Aquacultural Engineering 4 (1985) 175-190

Influence of Nitrogen Availability on the Biochemical Composition of Three Unicellular Marine Algae of Commercial Importance Susan D. Utting
Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Experiment Station, Conwy, Gwynedd LL32 8UB, UK

A BS TRA CT

Chaetoceros calcitrans (Paulsen) Takano, Tetraselmis suecica (Kylin) Butch. and Isochrysis galbana Parke were grown in artificial seawater containing either normal (9.8 mg atoms litre -1) or reduced (0.613mg atoms litre -1) nitrogen. When grown in the nitrogen-deficient medium protein decreased and carbohydrate increased in all three species; lipid, however, increased in Chaetoceros and Isochrysis but decreased in Tetraselmis. The biochemical composition o f Tetraselmis and lsochrysis sampled during the post-exponential phase of growth from normal medium was similar to that o f cells growing exponentially in nitrogen-deficient medium. Salinities of 10-35%o did not affect protein levels in Tetraselmis or Isochrysis but carbohydrate increased and lipid decreased in Isochrysis over the salinity range. Temperatures o f 18-25C had little effect on the cell composition o f either species. The energy potentially available from a unit volume o f Chaetoceros and Isochrysis cells from normal medium was similar to that from cells grown in nitrogen-deficient conditions in both species. Tetraselmis had 34% more energy available when grown in normal compared to nitrogen-deficient medium.

INTRODUCTION Unicellular marine algae are c o m m o n l y grown as a food source for commercially valuable shellfish. Early investigations led to the selection 175 Crown Copyright, 1985.

176

s. D. Utting

o f highly suitable food species for bivalves (Walne, 1974) and more recently significant improvements in algal production have been made at the Fisheries Experiment Station, Conwy, through changes in culture techniques (Helm e t al., 1979; Laing, 1979; Helm and Laing, 1981 ). The biochemical content o f algae can vary with age o f the culture and with changes in the environmental conditions (Lewin, 1962; Stewart, 1974). Protein is the main component of cells harvested during the exponential phase o f growth but the protein is replaced by storage products with a carbohydrate or lipid base as nitrogen becomes limiting during post-exponential phases. Variations in the silicate concentration o f the culture medium, in temperature, salinity, light intensity and wavelength can also cause changes in the biochemical content o f marine phytoplankton (Morris, 1981 ; Shifrin and Chisholm, 1981 ). The standard procedure at Conwy is to harvest cultures in the exponential phase when protein content is high. However, there is some evidence to suggest that low protein diets are beneficial to oysters at

TABLE 1

Composition and Preparation of Artificial Seawater

g litre -I m g litre -~ pg litre -1

NaC1 MgSO4.7H20 KNO3 CaCl2.2H20 NaHCOa aNaH2PO4 .H20 NaSiO3.9H20

20 5 1 1 0.2 0.1 0-04

MnC12.4H20 HaBOa bFeCla Thiamine HC1 ZnCla

1-4
.0-62

COC12.6H20
Biotin Cobalamin CuC12.2H20

0.54 0-5 0.1

5 5 1 0-034

Distilled water 1 litre. Medium buffered with 1 g Tris (trishydroxymethylaminomethane)litre -1. Buffer prepared by dissolving 25 g Tris in 75 ml distilled water, adjusting pH to 7.1-7-3 with concentrated HCI and making the volume up to 100 ml. Medium autoclaved at 1.06 kg cm-2 for 20min. Vitamins added after remainder of medium has been autoclaved. a Added to medium after autoclaving to prevent precipitation. b Chelated with NaEDTA in solution containing 0-3 mg FeCla : 0-05 mg NaEDTA m1-1.

