Effects of nutrient and light limitation on the ... - Springer Link

Journal of Applied Phycology 2: 45-56, 1990. © 1990 Kluwer Academic Publishers. Printedin Belgium.

45

Effects of nutrient and light limitation on the biochemical composition of phytoplankton* P. J. Harrison, P. A. Thompson & G. S. Calderwood Department of Oceanography, University of British Columbia, Vancouver, B.C. V6T I W5, Canada Received 10 September 1989; revised 1 February 1990; accepted 1 February 1990

Key words: phytoplankton, biochemical composition, nutrients, lipids, carbohydrates, fatty acids

Abstract Three marine phytoplankters (Isochrysis galbana, Chaetoceros calcitrans and Thalassiosirapseudonana), commonly used in the culture of bivalve larvae, were grown in batch or semi-continuous cultures. Changes in protein, carbohydrate, lipid and some fatty acids were measured as growth became limited by nitrogen, silicon, phosphorus or light. Under N starvation (2 d) the % lipid remained relatively constant, while% carbohydrate increased and% protein decreased in all 3 species compared to cells growing under no nutrient limitation. Under Si starvation (6 h) there was no change in lipid, protein or carbohydrates. The amount of two fatty acids, 20: 5o3 and 22: 6co3 remained relatively constant under N, P and Si starvation, exept for a sharp drop in the cells of P-starved T. pseudonana. However, there were pronounced species differences with I. galbana containing significantly less 20: 5 co3 than C. calcitrans or T. pseudonana. Under light limitation the amount of lipid per cell showed no consistent trend over a range of irradiances for all 3 species. The amount of N per cell (an index of protein content) as a function of irradiance, was relatively constant for I. galbana and T. pseudonana, while the amount of N per cell was lower under low irradiances for C. calcitrans.These examples of changes in protein, carbohydrate, lipid and certain fatty acids under nutrient (N, Si or P) or light limitation, emphasize the importance of knowing the phase (e.g. logarithmic vs stationary) of the growth curve in batch cultures, since the nutritional value of the phytoplankters could change as cultures become dense and growth is terminated due to nutrient or light limitation. Introduction Phytoplankton are often used as a food supply in the aquaculture industry for hatchery grown herbivores such as larval or juvenile bivalves. Dense, large-scale batch or semi-continuous cultures are used to provide a relatively continuous food supply. Depending on how frequently these cultures are harvested, the growth rate of the culture

may slow down (i.e. enter senescence) due to nutrient limitation or light limitation when the cultures are very dense. Phytoplankton composition data from some previous studies are difficult to interpret because little care was taken to determine the growth phase of the phytoplankton before they were harvested. It is extremely important to determine the growth phase because large changes in algal biochemical composition can oc-

*Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.

46 cur on the time scale of hours to days as the cells enter stationary phase due to nutrient limitation. Improvements in the understanding of algal physiology, better culturing techniques, and superior technology, primarily in GC columns, has resulted in a more reliable body of recent literature (e.g. Volkman et al., 1989; Parrish and Wangersky, 1987; Ackman, 1986). The objective of this paper is to remind aquaculturalists of the precautions required when using batch cultures and to illustrate the changes in protein, carbohydrate, lipid and some fatty acids that can occur when the growth of three marine phytoplankters become limited by nitrogen, silicon, phosphorus or light. Materials and methods Culturing Unialgal cultures of Isochrysis galbana Green, (clone T-iso, and henceforth called Tahitian Isochrysis), Chaetoceros calcitrans (Paulsen) Tokano and ThalassiosirapseudonanaHasle and Heimdal (Hust., clone 3H) were obtained from the Northeast Pacific Culture Collection, Dept. Oceanography, University of British Columbia. Cultures were grown as nutrient- or light-limited cultures. Nutrient-limited cultures were grown in 6 L Erlenmeyer flasks containing 3.5 L of culture. Continuous irradiance of 300 #mol photon m- 2s-I was supplied with cool-white fluorescent tubes. Cultures were grown at 21 + 1.5 °C. Culture pH was maintained between 8.0 and 8.3 by bubbling with filtered (1 ilm) air and sparging with pure CO 2 every 0.5 h for 2 min. Additional stirring was applied by swirling the cultures when daily samples were taken. Culture medium was filtered-sterilized (0.45 ,m), nutrient enriched natural seawater from Departure Bay, Nanaimo, B.C. The natural seawater was enriched as described by Harrison et al. (1980), except that FeNH4 (SO4 ) 2 6H 20 was replaced with an equimolar amount of FeSO4 7H 2 0 and sodium glycerophosphate was replaced with Na 2H 2 PO4 H 2 0. Selenium was

