utting 1985 aquacultural-engineering

7/28/2019 Utting 1985 Aquacultural-Engineering

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Aquacul tural Engineer ing 4 (1985) 175-190

I n f l u e n c e o f N i t r o g e n A v a i l a b i li ty o n t h e B i o c h e m i c a lC o m p o s i ti o n o f T h r e e U n i c e ll u la r M a r i n e A l g a e o fC o m m e r c i a l I m p o r t a n c e

Susan D . UttingMinistry of Agriculture, Fisheries and Food, Directorate of Fisheries Research,

Fisheries Experiment Station, Conwy, Gwynedd LL32 8UB, UK

A B S T R A C TChaetoceros calcitrans (Paulsen) Takano, Tetraselmis suecica ( K y l i n )B u t c h . a n d Isochrysis galbana Parke were grown in ar t i f i c ia l seawaterc o n t a in i n g e i th e r n o r m a l ( 9 . 8 m g a t o m s l it re -1 ) o r r e d u c e d ( 0 . 6 1 3 m gatom s l i tre -1) n it rogen. When g row n in the n i t rog en-de f ic ient m ed iumprote in decreased and carbohydrate increased in a l l three spec ies ; l ip id ,however , increased in Chaetoceros a n d Isochrysis but decreased inTetraselmis.

T h e b i o c h e m i c a l c o m p o s i t i o n o f Tetraselmis and lsochrysis s a m p l e dd u r i n g t h e p o s t - e x p o n e n t i a l p h a s e o f g r o w t h f r o m n o r m a l m e d i u m w a ss imi lar to tha t o f ce ll s grow ing exp one nt ia l l y in n i t rogen-de f ic ient m ediu m.Sa l in i ti e s o f 10 - 35 % o d i d no t a f f e c t p ro t e i n le ve ls in Tetraselmis orIsochrysis bu t ca rboh ydrate increased an d l ip id decreased in Isochrysisover t he sa li n it y range. Tem pera t ure s o f 18 - 25 C had l i tt l e e f f e c t on t hece l l com po s i t ion o f e i ther species .

The energy po t en t i a l l y availab le f ro m a un i t vo l um e o f Chaetoceros a n dIsochrysis cells f ro m norm al m ed i u m was sim i la r t o t ha t f ro m cell s g row nin n i t rogen-de f ic ient condi t ions in both spec ies . Tetraselmis h a d 3 4 % m o r eenergy available w he n grow n in norm al com pare d to n i t rogen-de f ic ientm e d i u m .

INTRODUCTIONUnicellular marine algae are commonly grown as a food source forcomme rcial ly valuable shellfish. Early investigations led to the selection

175 Crown Copyright, 1985.


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176 s . D . U t t i n go f h i g h l y s u i t a b l e f o o d s p e c i e s f o r b iv a lv e s ( W a l n e , 1 9 7 4 ) a n d m o r er e c e n t l y s i g n i fi c a n t i m p r o v e m e n t s in al ga l p r o d u c t i o n h a v e b e e n m a d ea t t h e F i s h e ri e s E x p e r i m e n t S t a t i o n , C o n w y , t h r o u g h c h a n g e s i n c u l t u r et e c h n i q u e s ( H e l m e t a l . , 1 9 7 9 ; L a i n g , 1 9 7 9 ; H e l m a n d L a i n g , 19 81 ) .

T h e b i o c h e m i c a l c o n t e n t o f a l ga e c a n v a r y w i th a ge o f t h e c u l tu r ea n d w i t h c h a n g e s in t h e e n v i r o n m e n t a l c o n d i t io n s ( L e w i n , 1 9 62 ;S t e w a r t , 1 9 7 4 ). P r o t e i n is t h e m a i n c o m p o n e n t o f ce ll s h a r v e s t e d d u r i n gt h e e x p o n e n t i a l p h a s e o f g r o w t h b u t t h e p r o t e i n is r e p l a c e d b y st o r a g ep r o d u c t s w i t h a c a r b o h y d r a t e o r l ip id b a s e a s n i t r o g e n b e c o m e s li m i t in gd u r i n g p o s t - e x p o n e n t i a l p h a s e s .

V a r i a t i o n s i n t h e s i li ca t e c o n c e n t r a t i o n o f t h e c u l t u r e m e d i u m , i nt e m p e r a t u r e , s a l i n i t y , l i g h t i n t e n s i t y a n d w a v e l e n g t h c a n a l s o c a u s ec h a n g e s i n t h e b i o c h e m i c a l c o n t e n t o f m a r i n e p h y t o p l a n k t o n ( M o rr is ,1 9 8 1 ; S h i f r i n a n d C h i s h o l m , 1 9 8 1 ) .

