FURTHER
MEASUREMENTS
OF PRIMARY
USING A LARGE-VOLUME
PLASTIC
PRODUCTION
SPHERE
N. J. Antia, C. D. McAllistel; T. R. Parsons,
K. Stephens, and J. D. H. Strickland
Fisheries
Research
Board of Canada,
Biological
Station,
Nanaimo,
British
Columbia,
Canada
ABSTRACT
The experiment described by McAllister,
& (II. ( 1961)) in which a phytoplankton
bloom
was induced to occur in a free-floating
20-ft diameter thin transparent
plastic sphere has
been repeated. Daily measurements were made of nutrients, particulate
matter, and photosynthetic rates with less frequent assays for vitamins and dissolved organic matter. In situ
light was recorded by a bolometcr.
The experiment
was prolonged to 100 days to study
phytoplankton
decay, most of this period being in the dark.
The phytoplankton
consisted mainly of 6 species of diatom and one large dinoflagellate.
The mean composition of this crop at various stages of its development
is reported by ratios
involving
chlorophyll
a and particulate
organic carbon.
A detailed discussion is given of the findings of the experiment which, in general, confirmed those of the carlicr work and yielded, in addition, valuable new information.
The plant cells excreted 35-4OoJo of their organic matter during growth.
The CI~ method
of measuring photosynthesis
gave results ngreeing well with the production
of particulate
carbon.
The growth kinetics of the bloom were dominated by the constancy of the mean cell
division rates which were relatively
independent
of temperature
and light.
The rate of
photosynthesis
per unit chlorophyll
was also remarkably
constant and not proportional
to
light intensity, cells developing
a highly efficient
photosynthetic
mechanism with respect
to available radiant energy.
The mean chemical composition of a cell changed after depletion of nutrients from the
surrounding
water.
a and phosphorus
decreased.
The
The carbon, protein, chlorophyll
silicon and lipid contents remained nearly constant and the carbohydrate
increased.
During the decay period over half the particulate
phosphorus was remineralizcd
in 2
weeks. Silicon returned to solution more slowly but at a constant rate. There was no significant nitrification
even after 75 days. The consumption of oxygen occurred mainly from
the oxidation
of “dissolved”
organic matter and not from the interaction
of oxygen with
particulate
material.
The latter may have been important
as a surface for bacteria.
The
evolution rate of carbon dioxide was constant with time but procccdcd with a variable RQ,
which was around 0.5 immediately
after the bloom but increased to nearly 2 in the “old”
water present at the beginning and end of the experiment.
firmed and new information has come to
light , as a result of more detailed and more
McAllister, et al. ( 1961) have described
the study of primary production in coastal precise measuremcntsS The following acwaters using a large-volume plastic sphere count summarizes the more important obsome 20 ft in diameter suspended with its servations and deductions from this latest
center a constant 5.5 m beneath the sea work and gives, we believe, sufficient information for the time being about wellsurface. The growth kinetics and composistirred large-volume
cultures of coastal
tion of a mixed crop of phytoplankters
phytoplankton.
The
logical
extension of
were described.
this
type
of
program
would
be
to systems
In 1962 the work was repeated using improvements to the apparatus and tech- with mixed animal-plant populations or to
systems involving the use of a large open
niques and the experiment was prolonged
cylinder in which an unstirred column of
to 100 days (about 80 days after the initial
water
may be studied ( cf. Goldman 1962).
“bloom” of plants) to study decay procWe would again like to express our apcsses. Many of the observations made
during the first experiment have been con- preciation of the work done by L. D. B.
166
INTRODUCTION
PRIMARY
PRODUCTION
Terhune and others who helped in the
launching and maintenance of the apparatus and to acknowledge the assistance
given by John Yu in the analytical program, Dr P. R. Burkholdcr provided the
bacterium used for the biotin assays and
kindly furnished suggestions for its use.
PROCEDURE
AND
METIIODS
These were the same or similar to those
previously used ( McAllister,
et al. 1961)
with the exceptions now described.
General
The detrital level in the “bag” was much
lower than in 1961 but still appreciable.
Initially
the water was quite clear with
very little particulate organic matter present but after a day a slight haze formed
and the amounts of particulate iron and
carbon rapidly increased. This was caused,
we believe, by ferrous iron, dissolved from
piping and from the filtration equipment,
oxidizing and precipitating
as ferric hydroxide which acted as a “scavenger” for
colloidal organic matter,
The stirring in this experiment was continuous and very efficient and observations
by SCUBA divers showed that there was
no significant
settling of the particulate
material in the bag until at least 36 days
after the commencement of the experiment. The mean “turnover” time of the
water in the bag was estimated to be only
2-3 hr.
On the 28th day after filling, the sphere
was “blacked-out”
by attaching strips of
opaque black plastic over the top half and
was left in this darkened condition for the
rest of the experiment.
A little light cntered the bag by back-scattering into the
bottom part of the sphere but the amount
was considered to be negligible and insufficient to promote any plant growth.
Stirring was stopped on day 54 to allow
plant detritus to compact and thus promote decay. The contents were rcstirred
for 24 hr immediately prior to taking samples on days 79 and 99.
Determination
of “dissolved” materials
Inorganic
components:
No determinations were made of “soluble” iron, manga-
IN
A PLASTIC
SPHERE
167
nest, and copper, The particulate iron was
initially 0.5 pg-at/L
and increased somewhat due to corrosion of parts of the
stirrer but showed no obvious pattern. A
major change was made in the detcrmination of total carbon dioxide, which had
from pH
previously
been determined
changes. In this experiment the total carbonate was analyzed by means of a modified Van Slyke apparatus, using 5-ml samples and compressing the liberated gases
to 0.5 ml. The precision was found better
than * 0.3% of the amount present, i.e.,
about * 60 mgC/m”.
Organic components: All determinations
were made on HA-milliporc@ filtered water samples, stored deep-frozen with Hutner’s volatile preservative.
No dctcrmination
of total dissolved organic carbon was attempted but an indication of this was obtained by running
complete UV spectra ( 2,200-4,000 A) on
filtered water against redistilled water in a
lo-cm cell. A Bausch and Lomb Spectronic 505 instrument was employed.
Carbohydrates
were estimated directly
on filtered samples using a modified anthrone reaction, details of which will be
published later. By scrupulous attention to
cleanliness it was possible to detect as little as 50 mgC/m3 present as carbohydrate
in terms of a glucose equivalent.
Sclcctcd samples were examined for the
presence of glycollic acid using the 2:7 dihydroxynaphthalenc
reagent (Calkins 1943)
directly on filtered samples previously concentratcd by evaporation from 50 to 15 ml.