added at 10-9 M using Na2 SeO3 (selenite). Nutrient ratios in the culture medium were modified in order to ensure growth rate limitation by a specific nutrient. For P-limited cultures the ratio of N: Si: P (by atoms) in the culture medium was 555: 81: 1; for N-limited cultures it was 14: 26: 1 and for Si-limited cultures it was 110: 5.3: 1.All three species were grown at N- or P-limited medium while only C. calcitrans and T. pseudonana were also grown in Si-limited medium. For light-limited cultures, continuous light was provided by VitaliteR fluorescent bulbs at four irradiances: namely 6 or 14, 24 or 44, 80 or 125 and 200 or 225 #jmolphoton m- 2 s- . Cultures were grown at 17.5 + 0.5 C and stirred with a stir bar at 60 rpm. They were bubbled with a mixture of ca. 2% CO 2 and air to maintain culture pH between 8.2 to 8.5. Cultures were grown for at least 9 generations to allow them to adapt to the different irradiances, before they were harvested at mid-exponential growth phase to ensure that nutrient limitation did not occur. Both light- and nutrient-limited cultures used similar enrichment solutions but light-limited cultures used artificial seawater, rather than natural seawater. Growth rates were monitored by measuring in vivo fluoresence (Turner Designs R Model 10 fluorometer) and cell counts were determined once or twice a day using a Coulter CounterR model TAII. Growth rates were calculated according to the equation: ji = ln(F/FO)/tl - to, where Fl is the biomass at time I (t) and Fo is the biomass at time 0 (to).

Phytoplankton chemical composition was assessed by harvesting cells during the mid-logarithmic growth phase and comparing them to the early stationary phase (i.e. 6 h to 2 d after cells exhausted a particular nutrient (N, Si or P) from the culture medium (Fig. 1). Light-limited cultures were assessed at mid-log phase (i.e. no nutrient limitation) where growth rates were reduced by sub-saturating growth irradiances. Samples for total lipid were analyzed by the lipid charring technique of Marsh & Weinstein (1966) using tripalmitin as a standard. Samples for fatty acid (FA) determinations were collected

47 on precombusted GF/F filters, placed inside a petri dish and sealed in plastic bags filled with nitrogen. Prior to analysis, they were frozen at - 20 C, for periods of less than 3 weeks, or for longer periods at - 80 C. Samples were saponified and methylated as in Whyte (1988). Intra and intersample duplicates were prepared and analyzed. FAs were analyzed on a Hewlett-Packard 5890A gas liquid chromatograph fitted with a Supelcowax 10 fused silica capillary column (30 m x 0.32 mm I.D., 0.25 ,um film) and identified by comparison with saturated and PUFA-1 (Supelco Inc.) methyl ester standards in accord with Ackman 1986).

100

. 75 .5 50

a

X-

W 25

0 ML -N -N 2d 6d

ML -N -Si 6h 6h

ML -N -Si 6h 6h

Isochrysis Chaetoceros Thalassiosira Results Batch culture growth curve Figure 1 shows a idealized growth curve for any photoautotrophic microalgae. Growth rate may be terminated quite abruptly, usually by one of the macronutrients, C, N, Si and P. The abruptness in the termination of growth rate is partially related to the ratio of the minimum to the maximum amount of nutrient per cell and therefore the nutrients can be ranked Si > C > N > P, in order from most abrupt to the least abrupt. If more

Fig. 2. Amount of lipid (), carbohydrate (E) and protein (1), as % ash-free dry weight for three microalgae, Isochrysis, Chaetoceros and Thalassiosira harvested during mid-log phase (ML), and after 6 h or 2 d of N or Si starvation.

nutrients were added (see arrow in Fig. 1), the culture continues growing, but the growth rate slows down as the culture becomes light-limited. Therefore, if enough nutrients are added and the pH of the culture is maintained within species specific tolerance levels, the maximum yield of a culture is determined by the amount of light received. An exception to this generalization is 300

250 0

0) -J

° 200

0~

co o150

.5 a) 0J -

3 100

.2 50 o Time (days) Fig. 1. Idealized growth curve for a microalga showing: (1) lag phase, (2) exponential or log phase and (3) stationary or scenescent phase, where growth rate is terminated by nutrients or light. Arrow indicates the addition of more nutrients. The process of semi-continuous culturing (- .-) is also shown.

