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HALF-SATURATION CONSTANTS FOR UPTAKE OF NITRATE

AND AMMONIUM BY MARINE PHYTOPLANKTON1

Richard W. Eppley, Jane N. Rogers, and James J. McCarthy


Institute of Marine Resources, University of California, San Diego, La Jolla 92037

ABSTRACT
Uptake rate of nitrate and ammonium was studied as a function of nitrate or ammonium
concentration with cultures of 16 species of marine phytoplankton. Half-saturation con-
stants (the concentration supporting an uptake rate one-half the maximum rate) were
computed as a measure of the ability of a species to use low levels of nitrate and ammonium.
The half-saturation constant (K,) varied approximately in proportion to cell size and
inversely with specific growth rate. Small-celled oceanic species, such as Coccolithus
huxleyi, showed the lowest K, values (GO.5 PM) and Gonyaulux polyeclra the highest
(K, > 5 PM). The K, values are considered to be important characteristics of organisms
living in nitrogen-limited environments.
Some apparent success was achieved in predicting the competitive advantage of one
species over another by calculating specific growth rates as functions of nitrate and ammo-
nium concentration for species with known growth response to irradiance, temperature,
and daylength. For example, at about 20C and 16 hr daylength C. huxleyi should be able
to compete successfully with Skeletonema costatum when nitrate, ammonium, and irradi-
ance are low, Skeletonema costatum should grow faster than C. huxleyi only when irra-
diance and nitrate or ammonium are fairly high. Rock pool fonns appear to be relatively
unsuccessful in the ocean because of a sluggish response to irradiance and not because of
an inability to use low levels of nitrate and ammonium. The success of G. polyedru, often
the dominant dinoflagellate in local red water blooms, appears to be due to its vertical
migration from the sea surface in daylight to nitrate-rich water at 10-15-m depth at night,
rather than to any special ability to grow rapidly or use low levels of nitrate and ammonium.

INTRODUCTION also of value for ecological interpretations


Uptake rates of nitrate or ammonium by (Dugdale 1967). V, is best taken as the
marine phytoplankton give hyperbolas when maximum specific growth rate of the orga-
graphed against the nitrate or ammonium nism imposed by external conditions, rather
concentration in the environment ( Dugdale than from measured uptake rates uncoupled
1967; Eppley and Coatsworth 1968; Mac- from growth, and is thus a variable subject
Isaac and Dugdale 1969). Half-saturation to effects of irradiance, daylength, and tem-
constants (the concentration supporting half perature as well as species-specific prop-
the maximum uptake rate) can be calcu- erties of the organism.
lated for the hyperbolas using the Michaelis- The half-saturation constant, or K,, fol-
Menten equation: lowing the terminology of Epstein and
Hagen ( 1952), varies with temperature
v = v, S/(K, + S), ( Carter and Lathwell 1967), but we will
where v is rate and S is concentration, to assume that & is not influenced by irradi-
allow quantitative comparisons between ance or the other external factors influenc-
species. This constant (K,) reflects the ing the growth rate of photosynthetic orga-
relative ability of phytoplankters to use low nisms. For example, the same K, for nitrate
levels of nutrients and thus may be of was found for exponentially growing cells
ecological significance. and for N-depleted cells with different V,
Another characteristic of the uptake hy- values ( Eppley and Thomas 1969).
This account provides K, values for ni-
perbolas, the maximum velocity (V,), is
trate and ammonium uptake by several
species of marinc phytoplankton. These
1 This research was supported by U.S. Atomic
Energy Commission Contract No. AT( 11-l )GEN values are then used with existing data on
10, P.A. 20. the variation lof specific growth rate with
912
NlTHATE AND AMMONIUM UPTAKE 913

