Limnol. Oceanogr.

, 26(4), 1981, 635-648

The effect of environmental factors on phytoplankton growth:

Temperature and the interactions of temperature
with nutrient limitation1
G-Y& Rhee and Ivan]. Gotham
Environmental Health Institute, Division of Laboratories and Research,
New York State Department of Health, Albany 12201

Abstract
The combined stress of nutrient limitation and suboptimal temperature on growth was studied
with turbidostat and chemostat cultures of Scenedesmus sp. and Asterionella for-mosa. The
combined effects were greater than the sum of individual effects and were not
multiplicative. In N- and P-limited Scenedesmus sp. and A. formosa the cell quotas (4) of both
limiting and nonlimiting nutrients increased with decreasing temperature. At a given temperature
cell quotas of limiting nutrients ,also increased with the growth rate (p) and followed a saturation
function. Higher values of the minimum cell quota (9J at lower tem-peratures show that cells
require more nutrient with decreasing temperature. The change of q,, with temperature varies with
the type of limiting nutrient. This change for N and P in Scenedesmus sp. suggests that their
optimum ratio, the ratio at which one limitation changes over to the other, is higher at suboptimal
temperatures.
Cell quotas of nutrient-sufficient cultures (qm) for C, N, and P and cellular chlorophyll a
concentration increased with decreasing temperature. The quota of each nonlimiting nutrient
in nutrient-limited cultures had the same value as 9m. The rate of protein synthesis per unit RNA
decreased with temperature.
The highest apparent maximum N uptake was observed at 15°C for N-limited Scenedesnzus
sp. growing at 0.5*d-‘. The optimal growth temperature range, however, was 20”-25°C. The
highest apparent maximum P uptake in A. formosa was found at 19”-2O”C, when p = 0.4. d l.
These temperatures were also optimal for growth.