0

0

ML-N -N 2d 6d

ML -N -Si 6h 6h

ML-N -Si 6h 6h

Isochrysis Chaetoceros Thalassiosira Fig; 3. Caloric content of three microalgae, Isochrysis, Chaetoceros and Thalassiosira harvested during mid-log phase (ML), and after 6 h or 2 d N or Si starvation.

48 20

e

2.50 l

15

2.00

a -

10 o

1.50

D

C 1.00 z Li 0

° 0.50

0

z

0.00 0 X

15

250

200

150

100

50

photon m-

LIGHT (mol

2 - 1

s

)

Fig. 5. The amount of nitrogen per cell for three microalgae, Isochrysis (A), Chaetoceros (A), and Thalassiosira grown on NO3 () and Thalassiosiragrown on NH' (o). Cells were harvested at mid-log phase after at least 9 generations of growth. Where shown, bars represent + 1 pop. std. dev.

0

n 110 s0

i-

ML -N -P 2d 2d

ML-N-Si -P 6h 6h 2d

ML -N-Si -P 6h 6h 2d

Isochrysis

Chaetoceros

Thalassiosira

Fig. 4. Percentages of the two fatty acids, (A) 20: 5 r3 and (B) 22: 6wo3 in three microalgae, Isochrysis, Chaetocerosand Thalassiosiraharvested during mid-log phase (ML), and after 6 h or 2 d of N or Si starvation and under 2 days of P starvation. Error bars represent + 1 std. dev.

autoinhibition. In this case, a species produces a metabolite that inhibits its own growth when concentrations of the metabolite builds up in the culture medium. Nutrient and light limitation and autoinhibition can be avoided by semicontinuous culturing, in which the culture is diluted with new culture medium before the growth rate slows down due to nutrient or light limitation (Fig. 1).

microalgae is shown in Fig. 2. Under N starvation (2 d) the % lipid remained relatively constant, while /o carbohydrate increased and % protein decreased in all 3 species, compared to cells growing under no nutrient limitation (i.e. midlogarithmic phase cells). Under Si starvation (6 h) 6.00 i 5.00 0

_4.00 ' ..

0,

° 3.00 oC

n 2.00

a

The effect of nitrogen or silicate starvation on the amount of lipid, carbohydrate or protein in three

0

50

I

I

I

1.00I Effects of nutrient limitation on algal chemical composition

..

.

'.

A

100

LIGHT (mol

150

photon

I

200

250

m- 2 s- 1 )

Fig. 6. Amount of lipid per cell for three microalgae, Isochrysis (A), Chaetoceros(A), and Thalassiosiragrown on NO (). Cells were harvested at mid-log phase after at least 9 generations of growth.

49 there was no change in lipid, protein or carbohydrates compared to mid-log phase cells of Chaetoceros and Thalassiosira. There was a decrease in caloric content under N starvation in all three algal species, but no consistent change in caloric content occurred in the two diatoms under Si limitation (Fig. 3). The effect of N, P and Si starvation on two fatty acids, eicosapentaenoic acid, 20: 5c3, and docosahexaenoic acid, 22: 6co3, thought to be importance in the nutrition of bivalves, is shown in Fig. 4. The amount of 20: 53 remained relatively constant under N, P and Si starvation in all three microalgae, except for the sharp drop in the P-starved Thalassiosira culture. The fatty acid 22: 6co3 also remained relatively constant under N, P and Si starvation in all three phytoplankters. In addition to these nutrient affects, there were pronounced species differences, with Isochrysis containing significantly less 20: 5 co3 than Chaetoceros or Thalassiosira(t-test, p < 0.05).

growth irradiance (Fig. 5). In general N per cell exhibits a complex reponse to irradiance. In Isochrysis and Thalassiosira,the amount of nitrogen per cell was relatively constant while the amount of nitrogen per cell was lower under low light in Chaetoceros.For Thalassiosiragrown on NO3 the amount of nitrogen per cell was significantly less than cells grown on NH' (t-test, p < 0.05) at 61 #mol photon m- 2 s- . This trend persisted at

20.0 15.0

0

In

.T

Ut) o 10.0

1

.0

E

D

.