Influence of nitrogen availability on biochemistry of marine algae

177

certain stages of growth. Wilson (1979) found that Ostrea edulis L. larvae increased their grazing rate when fed lsochrysis galbana which were in the post-exponential phase of growth. Crassostrea virginica spat had a higher weight and glycogen content when fed Thalassiosira pseudonana with a carbon/nitrogen ratio of 7.5-10.7 (Flaak and Epifanio, 1978). In this investigation, which was the preliminary part of a study into the effects of diet composition on the growth of oyster larvae and spat (Utting, in preparation), some of the standard Conwy culture techniques were changed in an attempt to produce algae with a low protein content. Chaetoceros calcitrans (Paulsen) Takano, Tetraselmis suecica (Kylin) Butch. and Isochrysis galbana Parke were grown in normal medium containing 9.8 mg atoms N litre -1 and in a nitrogen-deficient medium with 0-613 mg atoms litre -1. Cells with significantly different protein content were found. In addition the effects of harvesting cultures at various stages of the growth cycle and the effects of differing salinity and temperature regimes were studied using two of the genera Tetraselmis and Isochrysis.

METHODS

Culture of algae
Stock cultures of Chaetoceros (Bacillariophyceae), Tetraselmis (Prasinophyceae) and lsochrysis (Haptophyceae) were maintained in Erdschreiber medium (FOyn, 1934) and subcultured monthly. The experimental medium, an artificial seawater (Table 1), was a modification of that formulated by Lewin and Busby (de Mort, 1970). To study the effect of nitrogen deficiency, algae were grown in the normal medium (9-8 mg atoms N litre -1) or in artificial seawater to which only 0-613 m g a t o m s N l i t r e -1 was added. Salinities over the range 10-35~oo were obtained by dilution of the artificial seawater with distilled water. Nutrient concentrations were thus reduced, especially at the lower salinities, but earlier studies showed that nitrogen at least would not have limited algal growth even at 10%o (S. D. Utting, unpublished work). Algae were transferred from Erdschreiber medium to artificial seawater one week before the start of the experiment. After the week of

178

s. D. Utting

conditioning, cells were inoculated into 250 ml fresh medium in 500 ml boiling flasks to give initial concentrations of 50 cells 14 -1 for Tetraselmis and 500 cells #1-1 for Chaetoceros and Isochrysis. Cultures were unialgal but non-axenic. Incubation temperature was 20C+ IC but extended in one trial to 18-25C in order to determine the effect of temperature on the biochemical composition of Tetraselmis and Isochrysis. Continuous illumination was provided by two 65 W warm-white fluorescent tubes. The incident light intensity at the outer surface of the culture vessel was 3 klux. Cultures were aerated at 1 litre min -1 with filtered air which was enriched with 2% v/v carbon dioxide. In most trials Chaetoceros was harvested after 2 days, Tetraselmis after 4 days and lsochrysis on the fifth day to ensure that cells were taken from a similar phase o f growth (S. D. Utting, unpublished data). When the effect o f culture age on the chemical composition of Tetraselmis and lsochrysis was being studied, samples of cells were taken from day 1 to day 13 o f the experiment.

Analytical

techniques

Cell concentration was determined on a model ZB Coulter Counter to which a volume distribution plotter was connected to measure cell volume. Dry weight of cells was found by filtering 10 ml samples of culture in duplicate under a vacuum not exceeding 0.35 kg cm -2 on to Whatman GF/C filter discs which had previously been ashed at 450C for 4 h. The discs were washed with 0.9% (w/v) isotonic ammonium formate, dried at 80C for 48 h and then weighed. Ash-free dry weight was obtained by subtraction after the discs had been left for a further 4 h at 450C in a muffle furnace. Subsamples of culture were taken in duplicate for the estimation of protein (10 ml), carbohydrate (10 ml) and lipid (5 ml). The algae were concentrated by centrifugation at 3000 rpm for 10 min and the supernatant was removed. Protein was measured by a modified microKjeldahl technique (Holland and Hannant, 1973) and protein content was assumed to be nitrogen concentration X 6-25. Cells for carbohydrate estimation were resuspended to a volume of 10 ml with distilled water and carbohydrate was determined by the anthrone method (Strickland and Parsons, 1968).