irradiance to compute the relation between copper-coated cadmium metal. We are


specific growth rate and nitrate or am- indebted to several workers involved in the
monium concentration at a low and at a evolution of this method, namely Morris
high irradiance relative to sunlight in the and Riley, Grasshof, and Wood, Armstrong,
euphotic zone. The calculations are rc- and Richards (see Strickland and Parsons
stricted to ZOC, 16 hr sunlight per day, and 1968). WC compensated for the slight varia-
to only four species for which data were tion in efficiency of reduction with nitrate
available. Finally, environmental conditions concentration in our columns by using ni-
suggested by the calculations to be favor- trate standards at several concentrations.
able for these species are compared with Ammonium was de termincd by Johnston’s
observed local distributions. ( 1966) modification of the bispyrazolone
We thank Miss E. Fuglister for computer method (Prochazkova 1964).
programming and statistical analysis of the A linear transform of the Michaelis-
data. Menten equation:
METHODS
s = v, (S/v) - K,
Uptake measurements was used to calculate K, and V, and their
Cultures were grown in an enriched sea- 95% confidence limits by a regression anal-
water medium with nitrite as the nitrogen ysis. In this transform, K, is given as the
source at 25 pM concentration. Growth of Y-intercept and V, as the slope of the re-
the crops was limited by N-depletion (ni- gression equation of S on (S/u). The (S/U)
trite no longer detected in the medium) vs. S plot was used in preference to other
when crop chlorophyll a reached about 50 linear transformations because it gave the
pg/liter. The cultures were then divided best spread of the experimental points.
into aliquots and enriched with nitrate or Only lo-14 values were obtained in each
ammonium at the beginning of the experi- experiment (5-7 concns with duplicate
ments. The aliquots were incubated for determinations), so sample size was small
15-120 min at 18C with fluorescent light at for this kind of statistical analysis and con-
about 7,500 lux (in the same incubator used fidencc limits are large. In some cases v vs.
for culturing ) , with occasional shaking, (v/S) transforms of the hyperbolas could
until the cells were harvested by filtration. be used, but only where the half-saturation
The cell-free filtrates were then analyzed constant was large compared to the lowest
for nitrate and ammonium and uptake was nitrate or ammonium concentration tested.
calculated as the difference between the In these cases, the K, and V, estimates and
concentration added and that remaining at their confidence limits were essentially the
the end of the experiment. Incubation times same as those reported. Lineweaver-Burk
were designed to give about 50% uptake of (l/v) vs. (l/S) plots were not considered
substrate at low substrate concentration or suitable for accurate determinations of K,
2 pmoles/liter uptake at higher levels. Up- and Vm for the reasons given by Dowd and
take of ammonium was linear with time, Riggs ( 1964). Since at least two replicate
but nitrate uptake showed a lag phase. The experiments were carried out with each
lag phase was avoided in these N-depleted organism, we tried combining data from
cells by a preincubation of 34 hr with 1 p~ different experiments as a means of increas-
added nitrate; subsequently uptake was ing sample size. However, the confidence
linear with time. An earlier value of K, for limits were not appreciably reduced, and
nitrate uptake by Ditylum brightwellii we consider that most of the variation is
(Eppley and Coatsworth 1968) was an experimental.
overestimate because we were unaware of
the lag problem. A revised value is given Experimental organisms
here. Since the half-saturation constants of
Nitrate was determined as nitrite, after nutrient uptake may be under genetic con-
reduction by passage through a column of trol and conceivably could be subject to
914 R. W. EPPLEY, J, N. ROGERS, AND J. J. MCCARTHY