5.0 A

0.0

--

-------

.......--*.....

20.0

n

15.0 ,A.

to t

-A. .

C04

The amount of nitrogen per cell (an index of the amount of protein) was measured as a function of I

I A

Effect of light limitation on algalchemical composition

IU

I

co

7

K' X

5

10.0

5.0

0-o _j L c.)

2

e

0.0

0

50

100

LIGHT (mol

0

50 LIGHT (mol

100

150

photon

m- 2 s-

200 1

)

Fig. 7. Cell volumes (m 3) as a function of irradiance for Thalassiosirapseudonana grown on NH' (o) or NO3 (e). Bar represents + I pop. std. dev. (n = 2). Symbols are 22 pop. std. dev. (n = 2) (from Thompson et al. 1989).

150

200 2

250

- 1

photon m- s

)

Fig. 8. Percentages of the two fatty acids: 20: 5w3 (A), and 22: 6o3 (B), for three microalgae, Isochrysis (), Chaetoceros(A), and Thalassiosiragrown on NO3 (). Cells were harvested at mid-log phase after at least 9 generations of growth. Bars represent the range of values obtained from duplicate cultures of Thalassiosiragrown at 44 mol photon m-2s-1.

50 higher irradiances (Fig. 5). The amount of lipid per cell showed no consistent trend over a range of irradiances for all three microalgae (Fig. 6). Growth irradiance also influences cell size (Fig. 7). Although the universality of the phenomenon is not known, the cell volume of Thalassiosira at low light (9 jmol photon m-

2

s - ) was only one-quarter the cell volume at

high light (87 mol photon m- 2 - 1). The effect of growth irradiance on two nutritionally important fatty acids is shown in Fig. 8A,B. The amount of 20: 5 o3 as a function of light intensity was relatively constant for all three microalgae. The amount 22: 6co3 was low but relatively constant over a range of growth irradiances for Chaetoceros and Thalassiosira,but minima in 20: 5o3 and 22: 6co3 were observed at 80-125 /mol photon m - 2 s-1; 22:6ro3 increased markedly as growth irradiance increased for Isochrysis (Fig. 8B).

Discussion Changes in cellular, N: Si: P The nutrient that will ultimately limit phytoplankton growth (provided the culture does not become too dense and the cells become light-limited) depends on the ratio of the macronutrients C, N, Si and P in the culture medium and the chemical composition of the particular phytoplankton grown under nonlimiting conditions. For example, Table 1 shows several commonly used culture media for mass culture of algae and the variation in the C: N: Si: P ratio in these media.

Table 2 shows the variation in the normal chemical composition for several algae. Therefore it is important to realize that one phytoplankter may become N-starved in a particular culture medium while another phytoplankter may become Pstarved in the same medium. For example, if species A has a N: P ratio of 12: 1 and species B's ratio is 20: 1, growth of these two species in a culture medium with a N: P ratio of 15: 1 would result in growth of species A being terminated by N limintation and species B would become P-limited. The normal chemical composition of microalgae (composition under non-limiting conditions of light, nutrients, and CO2) can be altered by nutrient and light limitation and changes in temperature. In a study on the effect of low growth irradiances on the N : P ratios of 4 phytoplankters, Wynne & Rhee (1986) found the N: P ratio increased in Dunaliella tertiolecta, Thalassiosira pseudonana and Prymnesium parvum, but not in Phaeodactylum tricornutum. The increase in N: P was due primarily to an increase in nitrogen per cell. The N: P ratio was also affected by light quality (Wynne & Rhee, 1986) and the light/dark cycle (Rhee & Gotham, 1980). Table 3 shows the ratio of the maximum (Qmax) and minimum