The limit of sure detection was estimated
to be about 100 mgC/m” as glycollic acid.
Vitamin Blz was bioassaycd using, as
assay organism, the marinc dinoflagellate
Amphidinium
carteri which has a known
requirement for this vitamin, The inoculum used was stripped of the vitamin by a
standard method, and vitamin standards
( cxtcrnal ) were prepared
with
Norittrcatcd synthetic sea water (3O%G)that was
appropriately cnrichcd with nutrients. After 15 days’ growth, under standard illumination, samples and standards were ccntrifuged to remove the plant cells, which
168
N. J. ANTL4,
C. D. MCALLISTER,
T. R. PARSONS,
were then extracted in a fixed volume of
90% acetone and the pigment extinctions
read on a lo-cm cell at 4,400 A (Beckman
DU Spectrophotometer).
The extinction
of standards was found proportional
to
ad&d BL2 up to a concentration of Eippg/
ml of vitamin,
Biotin was bioassayed using a marine
bacterium ( strain CSS ), with a known rcquircment for this vitamin, obtained from
Dr P. R. Burkholder.
The bioassay organism showed sensitivity to the growth
factor in the range 3-12 ppg/ml, best
working range being 5-10 ppg/ml.
The
filtered water samples were assayed at
three successive levels of dilution in order
to obtain biotin concentrations within the
range of sensitivity of the organism. External standards of the vitamin at three
concentration
levels were assayed with
each batch, using synthetic sea water,
Norit-treated
and appropriately
enriched
with nutrients.
The inoculum for each
batch was prcparcd from CCI. 24-hr agar
slants of the bacterium.
Aliquots of the
diluted and appropriately
enriched sea
water samples were measured into culture
tubes, inoculated with a loop full of inocuhim preparation, and incubated (without
agitation) at 23°C for 4 days. Turbidities
were spectrophotometrically
evaluated at
4,500 A. It is hoped to publish further details of the bioassay procedure used in a
later paper.
Determination of particulate matter
No “crude fiber” determinations
were
undertaken. Protein was calculated as 6.25
times the Kjeldahl nitrogen figure. The
“balance” between particulate phosphorus
and nitrogen formed and soluble phosphorus and nitrogen lost from solution was acceptable, This is in contrast with the 1960
experiment when the Kjeldahl nitrogen
results were lower than anticipated. There
was also better agrecmcnt this year between the Kjeldahl protein data and a few
spot checks of protein determined colorimetrically
on an acid hydrolysate using
Kceler’s method ( Strickland and Parsons
1961). We have no explanation for this.
Particulate silicon was not measured di-
K. STEPIIENS,
AND
J. D. I-1. STRICKLAND
rectly but was assumed to be equal to the
loss of reactive silicon from solution bctween day 10 and day 20.
Determination of plant pigments
The technique used was that described
by Strickland and Parsons ( 1961) but an
important modification
was made in the
Richards equations used to calculate the
amount of pigment.
The new equations
are given by Parsons and Strickland (In
press). All chlorophyll a data given here
are about 7570 of the values that would
have been obtained with the formula used
by Richards with Thompson (1952). This
fact should be borne in mind when comparing amounts and ratios, etc., in this report with results in the earlier experiment.
Chlorophyll
c is reported in mg/m” and
the SPU for carotenoids has been redefined to allow for the specific extinction
coefficients of fucoxanthin
and peridinin
being less than a half of the values for
most other carotenoids.
The unit used here for carotenoids is
such that one SPU of pigment dissolved in
1 L of 90% acetone has an extinction in a
I-cm cell of 100 at 4,800 A. All carotenoid
data are more than twice the values that
would have been obtained with the formula used by Richards with Thompson.
There was no evidence of incomplete
extraction of pigments by 90% acetone.
This would have been immediately
obvious with the detritus-free
high crop densitics encountered at the time of the plant
bloom.
Enumeration of phytoplankton cells
all weights were estiAs previously,
mated from cell counts converted to cell
volumes ($3) by a geometrical factor. The
assumption was made that the specific
gravity of the cells was close to unity. The
factors used were as follows: Skeletonema
costatum,
500; Chaetoceros
pelagicus,
1,200; Navicula sp., 5,500; Thalassiosira
aestivalis,
7,000; Stephanopyxis
turris,
22,000; Thalassiosira rotula, 35,000; Gyrodinium spirale ( ? ) , 120,000.
Cells were only counted if they were
clearly recognizable and appeared healthy.
PRIMARY
PRODUCTION
Badly misshapen cells and empty frustules
were not included and ccl1 counts therefort only measure the total plant biomass
up to about the 19th day of the experiment. Except for SMetonema thcrc was
no great variation in cell size for any spcties and no obvious changes in volume
took place from beginning to end of the
“bloom.” We do not claim any great precision for the: conversion factors given here
but errors in totnl cell weight will bc lessened by the fact that the biomass of the
whole crop was distributed
fairly evenly
between 5 or 6 individual
species. Relative changes in total cell weight should be
fairly precise as these were measured from
the changing ccl1 counts of 7 species and
thus depend little on the accuracy of each
conversion factor.
IN
A
PLASTIC
169
SPHERE
RESULTS
AND
DISCUSSION
The salinity of the water in the bag was
constant at 27.84%0 from the beginning to
the end of the experiment.
As previous,
the temperature of the water inside the
bag followed that of the surrounding sea
with a mean delay of 12-24 hr.
The data obtained during the present
are summarized
graphically
cxperimcnt
with confidence limits (95% probability)
shown where necessary. Ratios of metabolitcs, elc., collected in the tables, are reported with the number of significant figures considered justified by the precision
of the methods employed.
The initial level of nutrients in the water
was even greater in this cxpcriment than in
the previous study (cu. 54 pg-at. Si/L; 23
pg-at. NO,-N/L,
and 2.1 pg-at. PO‘I-P/L)
leading to the eventual production of a huge
diatom crop of more than twice the conMeasurement of radiant energy
ccntration encountered naturally in these
The illumination
in the sphere was
waters. The inoculum used to “seed” the
measured directly
using a pressurized
bag had a pigment concentration of 2.5 mg
thermopile bolometer with thcrmistcr com- chlorophyll u/m3 and was present in water
pensation for temperature
changes (the
containing 16 ,ug-at. N/L and 1.67 pg-at. P/L.
unmounted unit was obtained from the The initial chlorophyll a concentration in
Epply Co., R. I., U. S. A.). The instrument
the bag was 0.2 mg/m” corresponding to a
had an output of cu. 0.15 ly/min/mv
which
concentration of plant carbon of around 7
was measured by a continuous millivolt
mgC/m”.