TABLE 1. Experimental organisms

Coccolithus huxleyi BT-0 R. Guillard Sargasso Sea 1960


C. huxleyi F-5 E. Paasche Oslo Fjord 1959
Chaetoceros gracilis - W. Thomas Costa Rica Dome 1958
C yc2otella nana 13-1 R. Guillard Sargasso Sea 1958
Skeletonema costatum Skcl. R. Guillard Long Island Sound 1956
Leptocylindrus danicus FCRG 11 J. Jordan Off La Jolla 1968
Rhixosolenia stolterfothii FCRG 3 E. Paasche Off La Jolla 1966
R. rohusta FCRG 13 J. Jordan Off La Jolla 1968
Ditylum brightwellii - T. Smayda Narragansett Bay ( ?) <1963
Coscinodiscus cf. lineatus FCRG 12 J. Jordan Off La Jolla 1968
C . tc;aijesii Cos. R. R. Hohncs Off La Jolla 1965
Asterionella janonica - W. Thomas San Diego Bay 1963
Gonyaulax polyedra FCRG 8 J. Jordan Off La Jolla 1968
Gymnodinium splendens GY. B. Sweeney Off La Jolla <1951
Monochrysis tutheri Mono. M. Droop Rock pool <1953
Isochrysis galbana Iso. M. Parke Rock pool <1940
Dunaliella tertiolecta Dun. B. Foyn Oslo Fjord 1928
* The symbol < is used to mean before.

TABLE 2. Half-saturation constants for uptake (K, ) of nitrate and ammonium by cultured marine
phytophznkton at 18C. K, units in ,umotes,‘liter

Nitrate Ammonium
- - Cell
-c95% k95% diam*
Organism l-c.9 conf. limit Ku conf. limit (PI

Oceanic species
Coccolithus huxleyi BT-6 0.1 0.3.1 0.1 0.7 5
C. huxleyi F-5 0.1 1.6 0.2 0.9 5
Chaetoceros gracilis 0.3,O.l 0.5,0.2 0.5,0.3 0.5,0.3 5
Cyclotella nanu 13-1 0.3,0.7 0.4,o.s 0.4 0.3 5
Neritic diatoms
Skeletonema costatumz 0.5,0.4 0.4, 0.1 3.6,0.8,0.8 0.8,0.7,0.5 8
Leptoc ylindrus danicus 1.3, 1.2 0.5,O.l 3.4,0.9,0.5 1.4,0.2,0.4 21
Rhixosolenia stolterfothii 1.7 0.4 0.5,0.5 0.9,0.4 20
R. robustaf 3.5, 2..5 1.0, 1.0 5.6,9.3 2.0, 1.5 85
Ditylum brightwellii 0.6 1.7 1.1 0.6 30
Coscinodiscus lineatus 2.4,2.8 OS, 0.6 2.8,1.2 2.6, 1.0 50
C . wailesii 2.1,5.1 0.3, 1.8 4.,3,5.5 5.4,2.0 210
Asterionella japonica 0.7,1.3 0.3,0.5 1,5,0.6 1.2,O.B 10
Neritic or littoral flagellates
Gonyaulux polyedra 8.6, 10.3 -, 2.4 5.7, 5.3 0.6, 1.1 45
Gymnodinium splendens 0.9 1.1 1.0 47
Monochrysis lutheri E 0.3 0.5 0.4 5
Isochrysis galbana 0:1,0.1 0.2,0.2 -- - 5
Dunaliella tertiolecta 1.4 1.1 0.1 0.6 8
Natural marine communities (from MacIsaac and Dugdale 1969)
Oligotrophic GO.2 ( 6 expts ) Ol.l-0.6 ( 3 expts )
Eutrophic 21.0 ( 3 expts) I..3 ( 1 expt)
* Geometric mean diameter rounded off to the nearest micron.
t This notation means that -0.2 < KS <0.4. Negative KS values have no physical interpretations.
$ At 28C, KS for nitrate uptake was 1.0 + 0.5; at SC, it was 0.0 -C 0.5.
fi An oceanic species according to Cupp ( 1943).
NITRATE AND AMMONIUM UPTAKE 915

