Biomass production and variation in the biochemical profile (total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae

Aquaculture, 83 (1989) 17-37 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

17

Biomass Production and Variation in the Biochemical Profile (Total Protein, Carbohydrates, RNA, Lipids and Fatty Acids) of Seven Species of Marine Microalgae

M.J. FERNANDEZ-REIRIZ’, A. PEREZ-CAMACHO’, M.J. FERREIRO’, J. BLANCO’, M. PLANAS’, M.J. CAMPOS’ and U. LABARTA’

‘Institute Znvestigaciones Marinas, C.S.Z.C., Muelle de Bouzas, 6,36208 Vigo (Spain) ‘Znstituto Espaiiol de Oceanografia, Muelle de Animas, s/n, Apdo. 130, La Coruna (Spain)

(Accepted 27 October 1988)

ABSTRACT

Fernandez-Reiriz, M.J., Perez-Camacho, A., Ferreiro, M.J., Blanco, J., Planas, M., Campos, M.J. and Labarta, U., 1989. Biomass production and variation in the biochemical profile (total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae. Aqua- culture, 83: 17-37.

Seven species of marine microalgae (Paulooa lutheri, Zsochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, Phaeodactylum tricornutum, Rhodomonas sp. and Heterosigma akashiwo) were harvested at three phases in the growth curve and biochemical composition (total protein, carbohydrates, RNA, lipids and fatty acids) was determined. Carbohydrate and lipid contents increased with the development of the culture, while protein levels increased in the later phases of the culture in the case of diatoms and Rhodomonas sp. and decreased in I. galbana, P. lutheri and T. suecica. Saturated fatty acids and, to a lesser extent, monoethylenic fatty acids represent between 70 and 100% of the total fatty acids. Polyunsaturated fatty acids reached their highest values in the exponential phase in Rhodomonas sp. (30.99% ), in an early stationary phase in P. tricornutum (19.58%) and C. calcitrans (9.06%) and in a late stationary phase in I. galbana (12.89%), P. lutheri (4.24%) and T. suecica (18.58%). RNA levels never exceeded2.5%.

The daily production was calculated for each batch culture in three growth phases and was compared with the production of a semicontinuous culture maintained in the exponential phase. Rhodomonas sp., C. calcitrans and P. tricornutum showed a lower daily production under semicontinuous culture than in batch cultures, while I. galbana, P. lutheri, T. suecica and H. akashiwo showed the opposite. The daily production of each of the biochemical components also varied with the species.

INTRODUCTION

Marine microalgae constitute the main food source for bivalve molluscs. Considering that the success of a mollusc hatchery (expressed in terms of mor-

0044~8486/89/$03.50 0 1989 Elsevier Science Publishers B.V.

18 M.J. FERNANDEZ-REIRIZ ET AL.

tality, growth or fixation rates) depends on the availability of suitable nutrients (Walne, 1970; Watanabe and Ackman, 1974; Webb and Chu, 1982; En- right et al., 1986; Whyte, 1987), knowledge of the biochemical composition of the phytoplankton supplied as food is essential in order to establish an adequate diet for bivalve larvae and juveniles.

The specific nutrient requirements of mollusc larvae and spat are not com- pletely known. While some authors pointed out the role of carbohydrates and lipids (Enright et al., 1986 ) as well as some polyunsaturated fatty acids (Millar and Scott, 1967; Helm et al., 1973; Kanazawa et al., 1979; Ackman, 1982; Lang- don and Waldock, 1981; Langdon, 1982 ) , other authors (Webb and Chu, 1982 ) think that the importance of the food given to bivalve larvae and juveniles depends more on other nutrients (fatty acids, amino acids, monosaccharides, minerals and vitamins) than on the gross biochemical composition.

Although its biochemical composition is not the only factor to be considered when studying the nutritional value of a phytoplanktonic species, and other factors such as cell size, digestibility and even toxicity have been suggested as explanations for differences in the nutritional value among different species, knowledge of the biochemical composition of these species is essential in order to attain an optimal diet.

It has also been established that the biochemical composition of a given species of phytoplankton can be modified under different growing conditions (Webb and Chu, 1982; Fhbregas et al., 1985) and also changes according to the growth phases of the algae.