Detrital
carbon exceeded 300
recorder on shore, the signal being carried
mgC/m:’ and made direct measurement
by a submarine cable cmcrging from the of plant carbon impracticable
until day
neck of the bag. The bolometer was 10 when the plant carbon exceeded 100
mounted next to the BOD bottles fixed at mgC/m?. (This was still only 5-6% of the
the center of the bag and thus measured
final crop. )
the actual photosynthetically
active energy
Period of plunt bloom
reaching the bottles in which photosynthcThe consumption of nitrate nitrogen and
sis was being measured. The minimum il- reactive silicate is shown in Figure 1. The
lumination
that could be measured with
nitrate was dcpletcd before the end of day
certainty was about 0.002 ly/min.
15 although it showed a very small but sigThe bolometcr was situated at the cen- nificant increase in concentration thereafter
ter of the bag at the beginning of the for another week before completely disapexperiment and was subsequently raised so pearing. An initial concentration of 0.25
that the illumination
measured was the pg-at. N/L as nitrite was removed from
same as the mean illumination
encountered
solution in the first week. The concentraby a particle in random motion throughout
tion of soluble organic nitrogen changed
the bag. The height above the bag center
very little (around 5-9 lug-at. N/L) with no
at which this illumination
occurred could
obvious pattern and, unlike the 1960 experbe calculated theoretically knowing the at- iment, there was no very clear pattern in
tenuation coefficient of the sea water in the change of concentration of ammonia
the sphere.
nitrogen with time, although this was
170
N. J. RNTIA,
C. D. MCALLISTER,
T. R. PARSONS,
K. STEPHENS,
AND
J. D. H. STRICKLAND
25
ITRATE
/
TIME
FIG. 1.
Changes of nitrate
(DAYS)
and reactive
roughly at a minimum (0.3 lug-at. N/L) at
the peak of the plant bloom. The uptake
of reactive silicate will be seen to lag
markedly behind the uptake of nitrogen.
The concentration changes of total and
reactive phosphorus on filtered samples are
depicted in Figure 2. The reactive phosphate was never completely depleted although the minimum concentration reached
( 0.1 pg-at. P/L) was less than in the 1960
experiment.
The precision of “organic
phosphorus” results (the difference between total and reactive phosphorus) is
low but the data show a definite maximum
in concentration around day 18, which coincided with the peak of healthy plant cell
production,
Thcrc is obviously conversion
of soluble inorganic to soluble organic
phosphorus by the plants themselves. The
decrease at day 8 may be real and arise
from bacterial activity (see later).
The
values on day 18 of 0.7 lug-at. P/L (of soluble organic) and 0.1 pg-at. P/L (of soluble
inorganic) are typical of the data found in
the open waters of Departure Bay in summer ( Strickland and Austin 1960).
silicate
concentrations
during
the bloom.
The changes in concentration of vitamin
B12 may be summarized as follows. An initial level of ca. 3 ppg/ml ( day 8) dropped
to 0.2 (day 14) during the plant bloom,
and tended to rise thereafter to 1.5-2.0
ppg/rnl ( days 20-22) with cessation of vigorous plant growth, achieving the high
level of cu. 8 ppg/rnl on day 54. The pattern of changes observed is in accord with
current views on the ecological role of the
vitamin in marine systems, wix., that the
vitamin is consumed during a phytoplankton bloom by plankters with a B la-reyuircment and that it is subsequently regenerated by proliferating
bacteria (Provasoli
1958; P. R. Burkholder 1959). Although the
vitamin requirements of most of the algal
species that occurred in the bloom are not
known, it is significant that a dominant
species in the bloom, Skeletonemcz costatum,
with a known B12 requirement, followed a
pattern of changes in cell population in keeping with the above generalization. The vitamin concentrations
observed in the bag
system, except at the peak of the bloom,
are similar (5-10 plug/ml) to those reported
PRIMARY
PRODUCTION
IN
A PLASTIC
171
SPHERE
ORGANIC
I
OO
I
5
I
IO
I
15
TIME
FIG. 2.
Changes
of total, inorganic,
and organic
by Lewin ( 1954) and Droop ( 1954) for
coastal waters of northern temperate rcgions in late winter to early spring,
Unlike the changes in the amount of
vitamin Br2, biotin concentrations in the
bag suggested a steady accumulation of the
vitamin in the system from start to finish
of the experiment. An initial level of 22
,upg/rnl ( day 8) changed but slightly during the rise of the bloom (29 plug/ml, day
14). Around the peak of the bloom, however, the level rose to more than twice the
initial value (62 ,+g/ml, day 20; 52 ppg/rnl,
day 22). An ultimate very high level of
biotin concentration in the bag was indicated by the figure of 126 ,upg/rnl obtained
for day 54. As very limited numbers of
samples were assayed, the possibility is not
precluded that minor, but significant, fluctuations in the vitamin level may have
occurred during the periods not examined,
but a general trend does appear to bc indicated by the measurements made. Belser
( 1959) has noted the frequent occurrence
of biotin in sea water samples taken over
an extensive area and at a series of depths,
I
20
(DAYS)
phosphorus
concentrations
during
the bloom.
and Hutchinson and Setlow ( 1946) have
rcportcd biotin concentrations of the order
0.34.0 ppg/ml for the fresh waters of ccrtain inland lakes. Howcvcr, we have seen
no previous quantitative determinations of
dissolved biotin in sea water reported in
the literature and it is therefore not possible
to compare the vitamin levels observed in
the bag with those of marine systems clsewhere. Initial biotin levels observed (cn.
25 plug/ml) may well be representative of
the biotin concentration in coastal bottom
water during mid-spring
( April-May ) .
concentration
The changes in biotin
indicate that none of the predominant phytoplanktcrs present have an ecologically
significant
biotin requirement
under the
prevailing growth conditions, and, further,
that some of them may even produce and
contribute (most probably by cell autolysis ) a significant amount of the vitamin to
the system around the peak of a bloom. In
the lists, compiled by Saunders ( 1957) and
Provasoli ( 1958)) of representatives of the
various algal classes found to show growthfactor requirements, it is significant that no
172
N. J. ANTIA,
C. D. MCALLISTER,
T. R. PARSONS,
TIME
FIG.
3.
Changes
K. STEPHENS,
J. D. H. STRICKLAND
(DAYS1
in the concentration
diatom species examined showed a biotin
requirement.
