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Studies on the phytoplankton ecology of the Trondheimsfjord. I. The chemical composition of phytoplankton populations
J. eq.
mar. Biol. Ecol., 1973, Vol.
STUDlES
It, pp. 15-26; 0 North-Holland
ON THE PHYTOPLANKTON
ECOLOGY
TRONDHEIMSFJORD. THE CHEMICAL
COMPOSITION
Publishing Company
OF THE
I.
OF PHYTOPLANKTON
POPULATIONS
ARNE HAUG and SVERRE MYKLESTAD Norwegian
Institute
of Seaweed
Reseurch,
Trondheim,
Norway
and EGIL SAKSHAUG The University
of Trondheim
Biological
Station,
Trondheim,
Norway
Abstract: Samples of phytoplankton populations from the Trondheimsfjord, collected in 1970 and the first five months of 1971, have been analysed for carbohydrate, protein, lipid, and phosphorus. Lipid was in all cases less than 10% of the organic dry matter. The N/P ratio was remarkably constant, but the ratio protein/carbohydrate varied between wide limits. For samples consisting mainly of dinoflagellates, the protein/carbohydrate ratio was always low, due to a large amount of insoluble polysaccharides, probably corresponding to material in the cell walls. For diatoms, the carbohydrates may conveniently be divided into three fractions: 1) an acidsoluble glucan of the B-1, 3-linked type; 2) an alkali-soluble fraction giving a complex mixture of monosaccharides on hydrolysis and, 3) an insoluble glucan. The amounts of acid-soluble glucan varied from 7.7 to 36.5% of organic dry matter and these changes are the main cause of the variation of the protein/carbohydrate ratio of diatom samples. For diatom samples this ratio is a valuable indicator of the physiological state of the population. The variations observed in this study are discussed.
The Trondheimsfjord area has been quite extensively studied with respect toits phytoplankton ecology (Sakshaug, 1970, 1972). In the present work, variations of some of the main chemical components of the phytoplankton populations in this area have been studied. Phytoplankton samples were collected in 1970 and the first five months of 1971, at the time of the year when reasonably dense phytoplankton populations occurred. The chemical composition of phytoplankton has mainly been studied in culture. Parsons, Stephens & Strickland (1961) analysed samples of eleven different phytoplankton species harvested in the exponential growth phase. Among the four diatoms, the carbohydrate varied from 9.5 to 34 % of the organic dry matter, the protein from 36 to 60 %, while the amount of lipid in all cases was below 10 %. Several studies have indicated that from the exponential to the stationary growth phase the chemical composition of diatoms changes. Antia et al. (1963) found a marked increase in the carbohydrate content of phytoplankton grown in a large plastic sphere when the amount of available nitrogen was depleted, while the amount of protein as a percentage of algal weight decreased. Werner (1970), working with Cyclotdla crypticu and Hobson & Pariser (1971) working with C. nana and Thalussiosirujiuviatilis also found that nitrogen deficiency changed the composition of the cells.
ARNE HAUG, SVERRE MYKLESTAD AND EGIL SAKSHAUG
16
with Chaetoceros a&is
Work in our laboratory
var. willei (Myklestad
& Haug,
1972) showed that from the exponential growth phase to the stationary phase the composition changed and that the main cause of this change was an accumulation of glucan, probably ratio accordingly
a storage product decreased
in this ratio from 0.23-2.0
of the fl-1,3-linked
very markedly
during
type. The protein-carbohydrate the growth
period,
and variations
were observed.
Using these observations as a basis, we have studied the changes in chemical composition of natural phytoplankton populations, particularly the changes in the amounts of the different carbohydrate fractions, in the hope that these changes may provide valuable
information
about
the ecology
MATERIALS
of the phytoplankton
AND
in the fjord.
METHODS
Plankton samples were collected by horizontal towing of a net with mesh width 25 pm in the O-2 m layer outside of Trondheim harbour. The samples were stored in the dark and cold and transferred to the laboratory as fast as possible, usually within less than 1 h after collection. A small sample was removed for species and numbers determination. The main part of the sample was concentrated by filtration on porous filter paper (Schleicher & Schiill, 520 B, filtration time approximately 5 min), transferred to a round bottom flask, frozen, and freeze-dried. The freeze-dried samples were stored at about 4 “C, until used for analysis. Dry matter and ash content were determined by conventional 105 “C for 8 h and ashing at 480 “C overnight. Total carbohydrate,
methods, nitrogen
drying at (protein),
phosphate and lipid were determined as described previously (Myklestad & Haug, 1972). The carbohydrates were fractionated in the following way: the freeze-dried material (20 mg) was extracted twice with 0.1 N sulphuric acid (10 ml) and the amount of carbohydrate in the combined extracts determined (Fraction I). The residue was extracted twice with 0. I N sodium hydroxide and the amount of carbohydrate determined
(Fraction
glucose hydrate
II). The phenolsulphuric
acid method
(Dubois
et al., 1956) with
as a standard was applied in both cases. The amount of insoluble carbowas estimated as the difference between total and soluble carbohydrate
(Fraction
III).
Examination of the monosaccharide composition of the fractions by reduction, acetylation, and gas-liquid chromatography of the alditol acetates has been described previously (Myklestad, Haug & Larsen, 1972). RESULTS FRACTIONAL
EXTRACTION
OF CARBOHYDRATES
tn an investigation of C. afinis grown in culture in this laboratory, it was found that extraction of the freeze-dried material by 0.1 N sulphuric acid removed a poly-
STUDIES
saccharide
consisting
that the remaining
ON
mainly material
PHYTOPLANKTON
ECOLOGY
of glucose units (Myklestad, on hydrolysis
17
Haug &Larsen,
gave rise to a complex
1972) and
mixture
of mono-
saccharides with glucose forming only a minor component. Partial acid hydrolysis of the material removed by the dilute acid gave a mixture of oligosaccharides having mobilities by partial
in paper chromatography corresponding to the oligosaccharides obtained acid hydrolysis of laminaran from brown algae (Myklestad & Haug, 1972). TABLE I
Monosaccharide
composition matography
Date
of
fractions of phytoplankton as alditol acetates as ‘A total
Fraction
Rhamn.
Fuc.
samples determined alditol acetates.
Rib.
Arab.
Xyl.
Man.
by gas-chro-
Gal.
Glut.
1970 6th April
Alkaline
extract
7
10
12
32
19
4
12
3
27th April
Alkaline
extract
5
10
50
2
6
5
11
IO
I lth May
Acid extract Alkaline extract Residue
7
18
19
2
9
8
18
98 17 85
13
IO
13
6
32
i2
10th June
Alkaline
25th June
Acid extract Alkaline extract
6
95 90
6th Oct.
Acid extract Alkaline extract
4.5
95 90
extract
1971 30th Mar. 21st April
Acid extract Alkaline extract Acid extract Alkaline extract Residue
8
6
7
36
I8
15
9
I5
3
33
8
15
95 3 95 IO 85
Abbreviations: Rhamn.: Fuc. Rib. Arab.
Rhamnitol
penta-acetate.
: Fucitol penta-acetate. : Ribitol penta-acetate. : Arabitol penta-acetate.
Xyl. Man.: Gal. Glut.:
: Xylitol penta-acetate. Mannitol
: Galactitol Glucitol
hexa-acetate. hexa-acetate. hexa-acetate.
This indicates that in C. afinis it is possible to estimate the amount of the p-1,3-bound glucan which is functioning as storage polysaccharide in diatoms (Meeuse, 1962) by extraction of the material by dilute acid. This method of carbohydrate fractionation has been used in the present work on naturally occurring phytoplankton populations. The dilute acid extracts of several of the freeze-dried samples were hydrolysed and investigated by gas-liquid chromatography. The remaining material was further extracted by 0.1 N sodium hydroxide solution and the monosaccharide composition of this fraction after hydrolysis was investigated in the same way. In addition, in two cases the final residue was hydro-
18
ARNE
HAUG,
SVERRE
MYKLESTAD TABLE
Species composition
II
of net hauls, as percentage of cell numbers: diatoms given in Fig. 1.
