PHYTOPLANKTON
GROWTH AND COMPOSITION
IN
SHIPBOARD
CUL.TURES SUPPLIED WITH NITRATE,
AMMONIUM,
OR UREA AS THE NITROGEN
SOURCE1
R. W. Eppley, A. F. Carlucci, 0. Holm-Hansen, D. Kiefer,
J. J. McCarthy, Elizabeth Venrick,2 and P. M. Williams
Institute
of Marine Resources, University
of California,
P. 0. Box 109, La Jolla
92037
San Diego,
ABSTRACT
Coastal surface seawater off southern California
was enriched with phosphate, silicate,
vitamins Bla, BI, and biotin, a chelated trace metal mixture, and with nitrogen as nitrate,
ammonium, or urea, and the cultures were incubated on deck in daylight.
Natural phytoplankton present in the seawater served as inoculum.
Samples were taken every 6 hr,
once the cultures were established.
Diel periodicity
was noted in diatom cell division, in
nitrate and ammonium assimilation
rate, in phosphate assimilation
rate, and in photosynthetic rate measured at intervals under constant, artificial
irradiance,
but not in the rate
of chlorophyll
a synthesis. The chemical composition
of the crops was influenced
by the
nitrogen source used for growth and by diel periodicity
in assimilation rates. Most of the
vitamin B1 content of the crop was synthesized by the organisms, only a small proportion
being supplied initially
from the medium.
Some species known to require vitamins continued to grow after vitamin depletion from the mleclium, their requirements
apparently
satisfied by vitamins released by other species.
INTRODUCTION
Field studies of marine phytoplankton
growth are hindered by the advective
and turbulent movement of water masses,
by the patchy distribution
of phytolplankton, and by the relatively low concentration of plant cells found in the sea.
Because of these difficulties,
information
concerning the characteristics
of phytoplankton growth has mainly come froIm
culturing
expcrimcnts
in closed vcsscls.
Strickland advocated the USC of large
volume cultures to minimize artifacts rcsulting from wall effects and to assure
that sufficient plant material for analysis
be obtained from cultures supplied with
low nutrient levels as in the sea (McAllister et al. 1961; Antia et al. 1963; Strickl Supported
by U.S. Atomic Energy Commission Contract
AT( 11-l ) GEN 10, P.A. 20, by
Federal Water Quality
Administration,
Contract
16010 EHC, by National Science Foundation
RV
Alpha I-l&x
Program
(ship time), and by the
Marine Life Research Program of the Scripps Institution of Oceanography.
2 Marine Life Research Group, Scripps Institution of Oceanography.
LIMNOLOGY
AND
OCEANOGRAPIIY
land ct al. 1969). Phytoplankton
cultures
reared outdoors have the advantage of exposure to natural sunlight. Cult&&
originating from unfiltcrcd
scawatcr, suitably
enriched, provide information
on ecologically significant species, even though not
all species present may grow and those
that become dominant in culture may not
have been so in the initial water sample
(e.g., Menzel ct al. 1963; Trantcr and
Newell 1963; Pratt and Berkson 1959).
Earlier studies have compared nitrate
and ammonium (Strickland et al. 1969), but
the importance of urea in oligotrophic water as a nitrogen source for phytoplankton
growth has been discovered only recently
(Newell et al. 1967; McCarthy 1970). Laboratory cxpcriments indicate that ‘not all
species can use urea as a nitrogen source
( Guillard 1963; McCarthy 1971).
Earlier outdoor culture experiments suggestcd differences in the clemental composition (C : N ratio) and in the carbon :
chlorophyll
ratio of phytoplankton
depending
on the nitrogen
source used
( Strickland ct al. 1969). WC have studied
these ratios in large (200 liter) shipboard
741
SEPTEMBER
1971, V. 16(5)
742
TABLE 1.
-
EPPLF,Y,
CARLUCCI,
HOLM-HANSEN,
KIEFER,
McCARTIIY,
VENRICK,
AND
WILLIAMS
Nutrient
enrichments
in culture experiments.
