ON NITROGEN
DEFICIENCY
IN
TROPICAL PACIFIC OCEANIC PHYTOPLANKTON.
II.
PHOTOSYNTHETIC
AND CELLULAR
CHARACTERISTICS
OF A CHEMOSTAT-GROWN
DIATOM1
William
Institute
1% Thomas and Anne N. Do&on
of Marine Resources,
University
of California,
Scripps Institution
of Oceanography,
San Diego, La Jolla
02037
R-BSTRACT
Cells of the tropical Pacific diatom, Chaetoceros
gracilis, were grown in a nitrogenlimited chemostat at varying percentages
of the maximum
growth rate, harvested,
and
photosynthetic
and cellular parameters
measured.
Assimilation
ratio (photosynthesis
at
light saturation per unit chlorophyll)
increased with increasing growth rate. Cellular C :
chlorophyll
ratios also decreased with increasing growth rate, but carotenoid : chlorophyll
ratios showed no obvious trend. The C : N ratio decreased and chlorophyll:
cell increased
with increasing
growth rate. Steady-state
cell numbers were not constant at different
growth rates but decreased as the growth rates increased.
Growth rates secmcd to be
controlled
by internal supplies of nitrogen and the apparent half saturation constant, K’, ,
decreased with increasing-growth
rate.-
INTRODUCTION
A previous paper (Thomas 1970a) dcscribed the need for quantitative
asscssmcnts of nutrient
deficiency in marine
phytoplankton
populations.
Assimilation
ratios ( photosynthesis at light saturation :
unit chlorophyll)
and dark uptake of 14CCO2 in phytoplankton
from nutrient-poor
areas of the tropical Pacific Ocean wcrc
compared with similar values in algae
from nutrient-rich
waters. Assimilation ratios were lower in nutrient-poor
water
than in nutrient-rich
water, but the diffcrencc was not great. It was concluded that
algae in poor water were not extrcmcly
nitrogen deficient and that levels of ammonium wcrc high enough to maintain
them in a healthy state.
At that time we could best describe deficiency by qualitative
terms such as “cxtremc,” “borderline” or “incipient,”
or “not
deficient.”
Our purpose here is to cxprcss
nitrogen deficiency in quantitative
terms.
WC have grown cells of a tropical Pacific
diatom in a chemostat at various pcrcentages of the maximum growth rate unlimitcd by N deficiency. At each limited subl Contribution
Oceanography.
LIMNOLOGY
AND
from
the
Scripps
OCEANOGRAPHY
Institution
of
maximal growth rate we have expressed
assimilation ratio and various intracellular
parameters, such as C : N, C : chlorophyll,
and carotenoid : chlorophyll ratios, as functions of the growth rate.
A chemostat allows one to grow cells at
a constant dcgrce of deficiency.
Deficicncy is controlled by having one nutrient
present in the medium in limiting amounts
and pumping medium into the culture
vessel at a constant rate. After a few
days, a steady state is rcachcd so that cell
growth keeps up with the dilution rate and
the cell population remains constant. The
growth rate, p, is then equal to F/V, the
flow rate divided by the culture volume.
If F/V is less than p,,,, which is determined from exponential increases in cells
during batch culture under the same culture conditions and with the same medium, then deficiency can be expressed
quantitatively
as a certain pcrcentagc of
pmax. A good description
of chemostat
theory is given by Hcrbcrt ct al. ( 1956).
We are grateful to Dr. R. Lasker for
the use of the C-II-N analyzer. This investigation is part of the Scripps Tuna
Oceanography Rcscarch Program and was
supported by National Science Foundation
Grants GB-8618 and GA-27545.
515
JULY
1972,
V. 17( 4)
516
WILLIAM
MATERIALS
AND
H.
THOMAS
METIIODS
The diatom used in this work was Chae~OCCTOS gracilis;
its source
and tcmpcrature
and light requirements have been previously described (Thomas 1966). Axenic
stock cultures were maintained at 0.025
ly/min
continuous illumination
and 21C
in the medium of Sweeney and Hastings
(1957).
The chemostat was similar to that dcscribed by Capcron ( 1968). It consisted
of a 3-liter round-bottom
reaction vessel
with a glass water jacket to maintain a
temperature of 25C. The vessel lid had
ports for inoculation,
aeration, sampling,
overflow, and medium tubes. The overflow tube was adjusted so that the volume
of culture was 2.5 liters. By clamping off
this tube, air pressure forced culture suspension up through the sampling tube so
that a sample was taken from near the
bottom of the culture. The aeration tube
also extended to the bottom of the culture.