(Qmin) amount of nutrient per cell for N, Si and P. For a particular species, the amount of P per cell can vary by 3 to 70 times. The amount of N per cell is intermediate with a range of 1 to 6 and Si per cell is least variable with a range of 1 to 4. It is of interest to note that freshwater diatoms have an order of magnitude more Si per cell than marine diatoms (Conley et al., 1989). Therefore most microalgae can continue to grow for several

Table 1. N: Si: P (by atoms) in commonly used seawater culture media. Culture medium

N: Si: P (by atoms)

Reference

f/2 ESNW IMR MET 4 Aquil Enriched seawater SWM

24: 2.7: 1 25 : 4.8 : 1 10:10:10:1 15.5: 13.8: 1 10:1.2:1 10:1:1 20:5:1

Guillard (1975) Harrison et al. (1980) Eppley et al. (1967) Sch6ne and Sch6ne (1982) Morel et al. (1979) Vonshak (1986) McLachlan (1964, 1973)

51

cell generations after they run out of phosphate in the culture medium, but growth of diatoms stops quite suddenly after silicate becomes depleted in the medium. Results from nitrogen limitation are intermediate between Si and P limitation. If phosphate is added back to the depleted medium, cells can take it up 3 to 64 times faster than the rate required for maximum cell growth (Table 3) until the P debt has been overcome and the amount of P per cell reaches Qmax. Again, nitrogen is inter-

mediate and silicate uptake is relatively slower by Si-starved cells when it is added to the culture medium (Parslow et al., 1984).

Effects of temperature on chemical composition Although temperature experiments were not conducted in this study, temperature may be equally as important as nutrients and light in its effect on algal chemical composition. The effect of temperature on chemical composition appears to be species specific (Goldman, 1977; Yoder 1979). Temperature affects fatty acid composition, with a decrease in temperature resulting in an increase in essential fatty acids (Ackman etal., 1968; Mortensen et al., 1988).

Table 2. Examples from the litarature of variations in C: N: Si: P ratios (by atoms) for marine and freshwater phytoplankton. Species Diatoms Asterionellaformosa Asterionella glacialis Bacteriastrumfurcatum Chaetoceros affinis Chaetoceros convolutus Chaetoceros debilis Corethron criophilum Coscinodiscusgranii Fragilariacrotonensis Leptocyclindrus danicus Melosira binderana Nitzschia sp. 1 Phaeodactylum tricornutum Rhizosolenia alata Skeletonema costatum Stephanopyxis palmeriana Thalassionema nitzschioides Thalassiosiragravida Thalassiosirapseudonana Thalassiosirapseudonana Thalassiosira weisflogii Others Dunaliella tertiolecta Pavlova lutheri Pavlova lutheri Pavlova lutheri Pavlova lutheri Prymnesium parvum

C: N

C: P

N: P

Si: P

C: Si

N: Si

12 7.5 5.9

9.1 7.1

1.2

9.1 5.8 7.1 2.8

1.9 1.8 0.9 0.5

12.5

1.6

12.5

0.5

7.1 9.4 5.3 2.4 6.3

1.2 1.9 0.7 0.2 2.3

20 14.3

3.6 2.0

24 4.6 3.2 7.9 6.1

39

12

6.7

25 7.4 7 24.3 37 5.5 5.1 7.9 10.4 2.7

51

10

5.4

24

8.8 33

3.8

5.5 7.5

17

240

12 13.3 40 14 45 30

Reference

Rhee (1982) Brzezinski (1985) Brzezinski (1985) Rhee (1982) Brzezinski (1985) Harrison ert al. (1977) Brzezinski (1985) Brzezinski (1985) Rhee (1982) Brzezinski (1985) Rhee (1982) Brzezinski (1985) Wynne & Rhee (1986) Brzezinski (1985) Harrison et al. (1977) Brzezinski (1985) Brzezinski (1985) Harrison et al. (1977) Wynne & Rhee (1986) Brzezinski (1985) Brzezinski (1985)

Wynne & Rhee (1986) Tett et al. (1985) Terry (1980) Goldman (1986) Rhee (1982) Wynne & Rhee (1986)