Trace metals plus EDTA were added
ml stock solution (see Eppley et al. 1967b) per 200 liters of culture
-__.____
Nitrogen
source (pg-atom
N/liter)
KNO,
NH,CI
Urea
25
25
50
25
25
50
25
5
20
5
20
50
July
Phosphate as
KH,PO,
(N)
Experiment
5
I
Experiment
5
II
(ng/liter)
BL,
B,
Biotin
25
25
5
20
5
50
5
20
5
:;
5
20
5f
3-t
50
* Nitrogen added in small quantities
+ Added to NH.+ culture onlv.
4: Added to NO:- culture only.
Vitamins
Sodium
silicate
(W)
as lo-
(2-10
pg-atom/liter
cultures, inoculated with a natural community of phytoplankton,
enriched identically
with nutrients, but with nitrate, ammonium, or urea as the nitrogen source.
Further, by sampling the cultures at 6-hr
intervals WC found cvidcnce for diel pcriodicity in cell division, phosphate uptake,
nitrogen assimilation, and photosynthetic
rate which would be difficult to mIcasure
in situ because of advection. The species
composition and generation times did not
appear to be influenced by the nature of
the nitrogen source or by vitamin dcpletions during growth.
The latter observation implies that some species of the
phytoplankton
community produced vitamins used by others.
We arc grateful for the support of our
colleagues:
Mr. E. II. Renger, Mrs. G.
IIirota, Mrs. N. Wcarc, Mrs. P. Bowes,
Miss J. Rose, Mr. R. J, Linn, Mr. C.
Stearns, and Mr. G. Hamburg; Miss J.
Walker typed the manuscript.
We thank
Captains G. Coleman and R. Haines, their
o fficcrs and the crew of the RV Alpha
Helix, and Dr. W. F. Garcy.
METHODS
Culture vessels were four 200-liter polyethylene vats fitted with translucent lids,
wrapped with checsccloth, and continually
sprayed with surface seawater for cooling.
They were filled with nutrient-deplctcd
sot
G7$
culture)
at 6-hr intervals.
surface seawater that had been filtered
through 183-p netting to remove all but
the smallest zooplankton,
Two cxpcriments were carried out. Trace metals
wcrc added at the start to each culture,
in addition to the nutrients shown in Table I, as 10 ml/200 liters of the stock
solution given in Epplcy et al. ( 1967b).
Four cultures were studied in each cxperiment : one with all nutrient
additions
except the nitrogen source, the second
with nitrate, the third with ammonium,
and the fourth with urea. Samples were
taken daily for analysis until a reasonable
crop develo$pcd or until circumstances pcrmitted more frequent sampling.
Thcreafter samples were taken cvcry 6 hr. In
experiment I the intensive sampling began
late and the cultures were approaching the
stationary
growth phase; experiment
II
sampled cells in the logarithmic phase.
The cultures wcrc cxposcd to natural
sunlight.
Average irradiance within
the
vessels was estimated to bc about 20% of
sunlight. The temperature of the cultures
was between 20 and 25C; they were stirred
vigorously before each sampling but were
not othcrwisc mixed or aerated.
Analytical
methods
Samples to bc analyzed for soluble
through
substances were first filtered
combusted Whatman GF/C glass-fiber fil-
PIIYTOPLANKTON
o
GROWTII
IN
SIIIl’BOARD
743
CULTURES
o
NO;
NO;
A
NH.,+
NH,++
o
UREA
A
~1
UREA
O.lI
102L
’
I200
,600
JULY 13
0000
I
0600
I200
JULY 14
IS00
0000
I
Increase in the concentration
FIG.
1.
diatoms present in cultures enriched with
ent nitrogen sources; cxpcrimcnt
II.