T h e s t a n d a r d p r o c e d u r e a t C o n w y i s t o h a r v e s t c u l t u r e s i n t h e e x p o -n e n t i a l p h a s e w h e n p r o t e i n c o n t e n t is h i g h. H o w e v e r , t h e r e is s o m ee v i d e n c e t o s u g g es t t h a t l o w p r o t e i n d i e t s a r e b e n e f i c ia l t o o y s t e r s a t

T A B L E 1Com position and P repara tion of Artificial Seawaterg l i t r e - I m g l i t r e - ~ p g l i t r e - 1

NaC 1 2 0 M n C 1 2 . 4 H 2 0 1-4MgSO 4.7H20 5 HaBOa . 0 - 6 2KNO3 1 bFeC la 0.54CaC l2.2H20 1 Thiam ine HC1 0-5NaHCOa 0.2 ZnC la 0.1aNaH2PO4 .H20 0.1NaSiO3.9H20 0-04

COC12.6H20 5Biotin 5Cobalamin 1CuC12.2H20 0-034

Distilled w ate r 1 litre.Medium buffered wi th 1 g Tr i s ( t r i shydroxymethy lam inomethan e) i t re -1 .Buffer prepared by dissolving 25 g Tris in 75 ml disti l led water, adjusting pH to7.1-7-3 with co ncen trated HCI and making the volume up to 100 ml.M edium autoclaved at 1.06 kg cm -2 fo r 20m in .Vitamins add ed a fter rema inder of med ium has been autoclaved.a A dde d to m ediu m after autoclaving to prev ent precipitation.b Chelated w ith NaE DTA in solution containing 0-3 mg FeC la : 0-05 mg NaED TAm1-1.


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Influence o f nitrogen availability on biochemistry o f ma rine algae 177certain stages of growth. Wilson (1979) found that O s t r ea ed u l i s L .larvae increased their grazing rate when fed l s o c h r y s i s g a l b a n a whichwere in the post-exponential phase of growth. Crassostrea virginicaspat had a higher weight and glycogen content when fed Thalass ios i rap s e u d o n a n a with a carbon/nitrogen ratio of 7.5-10.7 (Flaak andEpifanio, 1978).

In this investigation, which was the preliminary part of a study intothe effects of diet composition on the growth of oyster larvae and spat(Utting, in preparation), some of the standard Conwy culture tech-niques were changed in an attempt to produce algae with a low proteincontent.

Ch a e t o cer o s ca l c i t r a n s (Paulsen) Takano, Te t r a s e l m i s s u ec i ca (Kylin)Butch. and I s o c h r y s i s g a l b a n a Parke were grown in normal mediumcontaining 9.8 mg atoms N litre -1 and in a nitrogen-deficient mediumwith 0-613 mg atoms litre -1. Cells with significantly different p rote incontent were found. In addition the effects of harvesting cultures atvarious stages of the growth cycle and the effects of differing salinityand temperature regimes were studied using two of the genera Tetra-s e l m i s and I s o c h r y s i s .

METHODSC u l t u r e o f a l ga eStock cultures of C h a e t o c e r o s (Bacillariophyceae), T e t r a s e l m i s (Prasino-phyceae) and l s o c h r y s i s (Haptophyceae) were maintained in Erdschreibermedium (FOyn, 1934) and subcultured monthly. The experimentalmedium, an artificial seawater (Table 1), was a modification of thatformulated by Lewin and Busby (de Mort, 1970).To study the effec t of nitrogen deficiency, algae were grown in thenormal medium (9-8 mg atoms N litre -1) or in artificial seawater towhich only 0-613 mga tomsNl it r e -1 was added. Salinities over therange 10-35~oo were obtained by di lution o f the artificial seawater withdistilled water. Nutrien t concent rat ions were thus reduced, especially atthe lower salinities, but earlier studies showed that nitrogen at leastwould not have limited algal growth even at 10%o (S. D. Utting,unpublished work).