Studies on changes in the biochemical composition of phytoplankton during the different growth phases are not very common (Sakshang and Holm-Han- sen, 1977; Fernandez-Reiriz et al., 1983; Murado et al., 1985; Fabregas et al., 1985, 1986; Utting, 1985). Published data are usually referred to a given moment of the culture (Parsons et al., 1961; Ansell et al., 1964; Langdon and Waldock, 1981; Webb and Chu, 1982; Utting, 1985; Ben-Amotz et al., 1985, 1987; Whyte, 1987 and some others). In the case of the fatty acid composition, most of the published data describe a particular point in the growth curve ( Ackman et al., 1968; Chuecas and Riley, 1969; Watanabe and Ackman, 1974; Scott and Middleton, 1979; Waldock and Nascimento, 1979; Langdon and Waldock, 1981; Webb and Chu, 1982; Lubzens et al., 1985; Ben-Amotz et al., 1987). Only very few authors (e.g. Chu and Dupuy, 1980) have followed changes in the fatty acid content during the different growth phases of a culture.

In this paper we present data on the biochemical composition (lipids, fatty acids, proteins and carbohydrates) of seven phytoplanktonic species sampled at three different moments in the growth curve, in order to establish for each species the most appropriate growth phase from the point of view of its nutritional and energetic value for bivalve larvae and juveniles. The effect of the culture system (semicontinuous or batch culture) on the cellular biochemical composition is also discussed.

BIOMASS PRODUCTION AND COMPOSITION OF MARINE MICROALGAE 19

Seven species of marine microalgae (Paulova lutheri, Haptophyta - Hapto- phyceae; Isochrysisgalbanu, Haptophyta - Haptophyceae; Tetraselmis suecica, Chlorophyta - Prasinophyceae; Chuetoceros caZcitruns, Chrysophyta - Bacil- lariophyceae; Phueoductylum tricornutum, Chrysophyta - Bacillariophyceae, Rhodomonus sp., Cryptophyta - Cryptophyceae; and Heterosigma akashiwo, Chrysophyta - Raphydophyceae) have been studied. Some of these species are commonly used as food for marine animals while others, potentially interesting, are not normally used.

MATERIAL AND METHODS

Algal cultures The algae were cultured in 2-l flasks, in a temperature-controlled chamber

at 15°C; only H. akashiwo, due to its specific requirements, was cultured at 20°C. Continuous illumination was provided at 60 PE m-l s-’ by photosyn- thetically active radiation, salinity was constant at 35 ppm and air was provided at 5 l/min. The culture medium has been described by Walne (1966).

All cultures began with an algal inoculum of 2000 cells/ml, except for P. Zutheri and I. galbana cultures which, due to the small cell size, began with an inoculum of 10 000 cells/ml. Cell growth rates were followed with a Coulter counter TA or by direct microscopic cell counts with a Neubaneur camera. Four culture flasks were maintained for each species. The first flask was harvested during the exponential growth phase, the second in an early stationary phase (with duplication rates of 0.1 divisions/day or less) and the third during the late stationary phase (5 days after the first zero or negative duplication). The fourth flask was maintained as a reserve culture. The position in the growth curve of some additional samples is explained in the results.

Analytical methods One sample from each flask was filtered through fiberglass Whatman GF/

C filters, washed with 0.5 A4 ammonium formate and used for dry weight determination. The rest of the culture was centrifuged at 2000 g, washed with ammonium formate, freeze-dried and stored at - 30’ C under inert nitrogen atmosphere for later analysis. The samples were later homogenized with water in an ultrasonic vibrator Sonifier 250.

Lipids were extracted following a modification of the method of Bligh and Dyer (1959). Lipids were first extracted with chloroform-methanol (1: 2) and after centrifugation the sediment was extracted again with chloroform-methanol (2 : 1); both supernatants were then washed with chloroform-methanol- water (8 : 4 : 3 ) (Folch et al., 1957). Total lipid weight was determined gravi- metrically by evaporating the solvent of 200 ~1 of purified extract onto pre- weighed aluminium planchets on a slide warmer (60-80°C) (Rouser et al.,

20 M.J. FERNkNDEZ-REIRIZ ET AL.

1967). About 8 mg of lipids were then saponified with KOH (0.8 iV in 90% ethanol).