0600
JULY 15
of all
differ-
tcrs. The methods for analysis of nitrate,
ammonium,
reactive phosphate, silicate,
dissolved organic C, N, and I’, dissolved
vitamins B12, B1, and biotin arc listed in
Strickland and Parsons (1968). Ammonium
was also analyzed according to Sol6rzano
(1969) in conjunction with urea analysis
( McCarthy 1970). Proccdurcs for particulate Chl a, ATP, C, N, and P were also
those in Strickland and Parsons (1968).
Lipid was detcrmincd by measurement of
org-C (Helm-IIanscn
ct al. 1967) and carbohydrate by the phenol-sulfuric
colorimetric procedure (Dubois ct al. 1956).
Particulate vitamin methods will bc published scparatcly. Photosynthetic capacity
was measured as the rate of 14C assimilation in a scparatc, water-cooled incubator
illuminated
with tungsten quartz iodine
1030
JULY II
I
,100
JULY !2
I
I200
JULY 13
0000
I
,200
JULY 14
0000
I
JULY 15
FIG. 2. Increase in chlorophyll
a concentration
with
time of cultures
enriched
with
different
nitrogen sources. Abrupt changes in slope in the
graphs for nitrate
and ammonium
cultures
appearcd to correspond
to the decline in vitamin
Blz concentration
in the media to < 1 ng/liter;
experiment
II.
lamps. A scintillation counter was used to
assess the radioactivity
of the phytoplankton on dried membrane filters (Wolfe and
Schclskc 1967). The assimilation rate of
15N-labclcd nitrate, ammonium, and urea
have been reported
( McCarthy
1971) ;
studies of diel variation
in chlorophyll
fluorescence are underway.
RESULTS
Grozoth
of
the
cldtures
Some measures of growth in experiment
II displayed diel periodicity, such as the
rate of incrcasc in diatom cell conccntration ( Fig. 1). Howcvcr,
chlorophyll
a
increase showed no cvidcnce of such pcriodicity in these cultures (Fig. 2).
744
EPPLEY,
CARLUCCI,
HOLM-HANSEN,
KIEFER,
MCCARTHY,
VENRICK,
TAULE 2. Specific growth rates (doublings
of cell number/day)
for the dominam
ties in the cultures supplied with different
sources of nitrogen; experiment
-
AND
WILLIAMS
phytoplankton
II, days 1-4
spa-
-ADoublings/day
Species
Nitrate
Leptocylindrus
danicus
Nitzschia spp.*
Chaetoceros spp.*
Cylindrotheca
(Nitzschia)
Hemiaulus
sinensk
Skeletonema costatum
Asterionella
japonica
Prorocentrum
micans
All diatoms
Chlorophyll
increase
Ammonium
0.95
1.52
1.45
1.13
closterium
Urea
0.96
1.36
1.13
1.10
0.70
0.88
I
0.63
1.16
1.40
-1.3
WI.3
-1.1
0.70
1.35
1.48
None
0.40
0.30-t
0.31‘1’
0
0
0.25t
0
0
1.34
1.48
1.21
1.10
1.16
1.44
Iz JO.9
0.63
1.33
1.18
0.29.t
Iz /O
* Not counted by species.
t These species divided on the 4th day of the experiment.
C$Species present but counts were erratic.
The diatom cells divided in the afternoon and early evening ( Fig. 1) as noited
earlier in small laboratory
cultures of
Skeletonema costatum (Jgrgenscn 1966))
Ditylum brightweZZii (Eppley et al. 1967a),
and other diatoms (Paasche 1968). Numbcrs of Prorocentrum
micarq
the only
abundant dinoflagellate
of these cultures,
were too low for an assessment of pcriodic cell division but the rate noted agrees
with that measured by Barker (1935). The
synchrony in diatolm cell division was only
partial since division rates were mostly in
TABLE 3. Chemical
useful in productivity
excess of one doubling/day,
that is, many
cells divided twice daily ( Table 2).
Little or no growth took place without
added nitrogen, as is characteristic of nutrient-depleted
surface waters of the eastern Pacific Ocean (Thomas 1970). Growth
rates (from ccl1 numbers ) for the various
taxa differed little between the cultures
(Table 2) and no sp&es selcctivc effect
of urea was seen.