Algae were transferred from Erdschreiber medium to artificial sea-water one week before the start of the experiment. After the week of


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178 s. D. Uttingc o n d i t i o n i n g , c e ll s w e r e i n o c u l a t e d i n t o 2 5 0 m l fr e s h m e d i u m in 5 0 0 m lb o i l i n g f l a s k s t o g i v e i n it ia l c o n c e n t r a t i o n s o f 5 0 c e l ls 1 4 - 1 f o r Tetra-s e l m i s a n d 5 0 0 c e l l s # 1-1 f o r C h a e t o c e r o s a n d I s o c h r y s i s . C u l t u r e s w e r eu n ia l ga l b u t n o n - a x e n i c . I n c u b a t i o n t e m p e r a t u r e w a s 2 0 C + I C b u te x t e n d e d in o n e t r i al t o 1 8 - 2 5 C i n o r d e r to d e t e r m i n e t h e e f f e c t o ft e m p e r a t u r e o n th e b i o c h e m i c a l c o m p o s i t i o n o f T e t r a s e l m i s a n d I s o -ch r ys i s . C o n t i n u o u s i ll u m i n at io n w a s p r o v i d e d b y t w o 6 5 W w a r m - w h i t ef l u o r e s c e n t t u b e s . T h e i n c i d e n t l i gh t i n t e n s i t y a t t h e o u t e r s u r fa c e o ft h e c u l t u r e v e s s e l w a s 3 k l u x . C u l t u r e s w e r e a e r a t e d a t 1 l it re m i n -1w i t h f i lt e r e d a ir w h i c h w a s e n r ic h e d w i t h 2 % v / v c a r b o n d i o x i d e .

I n m o s t t r i a l s C h a e t o c e r o s w a s h a r v e s t e d a f t e r 2 d a y s , T e t r a s e l m i sa f t e r 4 d a y s a n d l s o c h r y s i s o n t h e f i f t h d a y t o e n s u r e t h a t c e ll s w e r et a k e n f r o m a si m i la r p h a s e o f g r o w t h (S . D . U t t i n g , u n p u b l i s h e d d a t a ) .W h e n t h e e f f e c t o f c u l t u r e a ge o n t h e c h e m i c a l c o m p o s i t i o n o f Tetra-s e l m i s a n d l s o c h r y s i s w a s b e i n g s t u d i e d , s a m p l e s o f c e ll s w e r e t a k e nf r o m d a y 1 t o d a y 1 3 o f th e e x p e r i m e n t .

A n a l y t i c a l techniquesC e ll c o n c e n t r a t io n w a s d e t e r m i n e d o n a m o d e l Z B C o u l t e r C o u n t e r t ow h i c h a v o l u m e d i s t r ib u t i o n p l o t t e r w a s c o n n e c t e d t o m e a s u r e c e llv o l u m e .

D r y w e i g h t o f c e ll s w a s f o u n d b y f il te r i n g 1 0 m l s a m p l e s o f c u l t u r ei n d u p l i c a t e u n d e r a v a c u u m n o t e x c e e d i n g 0 . 3 5 k g c m -2 o n t o W h a t m a nG F / C f i lt e r d i s c s w h i c h h a d p r e v i o u s l y b e e n a s h e d a t 4 5 0 C f o r 4 h .T h e d is cs w e r e w a s h e d w i t h 0 . 9 % ( w / v ) i s o to n i c a m m o n i u m f o r m a t e ,d r i e d a t 8 0 C f o r 4 8 h a n d t h e n w e i g h e d . A s h - f r e e d r y w e i g h t w a so b t a i n e d b y s u b t r a c t i o n a f t e r th e d i sc s h a d b e e n l e f t fo r a f u r t h e r 4 ha t 4 5 0 C in a m u f f l e f u r n a c e .

S u b s a m p l e s o f c u l tu r e w e r e t a k e n i n d u p l i c a t e f o r t h e e s t i m a t i o n o fp r o t e i n ( 1 0 m l ) , c a r b o h y d r a t e ( 1 0 m l ) a n d l i p id ( 5 m l ) . T h e a l ga e w e r ec o n c e n t r a t e d b y c e n t r i f u g a t i o n a t 3 0 0 0 r p m f o r 1 0 m i n a n d t h e s u p e r-n a t a n t w a s r e m o v e d . P r o t e i n w a s m e a s u r e d b y a m o d i f i e d m i c r o -K j e l d a h l t e c h n i q u e ( H o l l a n d a n d H a n n a n t , 1 9 7 3 ) a n d p r o t e i n c o n t e n tw a s a s s u m e d t o b e n i t r o g e n c o n c e n t r a t i o n X 6 - 2 5. C e ll s f o r c a r b o h y d r a t ee s t i m a t i o n w e r e r e s u s p e n d e d t o a v o l u m e o f 1 0 m l w i t h di st il le d w a t e ra n d c a r b o h y d r a t e w a s d e t e r m i n e d b y t h e a n t h ro n e m e t h o d ( S t r i c k la n da n d P a r s o n s , 1 9 6 8 ) .