Fatty acid methyl esters were prepared by esterification with BF,-methanol (10% BF, in methanol) (W/U) and analysed on a gas chromatograph Varian, Vista 5000, equipped with a fused silica capillary column, SP-P 2330, 30 m length, 0.25 mm i.d., 0.20 mm standard film and split retention of 70: 1. The detector temperature was 250’ C, the injector temperature was 225 ’ C and the column temperature increased from 140°C to 210°C at a rate of 1.5”C/min, with 5 min of initial heating time and 0.34 min of final heating.

RNA was extracted following a modification of the method of Shibko et al. ( 1967). Homogenates were cooled to 0°C before adding a volume of 70% per- chloric acid (PCA); centrifugation was followed by two more extractions with 5% PCA. Sediments were hydrolysed with 1N NaOH for 15 h at 15 “C (Harris, 1967), RNA being hydrolysed to acid-soluble mononucleotides that stay in solution when the sample is again acidified with 70% PCA at 0’ C for proteins and DNA precipitation. After centrifugation, the sediment was washed twice with 5% PCA at 0°C. Ribose was determined in the supernatants by a modification of the orcinol method (Ogur and Rosen, 1950).

Protein was assayed as described by Lowry et al. (1951) after hydrolysis in 0.5 N NaOH for 24 h at 30’ C.

Total carbohydrates were quantified as glucose by the phenol-sulphuric acid method (Strickland and Parsons, 1968). C/N/H were analysed with an Ele- mental Analyzer Perkin-Elmer 240. Ash-free dry weight was determined by ashing at 550” C for 10 h after drying the samples at 110°C for 2 h.

RESULTS

Algal growth Cellular growth for each species is shown in Fig. 1. To simplify the study of

the algal growth curves, two regression lines were fitted to the growth data in the case of the flasks maintained through the stationary phase. A computer program applied to the data assumes that each curve can be divided into two growth phases, an exponential one fitted by a regression line and a stationary one with a constant cell concentration (horizontal line) (Fig. 2). These two lines show the least square sum not explained by the regression and the least square sum of deviations from the mean of Yin the stationary phase.

The species under study showed different growth dynamics. Some of them, P. tricornutum, C. calcitrans, T. suecica, and H. akushiwo, showed exponential and stationary phases with a short transition between them. Two linear functions can be fitted to the logarithms of the data; one fits the exponential growth data and its slope represents the growth rates, and a horizontal linear equation describes the stationary phase.

For other species a linear equation showing moments of rapid and moments


Paulova

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M.J. FERNtiNDEZ-REIRIZ ET AL. 22

LW

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of slow growth during the exponential phase does not fit so well. In these cases it is not easy to fit linear functions to the data, and the estimated exponential growth rates are smaller than the real exponential growth. However, from a practical point of view, we are interested in the estimated growth rate. 1. galbana, P. lutheri and Rhodomonas sp. show this type of growth curve.

Cell numbers during the stationary phase were quite constant, H. akushiwo being the only species to show an important fall in the number of cells at the end of this phase.

The exponential growth rates derived from the linear equations fitted to the data varied between 0.5 and 2.1 divisions/day. P. tricornutum showed the highest rate followed by T. suecica, P. lutheri, I. galbanu, C. calcitrans, Rhodomonas sp. and H. akushiwo. The estimated cell numbers during the stationary phase varied between 8.65x 10” cell/ml (I. gulbana) and 1.14~ lo5 cells/ml (H. akashiwo) .

Although linear functions were fitted to the growth curves only in the cases of the cultures harvested at the end of the stationary phase, these functions allow us to establish for each species the approximate position of the three samples.

The first sample (Pl ) was taken in all cases during the exponential growth phase; in I. galbana, P. lutheri, H. akushiwo and P. tricornutum it represents a late exponential phase whereas the first sample of C. calcitruns, T. suecica and Rhodomonus sp. was taken earlier in this phase.