Growth curves based on chlorophyll
a
(Fig. 2) differed from ccl1 number growth
curves in other rcspccts than the absence
composition
of the phytoplankton
grown on different
sources of nitrogen.
Ratios
studies (g :g). Cells were in stationary
growth phase in experiment
I, in log
phase in experiment
II
Ammonium
Nitrate
C:N
C:ATP
C : Chl a
N:Chl
a
C : Bls
C : I31
C : biotin
c : cell*
N : cell
ATP : cell”
Chl a : cell*
B12 : cell?
B1 : cell-l
Biotin : cclli’
* Units
t Units
I
II
5.9’
5.2
270
46
280
90
15
8.7 x 10”
5.8 x lo4
5.9 x 106
ND
8.8
6.8 x lo6
2.7 x 10”
6.8 x l@
220
42
0.82,
4.8
3.2
82
3.2
are picogram (lo-= g) per cell.
arc lo-lo g per cell.
I
Urea
II
3.9
260
112
4.3
230
58
7.5:
l@
6.0 x 10’
5.4 x 10”’
ND
1.4 14
x 10”
3.5 x lo”
1.6 x 10”
225
53
1.0
3.9
1.6
64
1.4
I
5.6
260
96
17
7.8 x lo”
6t.7 x 10’
5.9 x lo”
ND
II
7.0
290
77
11
7.7 x 1IY
4.1 x lo”
8.2 x 10”
154
22
0.53
2..0
2.0
38
1.9
Pl3YTOPLANKTON
GROWTH
IN
SHJJ?l3OARD
745
CULTURES
Initially, the doubling rate
of pcriodicity.
for chlorophyll
was significantly
greater
than that for cdl number in the culture
supplied with ammonium. Abrupt changes
in slope are evident in the chlorophyll
curves fo8110wing depletion of vitamin B12
from the scawatcr during experiment II.
Cell size was smaller in the urea culture,
as indicated by calculation of content per
cell, but this was not due to a disproportionate abundance of a single species.
Physiological
periodicity
Assimilation of nitrate and ammonium,
calculated from the dccrcase in these ions
in the medium, showed diel periodicity in
phase with photosynthetic
capacity (Fig.
3). The latter, as rate of carbon assimilation per weight of chlorophyll
a, was
measured in artificial light in a separate
incubator.
The phase and amplitude of
nitrate and ammonium uptake pcriodicity
was similar to that noted in Peru Current
phytoplankton
(Epplcy ct al. 1970). Urea
assimilation showed no such pcriodicity.
Phosphate uptake, calculated from its
decrease in the medium, showed pcriodicity in all three cultures (Fig. 4). Uptake rate was at a minimum bctwecn 1800
and 2400 hours and was greatest during
the day. Similar data for silicate showed
greater scatter, but uptake rate appeared
to bc greatest between noon and midnight-during
the period of cell division
(Coombs et al. 1967; Busby and Lcwin
1967; Epplcy et al. 1967a, Miiller-Haeckcl
1965 ) .
The ratio C : Chl a, which has been used
for calculating the phytoplankton
standing
stock as carbon from chlorophyll mcasuremerits, showed a regular periodicity in all
three cultures (Fig. 5). The ratio incrcascd
in daylight and decrcascd at night: This
rcsultcd from continuous
chlorophyll
a
synthesis, with carbon assimilation in daylight only. The nocturnal dccrcasc in the
ratio also includes a respiration component
acting to rcducc ccl1 carbon, but our analytical methods wcrc not sensitive enough
for its accurate measurement.
o~ooooooo
JULY
IS I JULY
14 I JULY
15‘0
Rate of nitrogen assimilation,
as the
FIG. 3.
decline in nitrogen concentration
in the medium
between 6-hr sampling intervals, and the rate of
photosynthesis,
as ,ug C assimilated per liter per
hour at constant irradiance.
Points for nitrogen
assimilation
are plotted at the midpoint
between
consecutive B-hr samplings; experiment
II.