1 I , I I I I I 4
5 IO0

NO; CONCENTRATION(yh4) FIG. 2. Ammonium uptake rate (u) vs. ammo-


nium concentration ( S ) for Rhixosolenia robusta
FIG. 1. Nitrate uptake rate (u) of Coscin~&cus (0 -0 ). K, was 9.3 ,u~ from the linear ( S/v) vs.
Zineutus vs. nitrate concentration (S) of the medium s plot (x-x).
(0 -o ). The half-saturation constant (2.8 PM in
this case) is given as the negative S-intercept of
the linear regression of ( S/U ) vs. S ( x-x ) . Vcloc- the second and third day after nitrite dcple-
ity units are pmoles/liter hr (,&r/hr ). tion from the growth medium. This was not
observed with other organisms and we have
no explanation for it.
selection under conditions of laboratory Graphs of nitrate uptake by Coscinodis-
culture, it seems important to identify the cus Zineatus ( Fig. 1) and ammonium uptake
organisms as completely as possible and by R. rohusta (Fig. 2) show convincing
to indicate the approximate date of their hyperbolas, as the K, was high compared
original isolation from the sea ( Table 1) . with nutrient concentrations supplied. Data
typical of species with low K, are shown for
RESULTS
S. costatum (Fig. 3). Here most of the
A list of the half-saturation constants points approach V, with no points at con-
(K,) is provided in Table 2 along with centrations less than K,, due to the limited
95% confidence limits. Whcrc only one sensitivity of the analytical methods. Mac-
value is recorded, data for two experiments Isaac and Dugdale ( 1969) also encountcrcd
were combined for the regression analysis. this problem. In this case the extrapolation
In most cases two values are listed and of K, depends entirely on the assumed
represent different experiments, usually linearity of the S vs. ( S/v) regression line.
with the same culture on successive days Some trends can bc seen in the K, data.
following nitrite depletion. The two values 1) Organisms with a high K, for nitrate
usually agreed fairly well, but poor agrcc- uptake usually have a high K, for ammo-
ment was sometimes found, as with Cos- nium uptake as well. 2) Large-celled spe-
cinodiscus wailesii with nitrate and Rhixo- tics tend to show high K, values. 3) Oceanic
solenia rohusta with ammonium. These species (all small-celled in these expcri-
larger organisms were particularly difficult ments, except R. robusta) show low K,
to work with; the cells of C. wailesii tended values. 4) Although we lack specific growth
to stick together and to the glass walls of rate data for most of these organisms (ex-
the vessel. With Skeletonema costatum and ccpt when grown in continuous light which
Leptocylindrus danicus ammonium uptake does not always permit maximum rates), it
experiments, a high K, (>3.0 PM) was appears that fast-growing species tend to
found on the first day and lower values on have lower K, values than slow growers. In
916 R. W. EPPLEY, J. N. ROGERS, AND J. J. MCCARTHY

costatum

L! briglrlwellii

C. huxleyi

0. tertiolecta
2 IO -2
3 s

-!
% / x
K
'r-3 /
22 -I
Skeletonema costatum

/
8 OC -
i -II III1 11 0.15
0
0 5 IO IRRADIANCE (lylminl

NO3 CONCENTRATION (JIM)


FIG. 4. Specific growth rate as a function of
irradiance for Coccolithus huxleyi ( Paasche 1967))
FIG. 3. Nitrate uptake rate (v) vs. nitrate con- Skeletonema costcctum ( Jitts et al. 1964 ) , Ditylum
centration (S) for Skeletonema co&z&urn ( 0-0 ). brightwellii (Paasche 1968) and DunaZieZLz tertio-
The ( S/V ) vs. S pIot ( x-x) indicates a K, about Zectu (Epplcy and Coatsworth 1966). All at 20-
0.4 ,uM nitrate. 21C. The langley is 1 cal/cm2.