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Inf luence of ni trogen availabil i ty on biochemistry of marine algae 179L i p id w a s e x t r a c t e d b y a d d i n g 2 m l o f 2 : 1 ( v : v ) c h l o r o f o r m / m e t h a -

n o l m i x t u r e a n d l ea v in g f o r 2 h a t 2 0 C in t h e d a r k . T o r e m o v e n o n -l ip id c o n t a m i n a n t s 0 - 4 m l o f 0 . 7 % ( w / v ) N aC 1 s o l u t i o n w a s a d d e d a n dt h e s a m p l e s l e f t o v e r n i g h t a t 4 C . T h e l o w e r p h a s e w a s th e n m a d e u pt o 2 m l w i t h c h l o r o f o r m a n d 1 m l o f th a t p h a s e r e m o v e d f o r t h ea n a l ys i s o f l i pi d b y t h e c h a r r in g m e t h o d o f M a r sh a n d W e i n st e in ( 1 9 6 6 ) .C h o l e s t e r o l w a s u s e d as t h e s t a n d a r d a n d t o t a l l ip i d w a s c a l c u l a t e d b ya s s u m i n g 8 0 # g c h o l e s t e r o l w a s e q u i v a l e n t t o 1 0 0 # g t o t a l li p id (B a r n e sa n d B l a c k s t o c k , 1 9 7 3 ) .

T h e b i o c h e m i c a l c o m p o s i t i o n h a s b e e n e x p r e s s e d a s a p e r c e n t a g e o ft h e t o t a l o r g a n i c c o n t e n t in s o m e c o m p a r i s o n s . T o t a l o rg a n ic c o n t e n tw a s e q u i v a l e n t t o t h e s u m ( in tag p e r 1 06 c e ll s) o f t h e p r o t e i n , c a r b o -h y d r a t e a n d l i p i d r e c o v e r e d i n t h e c h e m i c a l a n a l y s e s .

T h e i n o rg a n ic a n d t o t a l c a r b o n c o n t e n t o f c e lls w a s m e a s u r e d b yc o m b u s t i o n o f r e p l i c a t e 5 0 tal s u b s a m p l e s o f a lg a e a t 1 5 0 a n d 9 5 0 C ,r e sp e c ti ve l y , o n a B e c k m a n t o ta l o r g a ni c c a r b o n ( T O C ) a n a l y s e r m o d e l9 1 5 B . T h e s t a n d a r d c o n t a i n e d 1 0 0 tag c a r b o n m 1 -1 a n d w a s m a d e u p ind i st il le d w a t e r w h i c h h a d b e e n p h o t o o x i d i s e d f o r 4 h . O r g a n ic c a r b o nw a s f o u n d b y s u b t r a c t io n .

R E S U L T SE f f e c t o f nitrogen deficiencyC e l l s iz e a n d w e i g h t w a s g e n e r a l l y n o t a f f e c t e d b y a c h a n g e i n t h en i t ro g e n c o n t e n t o f th e c u l t u r e m e d i u m ( T a b l e 2 ) e x c e p t i n g th a t th ea s h - f r e e d r y w e i g h t o f C h a e t o c e r o s w a s h i g h e r i n t h e n i t r o g e n - d e f i c i e n tc u l t u r e s ( S t u d e n t ' s t - te s t , t = 2 - 8 4 , d e g r e e s o f f r e e d o m = 6 6 , p < 0 . 0 1 ) .T h e b i o c h e m i c a l c o m p o s i t i o n o f a ll s p e c ie s w a s , h o w e v e r , s i g n if ic a n t lya l te r e d . A t t h e l o w e r n it r o g e n c o n c e n t r a t i o n t h e p r o t e i n c o n t e n t p e rl 0 6 c e ll s w a s s i g n i f ic a n t l y l es s in a ll s p e c i e s a n d c a r b o h y d r a t e w a s h i g h e r( p < 0 - 0 0 1 ) . C h a e t o c e r o s a n d I s o c h r y s i s c e ll s c o n t a i n e d m o s t li p idw h e n g r o w n in n i tr o g e n -d e f ic i e n t m e d i u m ( p < 0 .0 5 a n d p < 0 . 0 2 ,r e s p e c t i v e l y ) w h i l e T e t r a s e l m i s c e ll s h a d a h i g h e r l ip i d c o n t e n t w h e ng r o w n in n o r m a l m e d i u m ( p < 0 - 0 0 1 ) . C o m p a r i s o n o f t h e m e a n v a l ue so f e a c h b i o c h e m i c a l p a r a m e t e r f o r e a c h s p e c ie s g r o w n i n n o r m a l o rr e d u c e d n i tr o g e n m e d i a g a v e t h e f o l lo w i n g t v al ue s :