Phytoplankton
chemical
composition
Certain ratios and variolus measures of
composition arc given in Tables 3, 4, and
5. These arc useful for comparing results
of growth on the different sources of nitrogen Cells grown on ammonium wcrc
relatively rich in nitrogen with low C : N
and high N : Chl a ratios. The ratios C :
Chl a and N : Chl a were higher in the
stationary phase cultures ( expcrimcnt I)
than in log phase cultures (experiment II),
implying cessation of chlorophyll synthesis
with continuing assimilation of carbon and
746
EPPLEY,
CARLUCCI,
IIOLM-IIANSEN,
KLEFER,
MCCARTHY,
VENRICK,
AND
WILLIAMS
160
t
40
o
20
0 I-
I200
1600
JULY 13
0000
I
0600
I200
JULY 14
1600
0000
I
0600
JULY I5
FIG. 4. Rate of phosphate
disappearance
from
culture media with time of day. Points arc plotted at the midpoint between 6-hr sampling periods. Phosphate assimilation represents the decline
in phosphate in the medium between consecutive
samplings; experiment
II.
nitrogen into the stationary phase. The
ratio C : ATP was fairly uniform throughout and averaged 265. The C : Chl c1ratios
of log phase cells varied with the nitrogen
sour-cc as nitrate < ammonium < urea.
Dissolved
orgunic N and P
Both dissolved organic nitrogen (DON)
and dissolved organic phosphorus ( DOP)
increased in cxpcrimcnt I (measured for
122 hr) and expcrimcnt II (measured for
96 hr ) , The DON increase was 3 pM and
the DOP increase 0.6 pM with all three
substrates, suggesting that the excreted
soluble organic matter was enriched in
phospho,rus relative to the phytoplankton
stock regardless of the substrate. Measurements of dissolved organic carbon
(DOC) generally increased with time but
quantification
was not possible due to an
unknown contribution of DOC from lcaching of the polyethylene tubs. There was
no periodicity
observed with DON or
DOP, nor was there any correlation of
DON and DOP with nitrate or phosphate.
,200
I600
JULY 13
0000
0600
NO;
A
NH4+
q
UREA
I
1200
JULY 14
.
I600
;
0000
!
0600
JULY IS
FIG. 5. Ratio of cell carbon to cell chlorophyll
a vs. time of day for phytoplankton
in cultures
with nitrate, ammonium, or urea as the nitrogen
source; experiment
II.
DISCUSSION
There is a vast literature on planktonic
algae ( particularly
laboratory
organisms
such as Chlorella),
the principles, methods, and results of which are often applied, but with questionable assumptions,
occasional misgivings, and variable success, to the study of productivity
in natural waters. Perhaps the greatest value o’f
detailed studies of the physiological
and
biochemical
processes of ocean phytoplankton is that they serve to bridge the
gap between experimental laboratory science and primary productivity
work.
Periodicity
in phytoplankton
growth
A prominent feature of the growth of
unicellular
algae under light/dark
illumination cycles is its periodicity (see Tamiya
1966; Pirson and Lorenzen 1966). In the
culturing
is a
laboratory,
synchronous
technique for isolating in time discrete
portions of the grolwth cycle of a cell. WC
kno$w of no published reports of periodic
ccl1 division
in natural
phytoplankton
(measured in situ) but many workers arc
aware of its likelihood in the sea. Our
observations reported here support the hy-
J?IIYTOl?LANKTON
TADLE 4.
Nitrogen
source
Chemical
Cells
(millions/
liter)
composition
represent
GROWTI
I IN
of the phytoplankton
net increase during
SHIPBOARD
grown
growth.
747
CULTURES
on different sources of nitrogen.