__~ Total
roman numerals refer to group of
Dinoflagellates
Diatoms Date
AND EGIL SAKSHAUG
Species
%
Total
Species
Others %
Total
Species _
1970 31st Mar.
6th April
11th May 14th May 27th May
90
80
95 39 92
C. socialis T. gravida C. debilis T. nitrsch. Skeletonema
(I) (I) 01) (11) (II)
24
C. socialis T. graoida C. debilis T. nitzsch. Skeletonema C. laciniosus
(1) 0) 01) (II)
3 7 35 10 8 7
C. curoisetus C. debilis
(II)
C. curcisetus C. debilis
(II)
(II) (111) (II) (II)
C. curuisetus C. debilis Skeletonema
(11)
(II) (II)
10th June
100
C. curoisetus
(11)
25th June
19
C. curcisetus
(II)
30 IO 7
49 44
47
3rd Sept.
3
C. brecis R. imbricata
20 Phaeocystis
5
25 14
61
53 26 13
8
19
23rd July
14th Aug.
10 Phaeocystis
11
12 24
G. tamarensis P. trochoideum
27 17
81
C. longipes P. trochoideum
50 10
100
C. longipes P. trochoideum Dinophysis spp.
55 22
P. trochoideum
47
53 97
I2
c. fusus
16
P. trochoideum
67
14th Sept.
6
76
c. fusus P. trochoideum
37 16
18 D. speculum
6th Oct.
10
64
c. fusus C. tripos
10
26 D. speculum
38
1971 25th Mar.
26
C. socialis (I) Skeletonema (II) Thalassiosira spp. (I)
18 3 2
74 Phaeocystis
30th Mar.
37
C. socialis (I) Skeletonema (11) Thalassiosira spp. (I)
31 3
63 Phaeocystis
1
STUDIES
ON
PHYTOPLANKTON
19
ECOLOGY
TABLE II (cont.)
Date
Total
Others
Dinoflagellates
Diatoms Percentage
Species
Species
Total
Per- Total centage
Species
1971 2nd April
17
C. socialis (I) Skeletonema (II) Thalussiosira spp. (1)
83 Phaeocystis
9 5
I
6th April
57
C. debilis C. similis Skeletonema Thalassiosira spp.
(11) (I) (II) (1)
I3 10 24 IO
43 Phaeocystis
14th April
60
C. debilis C. similis Skeletonema Thalassiosira spp.
(II) (I) (11) (I)
9 6 44 I
40 Phaeocystis
C. debilis C. similis Skeletonema
(11) (I) (11)
8 86
2lst
April
100
29th April
98
C. debilis Skeletonema
(II) (11)
4 88
6th May
100
Skeletonema
(II)
99
2
G. tamarensis
2
Abbreviations: C. C. C. C. C. C. R.
brevis curvisetus debilis laciniosus similis socialis imbricata
Skeletonema T. nitzsch.
Chaetoceros brevis Schiitt Chaetoceros curvisetus Cleve Chaetoceros debilis Cleve Chaetoceros laciniosus Schiitt Chaetoceros similis Cleve Chaetoceros socialis Lauder Rhizosolenia imbricata var. shrubsolei (Cleve) Schrijder Skeletonema costatum (Grev.) Cleve Thalassionema nitzschioides Grunow
T. gravida c. fusus C. longipes C. tripos G. tamarensis P. trochoideum D. speculum Phaeocystis
Thalassiosira gravida Cleve Ceratium fusus (Ehrenb.) Dujardin Ceratium longipes (Bailey) Gran Ceratium tripes (0. F. Miiller) Nitzsch Gonyaulax tamarensis Lebour Peridinium trochoideum (Stein) Lemm. Distephanus speculum (Ehrenb.) Haeckel Phaeocystis pouchetii (Hariot) Lagerheim
lysed in 80 % sulphuric acid (20 “C for 20 h), followed by hydrolysis in 1 N sulphuric acid (100 “C for 5 h) and examined. The results are given in Table I. The amounts of the different alditol acetates are estimated from the area of the peaks and should be regarded as semi-quantitative. From Table I1 showing the species composition, it follows that the samples collected on the 25th June and the 6th October 1970 mainly consist of dinoflagellates, while the rest of the samples predominantly contain diatoms. The fractionation into acid and alkaline soluble polysaccharides seems very convenient for diatom samples. The pattern previously observed for C. aj%s was repro-
ARNE
20
HAUG,
SVERRE
MYKLESTAD
AND
EGIL
SAKSHAUC
duced. After the extractions the residue contained predominantly glucose. The results, therefore, confirm that our extraction procedure separates the carbohydrates of diatoms into an acid-soluble gluean, an alkali-soluble polysaccharide fraction of complex monomer composition, and an insoluble glucan. For samples containing TABLE 111
Chemical
composition
of phytoplankton as percentage of fractions I, acid-soluble; IL, alkali-soluble;
N/P
P
Protein
Carbohydrate
atom --
Lipid
carbohydrate
Carbohydrate
Main components
Elements Date
organic dry matter: 111, insoluble.
Sum
Pr0t.j carb.