The implication
from the
present study that diatom blooms may contribute significantly to the biotin budget of
a marine bio-ecosystem needs verification
from culture studies. The exceptionally
high biotin level (cu. 125 ppg/ml) achieved
in the bag towards the end of the experiment may be attributed to production of
the vitamin by the prevalent bacterial flora
since in studies on vitamin-producing
bacteria in the sea, Burkholder (1959) has shown
that half of the 300 or more isolates examined in one experiment produced biotin.
The pigment curves given in Figure 3
differ from those reported in the earlier
experiment ( McAllister, et al. 1961). This
arises from the use of new formulae, as
mentioned earlier. Throughout the first 30
days, the absolute concentrations of chlorophyll a and of the carotenoids (expressed
as the new SPU ) remained about equal to
each other while the absolute quantity of
chlorophyll c remained equal to about half
the quantity of chlorophyll a. The ratios
chlorophyll a/c and chlorophyll a/total carotenoids did not increase with the decrease
in total pigment concentrations toward the
decline of the bloom, in contrast to reports
by other investigators.
We believe the
effect so reported to be an artifact brought
AND
of plant
pigments.
about by use of the uncorrected Richards
equations. Furthermore, the carotenoid to
chlorophyll
a ratio was remarkably constant after the peak of the bloom and the
ratios stayed at this level for the remainder
of the 100 days. These results, and those
reported last year, indicate that very little
information on the state of growth, decay,
nutrition, etc., of a coastal diatom crop can
be obtained from chlorophyll-carotenoid
ratios.
The maximum pigment concentration occurred 2-3 days before the maximum production of particulate matter. There was
subsequently a fairly rapid decrease of
pigment concentration
to about one-half
the peak values which then held steady for
10 days or more even under conditions of
nitrogen depletion and when the bag was
blackened out. This decrease did not result
from the settling out of plant cells and was
not accompanied by any obvious changes
in the complete spectrum of 90% acetone
pigment extracts. We suspect that death,
with rapid autolysis of pigment, occurred
to about half of the plant crop whereas the
remaining half entered a spore form or
some more resistant phase. This conclusion
was borne out, at least qualitatively, by the
microscopic examination of settled samples.
The net production of organic carbon in
PRIMARY
FIG.
PRODUCTION
4.
Net fixation
the bag has been estimated by three different methods and the results are given in
Figure 4. The particulate carbon was estimated directly by wet oxidation, correcting
for an initial blank of detrital carbon. The
loss of total carbon dioxide from the whole
system taken from day 3 as arbitrary zero,
was equated to organic production
and
finally the gain of oxygen by the system
was equated to organic production.
In the
latter case an RQ of 1.0 was assumed when
the gain was negative (LIP to day 10) and
PQ of 1.2 was used to convert oxygen increases to increases of organic carbon.
As in the previous year, respiration in the
bag as a whole initially
excccdcd photosynthesis for many days. This period of
net respiration is of some importance but
will be discussed later together with other
data on bacterial activity. If we consider
day 10 to mark the beginning
of the
“bloom” proper, i.e., when the predominant
metabolic activity in the sphere was plant
photosynthesis, and if we assume day 19
effectively terminated this period of vigorous growth, then the net production
of
organic carbon during this interval, as
measured by carbon dioxide change, was
2,600 mgC/m”, the production measured
by oxygen change was 2,500 mgC/m3, and
the production measured directly as oxidizable particulate carbon was about 1,600
IN A PLASTIC
of carbon
SPIIERE
173
in the bag.
mgC/m”. The latter figure is for oxidizable
organic matter expressed as carbon but the
true carbon value could scarcely have exceeded 1,750 mgC/m3 (see Parsons, et al.
1961) .
The oxygen and carbon dioxide methods,
which measure total net production
of
organic matter by photosynthesis,
gave
results in excellent agreement with each
other but at variance with the result for
particulate
organic matter, which
was
much lower. These data furnish striking
proof of the direct excretion of “soluble”
organic material from a coastal diatom
crop whilst in vigorous growth. The amount
excreted was close to 900 mgC/m”, or some
35% of the total net organic matter photosynthesized.
We have not yet been able to make a
systematic search for this excreted organic
matter. Absorption spectra in the ultraviolet region had a broad maxima at 2,600 A
and the initial water had a relatively high
extinction (ca. 0.4 on a lo-cm cell) at this
wavelength. The increase at 2,600 A during
and immediately after the bloom was relatively small, being a maximum of 0.08 at
day 22 which probably corresponded to the
autolysis of dead cells rather than excretion
during growth. At day 19 the increase was
only 0.03. The maximum dissolved carbohydrate also appeared on day 22, and was
174
N. J. ANTIA,
C. D. MCALLISTER,
T. R. PARSONS,
K. STEPHENS,
AND
J. D. IX. STRICKLAND
JSOD-
3000-
m
2500-
r
5
F
2000-
5
s
2
1500-
P
%
IOOO-
TIME
FIG.
5.
Production
mcasurccl
by gross oxygen
(DAYS)
evolution,
CL uptake,
and the formation
of particulate
carbon.
clearly of plant origin as no carbohydrate
was detected before about day 14 or after
day 27 ( when presumably microbiological
action had decomposed it). In terms of
“glucose equivalents”
about 300 of the
900 mgC/m” of excreted matter could be
accounted for as carbohydrate.
The anthrone reaction used for the determination
of soluble carbohydrates was very much
less sensitive towards pentoses and hexuranic acids than to hcxoses, so that although there was an indication that hcxuronates may have predominated early in
the bloom, hexoscs at the peak and pentoscs later, the evidence must be considcred as marginal. Had the hexoses present
on clay 22 been all galactose or mannose,
rather than glucose, the corresponding soluble organic carbon figure would have
been doubled but still could not a.ccount
for all of the excreted matter. In view of
the reports by Allen (1956) and Tolbert
and Zill ( 1956) of the excretion of glycollit acid in relatively large amounts from
some algae during active growth and the
rcccnt discovery by Fogg (personal communication)
of the presence of glycollate
in fresh and salt water containing diatom
blooms, we analyzed filtrates for this acid.
Only on day 22 was there any indication of
the possible presence of glycollate and this
was only equivalent to 100 mgC/m3.
As in the 1960 experiment, photosynthesis was measured each day in samples enclosed in BOD bottles placed near the
center of the bag and in a fluorescent light
incubator at an illumination
of 0.08 ly/min
of photosynthetic
radiation. The accumulative gross photosynthesis in the bag measured from oxygen evolution (assuming a
PQ of 1.2) and from C I4 uptake data arc
plotted in Figure 5. Ratios of the results
from oxygen and C 14 measurements ( both
in bag and incubator)
were again very
high, indicating that the one method must
have been overestimating organic production or the other badly underestimating
it.