52 Table 3. Ratios of the maximum amount of nutrient per cell or cell quota (Qmax) to the minimum cell quota (Qmin) and the

maximum uptake rate of the limiting nutrient (Vm) to the maximum growth rate (m) for marine and freshwater phytoplankters grown in cultures in which N, Si or P was the limiting nutrient. Species

Vm/#mb

Qmax/Qmina

N Marine Thalassiosirapseudonana

Si

P

N

Reference

Si

P

2.7 4 5 3

0.5 1

8

Thalassiosira weissflogii

5 15

3

50

1-2 1.3

Dunaliella tertiolecta 5 17 30 4

Pavlova lutheri 2 Phaeodactylum tricornutum Chaetoceros simplex Chaetoceros debilis Skeletonema costatum Thalassiosiragravida Freshwater Diatoma elongatum Asterionellaformosa Asterionella formosa Cyclotella meneghiniana Anabaenaflos-aquae Ankistrodesmusfalcatus Asterionellaformosa Fragilariacrotonensis Microcystis sp. Scenedesmus sp. Scenedesmus sp. Peridinium cinctum a

b

4 3-4 6

3 1-2 1-2

4 2

2 3 3

15 6 2 2 4

28 28 70 6 6 5 2 8 6 4

3

2 1.5 1.5

0.5

6 16 1.5

3

3

40 40 64 3 11 6

Paasche (1973) Eppley & Renger (1974) Goldman & McCarthy (1978) Parslow et al. (1984) Goldman & Glibert (1982) Laws & Bannister (1980) Goldman & Glibert (1982) Goldman & Peavey (1979) Droop (1975) Goldman (1979) Terry (1980) Goldman & Glibert (1982) Goldman & Glibert (1982) Harrison et al. (1977); Conway & Harrison (1977)

Kilman et al. (1977) Peterson Holm & Armstrong (1981) Tilman & Kilham (1976) Tilman & Kilham (1976) Gotham & Rhee (1981a, 1981b) Gotham & Rhee (1981a, 1981b) Gotham & Rhee (1981a, 1981b) Gotham & Rhee (1981a, 1981b) Gotham & Rhee (1981a, 1981b) Gotham & Rhee (1981a, 1981b) Rhee (1978) Elgavish et al. (1980)

Estimates from a range of dilution rates or over 24 to 72 h nutrient starvation. Estimates of Vm are made over an incubation period of 5 min or longer and at the lowest dilution r or the optimum starvation period.

Effects of nutrient starvation on chemical composition In this study, N starvation increased% carbohydrate, and decreased% protein, while% lipid remained relatively constant. These results agree with several previous studies. When Shifrin & Chisholm (1981) N-starved 11 diatoms, lipid content generally increased only slightly, while carbohydrate clearly increased. Thomas et al. (1984)

also found that N deficiency increased carbohydrate yield in mass cultures of Isochrysis. Nitrate deficiency generally stimulates the synthesis of triglycerides composed largely of unsaturated and monounsaturated fatty acids, while the polyunsaturated fatty acids and glycolipids decrease with the polar lipids (Suen et al., 1987). In this study and an accompanying study (Calderwood, 1989), Tahitian Isochrysis increased its monounsaturated levels by almost 50% within 2

53 days of N starvation and a proportional drop occurred in polyunsaturate levels, largely due to reduced proportions of some co3-polyunsaturates. The proportion of the nutritionally important fatty acids 20: 5 o3 was constant under N starvation and agreed with results of Ben-Amotz et al. (1985) on Isochrysis. No change in lipid, protein or carbohydrates per cell in Si-starved diatoms was observed in this study. One possible reason why we did not see the previously reported increase in lipids, is because cell composition was assessed after only 6 h of Si starvation. An increase in lipids due to Si limitation in Cyclotella cryptica and Chaetoceros gracilis has been observed by several workers (Werner, 1977; Enright et al., 1986; Vaulot et al., 1987; Roessler, 1988). The fatty acid composition of C. calcitransand T. pseudonanawere largely unaffected by nutrient deprivation in this study, except for a drop in 22: 6co3 in Si-starved C. calcitrans. Two recent studies (Enright etal., 1986; Mortensen etal., 1988) also observed a decrease in 20: 5(03 and 22: 6co3 in Si-starved C. gracilis. The effects of P starvation on fatty acid composition was species specific. Thalassiosiraexhibited a marked decrease in 20: 5o3 while Chaetoceros and Thalassiosira showed decreases in 22: 6co3 after 2 days of P starvation. This decrease under P starvation was larger than decreases under N or Si starvation. It is not surprising that P starvation would cause a decrease in long chained polyunsaturates since these fatty acids are found most commonly in the phospholipid fraction associated with cellular membranes. Aquaculturalists should ensure that microalgae are grown under P-replete conditions (i.e. N: P < 60: 1 by atoms for most species) in order to obtain maximum amounts of the nutritionally valuable polyunsaturated compounds such as 20: 5co3 and 22: 6o3.

