar to values obtained for T pseudonana.In addition, the effect of light intensity on the stoichiometry ofnitrite production can be seen by comparing surface and 10%o lightlevel samples at each station, as in Table II. The ratiojpo2/j o, issignificantly greater at the surface, in agreement with the trendfound in the culture work.A second field observation using '6N-labeled nitrate was under-

taken in April, 1979, within the coastal waters of San Diego. Atthis time, nitrate concentrations in surface waters were high (>7tLM). The rates of nitrate uptake and nitrite production were

15

CLza.

0E

-

IN0Z 5

D

0

0

0

0

0x

x

0 50 l00 150 200jN0o (nmole day'1/Lmole PN')

FIG. 9. Nitrite production as a function of nitrate uptake in the Ant-arctic Ocean in September to October, 1978. C, surface samples; x,samples from the 100Y light depth.

Table II. Efficiency of Nitrite Production from Nitrate in Field Samplesfrom the Antarctic and off Southern California

Samples were taken from the surface and the 10% light depth and wereincubated either in situ or under simulated in situ conditions.

StatiOn ;jO2/jfNO: jiO2/jNO. Efficiency at 10%o lo/Surface 1%o lo Efficiency at Surface

5 0.129 0.047 0.365 0.092 0.103 1.127 0.309 0.063 0.2019 0.318 0.277 0.8719 0.379 0.216 0.5723 0.075 0.017 0.2332 0.105 0.047 0.4532 0.114 0.043 0.38

X = 0.52SD = 0.32

95% CLa = 0.26 < X < 0.79Southern California 0.068 0.035 0.51

coastala Confidence limits.

consistent with the laboratory cultures of T. pseudonana (TableII).

Photolytic reduction of nitrate by sunlight (26) is also a possiblemechanism for the observed nitrite production, although uncer-tainties in the rate coefficient of such a reaction preclude calcu-lation of its importance in this work.

Finally, we have applied the T. pseudonana model to the datacollected from a 3-year study of nutrient distributions at oceano-graphic Station S, 15 km east of Bermuda Island. This study ledto Vaccaro and Ryther's (21) original suggestion that phytoplank-ton are an important source of nitrite production in the openocean. Figure 10 shows the seasonal distribution of the meannitrate, nitrite, and Chl a concentrations in the upper 100 m of thewater column. Most noteworthy in this figure are the paralleldistributions of the three parameters; mean nitrite concentrationsare highest when concentrations of nitrate and Chl are highest.This peak in nitrite concentration occurs in the late winter andearly spring when the water column is mixed most deeply and

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STEADY-STATE NITRITE PRODUCTION

concentrations found in Station S. If we have underestimatedX~, t jiiNo2 or overestimated jNo2 by a factor of 3, then the model will01 ,,, simulate the field data closely. Third, other biological factors mayI O be more important in determining the observed nutrient concen-Z z trations. Particularly important may be the effects of grazing and< associated production of ammonia upon the N metabolism of the

natural population and the effect of ammonia oxidation by che-moautotrophic bacteria.

T

eN0

Q.Z

Z

hLJ

-;

I

0

-3z

2LLU

M M J S N J M M J S N J M M J S N1958 1959 1960

FIG. 10. Top, seasonal distribution of mean nitrate, nitrite, and Chl a

in the upper 100 m at Station S 15 in the Sargasso Sea, from data ofMenzel and Ryther (16). Bottom, specific rates of nitrite assimilation andgross nitrite production calculated from the above data using equations Ito 6 (see text).

when primary production is maximal (14).To determine whether the measurements at Station S bear a

resemblance to the kinetics of nitrite production of T. pseudonana,we have introduced independent variables, nitrate, nitrite, andirradiance into equations 1 to 6. Nitrate and nitrite concentrationsare measured values for specific depths over the 3-year interval,and irradiance values were estimated from Beers' Law assuminga reasonable 24-h mean irradiance, 1 x 1016 quanta cm-' s-1, andextinction coefficient, 0.1 m-l. The calculations also accounted forthe rapid mixing which occurs in the upper mixed layer (definedby the vertical distribution of temperature), so that productionand assimilation within the mixed layer was independent of depth.The lower half of Figure 10 contains calculated specific rates ofgross nitrite production (jpo2) and specific rates of nitrite assimi-lation (jNO2). The values represent mean rates in the upper 100 mof the water columns. A comparison of nitrite concentrations inthe upper half of Figure 10 with rates jNo2 and jNo2 is somewhatdisappointing. Although JiiNO, is maximal in late fall and earlyspring when nitrite concentrations are highest, jNo, is also highestat this time, so that the model predicts a net assimilation of nitriteby phytoplankton during times of highest nitrite concentration. Infact, the model predicts that through most of the year nitrite willbe assimilated rather than produced. The only time when there isnet production of nitrite is during the summer months when nitriteconcentrations are extremely low relative to concentrations ofnitrate.