Influence of nitrogen availability on biochemistry of marine algae

179

Lipid was extracted b y adding 2 ml o f 2 : 1 (v : v) chloroform/methanol mixture and leaving f o r 2 h at 20C in the dark. To remove nonlipid contaminants 0-4 ml of 0.7% (w/v) NaC1 solution was added and the samples left overnight at 4C. The lower phase was then made up to 2 ml with chloroform and 1 ml o f that phase removed for the analysis o f lipid by the charring method of Marsh and Weinstein (1966). Cholesterol was used as the standard and total lipid was calculated by assuming 80 #g cholesterol was equivalent to 100 #g total lipid (Barnes and Blackstock, 1973). The biochemical composition has been expressed as a percentage of the total organic content in some comparisons. Total organic content was equivalent to the sum (in tag per 106 cells) of the protein, carbohydrate and lipid recovered in the chemical analyses. The inorganic and total carbon content of cells was measured by combustion of replicate 50 tal subsamples o f algae at 150 and 950C, respectively, on a Beckman total organic carbon (TOC)analyser model 915B. The standard contained 100 tag carbon m1-1 and was made up in distilled water which had been photooxidised for 4 h. Organic carbon was found by subtraction.

RESULTS Effect of

nitrogen deficiency

Cell size and weight was generally not affected by a change in the nitrogen content o f the culture medium (Table 2) excepting that the ash-free dry weight o f Chaetoceros was higher in the nitrogen-deficient cultures (Student's t-test, t = 2-84, degrees of freedom = 66, p < 0.01). The biochemical composition of all species was, however, significantly altered. At the lower nitrogen concentration the protein content per l 0 6 cells was significantly less in all species and carbohydrate was higher (p < 0-001). Chaetoceros and Isochrysis cells contained most lipid when grown in nitrogen-deficient medium ( p < 0.05 and p < 0.02, respectively) while Tetraselmis cells had a higher lipid content when grown in normal medium (p < 0-001). Comparison of the mean values of each biochemical parameter for each species grown in normal or reduced nitrogen media gave the following t values:

OO O

TABLE 2 The Structure and Biochemical Content of Chaetoceros calcitrans, Tetraselmis suecica and lsochrysis galbana Cultures Grown in Artificial Medium Containing 9.8 mg atoms N litre -1 or 0.613 mg atoms N litre -1 (mean values over number of cultures started)

Chaetoceros calcitrans Tetraselmis suecica

9.8mgatoms N litre -1 0.613mgatoms N litre -1 9.Smgatoms N litre -~ 0.613mgatoms N litre -~

Isochrysis galbana
0.613mgatoms N litre -1

9.8mgatoms N litre -1

10.57 0.021 9-48 0.025 0.017 40.42 4.812 7.435 2.612 13.66 34 21.282 29.124 4.49 17 0.269 450.68 179.965 0.258 464.75 53.159 144.453 17.912 15.39 17 3.29 0-312 3.11 0.306

10.104 0-035 0-027 69.21 12.161 5-479 5.889 7.60 28

9.829 0.034 0.027 67.41 8-506 8.150 7.489 10.75 28

Cell concentration /A-1 (x 106) Dry weight, mg per 106 cells Ash-free dry weight, mg per 106 cells Mean cell volume, pm 3 Protein,/ag per 106 cells Carbohydrate/ag per 106 cells Lipid, ttg per 106 cells Carbon/nitrogen Number of cultures

0.014 38.14 7-097

2.985 2.074 6.42 34

Influence of nitrogen availability on biochemistry of marine algae Chaetoceros t Degrees of freedom 66 66 66 t Tetraselmis Degrees of freedom 32 32 32 t Isochrysis

181

Degrees of freedom 54 54 54

Protein Carbohydrate Lipid

7.67 9.25 2-03

19-66 17-88 4-65

5.20 4-70 2.52

Carbon/nitrogen ratios ranged from 4-49 to 7-60 in algae from normal medium to 10.75-15-39 in nitrogen-deficient conditions as a result of the change in biochemical content.

Effect of culture age

Exponential growth finished on day 8 in Tetraselmis cultures and on day 6 in lsochrysis. Unfortunately no measurements could be made on 13 day old Isochrysis cultures because of a contaminant flagellate in the cultures. Protein decreased with age of culture in both species (Table 3). In Tetraselmis carbohydrate and lipid replaced the protein, with carbohydrate rising sharply between days 7 and 13. Lipid increased threefold in Isochrysis cells during the exponential phase but after an initial increase in carbohydrate from 22.8 to 34.5% of the total organic content, there was a decline in that fraction to 20L1% by day 7. Tetraselmis from nitrogen-deficient conditions was comparable in organic content to cells from 13 day old cultures, where nitrogen would be expected to be limiting, while cells from 1-4 day old cultures, with an expected high nitrogen content, compared with Tetraselmis grown in normal medium.