Growth limitation by nutrients such as
phosphate and inorganic nitrogen affects
the population dynamics of phytoplank-
ton in aquatic environments. However,
dynamics also change with season, lati-
tude, and depth because of the effects of
limiting factors other than nutrients.
These factors include temperature, light,
and daylength. It has been suggested that
combined effects of these factors may be
more important than the effect of any sin-
gle one (Rodhe 1948,1978). Temperature
stress is one of the most important factors
which may interact with nutrient limita-
tion in nature, since the phase transition
of lipids, the conformation of macromol-
ecules, and the kinetics of physicochem-
ical reactions are all profoundly affected
by it.
There is little information regarding the
effect of temperature on nutrient-lim-
ited growth or temperature-nutrient in-
teractions. Turbidostat studies by Mad-
l This work was supported by U.S. EPA (R-
804689) and U.S. NSF (DEB 75-19519).
635
dux and Jones (1964) showed that the
optimum growth temperature for Nitx-
schia closterium and Tetraselmis sp. was
different in culture media with “low”
and “high” levels of phosphate and ni-
trate. These workers did not demonstrate
that the low level was nutrient limiting;
their results suggest that it was not. Thus
it is not clear whether the observed effect
was due to the difference in nutrient
levels or to other differences such as in
the total ionic strength of the media.
Goldman (1979) reported difficulties in
using Droop’s kinetic equation for growth
at a suboptimal temperature for Mono-
chrysis lutherii.
Blackman (1905) suggested that sub-
optimal temperature for growth in nu-
trient-limited environments could be
treated as a limiting factor in the sense of
Liebig’s law of the minimum; in this view
growth is controlled by either tem-perature
or nutrient but not simulta-neously by both.
Baule (1918), on the oth-er hand,
expressed the combined effects
of limiting factors by a multiplicative for-
636 Rhee und Gotham
mula. Goldman and Carpenter (1974) de-
scribed the simultaneous effect of tem-
perature and nutrient with multiplicative
forms of the Arrhenius equation and
Monod’s growth function of nutrient-lim-
ited growth. Many environmental mod-
elers including Kiefer and Enns (1976) and
Nyhohn (1978a,b) have adopted sim-
ilar multiplicative expressions without
experimental evidence.
Multiplicative models assume essen-
tially that temperature affects only the
maximum growth rate. There is evi-dence,
however, that the half-saturation constant
of the Monod equation also changes with
temperature (Topiwala and Sinclair 1971;
Shelef et al. 1970; Thomas and Dodson
1974; Sawada et al. 1978; Ahlgren 1978).
Furthermore, the use of Monod’s model
under conditions of tem-perature stress
may require a term for maintenance rate.
Our study was designed to investigate
the simultaneous effects of nutrient lim-
itation and suboptimal temperature on
growth and nutrient uptake kinetics by
continuous-culture techniques. To pro-vide
baseline information, we also inves-tigated
temperature effects on nutrient-sufficient
growth. The use of continuous
culture in phytoplankton ecology has been
reviewed by Rhee (1980) and Rhee et al.
(1981).
We thank G. W. Fuhs for comments on
the manuscript, V. J. Bierman, Jr., for
helpful discussions, and T. C. Lederman
for assistance on some statistical analy-
ses. E. Kusel provided technical assis-
tance.
Muterials und methods
Axenic Asterionella formosa, obtained
from S. S. Kilham, and Scenedesmus sp.
were used as test organisms. Scenedes-
rr~us was grown in a modified Smith-
Wicdeman inorganic medium (Rhee
1973, 1974). The nutrient-sufficient tur-
bidostat medium contained 300 PM N and
10 PM P. For N-limited cultures, ni-trate
was reduced to 42 ,xM and KC1 adcled in
amounts equivalent to the ni-trate omitted.
For P-limited cultures, P was reduced to 1
JIM and N kept at 300
PM. Asterionella formosa was grown in the
medium of Guillard and Lorenzen (1972)
with Tris reduced to 2 PM. For P-limited
cultures of A. formosa, P was reduced to 2
PM, and NaNO, replaced with KN03 (250
PM) to supplement K, which had been
reduced by the de-
creased concentration of K,HPO,. The
medium for the turbidostats contained 1
mM KNO, and 50 PM P. The pH of all
cultures ranged from 7.3 to 7.6.
A turbidostat was made from a resin re-
action kettle with a l-liter working vol- .,
ume. A low-energy (0.8 mW) helium-
neon laser was used as the light source.
Population density was maintained at a
predetermined low level (<5 x 10” * m-l)
by the use of photocells. When the den-
sity exceeds the predetennined level, an
electric impulse to a peristaltic pump lets
Fresh medium from the reservoir enter
the culture vessel and simultaneously al-
lows an overflow of an equal volume of
the culture (Gotham and Rhee in prep.).
Some chemostats were Bio-Flow
models (model C30, New Brunswick Sci.
Co.) with a 1.3-liter working capacity.
The others were made from resin reac-
tion kettles with l-liter working volume
or Bellco spinner flasks.
Illumination was continuous, with
“cool-white” fluorescent lamps (Sylvan-
ia) and an average irradiance in culture
vessels of 17.1 W *m+. Temperature was
kept constant within +‘l”C by circulating
coolant at various temperatures through
stainless-steel cold fingers or glass coils
from cooler circulators (Neslab Instr.
Co.). Mixing was provided by Teflon-
coated magnetic bars on a nonheat-gen-
crating magnetic stirrer for culture ves-sels
made with resin reaction kettles.
Cultures were aerated at about 0.5
liter-min-l. Air was passed through sat-
urated zinc chloride, distilled water, a se-
ries of sterile bottles packed with glass
wool, and finally a 0.2-pm sterile Inem-
brane filter.
When the cultures had achieved a
steady state, aliquots were removed to
determine chlorophyll a, cell volume, and
nutrient uptake as needed. The rest of the
culture was concentrated by ccn-
 Temperature-nutrient
trifugation, quickly frozen in a Dry Ice-
acetone bath, and stored at -20°C for fur-
ther analyses. The supernatant from the
harvest was gently filtered through a pre-
washed 0.45~pm membrane filter. Resid-
ual substrate in this filtrate and the sub-
strate concentration in the reservoir
medium were determined with an
AutoAnalyzer.
Cell quotas of C, N, and P were di-rectly
determined by cell analyses (Rhee 1973,
1978). Chlorophyll a was measured in vivo
with a Turner fluorometer (Turn-er Assoc.)
and expressed in arbitrary units. RNA-P
and protein-N analyses were also
described previously (Rhee 1973, 1978).
For nitrate and phosphate uptake stud-
ies, aliquots (50 ml) of steady state cul-
tures growing at a fixed dilution rate were
delivered into a series of 125-ml Erlen-
meyer flasks. These flasks contained var-
ious concentrations of nitrate or phos-
phate (32P as a tracer). The flasks were
shaken in a constant-temperature shaker-
bath (New Brunswick Sci. Co.) at the same
temperature conditions as the che-mostat
cultures. The highest concentra-tion of
residual N in N-limited Scenedes-mus sp.
was about 0.5 PM at 13.5”C; that of
residual P in P-limited A. formosa was 1.44
PM at 10°C and co.27 PM at other
temperatures. The higher the tempera-
ture, the lower was the concentration of
residual limiting nutrient.
Nitrate uptake was measured at 15-min
intervals and phosphate uptake at 3-min
intervals. The uptake rate at a given con-
centration was determined from the ini-tial
linear portion of the curve for resid-ual
nitrate or cell 32P vs. time. Kinetic
constants were calculated from an Eadie-
Hofstee plot of the results.
Cells were counted in a hemacytome-
ter, and cell volume was measured by a
Coulter counter (model TA II, Coulter
Electronics).
Statistical analyses of data followed So-
kal and Rohlf (1967).
Results
Effects of temperature on nutrient-suf-
ficient growth-The effects of tempera-
interactions 637
Fig. 1. Growth rate of Scenedesmus sp. (0) and
Asterionella formosa (0) under nutrient-sufficient
conditions as a function of temperature. Slope is
0.078 k 0.01 for the former and 0.077 2 0.01 for the
latter.
ture on nutrient-saturated growth were
examined in turbidostat cultures. In both
species the growth rate under nutrient-
sufficient conditions (hT) was a linear
function of temperature (T) in the sub-
optimal temperature range; and the slopes
of the curves for both species were
identical (Fig. 1). This linear relationship
can be expressed as
A,~ = O.O78(T - T,,)
where To is the intercept of the temper-
ature axis (see discussion). However,
these linearities and identical slopes
were coincidental, since for other spe-
cies the temperature-growth rate rela-
tionships appear to be nonlinear and
species-specific (see discussion).
Under nutrient-sufficient conditions
the intracellular concentrations, or cell
quotas, oft, N and p (+c, %nN, and 9d
increased with decreasing temperature
in Scenedesmus, and the inverse of these
values seems to be linearly related to
temperature within the suboptimal tem-
perature range examined (Fig. 2). For A.
formosaT 4mN and 9mP showed the same
relationship to temperature (Fig. 3). These
were the maximum values of the cell
quotas at each temperature.
Temperature does not seem to influ-
ence the carbon fixation rate per cell
(hT-9,,J in Scenedesmus sp. The rate,
judged by the F-test (P < 0.75), was con-
stant at-all temperatures investigated
638 Rhee and Gotham