The second and third samples (P2, P3) represent an early and late stationary phase. I. galbunu was sampled on days 4 and 10 from the beginning of the stationary phase, P. lutheri on days 1 and 10, P. tricornutum on days 2 and 6, C. calcitrans on days 2 and 7, Rhodomonas sp. on days 3 and 13, in the case of


T. suecicu the second sample was taken during the end of the exponential growth and the third sample after 4 days of the stationary phase, and for H. akashiwo the second and third samples represent 3 and 6 days of the stationary phase.

Coulter counter analysis of cell size, reported for three species, showed that T. sue&a and Rhodomonus sp. cell size decreases during the stationary phase. H. akashiwo cell size increases during the linear phases and decreases after- wards. In the case of H. akushiwo, the total cell volume in the culture is controlled mainly by the concentration of the cells and not by cell size, as can be deduced from the high correlation coefficient (r = 0.99) between total cell volume and cell concentration. Correlation coefficients for the other two species (r=0.46 and r=0.35) show that cell size controls the total volume of cells in the culture in some way.

Proximate cellular composition Table 1 and Fig. 3 illustrate the chemical composition (protein, carbohy-

drate, lipids and RNA) of the seven unicellular marine species assayed during three periods of the growth cycle.

P. Zutheri and I. gulbanu show a similar pattern of changes in their chemical parameters. The percentage of proteins decreased with the development of the culture, with maximum values during the exponential phase (43.00% and 39.97% respectively). Carbohydrate and total lipid increased according to the development of the culture up to levels of 53.10% and 48.35% for carbohydrates and 37.83% and 36.16% for lipids.

In H. akashiwo, maximal values of protein (27.27% ), carbohydrate (16.55% ) and lipid (49.96% ) were found during the exponential phase, with values clearly higher than the levels reached during the stationary phase.

With regard to Rhodomonas sp., the highest levels of protein were found in Rhodomonas 2, 44.64%. Lipid and carbohydrate increased with growth and reached maximal values in the late stationary phase, 28.94% and 40.24% respectively.

In the case of T. suecica, protein levels decreased from the exponential phase (40.85% ), carbohydrate accumulated during growth and accounted for 43.23%, while the percentage of lipids decreased after the exponential phase from 24.40 to 14.83%.

Both diatoms, P. tricornutum and C. calcitruns, showed a continuous increase in the percentages of proteins up to 17.51% and 14.01%, respectively. Carbohydrate reached 25.0 and 11.32% and lipids 38.75 and 10.35%. It is nec- essary to note the relatively high content of an unknown fraction in both diatoms, already reported by other authors (Ben-Amotz et al., 1987) for some phytoplanktonic species.

RNA levels, quantified for each species, never exceeded 2.5% of the organic weight of the algae and were quite similar during the different growth stages.

Variations in the carbohydrate/protein ratio (Table 2) showed an increase

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44.1

7 S

tati

onar

y phas

e 42

.27

C. c

akitr

ans

1 C

. cak

itran

s 2

C. c

akitr

ans

3

Pre

sen

t pape

r E

xpon

enti

al, 20

°C

Pre

sen

t pape

r E

arly

stat

ion

ary

Pre

aen

tpap

er

Lat

e sta

tion

ary

Par

son

s et a

l., 19

61

utt

ing,

1985

9.

8 mg N

/I

utt

ing,

1985

0.

613 m

g N/I

B

en-A

mot

z et a

l., 19

87

Wh

yte,

1987

E

xpon

enti

al

phas

e W

hy&

198

7 S

tati

onar

y phas

e

27.9

5 40

.85

37.0

1 14

.59

38.2

9 16

.42

68.2

4 75

.90

69.3

3 2.

60

46.5

3 11

.49

47.5

2 14

.01

48.6

1

36.1

0 31

.25

37.5

4

65.6

3 24

.40

23.9

0 16

.96

37.3

0 14

.83

118.

78

3.81

14

.46

18.6

4 34

.38

43.4

7 17

9.97

12.

60

53.1

6 8.

30

7.10

14

.50

12.3

0 15

.40

9.08

10

.35

0.17

11

.76

0.67

10

.35

1.01

8.

89

79.5

4 9.

58

7.10

4.