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TABLE2

TSuueanBohmicCenoChooccraTramissucanso

sgbCueGow

inAcaMedumCann98mgaomsNe-1o06mgaomsNe-1(menvuonmbocuue

saed

OOO

Cooccan

Tamissuc

Isohysgbn

98mgoms06mgoms98mgoms06mgoms9Smgoms06mgoms

Nr-1

Nr-~

Nr-1

Nr-1

Nr-~

Nr-1

Cconao

/A-1(x1

15

Dywgmgp1cs00

Ahedywg

mgp1cs

00

Mencvumepm3

31

Poenagp1cs

70

Cb

aeagp1

cs

29

Lpdgp1cs

20

Cbnon

64

Nmbocuue

3

94

32

31

11

98

00

03

03

00

00

00

02

02

00

00

44

46

47

62

64

48

19

51

11

85

74

22

14

54

81

26

21

19

58

74

16

44

13

76

17

3

1

1

2

2


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Influence of nitrogen availability on biochemistry of marine algae 181Chaetoceros Tetraselmis Isochrysis

t Degrees f t Degrees f t Degrees ffreedom freedom freedomProtein 7.67 66 19-66 32 5.20 54Carbohydrate 9.25 66 17-88 32 4-70 54Lipid 2-03 66 4-65 32 2.52 54

Carbon/nitrogen ratios ranged from 4-49 to 7-60 in algae fromnormal medium to 10.75-15-39 in nitrogen-defic ient conditions as aresult of the change in biochemical content.E f f e c t o f c u l tu r e a g eExponential growth finished on day 8 in Tetraselmis cultures and onday 6 in lsochrysis. Unfortunately no measurements could be made on13 day old Isochrysis cultures because of a contaminant flagellate inthe cultures.

Protein decreased with age of culture in both species (Table 3). InTetraselmis carbohydrate and lipid replaced the protein, with carbo-hydrat e rising sharply between days 7 and 13. Lipid increased threefoldin Isochrysis cells during the exponential phase but after an initialincrease in carbohydrate from 22.8 to 34.5% of the total organicconten t, there was a decline in that fraction to 20L1% by day 7.

Tetraselmis from nitrogen-deficient conditions was comparable inorganic con ten t to cells from 13 day old cultures, where nitrogen wouldbe expected to be limiting, while cells from 1-4 day old cultures, withan expected high nitrogen conten t, compared with Tetraselmis grown innormal medium.T h e e f f e c t s o f s a l i n it y a n d t e m p e r a t u r eSalinity, over the range 10-35700, and culture temperatures of 18-25C,had less of an ef fect on the chemical composi tion of Tetraselmis andlsochrysis (Table 4) than had nitrogen depletion or harvesting cells atdifferen t stages during the growth cycle (Table 3).

The optimum salinity for Tetraselmis, measured by the highestpro tein/carbohydra te ratio, was 25~'oo. The biochemical composit ion of


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70

b3

TABLE3

EeoNoDcea

AoCueohPoenCb

aeaLpdCeaaPcaoh

TaOgcMaeaoTraemssucaasohysgbCue

Se

Ccomn(%

ooaogc

coen)

Nronconrao

(maomNre1

Aocuued

(98maomNre-1nay

98

06

1

4

7

1

Traemssuca

Isohysgb

Poen

Cb

ae

Lpd

Poen

Cb

ae

Lpd

71

27

71

65

63

95

60

12

11

24

16

83

71

15

13

57

32

61

40

47

23

38

28

35

21

20

30

11

21

34

26

57

14


9/16

T

4

EeoSny(13oaTmpu(12CothPenCb

eaLpdCenaaPna

othTaO

cMaeaoTamscsysb

%

Se

Cm

Sny%

Tmaue

(%oa

ogce1

12

2

3

3

1

2

22

g 5

Tamsc

en

57

50

67

64

62

51

63

63

5367

~

Cb

e

31

31

20

25

32

30

29

26

3328

~

Lpd

12

19

13

12

96

12

79

54

15

76

~

Isysb

Pen

56

46

48

40

48

52

63

51

48

-

~.

Cb

e

26

23

25

31

29

37

27

24

37

-

Lpd

28

21

27

29

23

12

19

15

25

-

o ta


10/16

184 S. D. Utting2 6

gu

- J

1 8 I2 0S a t i n t y % .

I~ 0

! 3 5tJ

o~o, Z5Lg

.t 3 I200 t ,O2 0S a l i n i ty , % .

Fig. 1. The relationship between thelipid content of lsochrysis galbana cellsand the salinity of the artificial seawatermedium, y= 26- 45- 0.1 8x (r=- 0.87 ,

degrees of freedom = 4, p < 0-05).