fractions
1
11
III
17.0 30.5 36.5 29.0 21.0 19.9 25.0 16.0 13.0 24.5 25.8 11.6 8.5 7.7 10.7 7.9 12.1
7.5 10.0 13.2 15.0 11.0 7.5 8.5 10.0 8.5 11.8 10.0 10.x 5.3 6.8 6.9 4.0 7.1
5.5 10.0 5.8 6.5 3.0 5.1 8.5 15.5 8.7 3.7 35.6 52.2 35.5 44.9 44.6 41.8
11.6 14.4 19.2 30.0 14.8 22.6 14.2 14.3
16.3 24.0 24.4 22.5 21.0 32.1 17.0 9.5
7.6 3.1 2.9 6.5 6.7 4.3 10.8 3.7
1970 2313 31/3 614 1314 20/4 27j4 415 111s 1415 271.5 1016 2516 23/l 14,‘8 3/9 I4/9 6110
0.49 0.78 0.67 0.96 0.74 0.83 0.76
1.0 7.2 4.8 7.2 7.4 9.5 6.5 6.4 6.8 7.8 3.9 5.6 5.3 6.6 5.2 4.2 5.3
16.7 16.3 15.4 17.2 15.4 16.7 16.5 15.8 16.1 16.7 17.6 15.8 17.4 15.2 15.4 16.4 15.4
44.0 45.0 30.0 45.0 46.0 59.5 40.5 40.0 42.5 49.0 24.5 35.0 33.0 40.5 32.5 39.0 33.0
30.0 48.0 55.5 50.5 45.0 32.5 42.0 37.0 37.0 45.0 39.5 58.0 66.0 50.0 62.5 56.5 61.0
5.0 8.0 6.0 6.0 7.5 7.5 4.5 4.0 4.5 8.0 3.5
79 101 91.5 101.5 98.5 99.5 87.0 81.0 84.0 102.0 67.5
3.0
102.0
4.5
99.5
2.0
96.0
1.13 0.93 0.51 0.52 0.39 0.67 0.88 0.98
7.9 6.8 4.0 4.6 3.2 4.8 5.5 6.6
15.4 16.0 17.2 19.3 18.1 15.8 13.9 14.7
49.5 42.5 25.0 29.0 20.0 30.0 34.5 41.0
35.5 41.5 46.5 59.0 41.5 59.0 42.0 28.0
5.0 6.5 6.0 6.5
90.0 90.5 77.5 94.5
5.5
94.5
4.0
73.0
0.92 0.97 0.69 0.92 1.06 1.25 0.85 0.89 0.93
1.03
1.46 0.94 0.54 0.89
1.02 1.83 0.96
1.08 1.15 1.09 0.62 0.60 0.50 0.81 0.52 0.69 0.54
1I.0
1971 2513 3013 2j4 6/4 1414 2114 2914 615
1.39
I .02 0.54 0.49 0.48 0.51 0.82 1.46
predominantly dinoflagellates, on the other hand, the extraction procedure does not give very useful information since the composition of the acid- and alkali-soluble material was similar, with glucose as the main component. SPECIES
CO~~POSITION
The species composition of the samples is given in Table El. All samples collected after the 1lth May 1970 have been counted; of those collected before that date only
STUDlES
ON
PHYTOPLANKTON
ECOLOGY
21
two were examined for their species composition. The species composition is given as cell numbers, and only the quantitatively more important species are recorded in the Table. When comparing the distribution of the species with the chemical composition (Table III), the greatly differing cell size must be taken into account. As an example, it may be mentioned that Pftaeocystis (Haptophy~eae) forms a very much lower percentage of the total cell volume than of the cell number, while the opposite is the case with most dinoflagellates. As discussed in detail elsewhere (Sakshaug & Myklestad, 1973), the phytoplankton population in the spring season (March-June) reaches two peaks, the first spring bloom reaching its maximum in the first week of April and the second taking place in May. The succession of species may be described by dividing the diatom species occurring in this period into three groups: 1) species reaching maximum during the first spring bloom and being insignificant later in the season (e.g. Chaetoceros soc~alj~; for taxonomic authorities, see Table II); 2) species dominating during the second spring bloom, but also occurring in significant amounts during the first (e.g. C. debilis, C. curvisetus, and Skeletonemu); and 3) species being most important in the period between the two blooms (e.g. Chaetoceros Zaciniosus).The group to which the different species belong is indicated in Table II. Fig. 1 gives the number of diatom cells/l of the three groups estimated by quantitative sampling in 1970 fromstation 15 in the Trondheimsfjord (for details, see Sakshaug & Myklestad, 1973) and in 1971 from the Trondheim harbour area. The plankton blooms show the samegeneral development in the two areas (Sakshaug & Myklestad, 1973); the results from Station 15 may also be taken as representative of the Trondheim harbour. The significance of these observations will be discussed later.
CHEMICAL
CO~JPOSITIO~
The composition in terms of organic dry matter of the plankton samples are given in Table ICI. Very marked variations in the amounts of the main components were observed. In all cases, however, protein and carbohydrate form the main part of the organic matter, lipid being a retatively minor component (< 10 %). The sum of protein, carbohydrate, and lipid accounts in most cases for more than 90 % of the organic dry matter. Somewhat arbitrary standards have been used for the determination of the three main components and this, together with the fact that compounds such as nucleic acids have not been determined, may cause some deviation from a sum of 100 7:. For some of the samples, however, (e.g. 10th June, 1970) the results indicate that the organic matter contains some components not accounted for in our procedure. The same observation was made in a study of our laboratory of C. c-Q&is (Mykiestad & Haug, 1972) and has also been reported for other diatoms grown in culture (Parsons, Stephens & Strickland, 1961). Marked variations in the distribution of carbohydrate between the three different fractions are evident from the results in Table 111.These variations are partly caused
22
ARNE
by variations
HAUG,
SVERRE
MYKLESTAD
AND EGIL SAKSHAUG
in species, and partly reflect differences
will be discussed
in physiological
state. This point
in more detail below.
DISCUSSION It is well known plankton position
that
net hauls
do not give a representative
sample
of phyto-
populations. It should, therefore, be kept in mind that the chemical comgiven in Table III may deviate somewhat from that of the total phytoplank-
ton population in the area, due to selective sampling. We have chosen this method of collection, however, in order to obtain sufficiently large samples for analysis. Nevertheless, the same species dominated both the net hauls and the quantitative samples (Sakshaug & Myklestad, 1973). All the results were calculated as percentages of organic dry matter. The ash content of the samples was not considered to have any biological significance, being dependent upon the removal of sea water before freeze-drying. The ratio between nitrogen and phosphorus was found to be remarkably constant and independent of whether diatoms or dinoflagellates were the predominant organisms in the samples. In the 1970 samples the range of the nitrogen-phosphorus atomic ratio was 15.2-17.6, while a somewhat wider range, 13.9-19.3 was found in 1971. Harris & Riley (1956) in a study of phytoplankton populations from Long Island Sound found a range of 13.0 to 20.0. Ryther & Dunstan (1971) discussed the nitrogen-phosphoros ratios of both natural phytoplankton populations and cultures, and reported results from the literature ranging from 3 to 30, and which indicated a correlation between the ratio in the medium and that in the organisms. For C. afinis we found in this laboratory (Myklestad & Haug, 1972) ratios between 10 and 25 for algae grown in a medium with a 24.4 N/P ratio. With a nitrogen-phosphorus ratio in the medium of 122, the ratio in the organisms was found to be 59.5. These observations make it unlikely growth of the natural phytoplankton would
most probably
that phosphorus was the limiting factor for populations analysed in this study, since this
have led to higher
values
of the nitrogen-phosphorus
ratio.
In contrast to the remarkably constant values of the nitrogen-phosphorus ratio, the protein-carbohydrate ratio varied markedly from 0.48 to 1.83. A closer inspection of the fractionation pattern of the carbohydrate content reveals a marked difference between those samples rich in dinoflagellates and those containing predominantly diatoms. In the former, the amount of insoluble carbohydrate, as defined by our extraction procedure, was always high, accounting for between 35-52 % of the organic matter, while the corresponding figure was usually below 10 y0 for samples without significant amounts of dinoflagellates. It is well known that dinoflagellates have a cell wall consisting of an insoluble glucan, usually described as cellulose (Fritsch, 1961). Recent work (Nevo & Sharon, 1969) has cast some doubt upon the assumption that this polysaccharide is identical to cellulose from higher plants; its insolubility and lack of reactivity is, however, in agreement with the fractionation pattern observed
STUDIES
in this work. The gas-chromatographic soluble
carbohydrates
ON PHYTOPLANKTON
investigation
(Table
ECOLOGY
of the monosaccharide
I) also reveals a significant
23
composition
difference
between
of the dino-
flagellates and diatoms. Diatoms were characterized by an alkali-soluble carbohydrate fraction of complex monomer composition, but this fraction was absent in dinoflagellate-rich samples, glucose being the predominating component of the small, alkali-soluble fraction then obtained. When considering the protein-carbohydrate ratio as a parameter of possible ecological significance as suggested by Myklestad & Haug (1972) it is, therefore, necessary to distinguish between samples dominated by dinoflagellates and by diatoms. Samples of dinoflagellates have a low protein-carbohydrate ratio due to the high content of insoluble cell wall material; diatom samples, on the other hand, have a highly variable protein-carbohydrate ratio, the variation being mainly caused by variations in the acid-soluble glucan fraction, as previously observed for cultures of C. a$bzis (Myklestad & Haug, 1972). The culture experiments indicated that the protein-carbohydrate ratio could give valuable information about the physiological state of the diatom population. A high protein-carbohydrate ratio (low glucan content) indicates a rapidly growing population while depletion of available nutrients, leading to a stationary phase in the growth of the population, is indicated by a rise in the glucan content and a corresponding decrease in the protein-carbohydrate ratio. If we compare the changes in the phytoplankton populations as illustrated in Fig. 1. and the variations in the protein-carbohydrate ratio given in Table III, we find that the development of the first spring bloom in both years was characterized by a rapid decrease in the protein-carbohydrate ratio. The chemical changes resembled those observed for a batch culture (Myklestad & Haug, 1972). The concentration of nitrate in the surface layers rapidly decreased in the same period, as shown in Fig. I (which also gives some data on the nitrate concentrations). As will be discussed in more detail elsewhere (Sakshaug & Myklestad, 1973) these observations support sumption that the main source of nutrients in the first bloom is the amount in the water masses when the bloom starts. The events following
the first spring bloom
were somewhat
different
the aspresent
for the two
years. In 1970, the protein-carbohydrate ratio began to increase again by the 13th April, reaching a maximum on the 27th April. Fig. 1 shows an increasing population of diatoms of Group II on this date. In the following period when the proteincarbohydrate ratio varied z 1.0, the population consisted mainly of diatoms of Group II. This remarkably constant chemical composition was probably caused by a continuous supply of nutrients by river water and the entrainment of nutrient-rich sea water from deeper layers, keeping the population in a state of relatively rapid cell division. The sample collected on the 10th June had a low protein-carbohydrate ratio, indicating a change in physiological state in the direction of a stationary growth phase, probably due to a diminished supply of river water. In 1971, the protein-carbohydrate ratio remained low for a longer period, any
ARNE
24
HAUG,
SVERRE
MYKLESTAD
AND EGIL SAKSHAUG
notable increase first being observed on the 29th April. This increase continued but, due to heavy grazing by an increasing zoopiankton population, the sampling was stopped after the 6th May. The last three samples collected in 1971 consisted mainly
210s521os52104
20 30 March
1NO;
10
20
10
30
April
30
20
10
20
30
June
May
..-)-_
6.6;3.7;06;0;
1.16;
0.b ;
0.L;
1.1 ;
2.0
i___
2 105 5
2
-?b--&- -------ii&r& 20 10
March
April
May
Fig. 1. Number of diatoms (cells/l) in the different successional groups (see text, p. 21 and Table II), determined by quantitative sampling, in 1970 at Station 15 in the Trondheimsfjord (geometric mean in the upper 20 m) and in 1971 in the Trondheim harbour area (maximum numbers for 0 or 2 m).