The effects of salinity and temperature

Salinity, over the range 10-35700, and culture temperatures of 18-25C, had less of an effect on the chemical composition of Tetraselmis and lsochrysis (Table 4) than had nitrogen depletion or harvesting cells at different stages during the growth cycle (Table 3). The optimum salinity for Tetraselmis, measured by the highest protein/carbohydrate ratio, was 25~'oo. The biochemical composition of

70
b3

TABLE 3 Effect of Nitrogen Deficiency and Age of Culture on the Protein, Carbohydrate and Lipid Content, as a Percentage of the Total Organic Material, of Tetraselmis suecica and lsochrysis galbana Cultures

Species

Cell component (% of total organic content) 9.800

78.1 9-5 12.6 51-7 23.3 25.0 35.2 33.8 31.0 67-1 22.8 10.1 24.7 67-0 8.3 73.1 19.2 7.1

Nitrogen concentration (mg atoms N litre-1) 0.613 1 4

67.5 18.1 14.5 46.0 34.5 20.1

Age of culture, days (9.800 mg atoms N litre -1 initially) 7

60.3 27.4 12.3 41.7 20-1 30.4

13
25.6 57.7 15.4

Tetraselmis suecica

Protein Carbohydrate Lipid

Isochrysis galbana

Protein Carbohydrate Lipid

TABLE 4 Effect of Salinity (10-35~/oo) and Temperature (18-25C) on the Protein, Carbohydrate and Lipid Content, as a Percentage of the Total Organic Material, of Tetraselmis suecica and Isochrysis galbana

Species 10
54.7 32.1 13.2 52-6 22.6 24.8 49.6 27.3 23.1 48.8 29.5 21-7 47-0 30.1 22.9 48-8 28.9 22-3 51.0 34.1 14.9 62.7 24.0 13.3 65.4 22.5 12-2 60.2 30-2 9-6 58-1 30.0 11.2 50.2 30.7 19.2

Cell component (% of total organic content) 15 20 25 30 35 18

69-3 22.9 7.9 60.3 24.7 14.9

Salinity (%o)

Temperature (C) 20
66.3 28.6 5.4 56-1 27-4 16-5

ga

22
55.3 34-3 10.5 40.8 38-7 20.5

25
69.7 22.8 7.6 -

55
~ ~" ~.
~.

Tetraselmis suecica

Protein Carbohydrate Lipid

Isochrysis galbana

Protein Carbohydrate Lipid

o~

ta~

184
26

S. D. Utting

! 35
tJ

g
u

o~ o, Z5
L -J

g
18
I
I
.t3

20 Satin ty % .

~0

200

I 20 Salinity, % .

t,O

Fig. 1. The relationship between the lipid content of lsochrysis galbana cells and the salinity of the artificial seawater medium, y = 2 6 - 4 5 - 0 . 1 8 x (r=-0.87, degrees of freedom = 4, p < 0-05).

Fig. 2. The relationship between the carbohydrate content of Isochrysis galbana cells and the salinity of the artificial seawater medium, y = 22-32 + 0.261x (r = 0-826, degrees of freedom = 4,