Fig. 2. The relationship between thecarbohydrate content of Isochrysis gal-bana cells and the salinity of the artificialseawater medium, y = 22-32 + 0.261x(r = 0-826, degrees of freedom = 4,

p < 0 . 0 5 ) .

T e t r a s e l m i s began to change o nly at 10 and 15~'oo but not to the sameexten t as occurred with nitrogen deficiency.

Protein in I s o c h r y s i s was similar over the salinities tested but lipiddecreased, r =- -0 .8 7 , degrees of fr eed om = 4, p < 0.05 (Fig. 1), andcarbo hydra te increased, r = 0-826, degrees of freedom = 4, p< 0.05(Fig. 2) at the higher salinities.

The biochemical content of T e t r a s e l m i s showed little variationbetween temperatures of 18 and 25C although at 22C protein was lessbut carbohydrate and lipid were more than might have been expectedfrom the measure ment s on cells grown at 20 and 25C. Protein decreasedin I s o c h r y s i s at the higher temperatures with a corresponding increasein carbohydrate and lipid. At 25C no growth occurred and the culturesdied within 3 days.Variation in biochemical conte ntRelationships between biochemical components were found in bothT e t r a s e l m i s and l s o c h r y s i s . As protein decreased ca rbohydr ate increasedin T e t r a s e l m i s , r = --0-977, degrees of freedom = 14, p < 0-001 (Fig. 3),while in I s o c h r y s i s lipid, r = --0-829, degrees of fr eedom = 12, p < 0.001(Fig. 4), and carbohyd rate, r =- -0 -5 48 , degrees of freedom = 12,p < 0.05 (Fig. 5), increased. There was no correl ation between carbo-


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Inf luence of ni trogen availabil i ty on biochemistry of marine algae 185- ~ 8 0

c

2 0 t,0 60C a r b o h y d r a t e , % o f total organ ic con ten tI80

F i g . 3 . T h e r e la t io n s h i p b e t w e e n p r o t e i n a n d c a r b o h y d r a t e c o n t e n t o f Tetraselmissuec i ca ,y = 8 9 - 6 2 - 1 - 0 2 4 x ( r = - 0 . 9 7 7 , d e g r e e s o f f r e e d o m = 1 4, p < 0 . 0 0 1 ) .

80

~ 6 co

t,C

2c

F i g . 4 .

8 O

u

~608

- . < .

I I I I1 0 2 0 3 0 4 0

Lipid,% of total organic contentThe r e l a t i onsh i p be t w een p r o -

t e i n a n d l i p i d c o n t e n t o f Isochrysisgalbana, y = 75-61 - 1-199x ( r = - -0-8 29,

deg r ee s o f f r eedo m = 12 , p < 0 . 001) .

t~

ZO 1 i 1I0 20 30 40

C a r b o h y d r a te , % o f t o t a l o r ga n ic c o n te n tFi g . 5 . Th e r e l a t ionsh i p be t w een p r o -t e in a n d c a r b o h y d r a t e c o n t e n t o fIsochrysis galbana, y = 73 . 46 - 0 - 84 3x( r = - 0 - 5 4 8 , d e gr ee s o f f re e d o m = 1 2,p < 0 .0 5 ).

h y d r a t e a n d l ip id w i t h e i t h e r s p e c ie s o r b e t w e e n p r o t e i n a n d l ip id inT e t r a s e l m i s .

T h e o n l y d a t a f o r C h a e t o c e r o s w e r e f r o m t h e n i t r o g e n s t u d y ( T a b l e2 ) . I n n o r m a l m e d i u m p r o t e i n w a s 5 8 % a n d c a r b o h y d r a t e 2 5 % o f t h eo r g a n i c c o n t e n t o f ce l l s b u t w h e n n i t r o g e n w a s l im i t in g t h e r e s p e c t iv ev a l u e s w e r e 3 2 % a n d 5 0 % . T h e s e d a t a w e r e s im i l a r t o re s u l t s fo rT e t r a s e l m i s ( F i g . 3 ) .


12/16

186 S. D. UttingE n e r gy c o n t e n tT h e e n e r g y c o n t e n t , a s j o u l e s t h e o r e t i c a l ly a v a il ab l e i f p r o t e i n , c a r b o -h y d r a t e a n d l ip id w e r e c o n v e r t e d i n t o e n e r g y w i t h 1 0 0% e f fi c ie n c y , w a sc a l c u l a t e d f o r C h a e t o c e r o s , T e t r a s e l m i s a n d l s o c h r y s i s g r o w n i n n o r m a la n d n i t r o g e n - d e f ic i e n t m e d i a w h e r e t h e m o s t s ig n i fi c an t d i f f e r e n c e s inb i o c h e m i c a l c o n t e n t w e r e fo u n d ( T a b le 5 ).