of Skeletonema; it is interesting to note that. the changes in protein-carbohydrate ratio were contrary to those observed for ordinary batch cultures by Myklestad & Haug (1972) and also contrary to the development during the first spring bloom. In this period analysis of the water showed an increase in the amount of nitrate
STUDIES
ON PHYTOPLANKTON
ECOLOGY
25
present so that both the protein-carbohydrate ratio of the plankton and the water analysis indicate that the population was in a state of rapid ceil division due to a continuous supply of nutrients. The decreasing number of cells in the Iast part of this period was probably due to the heavy grazing (Sakshaug & Myklestad, 1973). A closer inspection of the three samples of the Skeletonema population reveals that the changing protein-carbohydrate ratio was only partly due to a change in the content of acid-soluble glucan. A very marked decrease in the alkali-soluble fraction was also observed. The aIkali-soluble fraction of the sample collected on the 21st April was remarkably high (32 “/;;). In 1970 the highest amount of the alkali-soluble fraction was 15 y<, and this sample contained a noticeable amount of Phmocystis in addition to diatoms. This was also the case for the samples from the 25th March to the 14th April, 1971, which contained from 16-24 7: alkali-soluble fractions. Pure diatom samples, as observed in 1970 and obtained in culture experiments (Myklestad & Haug, 1972, and unpubl. results), usually contain less than 10 “/ of this fraction. At present, we can offer no expIanation for the remarkably high content of alkalisoluble polysaccharides in the sample collected on the 21st April, 1971, nor for the decrease observed in the subsequent samples. Further information on the polysaccharides of diatoms is needed, and work is in progress in our laboratory, Apart from its informative value concerning the physiological state of a diatom population, the protein-carbohydrate ratio is also of interest in connection with the nutritive value of plankton and the determination of productivity. A larger percentage of the assimilated carbon will be incorporated into protein in a population characterized by rapid cell division (e.g., as in the beginning of the first spring bloom) than in a population growing in a nutrient depleted medium where glucan is themain product, as in the latter part of the first spring bloom. The same amount of assimilated carbon may, therefore, lead to very different results from a nutritive point of view, depending upon the physiological state of the phytoplankton population.
REFERENCES ANTIA, N. J., C. D. MCALLISTER,T. R. PARSONS,K. STEPHENS & J. D. H. STRICKLAND,1963. Further measurements of primary production using a large-volume plastic sphere. Limnol. Oceanogr., Vol. 8, pp, 166-183. DUBOIS, M., K. A. GILL~S, J. K. HAMILTON, P. A. REBERS& F. SMITH, 1956. Co~orimetric deter-
mination
of sugars and related substances.
FRITSCH, F. E., 1935. The structure
Cambridge,
Ana&.
and reproduction
Chenr., Vol. 18, pp. 350-356. 1. Cambridge University Press,
of algae, Vol.
791 pp.
HARRIS, E. & G. A. RILEY, 1956. Oceanography of the Long lsland Sound, 1952-1954. VIII. Chemical composition of the plankton. Bull. Bingham Oceanogr. COO., Vol. 15, pp. 315-323. HOBSON,L. A. & R. J. PARISER,1971. The effect of inorganic nitrogen on macromolecular synthesis by Thafassiosirapuciintilis Hustedt and Cycfotefla nana Hustedt grown in batch culture. J. exp. mar. Biof. EC&., Vol. 6, pp. 71-78. MEEUSE,B. J. D., 1962. In, Physiolo.qy and biochemistry of aigae, edited by R. A. Lewin, Academic
Press Inc., New York, pp. 289-311.
26
ARNE
HAUG,
SVERRE
MYKLESTAD
AND
EGIL
SAKSHAUG
MYKLESTAD, S. & A. HAUG, 1972. Production of carbohydrates by the marine diatom Chaetoceros afinis var. wiDei (Gran) Hustedt. 1. Effect of the concentration of nutrients in the culture medium. J. exp. mar. Biol. Ecol., Vol. 9, pp. 125-136. MYKLESTAD, S., A. HAUC & B. LARSEN, 1972. Production of carbohydrates by the marine diatom Chaetoceros afinis var. willei (Gran) Hustedt. II. Preliminary investigation of the extracellular polysaccharide. J. exp. mar. Biol. Ecol., Vol. 9, pp. 137-144. NFVO, Z. & N. SHARON, 1969. The cell wall of Peridinium westii, a non-cellulosic glucan. Biochem. Biophys. Actu, Vol. 173, pp. 161-175. PARSONS, T. R., K. STEPHENS & J. D. H. STRICKLAND, 1961. On the chemical composition of eleven species of marine phytoplankters. J. Fish. Res. Bd Can., Vol. 18, pp. 1001-1016. RYTHER, J. H. & W. M. DUNSTAN, 1971. Nitrogen, phosphorus and eutrophication in the coastal environment. Science, N.Y., Vol. 171, pp. 1008-1013. SAKSHAUG, E., 1970. Quantitative phytoplankton investigations in near-shore water masses. K. norske Vidensk. Selsk. Skr. 1970, Vol. 3, pp. 1-8. SAKSHAUG, E., 1972. Phytoplankton investigations in Trondheimsfjord, 1963-1966. K. norske Vidensk. Selsk. Skr. 1972, Vol. 1, pp. l-56. SAKSHAUG, E. & S. MYKLESTAD, 1973. Studies on the phytoplankton ecology of theTrondheimsfjord. III. Dynamics of phytoplankton blooms in relation to environmental factors, bioassay experiments and parameters for the physiological state of populations. J. exp. mar. Biol. Ecol., Vol. I I, pp. 157-188. WERNER, D., 1970. Productivity studies on diatom cultures. Helgoliinder wiss. Meeresunters., Vol. 20, pp. 97-103.
mar. Biol. Ecol., 1973, Vol.