81

20.7

0 21

.57

26.9

5

61.8

6 12

.30

19.7

0 1.

29

51.7

0 37

.39

61.1

2 1.

56

44.5

6 43

.23

98.3

2 1.

67

6.59

19

.69

34.0

9

18.2

8 9.

20

10.5

5 29

.12

9.50

17

.91

67.0

0 19

.20

18.1

0 27

.40

57.7

0

43.6

2 26

.51

15.4

6

0.88

2.

42

1.45

11

.32

2.00

8.

36

15.6

8 9.

17

2.07

2.

61

49.2

0

0.18

0.

07

1.59

0.3

3 1.

87 0

.23

15.0

0 2.

98

7.43

2.07

0.

30

2.96

16

0.3

2.55

2.

56

3.60

16

3.9

3.83

2.

63

6.32

22

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4.49

15.3

9

0.00

5 0.

93

1.33

7.

5 0.

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0.98

2.

85

14.1

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13

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TA

BL

E

2

Car

boh

ydra

tes/

Pro

tein

s an

d C

/N

rati

os

for

the

seve

n

spec

ies

stu

died

P.lu

ther

i I.

gal

bana

H

. ak

ashi

wo

T.

suec

ica

Rho

dom

onas

sp

. P

. tr

icor

nutu

m

C.

calc

itra

ns

1 2

3 1

2 3

1 2

3 1

2 3

1 2

3 1

2 3

1 2

3

CH

/P

0.55

0.

36

3.31

0.

38

1.52

3.

63

0.60

0.

65

0.87

0.

30

2.56

2.

63

0.45

0.

65

1.04

0.

28

0.42

1.

43

0.93

0.

98

0.60

C/N

6.

35

5.67

16

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6.04

11

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18.7

2 3.

08

1.50

1.

34

2.96

3.

60

6.32

1.

02

5.03

6.

88

2.00

1.

21

5.31

1.

33

2.85

3.

97

r 0.

999

0.99

8 -

0.70

0 0.

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0.93

1 0.

956

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11


I Pavlova

120

100

60

60

40

20

0 6 11 20

100

60

60

40

20

0

% Chaetoceros

40

30

20

10

0 7 11 26 5 6 12

a Tetraselmls

x tsocht-ysis

7 14 26

% Rhodomonss

120

100

60

60

40

20

0 10 16 26

s Phaeodactylum

60

60

40

20

0

60

60

40

20

0 7 14 25 Day

Fig. 3. Biochemical composition (% organic matter) of the seven phytoplanktonic species studied. The days of harvesting are given for all the species.

with the age of the culture, related to an increase in the levels of carbohydrate as well as to a decrease in the proteins. In diatoms, however, with proteins increasing during the stationary phase, there were only slight differences among the values of this index although, in the case of C. calcitruns, the ratio decreased slightly from the exponential to the stationary phase. The C/N ratio showed the same pattern as the carbohydrate/protein ratio. There is a positive corre-

TA

BL

E

3 %

Fat

ty a

cid

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posi

tion

(a

s pe

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of t

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in s

even

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cies

of

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Fat

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P.

luth

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L.

galb

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H.

akas

hiw

o T

. su

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a R

hodo

mon

as

sp.

P.

tric

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12

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14:o

35

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19.7

4 15

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23.2

4 17

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02

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35

0.5

20.2

4 32

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29.8

9 16

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12.6

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36

16.3

1 16

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23.2

1 16

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4 36

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40.9

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.49

40.8

4 35

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62.4

6 62

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61.4

4 50

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48.0

8 32

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17.3

3 30

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26.4

21

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14.0

6 25

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26.8

7 26

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11.6

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0.91

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2.45

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77

3.32

2.

04

0.73

1.

22

1.52

1.

39

5.47

1.

74

0.51

5.

39

1.11

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16

18:

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64

10.3

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82

5.02

2.

49

2.71

0

2.96

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31

13.6

3 24

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35.6

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8.77

12

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18.1

6 19

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3.95

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2.19

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1.27

0

0 1.

30

1.62

2.

53

1.24

2.

61

3.52

2.

56

1.61

1.

27

0.49

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0.34

0.

68

18:

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0

0 0.