P r o t e i n c o n t a i n e d 4 7 - 7 4 % o f th e e n e r g y i n c e ll s g r o w n i n n o r m a lm e d i u m w i t h l ip id a c ti n g a s t h e s e c o n d m o s t i m p o r t a n t s o u r c e o fe n e rg y . In c e lls f r o m n i t r o g e n - d e f i c ie n t c u l t u r e s t h e a m o u n t o f e n e r g ya v a il ab l e i n p r o t e i n w a s v e r y si m il a r b e t w e e n s p e c ie s (2 8 - 3 3 % ) .C h a e t o c e r o s a n d T e t r a s e l m i s h a d m o r e e n e r g y b o u n d u p i n t h e c a r b o -h y d r a t e f r a c t i o n w h i l e l s o c h r y s i s h a d t h e m a j o r q u a n t i t y o f t h e e n e rg yin l i p id .

E x p r e s s e d o n a p e r u n i t v o l u m e ba s is C h a e t o c e r o s a n d I s o c h r y s i sg r o w n in t h e n i tr o g e n - d e f i c i e n t m e d i u m c o n t a i n e d r e s p e c t iv e l y 8 a n d6 % m o r e e n e r g y t h a n w h e n g r o w n in n o r m a l m e d i u m . H o w e v e r , T e t r a -s e l m i s c o n t a i n e d 3 4 % m o r e e n e r g y i f c u l t u r e d a t th e n o r m a l r a t h e r t h a nt h e r e d u c e d c o n c e n t r a t io n o f n i tr o g en .

TABLE 5Energy Content (J x 10 9 cell -1) o f Chaetoceros calcitrans, Tetraselmis suecica andIsochrysis galbana Gro wn in Normal Medium and N itrogen-deficient Medium.Species Nitrogen co nte nt

o f m e d iu m( m g a t o m slitre -1}

Potential energy (J 10 9 cell -1)Protein Carbohydrate Lip id Total

Chaetoceros 9.8 169.4 (56) a 52.5 (17) 81.6 (27) 303.5calcitrans 0.613 114-7 (33) 130.7 (38) 10 2 -8 30) 348.2Tetraselmis 9-8 4220.2 (74) 374.2 (6) 1146.0 (20) 5814.2suecica 0 .613 1268 .4 (28) 2539 .7 (56) 704 .9 (16) 4513 .0Isochrysis 9.8 290-1 (47) 96.3 (16) 231.9 (38) 618-3galbana 0.613 203.5 (32) 143.2 (22) 294.7 (47) 640-9a Figures in parentheses give the percentage of tha t fract ion to the total energyavailable.


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Influence o f nitrogen availability on biochemistry o f marine algae 187DISCUSSION

The most successful method of decreasing the protein content of expo-nentially-growing algae was by reducing the initial concentration ofnitrogen in the culture medium from 9.8 to 0-613 mg at om sl it re -1(Table 2). A similar effect was obtained by harvesting cultures in thepost-exponential phase when nitrogen would be expected to be limitinggrowth (Table 3). In both cases the change in organic composition ofall species under nitrogen-deficiency may have been caused by a reduc-tion in the rate of protein synthesis with the result that non-nitrogenousproducts, such as carbohydrate and lipid, accumulated.

Salinities of 10-35%o and temperatures in the range 18-25C did notcause significant differences in the protein con ten t of either T e t r a s e l m i sor I s o c h r y s i s cells (Table 4). Although protein in I s o c h r y s i s was similarover the salinities tested, there was a significant decrease in cellular lipid(Fig. 1) and a corresponding increase in carbohydrate (Fig. 2) possiblyto main tain the osmotic balance in the cells. Temperatures below 18Cwere not studied because it is doubt ful if a reduction in temperaturewould increase the non-proteinaceous fraction of the cells (Steeman-Nielsen and J~rgensen, 1968: Morris, 1981). Results for I s o c h r y s i sindicated that only by growing this species at temperatures close tothe limits of tolerance could a decrease in protein be effected (Table 4).Instability in the growth of I s o c h r y s i s at temperatures above 22C, asfound here and by Ukeles (1961), would make temperature manipula-tion an unsatisfactory way of reliably producing low protein cells ofthis species.