p < 0.05). Tetraselmis began to change only at 10 and 15~'oo but not to the same extent as occurred with nitrogen deficiency. Protein in Isochrysis was similar over the salinities tested but lipid decreased, r = - - 0 . 8 7 , degrees of freedom = 4, p < 0.05 (Fig. 1), and carbohydrate increased, r = 0-826, degrees of freedom = 4, p < 0.05 (Fig. 2) at the higher salinities. The biochemical content o f Tetraselmis showed little variation between temperatures of 18 and 25C although at 22C protein was less but carbohydrate and lipid were more than might have been expected from the measurements on cells grown at 20 and 25C. Protein decreased in Isochrysis at the higher temperatures with a corresponding increase in carbohydrate and lipid. At 25C no growth occurred and the cultures died within 3 days.
Variation in biochemical content Relationships between biochemical components were found in both Tetraselmis and lsochrysis. As protein decreased carbohydrate increased in Tetraselmis, r = --0-977, degrees of freedom = 14, p < 0-001 (Fig. 3), while in Isochrysis lipid, r = --0-829, degrees of freedom = 12, p < 0.001 (Fig. 4), and carbohydrate, r = - - 0 - 5 4 8 , degrees of freedom = 12, p < 0.05 (Fig. 5), increased. There was no correlation between carbo-

Influence of nitrogen availability on biochemistry of marine algae

-~ 80

185

I 20 t,0 60 80 Carbohydrate,% of total organic content

Fig. 3. The relationship between protein and carbohydrate content of Tetraselmis suecica,y = 89-62 - 1-024x (r = -0.977, degrees of freedom = 14, p < 0.001).

80
u

8O

~6c
o

~60 8

t,C

-.<.
t~

2c

I 10

I 20

I 30

I 40

Lipid,% of total organic content

1 40 Carbohydrate, % of total organic content

I0 20 30

ZO

The relationship between protein and lipid content of Isochrysis galbana, y = 75-61 - 1-199x (r = --0-829, degrees of freedom = 12, p < 0.001).

Fig. 4.

Fig. 5. The relationship between protein and carbohydrate content of Isochrysis galbana, y = 73.46 - 0-843x (r = - 0 - 5 4 8 , degrees of freedom = 12,

p< 0.05).
h y d r a t e and lipid with e i t h e r species or b e t w e e n p r o t e i n and lipid in

Tetraselmis. T h e o n l y data f o r Chaetoceros were f r o m the nitrogen s t u d y (Table 2). In n o r m a l m e d i u m p r o t e i n was 58% and c a r b o h y d r a t e 25% o f the organic c o n t e n t o f cells b u t w h e n n i t r o g e n was limiting the respective values were 32% and 50%. T h e s e data were similar to results for Tetraselmis (Fig. 3).

186

S. D. Utting

Energy content
The energy content, as joules theoretically available if protein, carbohydrate and lipid were converted into energy with 100% efficiency, was calculated for Chaetoceros, Tetraselmis and lsochrysis grown in normal and nitrogen-deficient media where the most significant differences in biochemical content were found (Table 5). Protein contained 47-74% of the energy in cells grown in normal medium with lipid acting as the second most important source of energy. In cells from nitrogen-deficient cultures the amount o f energy available in protein was very similar between species (28-33%). Chaetoceros and Tetraselmis had more energy bound up in the carbohydrate fraction while lsochrysis had the major quantity of the energy in lipid. Expressed on a per unit volume basis Chaetoceros and Isochrysis grown in the nitrogen-deficient medium contained respectively 8 and 6% more energy than when grown in normal medium. However, Tetraselmis contained 34% more energy if cultured at the normal rather than the reduced concentration of nitrogen.

TABLE 5 Energy Content (J x 10 9 cell -1) of Chaetoceros calcitrans, Tetraselmis suecica and Isochrysis galbana Grown in Normal Medium and Nitrogen-deficient Medium.
Species Nitrogen content of medium (mg atoms litre -1} 9.8 0.613 9-8 0.613 9.8 0.613 Potential energy (J Protein Carbohydrate
1 0 9 cell -1)

Lipid

Total

Chaetoceros calcitrans Tetraselmis suecica Isochrysis galbana

169.4 (56) a 114-7 (33) 4220.2 (74) 1268.4(28) 290-1 (47) 203.5 (32)

52.5 (17) 130.7 (38) 374.2 (6) 2539.7 (56) 96.3 (16) 143.2 (22)

81.6 (27) 102-8(30)

303.5 348.2

1146.0 (20) 5814.2 704.9 (16) 4513.0 231.9 (38) 294.7 (47) 618-3 640-9

a Figures in parentheses give the percentage of that fraction to the total energy available.

