SOME INTERACTIONS
OF TEMPERATURE,
LIGHT
INTENSITY,
AND NUTRIENT
CONCENTRATION
DURING
THE CONTINUOUS
CULTURE
OF NITZSCHIA
CLOSTERIUM
AND TETRASELMIS
SP.l
William S. Maddux2 and Razjmond F. Jones
Biology
Department,
Princeton
University,
Princeton,
New Jersey
ABSTRACT
A continuous culture apparatus was used to measure growth rates of Nitxschk cbsterium
and T&w.seZmis sp. at different temperatures and light intensities and at two different levels
of nitrate and phosphate cnrichmcnt
of artificial
senwntcr.
A set of symmetrical
relationships was found between light, temperature,
and nutrient concentration
in which the intcraction of any two of the factors was modified by varying the third. The optimum temperatures yielding maximum growth rates in a daily light-and-dark
cycle were lower when a
medium with nitrate and phosphate concentrations
similar to those found in natural waters
was used than when a medium having higher concentrations
of these substances was used.
The results of batch culture cxncriments
with high nutrient
concentrations
may not bc
applicable, thcrcfore, to natural situations.
Estimation of cell density with conventional laboratory photometers is not reliable
at cell concentrations below 10-100 cells/
mm3. To obtain growth curves and growth
rates, standard techniques require the use
of nutrient concentrations and cell concentrations much higher than those normal
in nature. With the equipment described,
the lower limit of workable cell concentration is not set by the sensitivity of the
monitoring device but rather by the adhesion of the organisms and their extracellular
products to the walls of the culture chambcr. The tendency of the organisms to
adhcrc or to produce film-forming products
varies with the species and culture conditions.
With Nitzschia
and Tetrawlmis
under the conditions used, it was possible
to work at concentrations
as low as 10
cells/mm”, although about 20-30 cells/mm3
was bcttcr. In the range from 10-100 cells/
mmR, growth rate was not measurably dependcnt upon concentration.
INTRODUCTION
Since the development of improved artificial media (Provasoli, McLaughlin,
and
Droop 1957 ) , a number of planktonic
algae have been grown axenically in fully
defined artificial seawater, and nutritional
studies have been carried out in test-tube
and flask cultures. Batch culture studies
usually reveal absolute requirements and
qualitative estimates of the effects of nutrient levels and of physical factors on rates
of growth and reproduction.
Such studies
are complicated by the uptake of material
from the medium, by the production
of
metabolites, and by crowding. Observations
so made seem applicable to conditions obtaining in nature only during the late stages
of blooms.
The continuous culture technique described here offers the following
advantages over batch culture methods: 1) concentration dependent factors arc controlled;
2) growth rates can be measured within
short periods of time; 3) the measurement
operation does not interrupt the culture
regime; 4) studies can be carried out at
low cell concentrations.
MATERIALS
AND
METHODS
The axenic clone of Tetraselmis sp, (cx
Pbztymonas) was obtained from Dr. R. R.
L. Guillard of the Woods Hole Oceanographic Institution, who isolated the clone
designated BT-2 from water taken in the
Sargasso Sea. The axenic clone of Nitzschia
dosterizcm ( Ehrenb. ) Smith was isolated
1 These studies were aided by Contract
NR
108-448 between the Office of Naval Research and
Princeton University.
2 Present address:
Woods Hole Oceanographic
Institution,
Woods Hole, Massachusetts.
79
80
WILLIAM
S. MADDUX
AND
RAYMOND
F. JONES
row of “cool white” fluorescent
continuously shaken.
The continuous
FIG. 1. Section
solenoid valve.
through
culture
vessel
and
from water collected at Sandy Hook, New
Jersey, by one of us ( WSM ).
The fully defined artificial seawater of
Jones ( 1962) was used except that the concentration of tris (hydroxy-methyl)
amino
methane used to buffer the pII at 8.0 was
reduced to 0.008 M. Thiamine, vitamin B12,
and biotin were always included in the
medium even when they were not required.
The culture apparatus was sterilized by
The medium was sterilized
autoclaving.
using a Selas porcelain filter. The filter was
left attached to the medium reservoir so
that more medium could be added.