general, phytoplankton growth rates de- earlier observations that N-deficient cells
crease with increasing cell size (Williams have high rates of nitrogen uptake (Fitz-
1965; Eppley and Sloan 1966). The rock gerald 1968; Syrett 1962).
pool and estuarine species, ‘Monochrysis
lutheri, Isochrysis galbanu, and Dunaliella DISCUSSION
tertiolectu, show low K, values in spite of It is difficult to be certain that the K,
their abundance in rock pools which one for growth is accurately measured by such
would expect to be rich in nitrogenous short-term uptake experiments. For ex-
nutrients. Other characteristics possibly ample, Droop (1968) calculated a 20-fold
limiting their distribution are discussed greater K, for short-term uptake of vitamin
later. Br2 by M. Zutheri than for growth in a
Uptake rates of cultures grown on nitrite chemostat culture; variation of growth rate
to N-depletion were high compared with with the nutrient concentration in the cell,
growth rates, both for nitrate and ammo- independent of that in the medium, was
nium. For example, the specific growth rate noted in Droop’s vitamin study, in phos-
during culturing was 0.7-0.9 doublings/ day phate-limited cultures of Phaeodactylum
for R. robustu, but the corresponding rates tricornutum ( Kuenzler and Ketchum 1962))
calculated from rates of short-term uptake and in nitrate-limited chemostat cultures of
and the nitrogen content of the cells would I. galbana (Caperon 1968). However, K,
have been 8 and 13 doublings/dav for for uptake in our experiments agreed well
nitrate and ammonium uptake, respectively. with independent measurements of K, cal-
Similarly, the observed specific growth rate culated from exponential growth rates of
of Gonyaulux polyedra was 0.4-0.5 dou- batch cultures limited by nitrate (Eppley
blings/day, although 1.5-4 doublings/day and Thomas 1969) and are consistent with
were caldulated from uptake rates.- Thus the values observed with 24-hr incubations
these experiments to determine K, give no of samples of natural communities of oce-
useful information on exponential -growth anic and neritic phytoplankton based on
rates of N-sufficient cells but only confirm uptake of l”lV-labeled nitrate and ammo-
NITRATE AND AMMONIUM UPTAKE 917

nium ( MacIsaac and Dugdale 1969). A according to these calculations, both neritic
more detailed discussion is given elsewhere diatoms are favored by high irradiance and
( Eppley and Thomas 1969). nitrate. DunaZieZZa tertiolecta generally
If these measurements are to be of any fares poorly because of its relatively slug-
ecological value, other than emphasizing gish response to irradiance. Apparently this
the obvious fact that oceanic phytoplankton is not an artifact of poor measurements since
possess mechanisms for using very low Jitts ct al. ( 1964) found a light response
levels of nitrogenous nutrients, they must curve for D. tertiolecta similar to ours.
help explain observed phytoplankton species Another rock pool flagellate, M. Zutheri,
distribution, for the uptake K, values have with a maximum specific growth rate of 1.2
little meaning for ecology in themselves. doublings/day at 2OC, has a growth rate vs.
Hence we have used the values to calculate irradiance response curve like that for D.
specific growth rates of different species in tertiozecta (Jitts et al. 1964). Thus our
idealized conditions that we think manifest comments for D. tertiolecta may apply also
the essential features of natural waters. For to M. Zutheri.
this we needed not only the K, for nitrogen The calculations suggest further that C.
uptake but measurements of specific growth huxleyi is the best competitor with any
rate, with adequate nutrients, as influenced ammonium level and low irradiance (Fig.
by irradiance, daylength, and temperature. SC). Because of its low K, for ammonium,
Few studies have been extcnsivc enough D. tertiolecta would grow faster than the
for such purposes, and none are above two diatom species at low ammonium con-
criticism. In what follows we have used centration (<0.5 PM) and low irradiance.
data for an oceanic (ubiquitous) species, At higher irradiance ( Fig. SD), the diatoms
CoccoZit7zus huxleyi clone F (Paasche 1967), would grow faster than C. huxleyi only if
the coastal diatoms D. brightwellii (Paasche ammonium is >1.5 PM for S. costatum or
1968) and S. costatum (Jitts et al. 1964), >3 PM for D. brightwellii. Such levels are
and a rock pool form, D. tertiolecta (Epplcy high but are often found in polluted coastal
and Coatsworth 1966). Specific growth rate water and in declining phytoplankton
data are given in the papers cited, or can blooms. We noted numerous D. bright-
be calculated from them, as a function of wellii when nitrate was very low in a declin-
irradiance and temperature with 16-hr light ing bloom of Ceratium furca in November
and 8-hr darkness each 24-hr day. 1967 off Huntington Beach, California.
Variation in specific growth rate (p) with When irradiance is about 0.05 ly/min and
nitrate and ammonium concentration ( S) temperature 20C (Fig. 4), C. huxleyi, S.
was calculated from K, values (Table 2) costatum, and D. brightwellii have essen-
and p,,%taken from growth rate vs. irradi- tially the same pnZ; only at this irradiance
ance curves (Fig. 4) given in the papers would the K, values alone determine the
cited above: supposed competitive advantage of one
species over the others.
According to the laboratory results, D.
Results of the calculations are shown in tertioZecta (a rock pool form) would be an
Fig. 5. effective competitor of diatoms for nitrate
At low irradiance (0.025 ly/min), the or of C. huxleyi for ammonium only with
calculations suggest that C. huxleyi is the bright light, long days (see Eppley and
better competitor and D. tertiolecta the Coa tsworth 1966) and appreciable ammo-
poorest at all nitrate concentrations ( Fig. nium levels. It would probably be an effcc-
5A). With higher irradiance (Fig. 5B) tive competitor where its broad salinity
( 0.1 ly/min-about 20% of full sunlight) tolerance ( McLachlan 1960; Eppley 1963)
the situation is complex, with C. huxleyi would be advantageous. Shallow rock pools
growing fastest with nitrate up to about probably best meet these criteria. The tem-
0.8 j-N; with higher nitrate, S. costatum perature optimum of this organism ( 34C,
becomes the better competitor, In fact, Eppley 1963 ) is also well suited for such
918 Il. W. EI’PLEY, J. N. ROGERS, AND J. J. McCARTIIY