Influence of nitrogen availability on biochemistry of marine algae

DISCUSSION

187

The most successful method of decreasing the protein content of exponentially-growing algae was by reducing the initial concentration of nitrogen in the culture medium from 9.8 to 0-613 m g a t o m s l i t r e -1 (Table 2). A similar effect was obtained by harvesting cultures in the post-exponential phase when nitrogen would be expected to be limiting growth (Table 3). In both cases the change in organic composition of all species under nitrogen-deficiency may have been caused by a reduction in the rate of protein synthesis with the result that non-nitrogenous products, such as carbohydrate and lipid, accumulated. Salinities of 10-35%o and temperatures in the range 18-25C did not cause significant differences in the protein content of either Tetraselmis or Isochrysis cells (Table 4). Although protein in Isochrysis was similar over the salinities tested, there was a significant decrease in cellular lipid (Fig. 1) and a corresponding increase in carbohydrate (Fig. 2) possibly to maintain the osmotic balance in the cells. Temperatures below 18C were not studied because it is doubtful if a reduction in temperature would increase the non-proteinaceous fraction of the cells (SteemanNielsen and J~rgensen, 1968: Morris, 1981). Results for Isochrysis indicated that only by growing this species at temperatures close to the limits of tolerance could a decrease in protein be effected (Table 4). Instability in the growth of Isochrysis at temperatures above 22C, as found here and by Ukeles (1961), would make temperature manipulation an unsatisfactory way of reliably producing low protein cells of this species. The response of the algae to low nitrogen stress was best observed in Tetraselmis and Isochrysis cultures grown over a period of up to 13 days (Table 3). In both species the amount of protein decreased but Tetraselmis stored primarily polysaccharides in conditions unfavourable for cell division whereas the carbohydrate pool in 4 day old Isochrysis was converted into lipid by day 7. Carbohydrate has been found to act as an intermediate reserve in some algae (Antia et al., 1963; Marker, 1965; Werner, 1970) because time is required after nitrogen becomes limiting for the enzymes essential for lipid synthesis to be produced (Fogg, 1956). Consequently in any conditions where protein decreased in Isochrysis both lipid and carbohydrate might be expected to increase (Figs 4 and 5). In similar conditions, Tetraselmis, a member of the Prasinophyceae which store polysaccharides and

188

S. D. Utting

presumably do not produce enzymes to synthesise lipid, might be expected to increase only the carbohydrate content (Fig. 3). Indeed, Tetraselmis cells from cultures which had been in the stationary phase for 3 weeks maintained an organic content of 26% protein, 64% carbohydrate and 9% lipid (S. D. Utting, unpublished data). Chaetoceros in nitrogen-deficient cultures increased the carbohydrate content more than the lipid (Table 2). The protein/carbohydrate ratio of 2.3 observed in normal medium contrasted with a ratio of 0.6 when nitrogen was reduced. Myklestad and Haug (1972) found a similar decline, from 2 to 0-23, in the protein/carbohydrate ratio o f Chaetoceros affinis when nitrogen was limiting and glucan, a complex polysaccharide, was synthesised. Manipulation of the nitrogen concentration of the culture medium was found to be a simple technique to effect significant differences in the protein, carbohydrate and lipid content of three species of marine phytoplankton. In two of the species, Chaetoceros and Isochrysis, the change in biochemical content altered the energy potentially available from a unit volume o f cells by less than 10% (Table 5). Tetraselrnis, however, contained 34% more energy, expressed on the same basis, when grown in normal medium primarily due to higher lipid levels (Table 2). The technique could be of use in commercial hatcheries if feeding trials prove that low protein algae sustain better growth of young oysters than high protein algae. Any methods of improving the growth and development rates o f young oysters, thereby reducing the duration o f dependence on cultured algae, must be of financial benefit.

REFERENCES Antia, N. J., McAllister, C. D., Parsons, T. R., Stephens, K. & Strickland, J. D. H. (1963). Further measurements of primary production using a large-volume plastic sphere, Limnol. Oceanogr., 8, 166-83. Barnes, H. & Blackstock, J. (1973). Estimation of lipid in marine animals and tissues: detailed investigation of the sulphophosphovanillin method for 'total' lipids, J. exp. mar. Biol. Ecol., 12, 103-18. Flaak, A. R. & Epifanio, C. E. (1978). Dietary protein levels and growth of the oyster Crassostrea virginica, Mar. Biol., 45, 157-63.