The inocula were preconditioned
by
growing them for at least four transfers
in a medium of the same composition as
that which was to be used during the
experiments. A light cycle of 14 hr light10 hr dark was imposed on the culture room
during this time. All of the inocula were
grown at 18 5 2C in Erlenmeyer flasks,
which were illuminated
from above by a
culture
tubes, and
apparatus
The continuous culture apparatus employed is of the turbidometric
or photometric type (Myers and Clark 1944) in
contrast to the chemostatic or biogenic systems used by Novick and Szilard (1950)
or Monod ( 1950).
The growth chamber ( Fig. 1) is a l-liter
Pyrex flask provided with an overflow tube
in one neck which permits the escape of
a compensating volume of culture when
diluting medium is added. The culture
flask is immersed in a water bath constructed of Lucite and in which the temperature of the circulating water is controlled. The culture is magnetically stirred
and is aerated through a fritted glass tube
which also serves for adding fresh medium.
The medium supply is held in a constanthead reservoir (an 8-liter Pyrex bottle)
above the culture flask, to which it is connected by a siphon provided with a solenoid-actuated Pyrex valve ( Fig. 2). The
only materials in contact with the medium
are Pyrex glass and silicone tubing used for
connections.
Light for photosynthesis is provided by
four “cool white” fluorescent tubes placed
SOLENOlD
II
*COOL
FIG.
culture
WHITEa’
FLUORESCENT
2. Diagrammatic
apparatus.
:
:
n
TUBES
scheme
of
continuous
CONTINUOUS
FIG. 3.
CULTURE
OF MARINE
ALGAE
81
Cross section of apparatus showing arrangements of lights and water bath.
in pairs on either side of the Lucite bath
(Fig. 3). The plane side of the water bath
provides a good optical match to the spherical flask both for the photosynthetic illumation and for the collimated monitoringlight beam. The light from the fluorescent
tubes is attenuated by inserting layers of
metal mesh screening between the light
tubes and the water bath.
The monitoring-light
source is a 6.3-v
tungsten filament lamp, operated at 4.5 v,
which greatly reduces aging of the lamp
without seriously lessening emission in the
red portion of the spectrum. The bulb is
mounted at the focus of a parabolic flashlight reflector, and the resultant parallel
beam passes through a near infrared KodakWratten No. 88A filter before being reflected through the culture flask, A lens
converges the light onto the active surface
of a cadmium sulfide photoconductive
cell
after passage through the culture and water
bath, with another infrared filter interposed
just after the lens. A second cadmium sulfide cell is placed facing the light source
st filter to serve as a reference in
order to reduce the effect of possible fluctuations in the intensity of the light source.
The use of the red filters precludes any
photosynthetic
effect by the monitoring
lights, eliminates errors due to pigment
changes, and has the added feature of
further reducing the small amount of light
from the fluorescent tubes which might be
scattered or reflected into the monitoring
optics system. Because the “cool white”
tubes radiate very little energy above 700
rnp which could be detected by the CdS
cell, the switching
on and off of the
fluorescent lamps dots not affect the control system.
The two photoconductive
cells arc connected in an a-c bridge, the output of
which passes to an amplifier and phase
detector ( Fig. 4), which activate a relay
to apply 60-cycle line a-c to the solenoid
valve whenever the turbidity of the culture
rises above the level determined by the
setting of the bridge potentiometer.
The
opening of the solenoid valve permits dilut-
82
W [LLIAM
S. MADDUX
AND
RAYMOND
F. JONES
B+
275V
Isolation
FIG.
4. Schematic diagram of culture density control circuit.
An unbalance of the bridge circuit
results in an a-c signal that is amplified by the two r-c coupled triodc stages (one 12AU7) and then is
compared with a reference a-c voltage across the 270 K resistor which is in parallel with a neon glow
lamp (NE 2). The reference voltage alone is insufficient
to cause discharge of the lamp. If the light
passing through the culture suspension is reduced, the error signal is in phase with the reference signal
and the neon bulb lights. The neon bulb is cemented to the face of a CdS photoconductive
cell connected
in the grid circuit of the second 12AU7 which has the relay connected to its cathodes. The second neon
lamp is always “on,” but is so arranged that it does not illuminate
the CdS cell directly but serves to
prevent “dark effect” in the control lamp, assuring a closely reproducible
firing voltage.
(The CdS cell
and the neon lamps are wrapped in a light-tight
covering of aluminum foil. ) The dark resistance of the
CdS cell is about 12 megohms, but when illuminated
by the neon bulb its resistance drops to a few
hundred thousand ohms. R* is chosen to have a resistance of Wo of the CdS cell’s light resistance.