Ammonia was removed from the air by
bubbling it through 1 N H$Oh followed
by a water wash; the air stream was stcrilized with a membrane filter placed in the
lint. A bank of “cool white” fluorescent
lamps provided continuous illumination
at
intensities of 0.05 ly/min (as dctcrmined
by a submersible photoelectric
cell calibrated against a radiometer) at the center
of the culture, which was above that found
previously to saturate growth of this alga
(Thomas 1966). Medium was pumped into
the culture through silicone rubber tubing
with a peristaltic
pump and mixed by
aeration and magnetic stirring. Cells were
counted with a Coulter counter (model B).
Seawater low in nitrogen was collected
offshore and enriched with 10 PM KzHP04,
100 PM NazSiOs, 0.2 mg/liter
Fe (supplied as the citrate: Rodhe 1948), trace
metals, and vitamins at the concentrations
given by Guillard and Ryther (1962) for
their medium “F,” and 10 PM
N as
(NH&Sod.
All nutrients except N were
present in excess, so that N was limiting.
Medium was prepared in 40-liter amounts
and filter sterilized with a PI-1 Millipore
AND
ANNE
N.
DODSON
filter into two 20-liter Pyrex aspirator
bottles connected to the pump in parallel
so that medium was pumped from both
bottles.
For each experimental run, the chcmostat was filled to the 2.5-liter volume and
then inoculated and allowed to grow as a
batch culture (without pumping but with
aeration) for several days. When the culture was in the exponential
phase of
growth (as determined
from successive
counts), cells were harvested for mcasurement of photosynthesis and cellular parameters (see below ) . The culture was
then pumped back to volume and batch
growth continued for 24 hr. At this point
some submaximal growth was selected and
pumping started at that rate (pumping
rate divided by the volume of culture
gave the submaximal
growth rate attained). The population in the chemostat
was followed for several more days until
equilibrium
between dilution and growth
(constant cell numbers) occurred, and then
harvested again. Batch culture conditions
were again maintained for 24 hr and the
process repeated at some other submaximal growth rate. Several steady states
were obtained with each batch of medium
(during each experimental run), At the
end of the last experimental run, the culture was allowed to reach a state of cxtremc deficiency (a growth rate of 0) by
growing it as a batch culture until numbers no longer increased and the same
measurcmcnts were repeated. Also during
the final run, two additional harvests were
made from batch culture growth at maximum growth rates.
We harvested 75% of the chemostat
each time. Five milliliters of cell suspension were diluted into 45 ml of low-N seawater for photosynthesis
mcasuremcnts,
inoculated with 1 @i of 14C-Na&0,? solution, and incubated in duplicate at the
following light intensities: 0.10, 0.07, 0.06,
0.045, 0.0325, 0.0175, and 0.0075 ly/min.
Dark uptake was measured in cultures
covered with aluminum foil. Total COa
was calculated from pH and alkalinity
after the addition of 25 ml of 0.0100 N HCl
PHYTOPLANKTON
TABLE
1.
Growth
rates and photosynthetic
NITROGEN
characteristics
Growth rate
(% of /&ax
for run)
517
DEFICIENCY
of chemostat-grown
Chaetoceros
Photosynthesis at
light saturation
(pg C liter-1 hr-1)
gracilis
Assimilation ratio
(pg C fig Chl .-I hr-I)
Harvest
Culture
condition
Growth rate, b
(doublings/day)
1
2
Batch
N-limited
3.72
2.32
Run 1
100
62
57.8
71.0
:
N-limited
0.57
1.21
32
15
41.6
25.7
2.49
3.20
1
2
3
4
Batch
N-limited
N-limited
N-limited
2.42
0.20
0.40
0.81
Run 2
100
8
16
33
45.1
2.2
27.0
48.1
3.34 (?)
1.03
2.81
3.20
1
2
3
Batch
N-limited
Batch
Batch
N-limited
2.87
1.68
2.73
2.64
0.00
Run 3
100
59
95
92
0
155.5
137.7
110.1
151.4
1.6
to 100 ml of diluted culture ( Strickland
and Parsons 1968). The temperature of the
cultures increased 4C during 3 hr of incubation in a constant temperature room.