Influence of nitrogen availability on biochemistry of marine algae

189

Fogg, G. E. (1956). Photosynthesis and formation of fats in a diatom. Ann. Bot. (N.S.), 20, 265-85. F~yn, B. (1934). Lebenszyklus, Cytologie und Sexualit~'t der Chlorophycee Cladophora suhriana Kfitzing, Arch. Protistenk., 83, 1-56. Helm, M. M. & Laing, I. (1981). Cost-effective culture of marine unicellular algae. In: Energy Conservation and Use o f Renewable Energies in the Bio-industries, ed. F. Vogt, Pergamon Press, Oxford and New York, pp. 247-59. Helm, M. M., Laing, I. & Jones, E. (1979). Culture of algae for larval fish and shellfish rearing, Part 1, The development of a 200 litre algal culture vessel at Conwy, Fish. Res. Tech. Rep., M A F F Direct. Fish. Res. Lowestoft, 53, 1-7. Holland, D. L. & Hannant, P. J. (1973). Addendum to a microanalytical scheme for the biochemical analysis of marine invertebrate larvae, J. Mar. Biol. Assoc. UK, 53,833-8. Laing, I. (1979). Culture of algae for larval fish and shellfish rearing, Part 2, Recommended procedures for the culture of Chaetoceros calcitrans, Fish. Res. Tech. Rep., M A F F Direct. Fish. Res. Lowestoft, 53, 8-12. Lewin, R. A. (1962). Physiology and Biochemistry of the Algae, Academic Press, New York. Marker, A. F. H. (1965). Extracellular carbohydrate liberation in the flagellate lsochrysis galbana and Prymnesium parvum, J. Mar. Biol. Assoc. UK, 45,755-72. Marsh, J. B. & Weinstein, D. B. (1966). 'Notes on methodology'. Simple charring method for determination of lipids, J. Lipid Res., 7,574-6. Morris, I. (1981). Photosynthetic products, physiological state and phytoplankton growth. In: 'Physiological bases of phytoplankton ecology' (ed. Trevor PlatO, Can. Bull. Fish. Aquat. Sci., 210, 83-102. Mort, C. L. de (1970). The culture and biochemical analysis of some estuarine phytoplankton species, PhD thesis, Oregon State University, Corvallis. Myklestad, S. & Haug, A. (1972). Production of carbohydrates by the marine diatom Chaetoceros affinis vat. willei (Gran) Hustedt. I. Effect of the concentration of nutrients in the culture medium, J. exp. mar. Biol. Ecol., 9, 125-36. Shifrin, N. S. & Chisholm, S. W. (1981). Phytoplankton lipids: interspecific differences and effects of nitrate, silicate and light-dark cycles,,/. Phycol., 17,374-84. Steeman-Nielsen, N. & J~brgensen, E. G. (1968). The adaptation of plankton algae. 1. General part,Physiol. Plant., 21,401-13. Stewart, W. D. P. (1974). Algal Physiology. and Biochemistry, Blackwell Scientific Publications, Oxford. Strickland, J. D. H. & Parsons, T. R. (1968). A practical handbook of sea water analysis, Bull. Fish. Res. Bd. Can., 167,227-30. Ukeles, R. (1961). The effect of temperature on the growth and survival of several marine algal species, Biol. bull. mar. biol. Lab., Woods Hole, 120, 255-64. Walne, P. R. (1974). Culture o f Bivalve Molluscs. 50 Years' Experience at Conwy, Fishing News (Books) Ltd, Whitefriars Press Ltd, London.

190

S. D. Utting

Werner, D. (1970). Productivity studies on diatom cultures, Helgolfinder wiss. Meeresunters., 20, 97-103. Wilson, J. H. (1979). Observations on the grazing rates and growth of Ostrea edulis L. larvae when fed algal cultures of different ages. J. exp. mar. Biol. Ecol., 38, 187-99.