4
NITRATE CONCENTRATION
(PM)

b
C

0 l+F? 0I I I I
0 I 2 0 I 2
AMMONIUMCONCENTRATION(PM)
FIG. 5. Calculated speciFic growth rates vs. nitrate and ammonium concentration at two irradiance
1eveIs (approx l/20, A and C, and l/5, B and D, of surface sunlight irradiance for wavelengths of 400-
700 nm). a = Coccolithus huxleyi, b = Ditylum brightwellii, c = Skeletonema costatum, and d = Dun-
aliella tertiolecta.

habitats. The other species of Fig. 5 would on the basis of differences in their tempera-
presumably be excluded from rock pools turc and salinity optima.
because of low tempcraturc optima and GonyuuZux ~~oZye&a, often the predom-
narrow salinity tolerances. Ryther ( 1954) inant species during local red tides, has a
could account for seasonal changes in the unique strategy that may account for its
relative abundance of diatoms and uni- success despite a low growth rate (about
cellular Chlorophyta in Long Island bays 0.5 doublings/day at 20C and optimal ir-
NITRATE AND AM :MONIUM UPTAKE 919

radiance, in the laboratory) and very high may have widespread importance. Physio-
K, values measured for both nitrate and logical characteristics of phytoplankton
ammonium: its diurnal migration, This measurable with existing methods may be
species, and several other local dinoflagcl- just as important to species distribution as
late species ( Holmes, Williams, and Epplcy the more exotic features remaining for
1967) swim to the surface in daylight, future study.
where nutrients are usually very low, and
to a depth of lo-15 m at night (Eppley, IUZFERENCES
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high nitrate levels were found at shallow of lsochrysis galbnna to nitrate variation at
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CARTFS, 0. G., AND D. V. LATITWIXL. 1967. Ef-
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length and light intensity on the growth of
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920 R. W. EPPLEY, J. N. ROGERS, AND J. J. McCARTIIY

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