5-

1-

1-

I ,,,I,,,, I,

)-

I-

,-

a
=
,-
8 4
:

I I I I I I

1c )- 10 20
Temperature (“C 1
Fig. 3. Reciprocals of cell quotas for N
and P in
uutrient-sufficient (e) and P-limited (0)
I-.
Aslerio-,zeZZaformosa at various temperatures. In P-
limited cultures, cells were growing at a fixed dilution
rate (0.4.d-‘).

temperature range, from 20” to 25°C

L I I 1
I I
10
Temperature (“C)
Fig. 2. Reciprocals of cell quotas for C, I’, and
N in nutrient-sufficient (e) and nitrate-limited (0)
Scenedesmus sp. at various temperatures. In N-lim-
ited cultures, cells were growing at a fixed dilution
rate (0.5 *d-l). Quotas for nutrient-sufficient cultures
are denoted in text as c/,,~, qmN, and qmIa.
(Figs. 1, 2). However, the carbon fixation
rate per unit of chlorophyll a decreased at
low temperatures, because the chlo-
rophyll a content increased (Fig. 4).
Scenedesmus sp. had a wide optimal
(growth above 25°C was not examined).
For A. formosa the optimal temperature
range was very narrow, between 19” and
I I
20°C (Fig. 1). Viable populations could not
be maintained above 20°C.
L
20
The effects of temperature on nutrient-
limited growth-The effects of nutrient-
temperature interactions were studied by
two different approaches: growing cells in a
chemostat at various temperatures at
various dilution rates or at a fixed dilution
rate.
The growth rate at various dilution rates
at various temperatures was a hy-perbolic
function of cell N quotas, 9N, for N-limited
Scenedesmus sp. at optimal as well as
suboptimal temperatures (Table 1). The
superimposed curves for 20” and
 Temperature-nutrient interactions 639
Table 1. Growth rate, p (day-‘), and cell N quo-

ta, qN (X lo-’ prnol.cell-l), for N-limited Scene&s-

mus sp. at various temperatures.
0 11°C 16°C 20°C 25°C

0 CL (Ih P YN F (Ih /I (I\

0.24 2.59 0.26 1.12 0.33 0.56 0.31 0.53

0 0.37 2.96 0.35 1.26 0.47 0.69 0.39 0.61
0.47 3.37 0.50 1.33 0.50 0.88 0.49 0.71
0 0.50 4.56 0.50 1.37 0.59
0.83 0.50 0.91
0
0.52 4.66 0.55 1.49 0.71
0.89 0.70 0.80
II I I I 0.31 2.80 0.58 1.54 0.77
10 15 20
0.95 0.74 0.84
Temperature (“C)
0.83 2.45 0.82 1.03
0.84 2.34 0.88 0.97
1.13 1.97
1.13 1.72
Fig. 4. Carbon fixation rate per unit chlorophyll
a (expressed in arbitrary units) at various temper-
atures. the growth rate when q is infinite (Table
2). Equation 3 can be considered as a spe-
25°C represent N-limited growth in the cial case of Eq. 2 when K, = qo.
P’~ is always larger than the true max-
optimal temperature range. The curves imum growth rate ,z,,, , since in reality q
for 16” and 11°C are growth responses has a finite maximum value at qnr and
under the double stress of N limitation cannot be infinite. The difference be-
and suboptimal temperature. All these tween b,2 and plrn in Eq. 3 is related to
curves can be described by the three-pa- storage capacity of limiting nutrient in
rameter growth equation. relation to the minimum cell quota or
P - 4 -40 (2) qO:qm ratio (Droop 1973, 1974; Goldman
--
P’rvl KJ + (4 - 40) and McCarthy 1978) by the equation
or by the simple two-parameter equation Pm = P’,,l[l - hhJ1. (4)
of Droop: In this equation Droop defined qn, as the
(3) cell quota at which a nutrient ceases to
limit growth and there can be a range of
values for q at ~1-n~Goldman. and Mc-
where q. is the minimum cell quota, 1;6 Carthy, on the other hand, suggest a sin-is the
half-saturation constant, and p’, is gle value. Our results do not show wheth-

Table 2. Kinetic constants for Eq. 2 and 3 and statisticsof their fit to data.