Effects of light limitation on chemical composition Changes in total lipid per cell as a function of irradiance was relatively constant, but some spe-

cific fatty acids did vary with the growth irradiance, particularly 22: 6co3 in Isochrysis. Other work has shown that 22: 6o)3 may be redeployed to 14: 0 in low light grown Isochrysis (Thompson etal., in press). Indications of a minimum in 20: 5o3 and 22: 6ct3 at intermediate irradiances were seen in Thalassiosira and Chaetoceros. Recent results by Mortensen et al. (1988) on Chaetoceros gracilis demonstrated only small changes in fatty acid composition over a range of growth irradiances (83 to 1395 mol photon m - 2 s- ) which could be considered saturating to photoinhibitory. Using the same species, Thompson et al. (in press) found large changes in specific fatty acids over a range of growth irradiances (6 to 225/ mol photon m-2 S-) which represented growth-limiting to saturating light. For Chaetoceros simplex the proportion of 20: 5o3 was correlated with and increased by a factor of three with increasing irradiance (Thompson et al., in press). Whether variation in growth irradiance can be used to improve the nutritional value of phytoplankton that are used as food by the oyster, Crassostreagigas, is currently being tested in our laboratory. The change in the amount of essential fatty acids with growth irradiance appears to be species specific (Thompson et al., in press). Their results suggest that if larger amounts of essential fatty acids are required, species such as Chaetoceros simplex and Chaetoceros gracilis should be grown at lower growth irradiances, while Phaeodactylum tricornutum and Isochrysis galbana should be grown at growth saturating irradiances. Light quality can also affect chemical composition. In a review summarizing the work on a number of microalgae, Kowallik (1987) concluded that blue light generally increases protein content, while red light increases cellular carbohydrates. Growth irradiance also affects cell size. Since cells of Thalassiosirapseudonanagrown under low light are much smaller than high-light grown cells, this could have a significant effect on the total nutritive value of a diet if the animals are fed on fixed numbers of cells. Equal numbers of cells

54 containing one-quarter of the cellular volume may not have equivalent nutritive value to equal numbers of cells that are 4 x larger. Cell size changes may also affect filtration efficiency of herbivores which have a narrow cell size filtration tolerance.

Conclusions The growth of phytoplankton in large-scale batch cultures is usually terminated by nutrient limitation (N, P, Si) or slowed by light limitation when the culture becomes very dense. Since the chemical composition of the phytoplankton and hence, the nutritional value of the food for herbivores such as oyster larvae can change quickly as phytoplankton cultures become nutrient- or lightlimited, aquaculturalists must know when to harvest the phytoplankton in order to avoid these changes in food quality. Knowing the normal chemical composition of the nonlimited phytoplankter (e.g. C : N: Si: P by atoms) and the ratio of these elements in the growth medium, it is possible to determine which element will limit growth first, and what the cell yield will be when nutrient limitation commences. This has the added benefit of reducing the risk of culture crashes due to nutrient starvation. These examples of changes in protein, carbohydrate, lipid, or certain fatty acids under nutrient or light limitation, emphasize the importance of knowing how nutrient (N, Si or P) or light limitation affect the nutritional value of the phytoplankter, especially when it is used as a food source for bivalves and other herbivores.

Acknowledgements We acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada. GSC was supported by a graduate research assistantship from the Dept. Fisheries and Oceans, Canada and PAT was supported by fellowship (GREAT Award) from the British Columbia government.

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