Thus, when the neon bulb is “on,” the grid of the relay tube rises to about 25 v, which activates the
relay. The CdS cells used were obtained from the Lafayette
Radio Electronics
Co., cataloguc No.
MS 855.
ing medium to flow into the culture flask
until the turbidity
is reduced enough to
rebalance the bridge.
To test control accuracy, a Coulter
counter was used to measure the cell concentrations in effluents from cultures of
l’etrselmis
and Nitxschin.
Samples were
taken 6.5 hr after the beginning of the
light period on one day and 1 hr after the
lights went on two days later. No significant variation was seen between the
cell concentrations,
the variation of the
counts overlapping for the two samples in
each case (Table 1).
air,
When in use, pressure-regulated
washed with distilled water, flows through
sterile cotton filters to the aerator in the
culture flask and to the constant-head
bleeder in the medium reservoir. A cooling
controlled
device and thermostatically
heater coupled to the water bath surround-
ing the culture permit operating at temperaturcs ranging from 54OC. A small
pump provides mixing and circulation of
the water between the bath and heat exchanger. Temperature variation as mcasured in the thermometer well of the culture flask did not exceed + 0.2C from the
set point.
The growth recorded consists of a drum
which holds a standard 8% x 11-inch sheet
of paper and is rotated by a 24-hr electric
1.
TABLE
Sample
time
Organism
Carteria
Results of Coulter counter analysis
of culture effluents
sp.
Nitxschin
closterium
Number
readings
of
Mean
( cells/mm3)
SE
0900
1430
5
5
38.1
37.6
20.84
-co.87
0900
z
28.5
29.3
kO.77
kO.82
1430
CONTINUOUS
CULTURE
clock. A glass capillary pen is attached to
a nut which rides on a lead screw mounted
parallel to the drum and driven by a gearreduced synchronous motor. The motor
which thus drives the pen along the drum
is connected to the culture density controller in parallel with the medium control
valve solenoid so that the pen advances at
a constant rate during any time that the
valve is open. Assuming constant pressure
head and constant flow resistance in the
medium supply system, the pen movement
is proportional
to the volume of culture
admitted. A continual check and calibration is easily provided by collecting the
culture effluent in a graduated cylinder and
writing the overflow volume and time directly on the chart at the current pen position from time to time during an expcrimcnt.
Illumination
was measured by instrumental integration over 4pi steradians at
the center of the culture flask. The illumination readings given arc expressed in terms
of equivalent response to that of a Weston
Illumination
Meter Model 756, exposed to
unidirectional
illumination
from a small
source (a 4-cm-long exposed segment of
“cool white” fluorescent tube ) .
RESULTS
AND
83
ALGAE
/ P-------O
0
0
I
60
Light
I
120
in Lumens
I
I60
I
240
x 16’ per
I
300
Square
I
360
I
410
Meter
FIG. 5. Effect of light intensity on the growth
rata of Nitzschia
closterium
and Tetraselmis
sp.
at two nutrient Icvcls. At 10 mg-d.
N/liter
and
0.52 mg-at. P/liter
(dashed
line);
8.9 pg-at.
N/liter
and 0.42 pg-at. P/liter (solid linc).
Tcmperaturc constant at 1GC.
DISCUSSION
The ability of the continuous culture apparatus to work with low cell-density cultures was exploited in a series of experiments in which cultures of Tetmselmis and
Nitxschicz were grown at 8.9 pg-at. N/liter
(nitrate nitrogen) and 0.42 pg-at. P/liter
(phosphate phosphorus) concentrations. Preconditioned cultures were placed in the culture system and allowed to grow for several
days until their growth rates had stabilized.
Temperature or illumination
changes were
made and the cultures observed for several
days under the new condition.
For both Tetmselmk sp, and Nitzschia
closterium growth, as measured by increase
in cell number, occurred only in the light
period of the light cycle.
Effect of nitrate and
on the light
Cells of Tetraselmis
closterium were grown
OF MARINE
phosphate levels
response
sp. and Nitzschia
in continuous cul-
ture in the seawater medium containing
8.9 ,ug-at. N/liter and 0.42 pg-at. P/liter at
a constant temperature of 16C but at light
intensities that were varied between 860
and 4,196 lux. A similar set of experiments
were conducted with these two algae using
seawater containing 10 mg-at. N/liter and
0.52 mg-at. P/liter.