Sum of
squared
elT0r.S
cl,,?
/+*kSE or K* for Cj
Temp +SE or SE range (lO~s~mol N.cellk’) II (x10-9 /Lmol
(“C) N .cell-I)’ F§
4 - 40 11 0.596+0.005 236.3k17.3 35.1k4.4 6 2,418.4 63.7
& + ((1 - 90) 201&625 1.170~0.005 96.5k3.7 57.2k2.6 8 218.5 476.3
1.385+0.005 55.7k3.4 29.3k1.9 16 1,298.7 229.1
6
1 - (40/q) 11 0.901 - 0.068 + 0.165 183.6 - 13.0 + 15.0 - - 5,421.6 36.5
16 1.360 - 0.056 + 0.073 89.4 - 2.4 + 2.50 - - 8 373.4 242.4
20 & 25 1.534 - 0.130 + 0.176 47.6 - 2.2 + 2.5 - - 16 1,509.8 194.4
* /A’,, for Eq. 2 determined by optimization of variance ratio for regression y = cl,, + K,.~/(/L’,,, - CL)] and for Eq. 3 from x-intercept of
regression for l/q vs. p. SE for Eq. 2 is error of precision in /.L’,, for optimization of variance ratlo and for Eq. 3, error of x-llltercept of
regression. CL)] and in Eq. 3 from y-intercept of regression of l/cl VF. /L.
t q0 in Eq. 2was obtained from y-intercept of regression 4 = y0 + K,.[/.L/(~‘, -
SE of q0 in Eq. 2 and 3 was error of y-intercept.
$ K, of Eq. 2 was determined from slo e of optimized regression of q vs. ~I(/.L’,,, - CL) and its SE was error of slope.
B Variance ratio for predicted+bserve B in terms of 4.
640
Table 3. Minimum cell quota (c/J for N and P at various temperatures in Scenedesmus sp. either by
calculation or from batch cultures.
(103
pm01
. cell-‘) 20 18
NkSE 47.6”
(55.7)1 - (236.3)
PkSE 1.60 1.87kO.05
* From Table 2: Eq. 3.
t Values with SE were determined in batch cultures.
$ From Table 2: Eq. 2.
B From Rhec 1973, 1978.
er there is a single value or a range of
values.
At all temperatures examined, pfmT (re-
sulting from curve-fitting of Eq. 2 and 3) is
larger than fiT (measured in nutrient-
sufficient cultures), but we were unable to
determine whether Eq. 4 also held un-der
temperature stress because of the scatter
of g,, data (Fig. 2).
Equations 2 and 3 were fit to p-9 data at
various temperatures (Tables 1 and 2).
The variance ratios for estimating 9 from
Eq. 2 were greater than those from Eq.
3. This difference is reflected in the
greater sums of squared errors (SSE) for
the prediction of 9 by Eq. 3. To deter-mine
the goodness of fit of each model to the
data, we calculated an overall F-sta-tistic
(F*) from the ratio of the SSE for 9 for both
equations, corrected for sample size
minus the number of parameters.
The degree of freedom for F* was cal-
culated from the total sample size minus
the product of data sets and the number of
parameters. F*(2,2,j = 1.79, which is
significant only at 0.10 < P < 0.25 level.
Since this test of goodness of fit cannot
determine the better of the two models, we
can choose the simpler one, Eq. 3, to
describe the data.
Whether we fit Eq. 2 or 3 to the data, 9”
values increase with decreasing tem-
perature (Table 2). This increase, which is
also evident in batch cultures for N and P
(Table 3), means that the cells require more
nutrient at lower temperatures.
Thus multiplicativemodels, in which only
C-L,,is assumed to vary, are in error.
Expression of temperature-nutrient in-
teraction effects requires that 9, be tem-
Rhee and Gotham
Ten-q (“C)
17 16 15 12 11
95.1+8.3t - 125.3+ 16.8 - 183.6
- 2.41 kO.09 2.29+0.12 2.84d.19 -
perature-dependent. Table 3 indicates
that the temperature effects on 9. vary
with the type of limiting nutrient.
We investigated growth at a fixed dilu-
tion rate at various temperatures to show
one detailed cross-section parallel to the
qN axis in Fig. 5. Scenedesmus sp. was
grown at 0.5 and 0.45. d-l in N-limited and
P-limited chemostats, and A. formosa at
0.4 *d-l in P-limited chemostats at various
temperatures. In both species the steady
state cell number, X, decreased linearly
with decreasing temperature, whether P or
N was limiting (Fig. 6). Thus the rate of
population change (p. X) was also lower at
lower temperatures.
If temperature affects the growth rate in
an “either-or” manner, or in a thresh-old
fashion, regardless of the cells’ nutri-tional
status, the change P-X in chemo-stat
cultures must be predictable from
b,,,T determined in nutrient-sufficient tur-
bidostat cultures, since
(d ,Z .X)/dT = (d
/L.~~~.X,,)/dZT (5)
where X,, is the steady state cell number
in a chemostat at a fixed dilution rate and
optimal temperature. The plot of p .X and
t-hT‘xo, vs. temperature should then be
parallel. However, their slopes are dif-
ferent (Fig. 7). This indicates that growth
is affected by temperature and nutrient
limitations simultaneously, not in a
threshold manner (see below).
The effects of temperature on various
degrees of nutrient limitation, D, can be
found from the relationship between D
and the critical temperature, T,, the tem-
perature at which the growth rate is de-
 Temperature-nutrient interactions 641

15-
-XL
0 25
0 20
0 16
0 0’ a 11

IO-

I I I
1 2 3 1
Temperature PC)
qN (10-7pmolcs cell-‘)