STUDlES
It, pp. 15-26; 0 North-Holland
ON THE PHYTOPLANKTON
ECOLOGY
TRONDHEIMSFJORD. THE CHEMICAL
COMPOSITION
Publishing Company
OF THE
I.
OF PHYTOPLANKTON
POPULATIONS
ARNE HAUG and SVERRE MYKLESTAD Norwegian
Institute
of Seaweed
Reseurch,
Trondheim,
Norway
and EGIL SAKSHAUG The University
of Trondheim
Biological
Station,
Trondheim,
Norway
Abstract: Samples of phytoplankton populations from the Trondheimsfjord, collected in 1970 and the first five months of 1971, have been analysed for carbohydrate, protein, lipid, and phosphorus. Lipid was in all cases less than 10% of the organic dry matter. The N/P ratio was remarkably constant, but the ratio protein/carbohydrate varied between wide limits. For samples consisting mainly of dinoflagellates, the protein/carbohydrate ratio was always low, due to a large amount of insoluble polysaccharides, probably corresponding to material in the cell walls. For diatoms, the carbohydrates may conveniently be divided into three fractions: 1) an acidsoluble glucan of the B-1, 3-linked type; 2) an alkali-soluble fraction giving a complex mixture of monosaccharides on hydrolysis and, 3) an insoluble glucan. The amounts of acid-soluble glucan varied from 7.7 to 36.5% of organic dry matter and these changes are the main cause of the variation of the protein/carbohydrate ratio of diatom samples. For diatom samples this ratio is a valuable indicator of the physiological state of the population. The variations observed in this study are discussed.
The Trondheimsfjord area has been quite extensively studied with respect toits phytoplankton ecology (Sakshaug, 1970, 1972). In the present work, variations of some of the main chemical components of the phytoplankton populations in this area have been studied. Phytoplankton samples were collected in 1970 and the first five months of 1971, at the time of the year when reasonably dense phytoplankton populations occurred. The chemical composition of phytoplankton has mainly been studied in culture. Parsons, Stephens & Strickland (1961) analysed samples of eleven different phytoplankton species harvested in the exponential growth phase. Among the four diatoms, the carbohydrate varied from 9.5 to 34 % of the organic dry matter, the protein from 36 to 60 %, while the amount of lipid in all cases was below 10 %. Several studies have indicated that from the exponential to the stationary growth phase the chemical composition of diatoms changes. Antia et al. (1963) found a marked increase in the carbohydrate content of phytoplankton grown in a large plastic sphere when the amount of available nitrogen was depleted, while the amount of protein as a percentage of algal weight decreased. Werner (1970), working with Cyclotdla crypticu and Hobson & Pariser (1971) working with C. nana and Thalussiosirujiuviatilis also found that nitrogen deficiency changed the composition of the cells.
ARNE HAUG, SVERRE MYKLESTAD AND EGIL SAKSHAUG
16
with Chaetoceros a&is
Work in our laboratory
var. willei (Myklestad
& Haug,
1972) showed that from the exponential growth phase to the stationary phase the composition changed and that the main cause of this change was an accumulation of glucan, probably ratio accordingly
a storage product decreased
in this ratio from 0.23-2.0
of the fl-1,3-linked
very markedly
during
type. The protein-carbohydrate the growth
period,
and variations
were observed.
Using these observations as a basis, we have studied the changes in chemical composition of natural phytoplankton populations, particularly the changes in the amounts of the different carbohydrate fractions, in the hope that these changes may provide valuable
information
about
the ecology
MATERIALS
of the phytoplankton
AND
in the fjord.
METHODS
Plankton samples were collected by horizontal towing of a net with mesh width 25 pm in the O-2 m layer outside of Trondheim harbour. The samples were stored in the dark and cold and transferred to the laboratory as fast as possible, usually within less than 1 h after collection. A small sample was removed for species and numbers determination. The main part of the sample was concentrated by filtration on porous filter paper (Schleicher & Schiill, 520 B, filtration time approximately 5 min), transferred to a round bottom flask, frozen, and freeze-dried. The freeze-dried samples were stored at about 4 “C, until used for analysis. Dry matter and ash content were determined by conventional 105 “C for 8 h and ashing at 480 “C overnight. Total carbohydrate,
methods, nitrogen
drying at (protein),
phosphate and lipid were determined as described previously (Myklestad & Haug, 1972). The carbohydrates were fractionated in the following way: the freeze-dried material (20 mg) was extracted twice with 0.1 N sulphuric acid (10 ml) and the amount of carbohydrate in the combined extracts determined (Fraction I). The residue was extracted twice with 0. I N sodium hydroxide and the amount of carbohydrate determined
(Fraction
glucose hydrate
II). The phenolsulphuric
acid method
(Dubois
et al., 1956) with
as a standard was applied in both cases. The amount of insoluble carbowas estimated as the difference between total and soluble carbohydrate
(Fraction
III).
Examination of the monosaccharide composition of the fractions by reduction, acetylation, and gas-liquid chromatography of the alditol acetates has been described previously (Myklestad, Haug & Larsen, 1972). RESULTS FRACTIONAL
EXTRACTION
OF CARBOHYDRATES
tn an investigation of C. afinis grown in culture in this laboratory, it was found that extraction of the freeze-dried material by 0.1 N sulphuric acid removed a poly-
STUDIES
saccharide
consisting
that the remaining
ON
mainly material
PHYTOPLANKTON
ECOLOGY
of glucose units (Myklestad, on hydrolysis
17
Haug &Larsen,
gave rise to a complex
1972) and
mixture
of mono-
saccharides with glucose forming only a minor component. Partial acid hydrolysis of the material removed by the dilute acid gave a mixture of oligosaccharides having mobilities by partial
in paper chromatography corresponding to the oligosaccharides obtained acid hydrolysis of laminaran from brown algae (Myklestad & Haug, 1972). TABLE I
Monosaccharide
composition matography
Date
of
fractions of phytoplankton as alditol acetates as ‘A total
Fraction
Rhamn.
Fuc.
samples determined alditol acetates.
Rib.
Arab.
Xyl.
Man.
by gas-chro-
Gal.
Glut.
1970 6th April
Alkaline
extract
7
10
12
32
19
4
12
3
27th April
Alkaline
extract
5
10
50
2
6
5
11
IO
I lth May
Acid extract Alkaline extract Residue
7
18
19
2
9
8
18
98 17 85
13
IO
13
6
32
i2
10th June
Alkaline
25th June
Acid extract Alkaline extract
6
95 90
6th Oct.
Acid extract Alkaline extract
4.5
95 90
extract
1971 30th Mar. 21st April
Acid extract Alkaline extract Acid extract Alkaline extract Residue
8
6
7
36
I8
15
9
I5
3
33
8
15
95 3 95 IO 85
Abbreviations: Rhamn.: Fuc. Rib. Arab.
Rhamnitol
penta-acetate.
: Fucitol penta-acetate. : Ribitol penta-acetate. : Arabitol penta-acetate.
Xyl. Man.: Gal. Glut.:
: Xylitol penta-acetate. Mannitol
: Galactitol Glucitol
hexa-acetate. hexa-acetate. hexa-acetate.