Cells were then filtered on HA Millipore
filters, which were washed several times
with filtered seawater and fumed for 20
min with HCl. After drying, the radioactivity on the filters was assayed (Thomas
1970a). The radioactivity
added to each
subculture
was standardized
by liquid
scintillation
assay. At the highest light
intensity used, photosynthesis
was saturated, so assimilation ratios here reported
are micrograms of carbon fixed at light
saturation per microgram of chlorophyll
per hour.
Subsamples of the original harvest were
also filtered on glass-fiber paper, extracted
with 90% acetone and their pigment composition de termincd spectrophotomctrically
( Strickland and Parsons 1968). The cquations of Parsons and Strickland
( 1963)
were used to calculate carotenoid concentrations, and the absorptions of extracts
were measured before and after acidification to correct the chlorophyll
a values
for pheopigments ( Strickland and Parsons
1968; Lorenzen 1967).
Other subsamples were filtered through
2.36 (?)
18.70 (?)
5.89
5.79
6.02
7.59
0.14
incinerated
glass-fiber filters for carbon
and nitrogen analyses, performed by combustion of the filters and gas chromatography of nitrogen and carbon oxides using
a CIIN analyzer (Hewlett-Packard
F & M,
model 185).
Ammonium in the filtrates was determined by the method of Solorzano (1969).
The presence of very low amounts of nitrate-0.10
PM---in the original medium
was determined by Cd-Cu reduction to
nitrite (Wood et al. 1967).
RESULTS
AND
DISCUSSION
Data from three experiments arc given
in Tables 1 and 2. Maximum growth rates
( pMBX) as determined from batch culture
growth varied from 2.42 to 3.72 doublings
/day in each experimental run, but are
similar to those found previously (Thomas
1966). Because of this variation, all photosynthetic and cellular parameters are expressed as functions of the percentage of
pmnX in each run in Figs. 1 and 2.
Three of these parameters are mcasurable at sea: assimilation ratio (in an incubator or in situ at light saturation), C:Chl
ratio ( Eppley 1968)) and carotenoid:Chl
ratio. Most cellular parameters, such as
518
WILLIAM
II.
THOMAS
AND
ANNE
N.
DODSON
70
60
0
% MAXIMUM
GROWTH
50
RATE
% MAXIMUM
20
GROWTH
RATE
-
D
C
0
0
0
0
0
0
0
I1
I
I
% MAXIMUM
,
I
50
,I
GROWTH
I
RATE
I
.y
100
1
I
I
0
%MAXlMUM
I
I
50
I
----I
GROWTH
100
RATE
FIG 1. Photosynthetic and cellular paramctcrs as a function of the growth rate of Chaetoceros graB. Cellular C:Chl a ratio. C. Carotcnoid:Chl
a ratio.
cilis. A. Assimilation
ratio at light saturation.
D. Cellular C:N ratio.
PIIYTOPLANKTON
TAISLE
Cultwc
112uvest conditions
2.
Callrrlar
Cells/ml
(X 10:s)
characteristics
Chl (,ug
Cdl c
/liter)
(,ug/litcr)
1
2
3
4
Batch
N-limited
N-limited
N-limited
218
76.6
205
418
24.5
3.8 (?)
13.0
10.3
289
218
454
638
1
2
3
4
Batch
N-limited
N-limited
N-limited
170
143
282
310
13.5
2.4
9.6
15.0
257
126
231
237
1
2
3
4
5
Batch
N-limited
Batch
Batch
N-limited
109
181
203
109
354
26.4
23.9
18.3
20.0
11.2
( Cells
( Cells
( Cells
( Cells
( Cells
NITROGEN
of chemostat-grown
Carat
(&liter)
C:Chl
Run 1
11.8
yJ
(?)
61:8
Run 2
19.0
53.6
24.0
15.8
Run
broke
broke
broke
broke
broke
C: N ratio, arc difficult to measure in natural communities bccausc of detrital contamination of phytoplankton
samples.
Figure IA shows the assimilation ratio
as a function of growth rate in the chemostat (the questionable ratios for batch culturc in the first two runs arc excluded).