The effect of light intensity and nutrient
concentration on the overall doubling rate
of both species is shown in Fig. 5. It is
seen that the two species have a lower optimum light intensity for maximum growth
when grown at the lower concentrations of
nitrate and phosphate. Of the two organisms, Nitzschia appears to be the more susceptible to this light effect. As can be seen
from Figs. 6 and 7, both Tetrasdmis and
Nitzschia exhibit a reduction in rate of
growth during the latter part of the 14-hr
light period, particularly at the higher light
intensities.
84
WILLIAM
S. MADDUX
AND RAYMOND
I?. JONES
4
.8
i
E
s
E
-I
.8 Nitzschia
4196
hm
/:
11,’
,’ ’
,* I’
/’ , ,,’
3066
lux
Tetraselmis
.6 -
.6
3066
lux
0
Hour
of
Lqht
2
4
Period
How
FIG. 6. Effect of various levels of light on the
rate of growth of Tetradmis
sp. during the 14-hr
light period at 16C. At 10 mg-at. N/liter and 0.52
mg-at. P/liter
(dashed line);
8.9 ,ug-at. N/liter
and 0.42 pg-at. P/liter
(solid line).
Effect of nitrate and phosphate
the temperature response
6
8
of
Light
IO
I2
14
Penod
FIG. 7. Effect
of various levels of light on
the rate of growth of Nitzschia closterium -during
the 14-hr 1igYht period at 16C. At 10 mg-at. N/liter
and 0.52 mg-at. P/liter
(dashed line); 8.9 pg-at.
N/liter
and 0.42 pg-at. P/liter
(solid line).
on
Tetraselmis and Nitzschia were individually grown in the artificial seawater medium containing 8.9 pg-at. N/liter and 0.42
pg-at. P/liter under the continuous culture
conditions at temperatures from lO-30C
and at a light intensity of 860 lux, and their
growth was recorded over a period of days.
Another set of cultures was grown under
similar conditions except that the seawater
contained 10.0 mg-at. N/liter and 0.52 mgat. P/liter and the light intensity was increased to 1,883 lux.
The effect of the temperature and nutrient concentration on the overall doubling
rate of both species at the two light intensities is shown in Fig. 8. Here it is seen
that at the higher nutrient level and light
intensity the rate of growth is increased and
that for Nitzschia, at least, the temperature
for optimum growth increases from 16-23C.
By comparison with the results given in
Fig. 5, it will be noted that the maximum
growth rate for Nitzschia, equivalent to that
obtained under the high nutrients and light
.
0
5
Temp
FIG. 8.
20
15
IO
25
30
35
‘C
Effect of temperature
on the growth
rate of Nitzschia
closterium
and Tetraselmk
sp.
at two nutrient levels. At 10 mg-at. N/liter
and
0.52 mg-at. P/liter
at 1,883 lux (dashed line);
8.9 pug-at. N/liter
and 0.42 pg-at. P/liter
at 860
lux ( solid line ) .
CONTlNWOUS
CULTURE
OF MARINE
.8I
Hour
of
Light
Period
85
ALGAE
Nitzschia
Hour
of
Light
Period
FIG. 9. Effect
of various temperatures
on the
rate of growth
of Tetrasehnis
sp. during
each
14-hr light period. At 10 mg-at. N/liter
and 0.52
mg-at.
P/liter
at 1,883 lux (dashed line);
8.9
,ug-at. N/liter
and 0.42 ,ug-at. P/liter
at 860 lux
( solid line).
FIG. 10. Effect
of various
temperatures
on
the rate of growth of Nitaschin closterium during
each 14-hr light period. At 10 mg-at. N/Iitcr and
0.52 mg-at. P/liter
at 1,883 lux (dashed line);
8.9 pg-at. N/Iitcr
and 0.42 pg-at. P/liter
at 860
lux (solid line ) .
of 1,883 lux described here, is obtained by
increasing the light intensity from 1,883 to
3,229 lux when the temperature remains at
16C. The interrelationship
between light,
temperature, and nutrients is thus clearly
demonstrated. For Te-tr~selmis a similar relationship holds. However, for both species,
under each circumstance of temperature
an d nutrient
concentration,
the rate of
growth varied during the course of the 14hr light period of growth as shown in Figs.
9 and 10.