This indicates that in C. afinis it is possible to estimate the amount of the p-1,3-bound glucan which is functioning as storage polysaccharide in diatoms (Meeuse, 1962) by extraction of the material by dilute acid. This method of carbohydrate fractionation has been used in the present work on naturally occurring phytoplankton populations. The dilute acid extracts of several of the freeze-dried samples were hydrolysed and investigated by gas-liquid chromatography. The remaining material was further extracted by 0.1 N sodium hydroxide solution and the monosaccharide composition of this fraction after hydrolysis was investigated in the same way. In addition, in two cases the final residue was hydro-
18
ARNE
HAUG,
SVERRE
MYKLESTAD TABLE
Species composition
II
of net hauls, as percentage of cell numbers: diatoms given in Fig. 1.
__~ Total
roman numerals refer to group of
Dinoflagellates
Diatoms Date
AND EGIL SAKSHAUG
Species
%
Total
Species
Others %
Total
Species _
1970 31st Mar.
6th April
11th May 14th May 27th May
90
80
95 39 92
C. socialis T. gravida C. debilis T. nitrsch. Skeletonema
(I) (I) 01) (11) (II)
24
C. socialis T. graoida C. debilis T. nitzsch. Skeletonema C. laciniosus
(1) 0) 01) (II)
3 7 35 10 8 7
C. curoisetus C. debilis
(II)
C. curcisetus C. debilis
(II)
(II) (111) (II) (II)
C. curuisetus C. debilis Skeletonema
(11)
(II) (II)
10th June
100
C. curoisetus
(11)
25th June
19
C. curcisetus
(II)
30 IO 7
49 44
47
3rd Sept.
3
C. brecis R. imbricata
20 Phaeocystis
5
25 14
61
53 26 13
8
19
23rd July
14th Aug.
10 Phaeocystis
11
12 24
G. tamarensis P. trochoideum
27 17
81
C. longipes P. trochoideum
50 10
100
C. longipes P. trochoideum Dinophysis spp.
55 22
P. trochoideum
47
53 97
I2
c. fusus
16
P. trochoideum
67
14th Sept.
6
76
c. fusus P. trochoideum
37 16
18 D. speculum
6th Oct.
10
64
c. fusus C. tripos
10
26 D. speculum
38
1971 25th Mar.
26
C. socialis (I) Skeletonema (II) Thalassiosira spp. (I)
18 3 2
74 Phaeocystis
30th Mar.
37
C. socialis (I) Skeletonema (11) Thalassiosira spp. (I)
31 3
63 Phaeocystis
1
STUDIES
ON
PHYTOPLANKTON
19
ECOLOGY
TABLE II (cont.)
Date
Total
Others
Dinoflagellates
Diatoms Percentage
Species
Species
Total
Per- Total centage
Species
1971 2nd April
17
C. socialis (I) Skeletonema (II) Thalussiosira spp. (1)
83 Phaeocystis
9 5
I
6th April
57
C. debilis C. similis Skeletonema Thalassiosira spp.
(11) (I) (II) (1)
I3 10 24 IO
43 Phaeocystis
14th April
60
C. debilis C. similis Skeletonema Thalassiosira spp.
(II) (I) (11) (I)
9 6 44 I
40 Phaeocystis
C. debilis C. similis Skeletonema
(11) (I) (11)
8 86
2lst
April
100
29th April
98
C. debilis Skeletonema
(II) (11)
4 88
6th May
100
Skeletonema
(II)
99
2
G. tamarensis
2
Abbreviations: C. C. C. C. C. C. R.
brevis curvisetus debilis laciniosus similis socialis imbricata
Skeletonema T. nitzsch.
Chaetoceros brevis Schiitt Chaetoceros curvisetus Cleve Chaetoceros debilis Cleve Chaetoceros laciniosus Schiitt Chaetoceros similis Cleve Chaetoceros socialis Lauder Rhizosolenia imbricata var. shrubsolei (Cleve) Schrijder Skeletonema costatum (Grev.) Cleve Thalassionema nitzschioides Grunow
T. gravida c. fusus C. longipes C. tripos G. tamarensis P. trochoideum D. speculum Phaeocystis
Thalassiosira gravida Cleve Ceratium fusus (Ehrenb.) Dujardin Ceratium longipes (Bailey) Gran Ceratium tripes (0. F. Miiller) Nitzsch Gonyaulax tamarensis Lebour Peridinium trochoideum (Stein) Lemm. Distephanus speculum (Ehrenb.) Haeckel Phaeocystis pouchetii (Hariot) Lagerheim
lysed in 80 % sulphuric acid (20 “C for 20 h), followed by hydrolysis in 1 N sulphuric acid (100 “C for 5 h) and examined. The results are given in Table I. The amounts of the different alditol acetates are estimated from the area of the peaks and should be regarded as semi-quantitative. From Table I1 showing the species composition, it follows that the samples collected on the 25th June and the 6th October 1970 mainly consist of dinoflagellates, while the rest of the samples predominantly contain diatoms. The fractionation into acid and alkaline soluble polysaccharides seems very convenient for diatom samples. The pattern previously observed for C. aj%s was repro-
ARNE
20
HAUG,
SVERRE
MYKLESTAD
AND
EGIL
SAKSHAUC
duced. After the extractions the residue contained predominantly glucose. The results, therefore, confirm that our extraction procedure separates the carbohydrates of diatoms into an acid-soluble gluean, an alkali-soluble polysaccharide fraction of complex monomer composition, and an insoluble glucan. For samples containing TABLE 111
Chemical
composition
of phytoplankton as percentage of fractions I, acid-soluble; IL, alkali-soluble;
N/P
P
Protein
Carbohydrate
atom --
Lipid
carbohydrate
Carbohydrate
Main components
Elements Date
organic dry matter: 111, insoluble.
Sum
Pr0t.j carb.