420

0 0

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0 18

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50

1.94

1.

56

3.64

0

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43

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5.

54

3.41

5.

20

3.86

2.

97

1.38

1.

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0.36

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43

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27

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11

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0.74

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3.36

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94

20 : 2

n6

0 0

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0 0

0 0

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22 :

ln9c

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0 0

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0

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5 57

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66.5

5 59

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44.4

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4 50

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1 53

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kz

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3 36

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20.8

0 24

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6 25

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34.0

6 40

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1 33

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4 P

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55

12.8

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37

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45

18.5

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9.06

14

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1 19

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11.3

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57

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0.71

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33

4.6

1.66

1.

77

1.8

5.59

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19

3.18

24

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23.6

8 g g F


lation between these two indexes, except for H. akushiwo and C. calcitruns, and this correlation makes it possible to use the C/N ratio as an expression of the variations in the carbohydrate versus protein content during the growth of the algae.

Fatty acids The fatty acid composition of the total lipids is given in Table 3. The major

fatty acids present in P. lutheri, I. galbana and H. akashiwo were 14 : 0, 16 : 0 and 16 : ln7. In these three species the ratio of polyethylenic acids to saturated and monoethylenic was higher in the last stages of the culture (Table 3). I. galbana presented a higher level of polyethylenic acid than P. lutheri. Long- chain polyunsaturated fatty acids (n3 PUFA) were also maximum in the samples taken during the late stationary phase, 1.5% in I. gulbuna and 9.25% in P. Zutheri, with lipid levels of 36.16% and 37.80%, respectively. Polyethylenic acids as well as n3 PUFA levels were minimal in H. akushiwo.

In the case of Rhodomonus sp. the major fatty acids were 14 : 0,16 : 0,18: ln9 and 18: 3n3 but the levels of each of them changes with the age of the culture: 18 : 3n3 was predominant in Rhodomonas 1, 14: 0 and 16 : 0 in Rhodomonas 2 and 18: ln7 in Rhodomonus 3. This species showed an increase of saturated fatty acids and a decrease of polyethylenic fatty acids as the culture grew. The maximal value of n3 PUFA (10.12% ) was reached in Rhodomonus 1 with lipid levels of 27.86%.

The major fatty acids in T. suecica were 14 : 0,16 : 0 and 18 : 1 in the exponential phase, 16: 0,18: 1 in the early stationary phase and 16: 0,18: 1 and 18: 3 in the late stationary phase. As the culture grew, saturated fatty acids decreased and polyethylenic acids increased; the highest value of n3 PUFA (5.88% ) was reached in T. suecica 3, which had a lipid level of 14.83%.

In the diatoms (P. tricornutum and C. calcitrans) the major fatty acids were 14 : 0,16 : 0,16 : 1 and 18: 1. The values of saturated fatty acids were quite constant in both species during the growth of the algae, with the highest values of polyethylenic acids in the early stationary phase. At that moment, levels of n3 PUFA were 8.70% in C. calcitruns and 16.01% in P. tricornutum, with lipid levels of 10.35% and 33.75%, respectively.

Among the species studied, I. galbana, P. lutheri and T. suecica synthesized polyethylenic acids as the culture aged, and presented the highest percentages of these acids in the latest phases of the culture. This was not the case for diatoms, which showed maximum levels of polyethylenic acids in the early stationary phase, and for Rhodomonus sp. with highest levels during the exponential phase.

DISCUSSION

The results presented in this work show differences in the biochemical composition among species as well as among the different phases of the culture. It

30 M.J. FERNhDEZ-REIRIZ ET AL.

is not easy to compare data on the biochemical composition of phytoplanktonic algae. Differences in the culture conditions, in the analytical methods or in the growth phase sampled make it difficult to compare the results presented by different authors, and also to compare our results with those in the literature, as is shown in Table 1.

Changes in the biochemical composition (carbohydrate, protein and lipids) of the species under study along the different phases of the culture show two general trends; one is followed by the diatoms and Rhodomonus sp. and the other by the Haptophyceae and T. suecica.

Carbohydrate levels (expressed as percentages or as weight per cell, Figs. 3 and 4) increase as the culture develops in all the species studied except Het- erosigma. This trend corroborates the data by Utting (1985) for T. suecicu.