Units, pg/liter
Particulate
Particulate
c
N
Nitrate
Ammonium
Urea
None
ND
ND
ND
ND
Experiment
5,200
880
4,500
1,170
4,700
840
3OQ
21
Nitrate
Ammonium
Urea
15.4
18.6
15.9
3,400
4,200
2,451)
P
I (stationary
122
65
157
7.6
phase cultures)
18.4
58
17.5
40
18.0
49
0.30
7.0
Experiment
II (log phase cultures)
650
ND
12.6
998
ND
18.5
2
350
ND
8.5
32
pothesis that phytoplankton
cell division
is synchronized
in natural co8mmunitics
where illumination
cycles permit.
Periodicity in photosynthetic
carbon assimilation
has been known since 1957
(Doty and Oguri 1957). Periodicity in nitrogen assimilation has been reported also
( Goering et al. 1964; Epplcy ct al. 1970) ;
our results arc consistent. The pcriodicity
in assimilation of ammonium is not well
understood, but periodicity
in glutamic
dchydrogcnase activity was rcportcd for
synchronous cultures of Chkamydomonns
reinha&
(Katcs and Jones 1967) and
Coccolithus huxleyi ( Eppley et al. 1971).
Nitrate and nitrite rcductase also show
pcriodicity in activity in synchronous culturcs ( Eppley et al. 1971)) rcflccting control of assimilation via enzyme regulation
as well as by availability
of rcductants
that may bc formed in photosynthesis or
other photoprocesses. Nitrate assimilation
in green plants involves its reduction to
nitrite followed by a photosynthetic
reduction of nitrite to ammonium (Joy and
Hagcman
1966). Its light depcndcncc
varies among algal species, appearing to
bc absolute in the diatom D. brightwellii
(Epplcy ct al. 1967u) but only partial in
other algae (Grant and Turner 1969). Diel
curves of nitrate and ammonium assimilation in laboratory cultures (Kanazawa ct
al. 1970; Eppley ct al. 1971)) in natural
phytoplankton
off Peru (Eppley
ct al.
1970), and in thcsc experiments show a
vitamins
B 12
%
Biotin
0.006
0.0061
0.006
0.003
0.090
0.075
0~.07Q
0.013
0.009
0.008
0.008
0.001
0.005
0.003,
0.003
0.126
0.1201
0.061)
0.005
0.003
0.0039
Chl0
ATP
Values
rate decline bcforc nightfall
and an increase before sunrise, implying operation
of a more subtle control mechanism than
an on-off switch. This is true also of the
pcriodicity in photosynthetic capacity, with
at least two implications : 1) Such pcriodicity is related to biological rhythms in
gcncral and results of studies on rhythms
may contribute to understanding better the
primary production processes in the sea.
2) In productivity
measurements incubation periods must be selected with cog,nizance of physiological
periodicity
( Doty
ct al. 1965; Ncwhousc ct al. 1967).
Chlorophyll
a synthesis in laboratory
cultures of marine phytoplankton
exposed
to light/dark cycles is reported to bc pcriodic (Jergenscn 1966; Eppley ct al. 1967a;
TABLE
5.
plankton
Nitrogen
source
Gross
grown
chemical composition
of phytoon different
sources of nitrogen
Celluhr
dry wt*
(/.&liter 1
Pcrccnt of dry wt
Protcint
Carbohydrate
Lipid
Nitrate
Ammonium
Urea
None
Experiment
9,625
10,250
8,990
1,018
I
50
60
53
25
17
18
21
48
27
30
29
24
Nitrate
Ammonium
Urea
Experiment
6,815
9,340
4,935
II
53
60
38
18
17
28
26
28
21
* Particulate organic
t Nitrogen X 6.25.
carbon
X 2.2.
748
lU?PLEY,
CARLWCCI,
HOLM-IIANSEN,
Paasche 1968) or not (Paasche 1967) according to the organism studied. It was
not periodic in thcsc experiments with a
crop of mostly diatoms. But this cannot
bc rcgardcd as general for diatoms. Differences in ability to synthcsizc chlorophyll
continuously again reflect the character of
the cell’s regulatory mechanisms and are
best studied in the laboratory.
The periodicity WC foiund in phosphate
assimilation has n’ot been studied in marine phytoplankton.