These high ratios which we have always
found in local coastal waters become explicable when we consider the accumulative CILi curve in Figure 5 along with the
curve showing the increase of particulate
carbon, The agreement over the main
growth period is quite striking and the only
reasonable explanation for the discrepancy
between the CL4 and oxygen results would
PRIMARY
TABLE
PRODUCTION
Dny
12
14
Protein
c
C
0.5
0.64
2.05
1.95
A PLASTIC
1. Ratios inuohing
carbon
-_~--~
-~
_~--~~
Si
IN
Carbohydrnte
_C
0.2
0.25
--
and nitrogen
_-_~--
--
6.
~
C
C
C
G-
F
0.13
0.15
3.1
3.2
17
22.5
3.25
3.9
4.4
4.7
4.7
4.7
3.6
3.9
ca. 3
27.5
31
37
33
32
35
35
30
12.5
15.5
18
18
19
16
15
16.5
22
17
3
20
12-15
4.7
33
15-18
6k2
40 2 15
---
14.5
--
N
iY
(atoms)
sufficient to account for the production
of dissolved organic matter measured by
direct analysis.
The ratios of elements and metabolites
to carbon and chlorophyll
a at various
stages during the growth and decay of the
phytoplankton
crop are shown in Tables 1
and 2 for selected days. Results in general
confirm the data given in our previous
paper ( McAllister,
et al. 1961) but are
more precise and detailed and incorporate
more accurate nitrogen and chlorophyll
data. It is clear that some ratios are quite
different when the plants are growing in
water still containing excess nitrate than
when growing in water that has been defi-
3ooot
FIG.
_-
Lipid
1’:
0.97
0.93
1.6
1.9
0.6
1.1
0.31
0.32
20
0.87
1.45
1.0
0.38
24
1.35
1.15
0.34
27
1.30
1.1
0.40
30
1.30
0.9
0.25
36
1.75
0.8
0.18
47
1.60
0.45
< 0.05
54
ca. 2
0.7
Vigorously growing crop with excess NOad in water:
0.6
2.0
0.2
0.15
Unhealthy crop in NOa- clcpletccl water:
ca. 1
1.1
0.35
( 1960) :
Values suggested by StricEnd
0.8
__I~
seem to be that the excreted organic matter
produced during this bloom was labelled
by the Cl4 added to bottles and hence prcsumably originated from early products of
photosynthesis. In these coastal waters during diatom blooms the C14 method measures the net production of particulate mutter whereas the oxygen method measures
the gross total production of organic material. This inference may explain many
discrepancies observed by workers using
various methods of measuring photosynthesis and may well account for the observation made by Duursma (personal communication) that the primary production in
the North Sea, as estimated by Cl+ is in-
175
SPIIERE
Changes in the concentration of major metabolites.
176
N. J. ANTIA,
C. D. MCALLISTER,
T. R. PARSONS,
~
-
--
-__
C
Day
____-~___-__
12
14
16
18
20
24
27
30
36
47
54
Vigorously
Ratios involving
2.
_
TABLE
------.A
---
N
--
P
Chl. a
Chl. a
_-~~
37
23.5
25
37.5
49
52
Y”9
12
7.3
7.6
Chl. n
____.~
2.2
1.05
0.93
1.2
13
1.3
11’
1.55
14.5
2.1
16.5
2.25
59
16.5
1.7
114
29.5
3.9
70
22
growing crop with excess NO, in water:
25
10
1.5
Unhealthy
crop in NOa clcplctcd water:
2
Values sugges?f$l by Striokla~l
( 1960) :
30
7zk3
0.75 + 0.2
* Chlorophyll
n calculated by revised Richards
** New definition
of carotcnbid SPU.
0
0
0
0
CHLOROPHYLL
05
0
04
00
0
0
3020
(3
___-
$0
2:
220..
0
0
00
0
OI
0
- --
0
OPROTEIN
OCARBON
0
-1
0
02-
SILICON
=: IO-
a
-
40-
Ood
- - - i
-- -_0
CARBOHYDRATE
0
LIPID
0
4
0
“0
0
OO
30
5 15
z
:
p
0
2
0
000
IO
OOOO
0
OO
0
00
5
0
0
OO
00
000
i
IO
-LI
12
LI
16
14
16
-’
0
IO
--I2
DAY
FIG.
during
7. Changes
the bloom.
of mean cellular
14
DAY
AND
chlorophyll
__ .--
---
a*
-.~.
Protein
_-Chl. (I
Carbohydrntc
~Chl. a
76
45
47.5
7”:
70
89
103
103
184
138
7
5.9
15.5
40
48
59
75
71
45
51
48
60
100
-
J. D. I-I. STRICKLAND
-.Lipid
Chl. a
7”i
Cnrotenoids*
____
-Chl. a
11:s
19
17
27
20
10.5
3
-
1.0
0.92
0.90
0.99
0.96
1.1
1.15
1.15
1.15
1.2
1.15
6
3-7
0.95
60
lo-25
1.15
-
-~.--~-
*
esuntions.
cient in nutrients for some days. We have,
therefore, suggested approximate
values
for ratios to be used in both such circumstances. This practice is to be preferred to
the use of one very approximate set of
values for all phytoplankton crops irrespcctive of growth conditions (see Strickland
1960). Ideally similar sets of factors to
those given here should be determined for
several oceanic arcas. A start on this has
been made ( McAllister, et nl. 1960) and
some values for pure cultures are given by
Parsons, et al. ( 1961).
50-
K. STEPIIENS,
-I
I6
I6
composition
i
The concentrations of protein, carbohydrate (as glucose), and lipid (as stearic
acid) are shown for the growth and early
decay period in Figure 6. This graph and
the ratios in Tables 1 and 2 imply great
changes in cell composition
during the
phytoplankton
bloom. The method of expressing results as ratios ( e.g., Tables 1 and
2) is useful for practical applications but
can be misleading if one is interested in
changes in cellular composition, because
both the carbon and pigment composition
of a ccl1 may be changing along with the
other metabolites.