fractions
1
11
III
17.0 30.5 36.5 29.0 21.0 19.9 25.0 16.0 13.0 24.5 25.8 11.6 8.5 7.7 10.7 7.9 12.1
7.5 10.0 13.2 15.0 11.0 7.5 8.5 10.0 8.5 11.8 10.0 10.x 5.3 6.8 6.9 4.0 7.1
5.5 10.0 5.8 6.5 3.0 5.1 8.5 15.5 8.7 3.7 35.6 52.2 35.5 44.9 44.6 41.8
11.6 14.4 19.2 30.0 14.8 22.6 14.2 14.3
16.3 24.0 24.4 22.5 21.0 32.1 17.0 9.5
7.6 3.1 2.9 6.5 6.7 4.3 10.8 3.7
1970 2313 31/3 614 1314 20/4 27j4 415 111s 1415 271.5 1016 2516 23/l 14,‘8 3/9 I4/9 6110
0.49 0.78 0.67 0.96 0.74 0.83 0.76
1.0 7.2 4.8 7.2 7.4 9.5 6.5 6.4 6.8 7.8 3.9 5.6 5.3 6.6 5.2 4.2 5.3
16.7 16.3 15.4 17.2 15.4 16.7 16.5 15.8 16.1 16.7 17.6 15.8 17.4 15.2 15.4 16.4 15.4
44.0 45.0 30.0 45.0 46.0 59.5 40.5 40.0 42.5 49.0 24.5 35.0 33.0 40.5 32.5 39.0 33.0
30.0 48.0 55.5 50.5 45.0 32.5 42.0 37.0 37.0 45.0 39.5 58.0 66.0 50.0 62.5 56.5 61.0
5.0 8.0 6.0 6.0 7.5 7.5 4.5 4.0 4.5 8.0 3.5
79 101 91.5 101.5 98.5 99.5 87.0 81.0 84.0 102.0 67.5
3.0
102.0
4.5
99.5
2.0
96.0
1.13 0.93 0.51 0.52 0.39 0.67 0.88 0.98
7.9 6.8 4.0 4.6 3.2 4.8 5.5 6.6
15.4 16.0 17.2 19.3 18.1 15.8 13.9 14.7
49.5 42.5 25.0 29.0 20.0 30.0 34.5 41.0
35.5 41.5 46.5 59.0 41.5 59.0 42.0 28.0
5.0 6.5 6.0 6.5
90.0 90.5 77.5 94.5
5.5
94.5
4.0
73.0
0.92 0.97 0.69 0.92 1.06 1.25 0.85 0.89 0.93
1.03
1.46 0.94 0.54 0.89
1.02 1.83 0.96
1.08 1.15 1.09 0.62 0.60 0.50 0.81 0.52 0.69 0.54
1I.0
1971 2513 3013 2j4 6/4 1414 2114 2914 615
1.39
I .02 0.54 0.49 0.48 0.51 0.82 1.46
predominantly dinoflagellates, on the other hand, the extraction procedure does not give very useful information since the composition of the acid- and alkali-soluble material was similar, with glucose as the main component. SPECIES
CO~~POSITION
The species composition of the samples is given in Table El. All samples collected after the 1lth May 1970 have been counted; of those collected before that date only
STUDlES
ON
PHYTOPLANKTON
ECOLOGY
21
two were examined for their species composition. The species composition is given as cell numbers, and only the quantitatively more important species are recorded in the Table. When comparing the distribution of the species with the chemical composition (Table III), the greatly differing cell size must be taken into account. As an example, it may be mentioned that Pftaeocystis (Haptophy~eae) forms a very much lower percentage of the total cell volume than of the cell number, while the opposite is the case with most dinoflagellates. As discussed in detail elsewhere (Sakshaug & Myklestad, 1973), the phytoplankton population in the spring season (March-June) reaches two peaks, the first spring bloom reaching its maximum in the first week of April and the second taking place in May. The succession of species may be described by dividing the diatom species occurring in this period into three groups: 1) species reaching maximum during the first spring bloom and being insignificant later in the season (e.g. Chaetoceros soc~alj~; for taxonomic authorities, see Table II); 2) species dominating during the second spring bloom, but also occurring in significant amounts during the first (e.g. C. debilis, C. curvisetus, and Skeletonemu); and 3) species being most important in the period between the two blooms (e.g. Chaetoceros Zaciniosus).The group to which the different species belong is indicated in Table II. Fig. 1 gives the number of diatom cells/l of the three groups estimated by quantitative sampling in 1970 fromstation 15 in the Trondheimsfjord (for details, see Sakshaug & Myklestad, 1973) and in 1971 from the Trondheim harbour area. The plankton blooms show the samegeneral development in the two areas (Sakshaug & Myklestad, 1973); the results from Station 15 may also be taken as representative of the Trondheim harbour. The significance of these observations will be discussed later.
CHEMICAL
CO~JPOSITIO~
The composition in terms of organic dry matter of the plankton samples are given in Table ICI. Very marked variations in the amounts of the main components were observed. In all cases, however, protein and carbohydrate form the main part of the organic matter, lipid being a retatively minor component (< 10 %). The sum of protein, carbohydrate, and lipid accounts in most cases for more than 90 % of the organic dry matter. Somewhat arbitrary standards have been used for the determination of the three main components and this, together with the fact that compounds such as nucleic acids have not been determined, may cause some deviation from a sum of 100 7:. For some of the samples, however, (e.g. 10th June, 1970) the results indicate that the organic matter contains some components not accounted for in our procedure. The same observation was made in a study of our laboratory of C. c-Q&is (Mykiestad & Haug, 1972) and has also been reported for other diatoms grown in culture (Parsons, Stephens & Strickland, 1961). Marked variations in the distribution of carbohydrate between the three different fractions are evident from the results in Table 111.These variations are partly caused
22
ARNE
by variations
HAUG,
SVERRE
MYKLESTAD
AND EGIL SAKSHAUG
in species, and partly reflect differences
will be discussed
in physiological
state. This point
in more detail below.
DISCUSSION It is well known plankton position
that
net hauls
do not give a representative
sample
of phyto-
populations. It should, therefore, be kept in mind that the chemical comgiven in Table III may deviate somewhat from that of the total phytoplank-
ton population in the area, due to selective sampling. We have chosen this method of collection, however, in order to obtain sufficiently large samples for analysis. Nevertheless, the same species dominated both the net hauls and the quantitative samples (Sakshaug & Myklestad, 1973). All the results were calculated as percentages of organic dry matter. The ash content of the samples was not considered to have any biological significance, being dependent upon the removal of sea water before freeze-drying. The ratio between nitrogen and phosphorus was found to be remarkably constant and independent of whether diatoms or dinoflagellates were the predominant organisms in the samples. In the 1970 samples the range of the nitrogen-phosphorus atomic ratio was 15.2-17.6, while a somewhat wider range, 13.9-19.3 was found in 1971. Harris & Riley (1956) in a study of phytoplankton populations from Long Island Sound found a range of 13.0 to 20.0. Ryther & Dunstan (1971) discussed the nitrogen-phosphoros ratios of both natural phytoplankton populations and cultures, and reported results from the literature ranging from 3 to 30, and which indicated a correlation between the ratio in the medium and that in the organisms. For C. afinis we found in this laboratory (Myklestad & Haug, 1972) ratios between 10 and 25 for algae grown in a medium with a 24.4 N/P ratio. With a nitrogen-phosphorus ratio in the medium of 122, the ratio in the organisms was found to be 59.5. These observations make it unlikely growth of the natural phytoplankton would
most probably
that phosphorus was the limiting factor for populations analysed in this study, since this
have led to higher
values
of the nitrogen-phosphorus
ratio.