One expects a relationship of phosphate assimilation to the
timing of RNA synthesis and the synthesis
of polyphosphatc
storage products.
The
synthesis and significance of these compounds has been examined in detail only
in laboratory organisms (c.g., Senger and
Bishop 1969).
Crop periodicity
and composition
The phenomenon of diel periodicity has
implications for several kinds of analytical
measurements pertinent to the study of
marine phytoplankton.
If the various spcties undertook cell division at diffcrcnt
times of day not only would species cnumeration be affected but also the measures
of community structure and diversity derived from species counts. Such error is
unlikely to exceed a factor of 2 or 3 and
may be unimportant in view of the reliability of sampling and counting methods. The
problem seems less important than loss of
fragile spccics by poor preservatioa
or
problems resulting From patchiness.
Although Fig. 1 shows only total diatom cell concentration over time, the seven
or eight most abundant diatom species
showed significant concordance in the timing of cell division in the cultures enriched
with nitrate and ammonium ( p < 0.05 and
p < 0.01; Kendall concordance test on the
percent increase of species). Increases in
individual
diatom species in the urea culture did not show significant synchrony,
probably
due to extreme variability
of
mrcr species.
Pcriodicity in chemical composition, for
example as reflcctcd in the C : C,hl a ratio
( Fig, 5)) results in an uncertainty in the
KIEFER,
McCARTIIY,
VENRICK,
AND
WILLIAMS
use of any standard ratio for calculating
crop size from Chl a. Apparently (Fig. 5)
the diel variation in the ratio is of the order
*20% for a crop growing on a given nitrogcn source. In cultures of such spccics as
Dunaliella
tertiolecta
(considered a rock
pool form) the ratio was more nearly constant because both photosynthesis and Chl
a synthesis wcrc restricted to daylight
(Eppley and Coatswo’rth 1966).
Species interactions
Specific gro’wth rates of the eight common taxa prcscnt in experiment II were
similar regardless of the nitrogen source
(Table 2). However, P. micans failed to
grow in the laboratory with urea as the
source oE nitrogen ( McCarthy 1971). Several alternative explanations may be offered for this apparent paradox: 1) The
P. micans clone of experiment II may have
had the capacity to utilize urea nitrogen
and hence may bc different from the isolate studied in the laboratory. 2) Bacteria
in the culture vessels may have hydrolyzed
urea and released ammonium. 3) Phytoplankters (which do grow in the laboratory with urea as their sole source of
nitrogen)
such as Cylindrotheca
(Nitxschia) ckosterium (Grant
ct al. 1967;
McCarthy 1971)) Ch,aetoceros spp. ( McCarthy 1971)) or S. costatum (Guillard
1963; McCarthy 1971) may have relcascd
nitrogen in a form such as ammonium
which was used by other spccics.
The same kinds of argument apply to
satisfying vitamin requirements.
Of the
taxa present in experiment II ( Table 1)
AsterioneZZa japonica and several isolates
of Nitzschia require no vitamins (Provasoli
1963; A. F. Carlucci, unpublished).
SkeZetonema costatum requires vitamin 1312but
releases B1 and biotin which arc used by
other species requiring them for growth
in mixed cultures in the laboratory (Carlucci and Bowes 1970a, b ). Probably some
species of experiment II produced vitamins
required by others. Vitamin B1 uptake
from the culture medium in experiment I
and II was only a small fraction of the
vitamin B1 content of the cells at harvest
PIIYTOPLANKTON
TABLE
GROWTH
IN
749
CULTURES
6. Vitamin budgets. Comparison of loss of vitamins from the medium with the final vitamin
content of the crop produced, with estimates of vitamin synthesis.
All values in r&liter
Loss from the medium
Nitrogen
source
B 12
B,
Biotin
B 12
I
II
4.1
17
0.7
Experimerzt
6.0
6.0
6.0
2.7
Nitrate
Ammonium
Urea
5.5
4.5
4.8
21
5
40
2.2
3.4
5.3
Experiment
5.0
3.0
3.2
Chemical composition of the crop
There were no dramatic differences in
chemical composition of cells grown on nitrate, ammonium, or urea ( Table 5). The
protein content in ammonium cultures was
slightly higher than that in the other cultures as shown by C : N ratios ( Table 3).