The growth curves for Nifzschiu and TetraseZmi.s show larger differences between
initial and final rates of growth (during
the actual light period of the day) under
the conditions of higher light intensity and
temperature.
In the experiments with increasing light intensity, it was evident that
at the lower concentrations of nitrate and
phosphate the organisms were particularly
susceptible to the increased light. Doty
and Oguri (1957) measured the Cl4 uptake
of samples of tropical Pacific water collected in the morning and in the evening
and found a 5.7-fold greater uptake by the
morning samples. Tamiya et al. (1953)
found that when Chlorella eltipsoidea was
grown on a light-and-dark
cycle the exponential growth rate was increased after
a period of darkness. During the darkness,
however, the chlorophyll
content of the
cells increased. The decrease in the growth
rate of synchronized and unsynchronized
ChZoreZla cultures during exposure to light
was more pronounced at lower temperatures (Sorokin and Krauss 1958, 1962).
These latter authors suggest that that photochemical damage of the photosynthetic
mechanism may be opposed by temperature-dependent
resynthesis.
A serious discrepancy has existed between tempcraturc data from field and laboratory results in the past (Braarud 1961).
For a number of forms, the temperature of
the sea during periods of abundance was
found to be much lower than the laboratory
optimum temperature for maximum growth
in batch culture. If the behavior of Tefrase2mi.s sp. and Nitxschicc closterium in con-
86
WILLIAM
S. MADDUX
tinuous culture is typical, then the anomalies observed by previous workers may
be explained as having been caused by the
use of unnaturally high nutrient levels.
The multiple interaction
displayed by
three factors-light,
tcmperaturc, and nutrients-reported
here is probably greatly
elaborated upon in natural situations where
many more factors arc involved. The concept of “limiting factors,” therefore, is of
doubtful value in dealing with ecological
problems likely to be encountered in the
oceans. Simple correlation between any one
factor and the growth rate of a natural
population is likely to be confounded by
variation among the others. The only recourse seems to be laboratory studies which,
under controlled conditions, can provide
the coefficients required in order to analyze
the respective roles of the factors affecting
the populations of the seas. The continuous culture technique offers great advantages which rccommcnd its use in such
work.
REFERENCES
1961. Cultivation
of marine orgaT.
nisms as a means of understanding
environmental influences on populations,
p. 291-298.
In Mary Sears, [ed.], Oceanography.
Publ.
Am. Assoc. Advan. Sci. 66.
‘BRAARUD,
AND
RAYMOND
F. JONES
DOTY, M. S., AND M. OGURI.
1957. Evidence
for a photosynthetic
daily periodicity.
Limnol.
Oceanog. 2: 37-40.
JONES, R. F.
1962. Extraccllular
mucilage
of
the red alga Po~phyridium
cruentum.
J. Cellular Comp. Physiol., 60: 61-64.
MONOD, J. 1950.
La technique de culture continue;
thboric
et applications.
Ann. Inst.
Pastcur, 79 : 390-410.
MYEN, J., AND L. B. CLARK.
1944. Culture conditions and the development
of the photosynthetic mechanism.
II. An apparatus for
the continuous culture of Chlorella.
J. Gcn.
Physiol., 28 : 103-l 12.
NOVICK, A., AND L. SZILARD.
1950. Experiments
with the chemostat on spontaneous mutations
of bacteria.
Proc. Nat. Acad. Sci. U.S., 12:
708-719.
PI~OVASOLI,
L., J. J. A. MCLAUGIILIN,
AND M. R.
DIIOOP. 1957. The development
of artificial media for marinc algae. Arch. Microbial.,
25 : 392-428.
SOROKIN,
C., AND R. w. KHAUSS. 1958. The
effects of light intensity on the growth rates
Plant Physiol., 33: 109-113.
of green algae.
-,
AND -.
1962. Effects of tcmpcraturc and illuminancc
on Chlorella
growth
uncoupled from ccl1 division.
Plant Physiol.,
37: 37-42.
TAMIYA,
H., K. SHIBATA,
T. SASA, T. IWAMURA,
AND Y. M~RIMUHA.
1953. Effect of diurnally intermittent
illumination
on the growth
and some cellular characteristics
of Chlorella,
p. 7%84. In J. S. Burlew,
[ed.], Algal culturc from laboratory
to pilot plant. Carncgic
Inst. Wash. Publ. 600.