Ratios were nearly constant at a value of
around 6.0 pg C pg Chl-l hr-l above 60%
of hax and dccrcascd to a value of 0.14
at p = 0. Our previous mean ratio in nutrient-poor water of the eastern tropical
Pacific was 3.15 (range 1.15-5.18) (Thomas
1970a). The mean corresponds to a growth
rate of 26% pmnX (range 6-53). Actual
values computed from two expcrimcnts
where pmax was measured in the field and
from 14C uptake and chlorophyll
values
were 33.4 and 22.6% (computed
from
Thomas 1970b), close to the mean value
of 26%. To the extent that C. gracilis is
representative of these natural communitics, we can bc reasonably confident in
assuming that the populations were growing at about a quarter of their maximum
potential rate. The phytoplankton
species
composition of these waters is not yet
known, In nutrient-rich
equatorial water
the mean assimilation ratio was 4.95 (range
3
)
)
)
)
)
519
DEFICIENCY
Chaetoceros
Cnrot:Chl
gracilis
Cell N
(pg/liter)
C:N by
atoms
NII, in
medium
(pg-atom
/liter)
-
23.5
4.5
9.7
12.3
0.96
1.18 (?)
0.75
1.19
41.8
69.7
38.2
53.1
8.04
9.75
13.32
13.99
6.1
2.2
12.7
17.1
0.45
0.92
1.33
1.44
20.7
11.4
17.6
19.2
14.5 (?)
12.9
15.3
15.3
0.23
0.24
0.35
0.11
26.2
21.9
24.8
24.3
15.5
0.99
0.92
1.36
1.22
1.38
-
8.0
13.9
10.3
18.4
0.07
0.84
0.18
0.68
0.21
3.53-6.19: Thomas 1970a), corresponding
to a growth rate of 55% p,,, (range 2984%)) or nearly twice those in nutricntpoor water. Extrcmc deficiency results in
ratios below 1.0, moderate or borderline
deficiency in ratios of about 2.55, while
nondcficicnt
cells have ratios close to 6.0.
Growth rates calculated from cell carbon and 14C uptake values were 24 times
higher than actual growth rates in the culturc calculated from the dilution rate. This
may be due to the higher light intensities
used to saturate photosynthesis, the slightly
higher temperature in the photosynthetic
bottles, and the fact that photosynthesis
was measured in diluted cultures, which
may have rcccived more light per cell. WC
feel that diluted cultures were more rcpresentative of cell concentrations in the sea
and that the measurements were more
comparable to the previous fieldwork than
if WC had used undiluted cell suspensions.
Figure 1B shows C:Chl ratios as a function of growth rate; values from run 3 are
not included bccausc C cstimatcs sccmcd
impossibly low. All the C:Chl values arc
less than 98, the mean value for phytoplankton in nitrate-free water off La Jolla
(Eppley 1968). If WC had used these lower
520
WILLIAM
H.
THOMAS
AND
ANNE
N.
DODSON
“‘:-
B
1.1---L..
1
% MAXIMUM
GROWTH
RATE
1
50
0
% MAXIMUM
GROWTH
A
100
RATE
D
r‘0
- I.0
X
d
II,,,,,,,,
50
”
% MAXIMUM
F.cG. 2.
GROWTH
100
RATE
Cellular parameters as a function
a per cell. B. Steady-state
ccl1 numbers.
constant, K’, .
% MAXIMUM
GROWTH
of the growth rate of Chaetoceros gracilis.
C. Steady-state
cell quota. ID. Apparent
RATE
A. Chlorophyll
half-saturation
PHYTOPLANKTON
NITROGEN
values in a previous cstimatc (Thomas and
Owen 1971) of phytoplankton productivity
in nutrient-poor tropical Pacific water, we
would have seriously underestimated productivity as compared with 14C productivity. In those calculations we used a value
of 98 taken from Eppley ( 1968); from the
present curve ( Fig. 1B ), a value of about
50 might have been more appropriate if
the crop was entirely C. gracilis. There
are apparently species differences in the
response of the C : Chl ratio to nitrogen
deficiency.
In batch cultures allowed to
become cxtremcly deficient, Hobson and
Pariser ( 1971) showed that the ratio in
Thahsiosiru
fluviatilis increased from 26.3
to 100, while in CycZoteZZa nana the ratio
increased from 50.-80 to as high as 1,000.