Fig. 6. Steady state cell numbers of Scenedes-

Fig. 5. Cell N quota (9J vs. growth rate at var-ious
temperatures in N-limited Scenedesmus sp. Curves
were drawn using kinetic parameters of Eq. 3 from
Table 2 for optimum temperatures and Eq. 2 for 11”
and 16°C.
mus sp. growing at a fixed dilution rate in P-limited
(A) and N-limited (0) chemostats as a function of
temperature. Dilution rate for P-limited culture was
0.45 *d-l and that for N-limited culture was 0.5 * d-l. h
is about 1.34-d-l.

termined solely by temperature without
nutrient effects. At this temperature p,T/ D
= 1. Thus if both sides of Eq. 1 are di-vided
by D and hT/D is equated to 1, the
relationship between D and T, can be
obtained:
hT/D = 0.078( T - T,)/D
T, = D/O.078 + T,,.
The plot of Eq. 7 is identical with that of
Eq. 1 due to the way in which T, was
derived, but it illustrates the relationship
between temperature and the degree of
nutrient limitation (Fig. 8). The linear
segment of the curve at suboptimal tem-
peratures represents the growth rate at
which limiting nutrients no longer exert
any effect. It thus describes the maxi-mum
rate of growth at each given tem-
perature. The more nutrient-limited a cell
is, or the lower the value of D, the lower T,
becomes. For example, N-lim-ited
Scenedesmus sp. growing at 1.21. d-l
becomes purely temperature-controlled
(6)
at 18”C, whereas at 0.68-d-l its growth
(7) rate is controlled solely by temperature at
11°C. Between T, and 2O”C, the tem-
perature at which growth is purely nu-
trient-controlled, the growth rate is reg-
ulated simultaneously by temperature
and limiting nutrients.
Cell quotas of limiting nutrients, 9N and
9, for Scenedesmus sp. and 9, for A.
formosa, increased with decreasing tem-
perature. This means that at suboptimal
temperatures the cells require more of

‘2.
%
2 500-
:
n
0
3.
2
t

10 20
Tcmpcro+“ra PC) Temperature (“C1

Fig. 7. Observed (p-X: 0) and theoretical (P,,~ Fig. 8. Schematic diagram of relationship be-tween
*Xop: 0) rates of population increase at various degree of nutrient limitation (D) and effects of
temperatures. temperature.
642 Rhee und Gotham
the limiting nutrients to grow at the same
rate than they do at optimal temperature
(Figs. 2, 3).
When reciprocals of the cell quotas of
both limiting and nonlimiting nutrients are
plotted against temperature, the re-
lationship appears to he linear within the
temperature range examined (Figs. 2, 3).
However, there is no a priori reason why
they should he linear. In fact, the rela-
tionship cannot be linear over the entire
suboptimal range, since cell quotas can-
not be infinite at the intercepts of the
temperature axis. When the cell quota
curves for limiting nutrients for nutrient-
limited culture are plotted together with
those of corresponding nutrients for nu-
trient-sufficient cultures, they seem to
converge at the critical temperature, ?Ic.
This temperature is about 9°C for N-lim-ited
Scenedesmus sp. growing at 0.5*d-’ and
about 12°C for A. Jormosa growing at 0.4.
d-l (Figs. 2, 3). The ccl1 quota
curves for nutrient-sufficient and P-lirn-ited
Scenedesmus sp. growing at 0.4-d-l
converge at about 8°C. These conver-
gences mean that the relative effect of
temperature becomes greater as the tem-
perature decreases and that it eventually
eliminates the effect of nutrient limita-tion
at and below the critical tempera-ture.
Cell quotas of nonlimiting nutrients,
including carbon for nutrient-limited cul-
tures, were the same as those of nutrient-
rich turbidostat cultures, qrn (Figs. 2, 3).
When Scenedesmus sp. was growing at
a fixed dilution rate in a chemostat, its
carbon fixation rate per cell, t-,~*yc, in-
creased at lower temperatures, since p
was constant at 0.5-d-l and qc increased
with decreasing temperature (Fig. 2).
However, the fixation rate was lower than
the rate for nutrient-rich turbidostat cells
(,x?,~~*qmT). This difference lessened as
temperature decreased and disappeared at
the critical temperature. For N-limited
Scenedesmus sp. growing at p = OS*d-I, T,
was about 9°C; the growth rate of the
nutrient-sufficient culture at this temper-
ature was 0.5.d-*. Ccl1 C concentrations in
both cultures were about the same (Fig.
2).
I I 1 I I
5 10 15 20 25
Temperature (“C)
Fig. 9. Cell volume of Scenedesmus sp. as a
function of temperature under nutrient-sufficient (A),
N-limited (0), and P-limited (0) conditions. N-and P-
limited cultures were growing at fixed dilu-tion rates of
0.5 * and 0.45 ad-‘.
Cell volume increased at suboptimal
temperatures in both chemostat and tur-
bidostat cultures of Scenedesmus sp. (Fig.
9). The increase was small, how-ever,
compared to the increase in cell quotas.
Therefore, even if cell quotas are
expressed per unit cell volume, they still
increase at lower temperatures. In A. for-
mosa the cell volume appeared to be lit-tle
affected by temperature at about 190 pm”,
yet cell quotas increased as temper-ature
decreased. Thus the higher cell quotas at
lower temperatures do not ap-pear to be
associated with increasing cell volume.
Although the individual cell volume
increased at lower temperatures in
Scenedesmus sp., the volume of the total
steady state population decreased with
decreasing temperature below 20°C (Fig.
10). Above 20°C the growth characteris-
tics, including steady state cell numbers
and cell quotas, were the same; but cell
volume appeared to decrease at these
higher temperatures, and thus the vol-ume
of the total steady state population
decreased.
Cell protein and RNA-In nutrient-suf-
ficient turbidostat cultures of Scenedes-
mus sp., cell RNA and protein concentra-
tions increased with decreasing
temperature (Fig. 11). However, the ef-
ficiency of protein synthesis, when ex-
 Temperature-nutrienk interactions