In nitrogen-starved
cells, the cellular material was high in carbohydrate and correspondingly
low in protein.
The lipid
content was fairly uniform in all cultures,
ranging from 21-30% of the total dry
weight.
A nitrogen budget was prepared (Table
7) based on measurements of nitrate, ammonium, and urea of the medium, dissolved organic nitrogen in the medium,
Nitrogen
buclgets. Comparison
of nutrient
phytoplankton
crop in experiments
66
46,
8.8
7.4
-0.5
-1.5
-1.8
105
1151
20
2.8
-0:8
-2.3
90
75
70
12
8.8
8.4
8.0
1.0
126
120
60
5.0
2.6
3.0
the medium with
Units in pug/liter
its increase
II
Nitrogen source loss from medium
Increase in dissolved org-N
Ammonium increase in medium
946
47
4
520
ND
0
Net loss of nitrogen from
Increase in particulate-N
895
880
520
650
I
in the
source for growth
Ammonium
I
medium
1.2
0.3
0
-1.4
B 12
Nitrogen
budget
Biotin
Biotin
loss from
I and II.
Nitrate
Nitrogen
B,
Bl
and particulate nitrogen. Only a few of the
inorganic nitrogen consumed appeared in
the dissolved organic nitrogen fraction, implying either little cxcrction (or leakage)
of nitrogenous substances from the cells or
else a stcady-state condition with loss and
uptake in equilibrium.
A level of ammonium (0.2-0.5 PM) persisted in the cultures
supplied with nitrate and urea. Possibly
ammonium rcleascd by nitrate reduction in
the dark or at very low irradiance may bc
involved, as has been shown for nitrite
( Vaccaro and Ryther 1960). A low level of
ammonium in the urea culture is not surprising but needs an adequate explanation,
since urea assimilation in Chlorella (Roon
and Lcvenbcrg 1968) and possibly other
algae involves not ureasc but rather an
ATP-requiring
enzyme system. Discrepancies between comparisons of net nitrogen
loss from the medium and the increase in
particulate nitrogen probably rcprcsent analytical error.
(Table 6). Synthesis by certain of the
phytoplankton is the most likely source of
this production. Vitamin B12, on the other
hand, showed net synthesis only with ammonium and nitrate in experiment I.
7.
Net synthcsis
Content of crop
Nitrate
Ammonium
Urea
None
TABLE
Sl3IPBOARD
Uren
II
I
II
1,110
42
-
92,5
ND
-
586
42
347
ND
1,068
1,170
925
990
538
840
346
350
750
EPPLEY,
CARLUCCI,
IIOLM-HANSEN,
Ratios involving
carbon, nitrogen, and
other constituents
in these cxpcriments
have been comp‘arcd with other reports on
the composition of phytoplankton
studied
in situ (Platt and Subba Rao 1970) or under conditions that simulate nature, such
as the Nanaimo plastic bag experiments
(McAllister
et al. 1961; Antia et al. 1963)
and the Scripps Institution
deep tank
( Strickland et al. 1969). Results arc similar but the average irradiance was higher
in the current experiments and this permitted higher C : Chl a and C : N ratios
(Table 3) than observed earlier. The finding of differences in these ratios consistent
with the source of nitrogen used for
an earlier suggestion
growth
confirms
(Strickland ct al. 1969) and may explain
in part the 3-fold difference in C : Chl a
ratio of phytoplankon observed earlier off
southern California between upwelling pcriods when nitrate was available and quiescent periods when ammonium and urea
would have been used for growth (Eppley
1968). The C : ATP ratio was unaffected
by the nitrogen source and agrees with the
value of 250 now used to convert ATP
mcasuremcnt to C (Holm-Hansen
1969;
Hamilton and Holm-Hansen 1967).
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