An idea of the meun change in chemical
composition of the pl.ant cells with time is
given by Figure 7, which shows the cellular composition as a percentage of wet
algal weight for silicon, carbon, chlorophyll
~1, protein, carbohydrate, and lipid. The
assumptions of a constant specific gravity
for the algal cells and of a constant mean
volume per cell throughout the bloom are
open to criticism. The cell densities would
almost certainly increase with age which
would tend to exaggerate the effects shown
in Figure 7. There must be a decrease in
mean diatom size during the 3 to 4 cell
divisions responsible for the major changes
shown in Figure 7. The effect would tend
PRIMARY
PRODUCTION
TIME
FIG. 8.
Changes
of algal weight
to counteract the effect of increased cell
density in the calculation of algal weight
but is not thought to be very significant.
The protein level per cell was initially
nearly constant but then commenced to
drop on day 14, even before all the nitrate
was depleted from solution, and the scnescent cells (day 19) contained a third or
less of their initial protein content. By contrast the phosphorus content (not shown)
decreased almost linearly from day 10 to
day 17, changing from 0.85%, which must
be considered a value for glutted cells, to
0.13%, a value which persisted until day 19
and was presumably the basic minimum
phosphorus content with which the cells
could function.
The amount of the “excess” phosphate in a cell was very roughly
proportional to the concentration of rcactive phosphate in the outside medium.
These results should be compared with
those obtained on cultures of Nitzschia
closterium described in the two classic
papers by Ketchum (1939a and 1939b).
There is marked general agreement. The
decrease in the mean chlorophyll a content
per ccl1 ( Fig. 7) was roughly parallel to
the decrease in nitrogen. Relatively spcaking, however, there was practically
no
IN A PLASTIC
177
SPHERE
(DAYS)
of the main phytoplankton
species.
change in the silicon and lipid contents of
the cells. Results for silicon are more nearly
constant when we allow for the algal
weight present as dinoflagellate
and indicate that the degree of silicification
of
growing cells is nearly constant, provided
that some reactive silicate is present in the
external medium. From day 10 to day 19
there was no indication that the cellular
silicon depended on cell concentration as
reported by Jergensen ( 1955). The constancy of the lipid level in the cells is surprising and shows that diatom cells do not,
as we had earlier supposed, appear to increase their fat content when growing
under nitrogen starved conditions. There
was a marked increase in the carbohydrate
level per plant ccl1 when nitrate was depleted from the outside medium. The carbohydratc more than doubled but even so
the total carbon per cell on day 19 decreased to less than 50% of the value measured in cells growing in water with 5 pg-at.
NO,-N/L
or more. Measurements of cell
volume are thus not satisfactory when we
wish to indicate the total organic carbon in
a phytoplankton crop and can be even less
satisfactory when attempting to estimate
the protein or carbohydrate content.
178
N. J. ANTIA,
C. D. MCALLISTER,
T. R. PARSONS,
.-
K. STEPHENS,
AND
J. D. H. STRTCKLAND
other species appeared unaffected. There
was no obvious “succession of species” in
5
4
J
0
this experiment.
Other algae noted were
Nitzschia delicatessima, C. dicipiens, Schroderella delicatulu, Rixosolenia fragilissima,
and a small Gymnodinium.
Although the
cell numbers of these were sometimes quite
large, all the species together contributed
only a few per cent of the total plant
biomass.
In Figure 9 we have plotted the accumulative photosyntheses measured by oxygen
evolution (gross photosynthesis assuming a
PQ of 1.2), the accumulative photosynthescs measured by Cl4 (nearly the same as
the amount of particulate
carbon until
about day 22) and the total algal weight.
The latter is shown by a heavy line with no
experimental points (to simplify presentation) as, on the scale used, all experimental
‘2
4
6
6
IO
12
16
18 20
22
24
TIME (DAYS)
data wcrc practically coincident with this
line. At the top of the curve is the total
FIG. 9. Photosynthetic
carbon fixation and increase of algal weight, showing radiation and temdaily ( 24 hr ) radiation recorded directly at
perature in the bag.
the depth at which the photosynthesis was
measured and at the bottom of the figure is
shown a plot of water temperatures inside
The relative increase in cell numbers for
the
bag.
each of the 7 major species present, ThalasThe most striking feature of this presensiosira rotula, Gyrodinium spirale, T. aestitation is the constancy of mean cell douvalis, Clzaetoceros pelagicus, Skeletonema
bling time ( 35 hr ) which was practically
costatum, Navicula sp., and Stephanopyxis
independent of radiant energy or temperaturris, is shown in Figure 8, with the
ture, provided that the latter was above
assumed algal weights (proportional to cell
about 10°C. This confirms the results of
number for each species) plotted on a logother work in progress in these laboratories
arithmic scale. The mean size of the Skeleusing pure cultures, which indicate that
tonema cells was only about a quarter of that
found in the 1960 experiment and hence cell division rate dominates phytoplankton
this species was not so significant a con- growth kinetics and is nearly constant for
any cell over a wide range of temperature
tributor to the total biomass as previously,
although
cell numbers exceeded 4.10G and light conditions provided that the cell
is subjected to relatively slow changes of
cells/L. The plots are remarkably linear,
temperature.
Shortage of nitrate may inallowing for errors in counting small numbers due to contagion effects. The algae duct drastic changes in the chemical composition of the cell (Fig. 7) but initially
had similar doubling times, ranging from
SO-40 hr, except for T. rotula which had a has much less effect on the division rate
( Fig. 8). The change in cell composition
doubling time of only 19 hr. The slowing
after day 14 is well illustrated by the deviain the growth rate of some species before
tion of the cell weight and Cl4 uptake
day 8-9 is most likely attributable to lower
curves in Figure 9. The gross photosynthcwater temperatures. After nitrate depletion
sis data had the least regularity.
Gross
the growth rate was significantly
reduced
in the case of T. rot&,
T. aestivalis, S. photosynthesis had to provide organic macostatum, and perhaps C. pelagicus but the tcrial for cellular substance, respiration, and
w
d
12
8
TOTAL
RAblANT
ENERGY
I
PRIMARY
TABLE 3.
PRODUCTION
10
11
12
::
15
16
17
18
19
20
21
22
23
179
SPHERE
Photosynthetic rates per unit pigment from Cl4 measurements (rates in mgC/hr/mg chlorophyll a)
._~
-~
.~
~~
Sphere
Day
IN A PLASTIC
Avcrnge
illumination
(ly/min
X 10-3)
5.5
9.2
7.1
5.1
4.4
1.7
1.9
1.3
0.3
0
0.5
0.9
0.8
0
(illumination
Maximum
illumination
(ly/min
X lo-“)
17
25
31
26
17
7
5.5
5.5
7
0
3.5
3.5
4.5
0
excretion. This apparent ability of phytoplankton cells to adjust their composition
over wide ranges to maintain a steady division rate relatively independent
of tcmpcrature, light, and external conditions
appea?s to bc of prime importance in algal
ecology and will bc the subject of further
communications.