643

. 0
0

0
I I I I I 0
a
10 20
Temperature (“C)
Fig. 10. Steady state population volume at var-ious
temperatures for Scenedesmus sp. growing at 0.5 *
d-l in N-limited chemostat.

--
I-
pressed as the rate of protein synthesis hnT B
h
*4 I,rolein) per unit weight of RNA, de- g
creased with temperature. ‘0
The concentrations of RNA and protein 5 O O 0
in N-limited Scenedesmus sp. growing at a a
fixed dilution rate also increased with 5
decreasing temperature, but at a given cT L I I
temperature the concentrations were lower .-10 15 20
than those in nutrient-rich cells of Temperature PC)
-
turbidostats.
Fig. 11. Cellular RNA P and protein N concen-
Effects of temperature on nutrient up- trations of nutrient-sufficient Scenedesmus sp.
take-steady state cells growing at a fixed
dilution rate at various tempera-tures were
used for short term nutrient uptake 10
experiments. These experiments were
carried out at the same temperature as the I
8

steady state cultures from which the cells .-

c

were taken. Nitrate uptake was =E 6E

investigated with N-limited Scenedes-mus 7-

sp. The apparent maximum uptake rate,
V’, (see Rhee 1980), varied with u 0 4t

temperature (Fig. 12). Although the stan- a

dard errors of some measurements over- - In
lapped, these results showed an apparent E. 1
optimurn temperature of WC, far below the 0
a
2
optimum temperature for growth, 2O”- T
0
25°C. It is not clear, however, whether -