In contrast to the remarkably constant values of the nitrogen-phosphorus ratio, the protein-carbohydrate ratio varied markedly from 0.48 to 1.83. A closer inspection of the fractionation pattern of the carbohydrate content reveals a marked difference between those samples rich in dinoflagellates and those containing predominantly diatoms. In the former, the amount of insoluble carbohydrate, as defined by our extraction procedure, was always high, accounting for between 35-52 % of the organic matter, while the corresponding figure was usually below 10 y0 for samples without significant amounts of dinoflagellates. It is well known that dinoflagellates have a cell wall consisting of an insoluble glucan, usually described as cellulose (Fritsch, 1961). Recent work (Nevo & Sharon, 1969) has cast some doubt upon the assumption that this polysaccharide is identical to cellulose from higher plants; its insolubility and lack of reactivity is, however, in agreement with the fractionation pattern observed
STUDIES
in this work. The gas-chromatographic soluble
carbohydrates
ON PHYTOPLANKTON
investigation
(Table
ECOLOGY
of the monosaccharide
I) also reveals a significant
23
composition
difference
between
of the dino-
flagellates and diatoms. Diatoms were characterized by an alkali-soluble carbohydrate fraction of complex monomer composition, but this fraction was absent in dinoflagellate-rich samples, glucose being the predominating component of the small, alkali-soluble fraction then obtained. When considering the protein-carbohydrate ratio as a parameter of possible ecological significance as suggested by Myklestad & Haug (1972) it is, therefore, necessary to distinguish between samples dominated by dinoflagellates and by diatoms. Samples of dinoflagellates have a low protein-carbohydrate ratio due to the high content of insoluble cell wall material; diatom samples, on the other hand, have a highly variable protein-carbohydrate ratio, the variation being mainly caused by variations in the acid-soluble glucan fraction, as previously observed for cultures of C. a$bzis (Myklestad & Haug, 1972). The culture experiments indicated that the protein-carbohydrate ratio could give valuable information about the physiological state of the diatom population. A high protein-carbohydrate ratio (low glucan content) indicates a rapidly growing population while depletion of available nutrients, leading to a stationary phase in the growth of the population, is indicated by a rise in the glucan content and a corresponding decrease in the protein-carbohydrate ratio. If we compare the changes in the phytoplankton populations as illustrated in Fig. 1. and the variations in the protein-carbohydrate ratio given in Table III, we find that the development of the first spring bloom in both years was characterized by a rapid decrease in the protein-carbohydrate ratio. The chemical changes resembled those observed for a batch culture (Myklestad & Haug, 1972). The concentration of nitrate in the surface layers rapidly decreased in the same period, as shown in Fig. I (which also gives some data on the nitrate concentrations). As will be discussed in more detail elsewhere (Sakshaug & Myklestad, 1973) these observations support sumption that the main source of nutrients in the first bloom is the amount in the water masses when the bloom starts. The events following
the first spring bloom
were somewhat
different
the aspresent
for the two
years. In 1970, the protein-carbohydrate ratio began to increase again by the 13th April, reaching a maximum on the 27th April. Fig. 1 shows an increasing population of diatoms of Group II on this date. In the following period when the proteincarbohydrate ratio varied z 1.0, the population consisted mainly of diatoms of Group II. This remarkably constant chemical composition was probably caused by a continuous supply of nutrients by river water and the entrainment of nutrient-rich sea water from deeper layers, keeping the population in a state of relatively rapid cell division. The sample collected on the 10th June had a low protein-carbohydrate ratio, indicating a change in physiological state in the direction of a stationary growth phase, probably due to a diminished supply of river water. In 1971, the protein-carbohydrate ratio remained low for a longer period, any
ARNE
24
HAUG,
SVERRE
MYKLESTAD
AND EGIL SAKSHAUG
notable increase first being observed on the 29th April. This increase continued but, due to heavy grazing by an increasing zoopiankton population, the sampling was stopped after the 6th May. The last three samples collected in 1971 consisted mainly
210s521os52104
20 30 March
1NO;
10
20
10
30
April
30
20
10
20
30
June
May
..-)-_
6.6;3.7;06;0;
1.16;
0.b ;
0.L;
1.1 ;
2.0
i___
2 105 5
2
-?b--&- -------ii&r& 20 10
March
April
May
Fig. 1. Number of diatoms (cells/l) in the different successional groups (see text, p. 21 and Table II), determined by quantitative sampling, in 1970 at Station 15 in the Trondheimsfjord (geometric mean in the upper 20 m) and in 1971 in the Trondheim harbour area (maximum numbers for 0 or 2 m).
of Skeletonema; it is interesting to note that. the changes in protein-carbohydrate ratio were contrary to those observed for ordinary batch cultures by Myklestad & Haug (1972) and also contrary to the development during the first spring bloom. In this period analysis of the water showed an increase in the amount of nitrate
STUDIES
ON PHYTOPLANKTON
ECOLOGY
25
present so that both the protein-carbohydrate ratio of the plankton and the water analysis indicate that the population was in a state of rapid ceil division due to a continuous supply of nutrients. The decreasing number of cells in the Iast part of this period was probably due to the heavy grazing (Sakshaug & Myklestad, 1973). A closer inspection of the three samples of the Skeletonema population reveals that the changing protein-carbohydrate ratio was only partly due to a change in the content of acid-soluble glucan. A very marked decrease in the alkali-soluble fraction was also observed. The aIkali-soluble fraction of the sample collected on the 21st April was remarkably high (32 “/;;). In 1970 the highest amount of the alkali-soluble fraction was 15 y<, and this sample contained a noticeable amount of Phmocystis in addition to diatoms. This was also the case for the samples from the 25th March to the 14th April, 1971, which contained from 16-24 7: alkali-soluble fractions. Pure diatom samples, as observed in 1970 and obtained in culture experiments (Myklestad & Haug, 1972, and unpubl. results), usually contain less than 10 “/ of this fraction. At present, we can offer no expIanation for the remarkably high content of alkalisoluble polysaccharides in the sample collected on the 21st April, 1971, nor for the decrease observed in the subsequent samples. Further information on the polysaccharides of diatoms is needed, and work is in progress in our laboratory, Apart from its informative value concerning the physiological state of a diatom population, the protein-carbohydrate ratio is also of interest in connection with the nutritive value of plankton and the determination of productivity. A larger percentage of the assimilated carbon will be incorporated into protein in a population characterized by rapid cell division (e.g., as in the beginning of the first spring bloom) than in a population growing in a nutrient depleted medium where glucan is themain product, as in the latter part of the first spring bloom. The same amount of assimilated carbon may, therefore, lead to very different results from a nutritive point of view, depending upon the physiological state of the phytoplankton population.
REFERENCES ANTIA, N. J., C. D. MCALLISTER,T. R. PARSONS,K. STEPHENS & J. D. H. STRICKLAND,1963. Further measurements of primary production using a large-volume plastic sphere. Limnol. Oceanogr., Vol. 8, pp, 166-183. DUBOIS, M., K. A. GILL~S, J. K. HAMILTON, P. A. REBERS& F. SMITH, 1956. Co~orimetric deter-
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FRITSCH, F. E., 1935. The structure
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HARRIS, E. & G. A. RILEY, 1956. Oceanography of the Long lsland Sound, 1952-1954. VIII. Chemical composition of the plankton. Bull. Bingham Oceanogr. COO., Vol. 15, pp. 315-323. HOBSON,L. A. & R. J. PARISER,1971. The effect of inorganic nitrogen on macromolecular synthesis by Thafassiosirapuciintilis Hustedt and Cycfotefla nana Hustedt grown in batch culture. J. exp. mar. Biof. EC&., Vol. 6, pp. 71-78. MEEUSE,B. J. D., 1962. In, Physiolo.qy and biochemistry of aigae, edited by R. A. Lewin, Academic
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26
ARNE
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SVERRE
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AND
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MYKLESTAD, S. & A. HAUG, 1972. Production of carbohydrates by the marine diatom Chaetoceros afinis var. wiDei (Gran) Hustedt. 1. Effect of the concentration of nutrients in the culture medium. J. exp. mar. Biol. Ecol., Vol. 9, pp. 125-136. MYKLESTAD, S., A. HAUC & B. LARSEN, 1972. Production of carbohydrates by the marine diatom Chaetoceros afinis var. willei (Gran) Hustedt. II. Preliminary investigation of the extracellular polysaccharide. J. exp. mar. Biol. Ecol., Vol. 9, pp. 137-144. NFVO, Z. & N. SHARON, 1969. The cell wall of Peridinium westii, a non-cellulosic glucan. Biochem. Biophys. Actu, Vol. 173, pp. 161-175. PARSONS, T. R., K. STEPHENS & J. D. H. STRICKLAND, 1961. On the chemical composition of eleven species of marine phytoplankters. J. Fish. Res. Bd Can., Vol. 18, pp. 1001-1016. RYTHER, J. H. & W. M. DUNSTAN, 1971. Nitrogen, phosphorus and eutrophication in the coastal environment. Science, N.Y., Vol. 171, pp. 1008-1013. SAKSHAUG, E., 1970. Quantitative phytoplankton investigations in near-shore water masses. K. norske Vidensk. Selsk. Skr. 1970, Vol. 3, pp. 1-8. SAKSHAUG, E., 1972. Phytoplankton investigations in Trondheimsfjord, 1963-1966. K. norske Vidensk. Selsk. Skr. 1972, Vol. 1, pp. l-56. SAKSHAUG, E. & S. MYKLESTAD, 1973. Studies on the phytoplankton ecology of theTrondheimsfjord. III. Dynamics of phytoplankton blooms in relation to environmental factors, bioassay experiments and parameters for the physiological state of populations. J. exp. mar. Biol. Ecol., Vol. I I, pp. 157-188. WERNER, D., 1970. Productivity studies on diatom cultures. Helgoliinder wiss. Meeresunters., Vol. 20, pp. 97-103.