Three factors may account for discrepancies between predictedrates of production and observed nitrite concentrations. First, it ispossible that the nitrite measured in February is produced veryrapidly and the conditions for such production by phytoplanktonwere not sampled during the 3-year study. Second, the quantitativebehavior of T. pseudonana may differ from that of natural popu-

lations. Relatively small changes in the value of the constants ofequations I to 6 effect significant changes in the behavior of thesystem of equations. This is particularly true at the low nutrient

Acknowledgments-We thank 0. Holm-Hansen for making possible the Antarcticwork and for continual encouragement and support, T. Enns for many helpfuldiscussions and use of the mass spectrometer, and R. Eppley for reading themanuscript. We also acknowledge the Bigelow Laboratory of Ocean Sciences forproviding facilities for the writing of the manuscript.

LITERATURE CITED

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2. BRANDHORST W 1958 Nitrite accumulation in the north-east tropical Pacific.Nature (Lond) 182: 679

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6. DIXON M 1953 The determination of enzyme inhibitor constants. Biochem J 55:170-171

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nitrogen in primary production. Limnol Oceanogr 12: 196-2069. EPPLEY RW, JL COATSWORTH 1966 Culture of the marine phytoplankton Dun-

aliella tertiolecta, with light-dark cycles. Arch Mikrobiol 55: 66-8010. EPPLEY RW, JL COATSWORTH 1968 Uptake of nitrate and nitrite by Ditylum

brightwellii, kinetics and mechanisms. J Phycol 4: 151-15611. KESSLER E 1959 Reduction of nitrate by green algae. Symp Soc Exp Biol 13: 87-

10512. KIEFER DA, RJ OLSON, 0 HOLM-HANSEN 1976 Another look at the nitrite and

chlorophyll maxima in the central North Pacific. Deep-Sea Res 23: 1199-120813. MADDUX WS, RJ JONES 1964 Some interactions of temperature, light intensity,

and nutrient concentration during the continuous culture ofNitzschia closteriumand Tetraselmis sp. Limnol Oceanogr 9: 79-86

14. MENZEL DW, JH RYTHER 1960 The annual cycle of primary production in theSargasso Sea off Bermuda. Deep-Sea Res 6: 351-367

15. MIYAZAKI T, E WADA, A HATTORI 1973 Capacities of shallow waters of SagamiBay for oxidation and reduction of inorganic nitrogen. Deep-Sea Res 20: 571-577

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17. SCHELL DM 1978 Chemical and isotopic methods in nitrification studies. In DSchlessinger, ed, Microbiology. Am Soc Microbiol, Washington DC, pp 292-295

18. SERRA JL, MJ LLAMA, E CADENAS 1978 Characterization of the nitrate reductaseactivity in the diatom Skeletonema costatum. Plant Sci Lett 13: 41-48

19. SERRA JL, MJ LLAMA, E CADENAS 1978 Nitrate utilization by the diatomSkeletonema costatum. I. Kinetics of nitrate uptake. Plant Physiol 62: 987-990

20. STRICKLAND JDH, TR PARSONS 1972 A Practical Handbook of Seawater Anal-ysis. Bulletin of the Fisheries Research Board of Canada 167: 2nd ed, 1-311

21. VACCARO RF, JH RYTHER 1960 Marine phytoplankton and the distribution ofnitrite in the sea. Joumal du Conseil. Conseil Permanent Intemational pourl'Exploration de la Mer, 25: 260-271

22. WADA E, A HATTORI 1971 Nitrite metabolism in the euphotic layer of the centralNorth Pacific Ocean. Limnol Oceanogr 16: 766-772

23. WADA E, A HATrORI 1972 Nitrite distribution and nitrate reduction in deep seawaters. Deep-Sea Res 19: 123-132

24. WADA E, T TsuJI, T SAINO, A HATTORI 1977 A simple procedure for massspectrometric microanalysis of '5N in particulate organic matter with specialreference to '5N-tracer experiments. Anal Biochem 80: 312-318

25. WILKINSON GN 1961 Statistical estimations in enzyme kinetics. Biochem J 80:324-443

26. ZAFIRIOU OC, MB TRUE 1979 Nitrate photolysis in seawater by sunlight. MarChem 8: 33-42

Plant Physiol. Vol. 66, 1980 389

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Steady-state Growth of the Marine Diatom Thalassiosira

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