the half-saturation constant for uptake, K,, - t

varies with temperature. The diffi-culty -9 I

arises from the limit of analytical detection I I I I
for nitrate.
10 20

The effect of temperature on P uptake Temperature CT>

 Fig. 12. Apparent maximum N uptake rate (VI,,,) for
N-limited Scenedesmus sp. growing at a fixed dilution
rate of 0.5 * d-’ at various temperatures.
644 Rhee and Gotham
was studied with P-limited A. formosa
growing at a fixed dilution rate. Its ap-
parent maximum uptake rate was highest
at the optimal growth temperature, 2O”C,
and decreased with decreasing tempera-
tures, unlike N uptake in Scenedesmus sp.
The rate (X lo-‘” pm01 . cell-l *min-l) was
2.68 at 2o”C, 0.45 at 13S”C, and 0.43 at
16°C. K, was about 0.34 PM and did not
seem to vary within the temperature range
13.5”-20°C. There could, however, be a
small variation in K,, because the lowest
substrate concentrations used for the
uptake experiments were the residual P
concentrations in the steady state cul-
tures, and these were higher at lower
temperatures, ranging from 0.13 to 0.01
/LM.
Discussion
The effects of nutrient-temperature in-
teractions are different from those of dual
nutrient limitation. Growth cannot be
limited by two nutrients simultaneously
(Rhee 1974, 1978; Droop 1974); the nu-
trient in shorter supply, in relation to the
optimal cellular ratio of the nutrients,
regulates growth.
For the simultaneous stresses of nu-
trient limitation and suboptimal temper-
ature, the combined effect is greater than
the sum of the individual effects. For ex-
ample, the N-limited growth rate at YN =
0.96 x 10h7 Fmol *cell-l is about 0.75. d-*
under optimal temperature conditions (Fig.
5). The temperature-controlled growth rate
at 16°C under nutrient-suffi-cient
conditions is about 1.05. d-l (Fig.
1). When qN = 0.96 x 1O-7 pmol.cell-l at
16”C, however, growth rate is zero (Fig. 5).
This occurs because cells require higher
cell quotas of limiting nutrients to maintain
their growth rate at lower temperatures.
This requirement is also indicated by
higher q,,N values at lower temperatures
(Fig. 5).
Conventional multiplicative growth
models (e.g. Nyholm 1978a,b) predict
growth rate under the simultaneous
stresses of nutrient limitation and tem-
perature by the equation
P = l&&w(4). (8)
This equation assumes implicitly that
temperature affects only pm. Our results
show, however, that change is taking place
not only in pm but also in yO, The increase
of y0 at a low temperature was also
reported in P-limited M. Zutherii (Goldman
1979). The use of multiplica-tive models is,
therefore, in error.
The temrlerature specificity of 4() also
suggests that information on nutrient-
limited growth under optimal tempera-
ture-or nutrient-sufficient growth at
suboptimal temperatures-may not be
sufficient to explain competitive interac-
tions as af&ted by temperature in nu-
trient-limited environments. The tem-
perature-dependent variability of 4. not
only is nutrient-specific, as shown in N-and
P-limited Scenedesmus sp., but also
appears to !be species-specific. Interspe-
cific differences in the increased require-
ment for a limiting nutrient per unit de-
crease of temperature may offset the
competitive advantage that exists at op-
timal temperatures.
The greater requirement for nutrients at
lower temperatures may reflect the cells’
need for more RNA to synthesize the same
amount of protein, as suggested by
Tempest: and Hunter (1965). Chohji et al.
(1976) showed that the efficiency of protein
synthesis, measured as the rate of protein
synthesis per unit weight of RNA,
decreases at lower temperatures. To in-
crease RNA synthesis, more protein or
enzymes may also be needed. The in-
creased RNA in Aerobacter aerogenes
(Tempest and Hunter 1965) and Candida
utilis (Brown and Rose 1969) was ac-
counted for by ribosomal RNA. The de-
creased yield (l/y) at lower temperatures in
our work may also be due to high
maintenance requirement of energy
(Tempest and Hunter 1965). Mainte-
nance ene.rgy-the energy required for
endogenous metabolism-can be consid-
ered as the consumption of biomass
through endogenous metabolism (Marr et
al. 1963).
An increase in cell C at lower temper-
atures has also been observed in other
algae (Goldman 1979). The rate of pho-
tosynthesis per cell, measured by the 14C
technique, however, does not appear to
change at low temperatures. Steemann
 Temperature-nutrient
Nielsen and Jorgensen (1968) reported
that in Skeletonema costatum a transfer
from 20” to 8°C initially decreased the
photosynthetic rate by a third, but when the
cells were adapted to the low tem-perature,
the rate at 8°C became practi-cally the
same as that at 20°C. Morris and Farrell
(1971) reported similar results. Jorgensen
(1968) had correlated this find-ing with the
high cell-protein levels at low temperatures
and suggested that al-gae adapt to
suboptimal temperatures by increasing the
concentration of enzymes.
Morris and Farrell found that their re-sults
supported this hypothesis. Morris and
Glover (1974), however, questioned it
because the rate of photosynthesis in
batch culture varied with changing phas-es
of growth. This change had not been
considered in interpreting the results of
earlier work.
WC found with steady state cultures that
at all temperatures examined the C fixation
rate per cell (~,,~*4~~~) remains constant,
while the concentration of pro-tein and
RNA increases with decreasing
temperature. These results seem to sup-
port Jorgensen’s hypothesis of tempera-
ture adaptation. When the C fixation rate is
expressed per unit chlorophyll a, how-ever,
it decreases with decreasing tem-perature
because cells at lower temper-atures
contain higher concentrations of the
pigment.
The effects of temperature on cellular
chlorophyll a content vary from species to
species. Morris and Clover (1974) found
that in Phaeodactylu,m tricornu-turn its
content was highest at 12°C and lower at
18” and 7”C, but in N. closterium it
decreased with temperature. Dwaaliel-la
tertiolecta, on the other hand, showed
increasing levels with decreasing tem-
perature on the fourth day of growth in
batch culture. Steemann Nielsen and Jar-
gensen (1968) reported that cellular chlo-
rophyll a in S. costatum was not much
different at 20” and 8”C, but decreased at
2°C. In Cryptomonas ovata (Cloern 1977),
the highest level was found at 20°C with
the level decreasing above and below this.
Although our present results are similar to
those for D. tertiolecta, it is difficult to
compare them with those of
interactions 645
other investigators because our cultures
were in temperature-adapted steady state
whereas other investigators used batch
cultures.
Although the cellular C:N:P ratio
of
nutrient-sufficient Scenedesmus Sp.
seems to increase with temperature, the
scatter of the data is too great to permit a
definitive answer. An increase in cel-lular
C:N ratios has been reported in ma-
rine phytoplankton at suboptimal tem-
peratures (Goldman 1977).
The greater the nutrient limitation, the
larger the decrease of temperature re-
quired for growth to be controlled purely by
temperature. Brown and Rose (1969)