Actual measurements of the C:Chl ratio in
tropical water by the method of Epplcy
(1968) would be of value in refining productivity estimates and in defining actual
growth rates of natural populations more
fully.
Carotenoid:Chl
ratios did not show any
particular
trend with changing growth
rate ( Fig. 1C). IDespite the promise of
such ratios in assessing deficiency (Yentsch
and Vaccaro 1958; Manny 1969), from the
present data one would have to discount
them as a means of quantifying
nitrogen
deficiency.
Such ratios have also been
shown not to change much with deficiency
by Antia ct al. ( 1963).
In run 3 absolute amounts of ccl1 carbon
and nitrogen were low, as evidenced by
improbably low C: Chl ratios. WC suspect
that cells broke during filtration of the C
and N samples (a different filter apparatus was used ) . Nevertheless, C : N ratios
could be calculated from analyses of the
C and N remaining on the filter. Deficiency was quite well delineated
by
changes in this ratio (Fig. ID), which
increased with decreasing growth rate.
Similar changes have been shown previously in batch cultures (Thomas 1964;
Holm-Hansen
1970; Hobson and Pariser
1971)) but such ratios are of little USC in
field assessments of deficiency because of
detrital contamination,
Chl: cell increased
DEFICIENCY
521
with increasing growth rate (Fig. 2A) and
is another measure of deficiency.
The main purpose of our work was to
establish varying degrees of nitrogen dcficicncy and to assess this deficiency in
terms of parameters that can be measured
in the field, but it is intcrcsting to compare our results with those of other investigators who have used algal chemostats
for different purposes. The paper by Herbcrt et al. (1956) is perhaps the best general explanation of chemostat theory. WC
arc using their notation. Implicit in this
theory arc the assumptions that yield, Y,
of cell material per amount of limiting
nutrient is constant with varying growth
rate, p, that the cell quota, Q (the reciprocal of Y), is also constant, and that there
is a constant cell concentration, X, in the
chemostat with varying p.
Such constancy dots not always occur
in bacterial (Herbert 1958) or especially
in algal chemostats (Droop 1966, 1968,
1970; Capcron 1968; Fuhs 1969). In our
experiments, cell numbers decreased with
increasing growth rate (Fig. 2B). Because
of possible filtration losses of cell N, nitrogcn:cell was calculated from the differcnce in NH4 inflowing and that remaining
in the medium. Nitrogen per cell-Q,
the
cell quota-increased
exponentially
with
increasing growth rate above a minimum
value of 0.26 pg-atom N X 10d7/cell (Fig.
2C). This minimum
value, 7~0, is the
amount of N necessary to maintain cell
integrity
without
growth; it was determined by plotting PQ against Q and extrapolating to zero PQ (Droop 1970). If
growth rate is plotted against Q, the rcsuits arc similar to those of Caperon, Fuhs,
and Droop, suggesting that the growth
rate is controlled by internal stores of the
limiting nutrient.
On the other hand, growth rate may be
controlled by the external supply of limiting nutrient. WC measured ammonium in
the medium ( S ) in runs 2 and 3, but unfortunately not in run 1. The ammonium
concentrations ranged from 0.11 to 0.88
PM, as compared with the value found
by analysis of the inflowing
medium of
522
WILLIAM
II.
TIIOMAS
11.22 ( SIz) . Most of the ammonium coming into the chemostat was used up by the
cells. Growth rate should be related to S
by the hyperbolic equation
( >
S
P = Pm= K+S
s
( Monod 1942)) where K, is a constant.
From S, p, and pm, we calculated an apparent constant, K’, and found, as had
Droop ( 1970), that the constant varied
with p ( Fig. 2D). Applying the square
root transformation of K’, vs. x/p (Droop
1970), we found that K, at x/p = 0 was
0.10 PM, which is very close to the K,
value found earlier from uptake expcriments with ammonium for this organism
( Eppley et al. 1969). Actually K, may not
be varying with p, but haz may vary.
The results would bc the same if pmnx
varied, except that K’,y would bc constant.
Our initial success in using equation ( 1)
in estimating phytoplankton
production in
oligotrophic waters of the eastern tropical
Pacific Ocean (Thomas and Owen 1971)
may not be due to our theoretical assumptions that external instead of internal supplies of S control production.
Rather, it is
highly probable that in such waters both
supplies are in equilibrium,
so that it
really dots not matter which is measured
to iakc production estimates.
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