The photosynthetic
rates per unit of
chlorophyll a have been collected in Table
3. CjL1 data are used as these provide the
most complete set of results of reasonable
precision.
(Much of the corresponding
gross photosynthesis data would be twice
or more the figures shown.) The rate of
photosynthesis per unit of particulate carbon can be calculated using the data in
Tables 2 and 3. The variations of photosynthetic rate on a unit carbon basis are
greater than those on a chlorophyll basis
because carbon to chlorophyll
ratios increase with time. All results in the incubator were obtained at a mean illumination
of about 80 X 10F3 ly/min of photosynthetic
radiation. The average illumination
in the
bag (as mean ly/min over a 24-hr period)
is also shown in Table 3. Values in the bag
rarely exceeded 510% of those in the incubator. The maximum illumination
recorded
in the bag for an appreciable period each
day is also reported. The highest of these
values did not exceed 40% of the incubator
shown)
_.~
Rate per unit
pigment
0.31
0.69
0.35
0.35
0.26
0.18
0.16
0.16
0.056
0.037
0.082
0.14
0.12
0.085
Incubator
80 X 10-S ly/min
Rate per unit
pigment
1.77
2.09
2.55
1.88
1.60
1.55
1.11
1.19
1.02
0.82
0.78
0.71
0.76
ca. 0.5
illumination
and most were less than 10%.
Photosynthetic rates per unit chlorophyll
a in the incubator are similar to those
found previously ( McAllister, et al. 1961))
allowing for the difference in formula used
for calculating pigment concentrations. The
ratios dccreascd from around 2 to just over
1 when the nitrate was depleted from solution and then dropped to around 0.7 after
the plant cells had existed in nitrogen deficient water for about one week. In general,
however, the values are roughly constant
on a pigment basis and a knowledge of the
chlorophyll
a content of water enables a
fair prediction to bc made of photosynthetic rates in field studies, especially if
the state of nutrition
of the plants is
known.
A remarkable observation is the high
level of photosynthetic activity in the center of the bag after the bloom commenced,
an activity which showed very little dependence on either mean or maximum illumination levels. The overall rate of photosynthesis per unit of chlorophyll
a was
lower in the bag than in the incubator but
generally not nearly as low as the ratios of
light intensities in the two environments
might lead one to suppose. The photosynthesis per unit pigment was very apprcciable even when no light was recorded by
the bolometer, i.e., less than 2 X lo-” ly/min.
180
N. J. ANTIA,
C. Il. McALLISTEH,
T. R. PARSONS,
An intensity of 2 x lo-” ly/min is much less
than that normally accepted as defining
the bottom of the euphotic zone on a 24-hr
basis ( e.g., Strickland 1958). The carbon
uptake at “zero” light intensity was not, we
believe, an artifact brought about by exposing the water briefly to light during
sampling and the only explanation we can
offer is that the cells developed an extremely efficient photosynthetic
apparatus
and may have stored “photosynthetic
capacity” before they were sampled. Light
gradients were very large in the bag on
days 19 and 23, due to the very heavy crop
density, and the illumination near the top of
the sphere would still have been quite considerable ( a minimum of 5 x 10B3 ly/min ) .
With the very efficient stirring used, most
of the cells in the bag would have been
exposed to light of this intensity for a few
hours during the day. This apparent ability of cells to store up energy during brief
exposures to light and reuse it later when
exposed to much lower light intensities is
supported by other (unpublished)
experiments in these laboratories and has considcrablc importance when interpreting
the
results of primary productivity
measurements.
Period of plant decay and nutrient
regeneration
The kinetics of phytoplankton
decay
were studied by measuring the changes of
concentration of reactive phosphorus and
silicate, the various dissolved nitrogen
compounds, and the oxygen and carbonate
in solution between day 25, when the
bloom was completely finished, and day
100 when the experiment had to be terminated.
The sphere was blackened to prevent
further photosynthesis on day 28. By about
day 40 some material had settled out, despite the vigorous stirring, and on day 55
all stirring was stopped to allow plant
detritus to settle, in the hope of promoting
Despite this, no significant
nitrification.
rcmincralization
of plant nitrogen occurred
in the 75-day decay period. On day 99 a
little nitrate (ca. 1.5 pg-at. N/L) had been
formed but none was detected earlier. The
K. STEPIIENS,
AND
J. D. II.
STRICKLAND
nitrite concentration on day 99 was 0.01
,ug-at. N/L. The ammonia level rose from
1 to about 3 pg-at. N/L and stayed roughly
constant. The dissolved organic nitrogen
level increased by 34 pg-at. N/L around
day 40 but subsequently decreased again.
In general, very little of the 23 pg-at. N/L
originally fixed in the phytoplankton
crop
returned to solution during a period cxceeding 2% months and there was no sign
of the successive formation of ammonia,
nitrite, and nitrate described by von Brand
and Rakestraw in their classic series of
papers ( 1939, 1940, 1941, and 1942). It is
clear, however, from these papers and from
the work of Carey ( 1938)) Spencer ( 1956))
and Watson (1960) that nitrification
processes are very dependent on the source of
water, growth factors, and bacterial inocula.
Regeneration of nitrate in the sea almost
certainly occurs erratically, and we have
still much to learn about the process
involved.
The exceptionally
large bloom
encountered during this experiment may
have had an adverse effect on the development of the correct bacterial flora for nitrification
( cf. von Brand and Rakestraw
1942). The present work serves to demonstrate how much delay may be expected
and illustrates how tenaciously a system of
plant cells and associated microflora will
conscrvc nitrogen once it has been converted into an organic particulate
form.
The regeneration of reactive phosphorus
(Fig. 10) was initially nearly linear with
time having a mean rate of about 0.13
pug-at. P/L/day.
The remineralization
appeared to be slowing down by day 40
when about 50% of the particulate phosphorus had returned to solution but unfortunately the contents of the bag then became heavily contaminated
with phosphorus and no further useful information
could bc obtained. The rapid initial liberation of much of the phosphorus into
solution confirms the observations of workers such as Cooper ( 1935), Hoffmann
(I956), and Golterman ( 1960).
The remineralization
of silicate from an
arbitrary zero at day 26 is also shown by
Figure 10 and was linear with time until
PRIMARY
FIG. 10.