Protein levels increase in the later phases of the culture in the case of diatoms and Rhodomonas sp. and decrease in the Haptophyceae and T, suecica. Whyte (1987) found a similar trend for a species of Chuetoceros and a slight increase for Isochrysis, and Utting ( 1985 ) showed a large decrease in Tetrusel- mis and a slighter decrease in Isochrysis. This, in general terms, agrees with our results.

The lipid content shows a tendency to increase with the development of the culture in I. gulbanu, P. lutheri and Rhodomonus sp., with maximal values in a late stationary phase. In the case of T, suecica, H. akashiwo, P. tricornutum and C. calcitruns, the highest levels of lipids are reached in an earlier stationary phase.

As regards the fatty acid composition, as a general trend, saturated and monoethylenic fatty acids represent between 70 and 100% of the total fatty acids (Table 3 ), saturated fatty acids being the most abundant.

The development of the fatty acid content during the culture shows a decrease in the levels of saturated fatty acids in the Haptophyceae, in T. suecica and H. akashiwo and a decrease during phases 1 and 2 followed by a variable increase during the later stationary phase (phase 3) in the diatoms. Rhodo- monas sp. shows an increase in the levels of saturated fatty acids during the early stationary phase followed by a slight decrease during the stationary phase.

Polyunsaturated fatty acids are present in very small quantities except in the case of the diatoms and Rhodomonas sp. where they reach levels of 6-30% of the total fatty acids.

Given the importance of the polyunsaturated fatty acids and especially the n3 PUFA for marine organisms, and the need to incorporate them into the diet (Langdon, 1980; Langdon and Waldock, 1981; Webb and Chu, 1982), the nutritional value of a given species depends on its fatty acid composition. As a diet for marine organisms, the more interesting species are those with adequate levels of polyunsaturated fatty acids, such as I. galbana, P. lutheri and T. sue- &a. The relative amounts of different groups of fatty acids are also important;

BIOMASSPRODUCTlONANDCOMPOSITIONOFMARINEMICROALGAE 31

m/d Pavlova

50

40

30

20

III

0 6 11 20

w-n Hetereslgma

800

600

400

200

0 I4 17 26

pglwll Chaetoceros

6

6

4

2

0 7 11 20

pglwll Tetraselmls

M/WI1 laochrysts

50

40

30

20

IO

0 7 14 26

wan Rhodbmonss

200

150

100

50

0 IO 10 26

pg/wn Phaeodactglum

6

7 14 25 Da!!

Fig. 4. Biochemical composition (pg/cell) of the seven species of microalgae under study. The days of harvesting are given for all the species.

the n6/n3 ratio is optimal around l/2 and l/3 (Webb and Chu, 1982) as is found in I. galbana, P. lutheri and Rhodomonus sp.

The fatty acid composition of the different species and the changes in the fatty acid pattern during the growth cycle of the algae (Table 3) allow us to select species with a balanced fatty acid composition and harvest each of them in the appropriate growth stage. In this sense, H. akashiwo which has no n3

TA

BL

E

4

Dai

ly p

rodu

ctio

n

of t

he

diff

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t sp

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s of

ph

ytop

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ases

(S

C =

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Spe

cies

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ay

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ls

( x

10’)

Div

./day

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ells

/day

P

rote

ins

Lip

ids

CH

P

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ll)

(pdc

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) (p

dcel

l) bd

dw

)

P.

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8 11

:

I. g

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7 14

20

SC

H.

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17

20

S

C

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18

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S

C

C. c

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5 8 12

SC

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25

SC

2845

35

45

3545

3920

83

80

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0

157

129 84

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785

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30

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85.3

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36.7

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30.4

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78.8

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63.2

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216.