apparently found a similar relationship in
C. utilis; they observed that the temper-
ature at which the population washed out
of a chemostat vessel decreased with de-
creasing dilution rate.
The relative growth rate (p/~~~,~)of cul-
tures maintained at a fixed dilution rate is
higher at lower temperatures because P,,~
decreases with temperature. We may
therefore suspect that the increased cell
quotas at lower temperatures (Figs. 2, 3)
could be related to the increasing relative
growth rate independent of temperature.
However, the increase of cell quotas is not
fully accounted for by the higher rel-ative
growth rates.
Either Eq. 2 or 3 appears to be acle-quate for
predicting p from 4 under the double stresses
of temperature and nu-tricnt limitation. The
parameters of these equations, however, have
little physio-logical meaning since they are
merely data-fitting terms. For example, p’,, is
an . asymptote value when 4 is infinite. On
the other hand, P,,~ is a true maximum
growth rate which can be measured ex-
perimentally. Growth rate alone, without
being related to pmL, cannot provide in-
formation on the degree of nutrient lim-
itation relative to the physiological max-
imum capacity for growth p/~,~. It is
therefore desirable to have an equation
that can predict t,c from 4 using measured
values of physiological characteristics of
species, such as p,,, and 40.
Our experiments did not show whether
the half-saturation constant for the Mo-nod
growth equation, K,, also changes
646 Rhee and Gotham
with temperature. In Oscillatoria agardhii
and Chlorella pyrenoidosa this constant
varied with temperature (Ahl-gren 1978;
Shelef et al. 1970). Topiwala and Sinclair
(1971) and Sawada et al. (1978) have
demonstrated that KS also changes in
Aerobacter aerogenes and Escherichia coli
and that an Arrhenius plot of the change is
linear. It might be possible to describe the
combined effects of nutrient and
temperature by express-ing the two kinetic
constants of the Mon-od equation, prIL and
KS, as temperature functions. Under
suboptimal tempera-tures, however, the
rate of endogenous metabolism increases,
so this term must also be included. For
example, the rate of growth under the
double stress in a chemostat can be
expressed as
dX= bdT)‘S .X -DX -M(T).X
dt K,(T) +S
(9)~,
where M is the rate of maintenance.
Therefore, at steady state
I] = /-‘w(~)‘~ -M(T).X. (10)
K,(T) + S
At low temperatures the contribution of A4
relative to D would increase, because M
increases with decreasing temperature
whereas D decreases.
In general, the change of growth rate
has been related to temperature by ex-
ponential functions of the Arrhcnius type
(Goldman 1979). However, growth char-
acterization by these relations has not al-
ways been successful, as more often than
not only a small portion of the curve is
linear. Our data could also be fitted by the
Arrhenius equation, but the resulting curve
would be of dubious utility. The
extrapolation of the temperature-growth
rate curve to T,, (Fig. 1) is strictly for
mathematical convenience. Growth may or
may not follow the linear slope below the
lowest temperature examined. In this
temperature range a different metabolic
pathway with different temperature char-
acteristics might operate. Such a possi-
bility is suggested by the finding that C
yanidium caldrium has different mini-mum
temperatures depending on wheth-
er nitrate or ammonia is the nitrogen
source (Rligano and Violante 1972). So-
rokin (1960) also reported changes in
temperature effects on a species at var-
ious light levels.
An increase of cell volume at subopti-
ma1 temperatures has been found in
Scenedesmus quadricauda (Komerek
and Ruzic‘ka 1969). At low temperatures
the coeno’bial differentiation of this or-
ganism is slow. The frequency-of-divi-sion
cycle also decreases with the change in
the phase of cell division. The yeast C.
utilis also increases in cell volume at
low temperatures (Brown and Rose 1969).
It is interesting that the optimum tem-
perature for uptake (15OC) is different from
that for growth (20”-25°C). Such dif-
ferences were also reported for C. pyre-
noidosa (Shelef et al. 1970) and Cod&m
fragile (Hanisak and Harlin 1978). These
differences may be explained by the fact
that optimum temperatures for enzyme
reactions are not necessarily the same as
those for growth (Innis and Ingraham 1978).
This uncoupling between growth and
nutrient uptake may be of ecological
significance, because the two different
temperature optima would effectively widen
the temperature range for survival. At
temperatures optimal for growth, the
increased rate of uptake would expedi-
tiously increase the cell quota which is
required for growth at a low temperature.
The measured apparent maximum up-
take rates, V’, in Fig. 12 are generally
higher than one might calculate from the
equation Vtm = P,?,~- 4mT with nutrient-
sufficient turbidostat data. This discrep-
ancy may be due to the differences in cell
quotas between nutrient-saturated tur-
bidostat cultures and N-limited chemo-stat
cultures. The feedback inhibition of uptake
(Rhee 1973, 1980) is much greater for the
turbidostat than for the chemostat cultures,
resulting in higher uptake rates in the
latter. As the chemostat culture ap-
proaches a, critical temperature (9”C),
however, the measured and calculated
values must be similar because the nu-
trient no longer exerts an effect on che-
mostat cultures. At this point the nutrient
 Temperature-nutrient
conditions of the chemostat are, in effect,
like those of nutrient-sufficient turbido-
stats. Indeed, at 10°C the measured and
calculated rates are demonstrably simi-lar:
2.83 and 3.03 X 10-l” pmol *cell-l* min-‘.
It is not clear why the calculated V’, did
not peak at 15”C, as the measured rates
did. Conceivably the intracellular
component of feedback inhibition is dif-
ferent in proportion between the nu-
trient-rich and nutrient-limited cells at
different temperatures.
For chemostat cells growing at a fixed
dilution rate, the uptake rate, v = ~‘4~)
increases with decreasing temperature
because p is constant and [IN increases at
lower temperatures. This trend of in-
creasing uptake rates also seems to con-
trast with the trend of V’, (Fig. 12). The
apparent discrepancy may result from
difficulties in the accurate measurement of
residual substrate levels at steady state.
When residual N levels are differ-ent,
calculated rates may not be compa-rable
with one another because of the Sensitivity
Of qN t0 Concentration changes when
substrate levels are low and of dif-
ficulties in maintaining an absolutely
steady dilution rate (Rhee 1980). Since
residual substrate levels are below the
half-saturation level for uptake, changes in
uptake rates are most sensitive at these
low substrate concentrations. The calcu-
lated rates are, therefore, actually values
at different external substrate concentra-
tions. It is important to remember that the
calculated uptake rate represents the rate
at the residual substrate concentra-tion in
the culture medium for steady state cells
with a specific intracellular nu-trien t
concentration.
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