Nutrient
regeneration
PRODUCTION
period,
IN A PLASTIC
liberation
stirring was stopped. Jgrgensen ( 1955) has
indicated that diatoms may have different
regeneration
rates according to species.
The mean rate of about 0.75 pg-at. Si/L/
day measured here was an average value
which is probably fairly rcprcscntative of
the behavior of a mixed diatom crop. Ry
day 55 some 40% of the plant silicon had
been remineralized and 53% had returned
into solution by day 100. A constant regeneration rate of silicon implies that dissolution was taking place at reaction sites on a
nearly constant surface. A very approximate calculation can be made of the surface area of the diatoms in the bag at maximum crop, This area turns out to be at
least as great and probably more than 10
times greater than the surface area of the
plastic walls of the container. (For even
large crops of moderate size diatoms a 20ft diameter vessel is necessary if the vessel
surface area is to be less than the area of
the particulate matter. ) Regeneration rates
at surfaces will depend upon the area of
the surface and the volume of water associated with the crop so that the rate quoted
above for silicate (at a mean temperature
of about 13°C) will have no absolute significancc. In situ marine values in coastal
181
SPIIERE
of phosphate,
silicate,
and carbon
dioxide.
waters may be expected to be no greater
than about % of this value.
The formation of carbon dioxide-carbon,
from an arbitrary zero at day 30, was also
linear with time until day 75 or later (see
Fig. 10) and this is most reasonably explained kinetically if we assume a constant
surface area of detritus covered with rcspiring bacteria (cf. Wood 1953; Jannasch
1954). This constancy of area precluded
the possibility that the substrate for rcspiration was the detrital matter itself, a supposition which is further borne out by the
fact that nearly 5,000 mgC/m3 were liberated as carbon dioxide by day 100 whereas
the total particulate
carbon in the bag
never cxcccdcd 2,000 mgC/m”.
Similarly
before the bloom commenced some 600
mgC/m” of respired carbon dioxide entered
the water with no significant decrease in
particulate carbon (Fig. 4). These observations imply that “dissolved” organic material is mainly responsible for oxygen consumption and carbon dioxide liberation.
Only a fraction of the total dissolved organics may be immediately
available to
bacteria although the presence of detrital
surfaces enables a greater fraction to be
attacked than would otherwise be the case
182
N. J. ANTIA,
C. D. MCALLISTER,
T. R. PARSONS,
(ref. Keys, Christensen, and Krogh 1935;
Kriss and Markianovich
1959). From the
present work it is unlikely that the consumption of oxygen in sea water is ever
caused to any great extent by the biological decomposition
of organic particulate
matter per se although the presence of particulatc matter may indicate the recent excretion of freshly formed reactive organic
matter. Plant and other detritus, however,
will act as a carrier for bacteria, the total
activity of which will be proportional
to
the surface area of particulate
material
present per unit volume of sea water.
The rate of evolution of carbon dioxide
was very near to that deduced from the data
of Waksman and Renn (1936) for coastal
waters and did not decrease appreciably
until the oxygen concentration dropped to
below 1 ml 02/L (NTP).
The kinetics of oxygen consumption were
anomalous. In Figure 10 the consumption
of oxygen has been expressed as an equivalent evolution of carbon dioxide-carbon,
from day 30 as arbitrary zero, assuming an
RQ of unity. It will be seen that more oxygen was consumed than carbon dioxide
evolved until about day 53 after which
more carbon dioxide was evolved than oxygen consumed. The latter state of affairs
appears to be characteristic of “old” water
as it will be seen to occur at the start of the
experiment (Fig. 4) when the bag was
initially filled with high nutrient, low pI1
water from the bottom of Departure Bay.
The initial RQ [ ( ACO,) / ( AO,) ] was as
low as 0.45 when the bloom had just finished, became unity at around days 50-56,
and then increased to nearly 2 in the “old”
water present at both the end and bcginning of the whole experiment. As the carbon dioxide evolution was so nearly linear,
it would appear that these changes of RQ
were associated with variations in the oxidation state of the substrate of dissolved
organic matter rather than with changes of
microflora.
The “new” water (immediate
post-bloom) in which the microorganisms
had such a low RQ contained most of the
900 mgC/m” of organic matter excreted by
the nlants
during
the bloom
and it mav not
K. STEPHENS,
AND
J, D. I-1. STRICKLAND
be a coincidence that this amount is the
same amount by which the two carbon
curves deviate in Figure 10.
Apparent
heterotrophic
uptake rates
were measured in the “new” water of day
33, using CL4 labelled organic compounds
(Parsons and Strickland 1962). The results
were very high being 0.66 mgC/m3/hr for
glucose and 0.61,0.35, and 0.14 mgC/m”/hr
for succinate, acetate, and citrate, respectively.
REFERENCES
ALLEN, M. B. 1956. Excretion
of organic compounds by Chlamydomonas.
Arch. Mikrobiol.,
24: 163-168.
BFXSEH, W. L. 1959. Bioassay of organic micronutrients in the sea. Proc. Nat. Acad. Sci.,
45: 1533-1542.
BUHKHOLDER, P. R. 1959. Vitamin-producing
bacFirst Intcrnat.
teria in the sea. Preprints
Oceanogr. Congress, pp. 912-913.
CALKINS, V. P. 1943. Microclctermination
of
glycollic
and oxalic acids.
Ind. Eng. Chem.
[Anal. Ed.], 15: 762-763.
CAREY, C. L. 1938. The occurrence and distribution of nitrifying
bacteria in the deep sea. J.
Mar. Res., 1: 291-304.
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COOPER, L. H. N. 1935. Liberation
in sea water by the breakdown
of plankton
organisms.
J. Mar. Biol. Ass. U. K., 20:
197-200.
in
DHOOP, M. R. 1954. Cobalamin requirement
Chrysophyccae.
Nature, 174: 520.
GOLDMAN, C. R. 1962. A method of studying
nutrient limiting factors in situ in water columns isolated by polyethylene
film.
Limnol.
Oceanogr., 7 : 99-10,l.
1960. Studies on the cycle
GOLTERMAN, H. L.
of elements in fresh water.
Acta Bot. Nccrl.,
9: l-58.
iibcr die
HOI~FMANN, C. 1956. Untersuchungen
Remineralization
dcs phosphors im plankton.
Kicl. Mecresforsch.,
12: 25-36.
HUTCHINSON, G. E., AND J, K. SETLOW. 1946.
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studies in Connecticut.
VIII.
The niacin
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lake.
Ecology, 27: 13-22.
JANNASCII, H. W. 1954. dkologishc Untersuchungcn der planktischen
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von Neapel.
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