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36.2

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98.8

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6.91

6 20

6.98

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13.5

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PUFA does not seem to be a very interesting species. Both Haptophyceae ac- cumulate n3 PUFA during the latest growth stages and have the highest levels in the stationary phase, 1.5% in P. lutheri and 9.25% in I. galbana. T. suecica also shows maximum values of PUFA during the stationary phase. All three of them are interesting species from the point of view of their utilization as food in marine cultures, but they must not be harvested during the early phases af growth. Rhodomonas sp., a species which is not usually used as food for marine organisms, has a high percentage of polyunsaturated fatty acids during the exponential growth phase (10.12% ) and, in this sense, can be considered as a potentially interesting species, In the case of the diatoms, the optimum fatty acid pattern is reached during the early stationary phase.

Changes in the biochemical composition of a culture during the different growth phases must be conditioned in a complex way by a simultaneous ex- haustion of the nutrients, as has already been reported by Antia et al. (1963) and Myklestand and Hung (1972)) and by a progressive accumulation of me- tabolites in the medium. The increase in the C/N ratio indicates that the con- sumption of nitrates can be the main cause of the relative decrease in the synthesis of protein and the increase in the synthesis of storage products (Moal et al., 1978; Yull-Rhee, 1978, Piorreck et al., 1984; Wikfors et al., 1984; Gang et al., 1986; Whyte, 1987).

The total efficiencies of each culture were also notably different in relation to the culture techniques: daily cell production in continuous or semicontinuous cultures is generally higher than the production attained in batch cultures. Since the increase in cell production is not always related to the increase in each of the biochemical components (Fermindez-Reiriz, 1982)) the culture technique seems to be an important factor if we try to obtain an adequate food source.

The daily production of a batch culture has been calculated from the cell concentration in each phase and from its proximate biochemical production in order to estimate the efficiency of these cultures. Considering that the optimal production of a semicontinuous culture is obtained by keeping it in the exponential growth phase and maintaining the highest cell numbers, the production of this type of culture has been calculated in the different species from the growth rates in the exponential phase, considering as maximal cell concentration the value obtained when this phase was sampled (Table 4). We assume that the production of a continuous culture is similar to or a little higher than the production of semicontinuous cultures, and so we do not discuss the details of that type of culture.

Table 4 shows again two groups of species according to their total daily production. C. cakitrans and P. tricornutum have a lower daily production of all chemical components under semicontinuous conditions than in batch cultures harvested in the stationary phase. 1. galbana, P. lutheri and T. suecica as well

34 M.J. FERNhDEZ-REIRIZ ET AL.

as H. akashiwo show the opposite. Rhodomonas sp. shows a similar daily production under both conditions.

If we consider each of the biochemical components (Fig. 5), we can observe different trends. In a semicontinuous culture the daily production of carbohydrates decreases in all cases except in H. akashiwo, protein production increases in I. galbanu, P. lutheri and T. suecica and decreases in Rhodomonas

MJhg Pavlova r9/&9 lsochrgsis

400 500

300 400

300 200

200

IO0 100

0 0 0112OSC Day 7 I42Osc Day

Heteroslgma

----I #I

Rhodomonas

----I

8 L9,ti9 Phseoasctglum

80

80

40

20

0 7 11 2OsC Day 5 8 12 9c Dog

w-8 Tetreselmls

1000

800

800

400

200

0 7 1423s Deg

Fig. 5. Daily production @g/day in 2-1 flasks) in semicontinuous (SC ) and batch cultures of each of the seven species studied. The days of harvesting are given for all the species.


sp., C. calcitrans and P. tricornutum. Lipid production is higher in semicontinuous cultures in all species with the exception of P. tricornutum which shows very low lipid production under semicontinuous conditions. There are also differences in the percentages of fatty acids. Given the importance of some fatty acids in mollusc nutrition (Langdon, 1980; Langdon and Waldock, 1981; Ack- man, 1982; Enright and Newkirk, 1986), these differences have a substantial effect on the nutritional quality of any species of phytoplankton.

All these remarks indicate that different culture techniques lead to important differences in the final composition and quality of a culture, and therefore to different food qualities when these algae are used as food for the larvae and spat of bivalve molluscs.

ACKNOWLEDGEMENTS

We are grateful to Dr. J. Franc0 and J.L. Garrido for their technical assis- tance in the GLC analysis and to C. Ferna’ndez-Campos for her technical help. This work was supported by the CICYT-CSIC-IEO, project ID number 87061.

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Biomass production and variation in the biochemical profile (total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae

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