The response of the algae to low nitrogen stress was best observedin Te t r a s e l m i s and I s o c h r y s i s cultures grown over a period of up to13 days (Table 3). In both species the amount of protein decreasedbut Te t r a s e l m i s stored primarily polysaccharides in conditions unfavour-able for cell division whereas the carbohydrate pool in 4 day oldI s o c h r y s i s was converted into lipid by day 7. Carbohydrate has beenfound to act as an intermediate reserve in some algae (Antia et a l . ,1963; Marker, 1965; Werner, 1970) because time is required afternitrogen becomes limiting for the enzymes essential for lipid synthesisto be produced (Fogg, 1956). Consequently in any conditions whereprotein decreased in I s o c h r y s i s both lipid and carbohydrate might beexpected to increase (Figs 4 and 5). In similar conditions, Te t r a s e l m i s ,a member of the Prasinophyceae which store polysaccharides and


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188 S. D. Uttingp r e s u m a b l y d o n o t p r o d u c e e n z y m e s t o s y n t h e s i s e l ip id , m i g h t b ee x p e c t e d t o i n c r ea s e o n l y t h e c a r b o h y d r a t e c o n t e n t ( F ig . 3 ). I n d e e d ,T e t r a s e l m i s c e ll s f r o m c u l t u r e s w h i c h h a d b e e n i n t h e s t a t i o n a r y p h a s ef o r 3 w e e k s m a i n t a i n e d a n o r g a n i c c o n t e n t o f 2 6 % p r o t e i n , 64 % c a rb o -h y d r a t e a n d 9 % l ip i d ( S. D . U t t i n g , u n p u b l i s h e d d a t a ) .

C h a e t o c e r o s i n n i tr o g e n - d e f i c i e n t c u l tu r e s i n c r e a se d t h e c a r b o h y d r a t ec o n t e n t m o r e t h a n t h e l ip id ( T a b le 2 ) . T h e p r o t e i n / c a r b o h y d r a t e r a t ioo f 2 .3 o b s e r v e d i n n o r m a l m e d i u m c o n t r a s t e d w i t h a r a t io o f 0 .6 w h e nn i t r o g e n w a s r e d u c e d . M y k l e s t a d a n d H a u g ( 1 9 7 2 ) f o u n d a s i m i l a rd e c l in e , f r o m 2 t o 0 - 23 , in t h e p r o t e i n / c a r b o h y d r a t e r a t io o f C h a e t o c e ro sa f f i n i s w h e n n i t r o g e n w a s l i m i ti n g a n d g lu c a n , a c o m p l e x p o l y s a c c h a r i d e ,w a s s y n t h e s i s e d .

M a n i p u l a t io n o f t h e n i tr o g e n c o n c e n t r a t i o n o f t h e c u l tu r e m e d i u mw a s f o u n d t o b e a s i m p le t e c h n i q u e t o e f f e c t si g n if ic a n t d i f f e r e n c e s int h e p r o t e i n , c a r b o h y d r a t e a n d l ip id c o n t e n t o f th r e e s p e c ie s o f m a r i n ep h y t o p l a n k t o n . I n tw o o f t h e s pe c ie s , C h a e t o c e r o s a n d I s o c h r y s i s , t h ec h a n g e i n b i o c h e m i c a l c o n t e n t a l t e re d t h e e n e r g y p o t e n t i a l l y a va il ab lef r o m a u n i t v o l u m e o f c e l ls b y l es s t h a n 1 0% (T a b l e 5 ). T e t r a s e l r n i s ,h o w e v e r , c o n t a i n e d 3 4 % m o r e e n e r g y , e x p r e s s e d o n t h e s am e ba sis ,w h e n g r o w n in n o r m a l m e d i u m p r i m a r i l y d u e t o h ig h e r l ip id le ve ls( T a b l e 2 ) .

T h e t e c h n i q u e c o u l d b e o f u se i n c o m m e r c i a l h a t c h e r i e s i f f e e d i n gt ri al s p r o v e t h a t l o w p r o t e i n a lg a e s u s ta i n b e t t e r g r o w t h o f y o u n go y s t e r s t h a n h i gh p r o t e i n a lg a e. A n y m e t h o d s o f i m p r o v i n g t h e g r o w t ha n d d e v e l o p m e n t r a te s o f y o u n g o y s t e rs , th e r e b y r e d u c i n g t h e d u r a t i o no f d e p e n d e n c e o n c u l t u r e d a lg a e, m u s t b e o f fi n a n ci a l b e n e f i t .

R E F E R E N C E SAntia, N. J., McAllister, C. D., Parsons, T. R., Stephens, K. & Strickland, J. D. H.(1963). Furth er me asurem ents of primary pro duct ion using a large-volume

plastic sph ere, Limnol. Oceanogr. , 8, 166-83.Barnes, H. & Blackstock, J. (197 3). Estimation o f lipid in marine animals andtissues: detailed investigation of the sulphophosphovanillin m eth od for ' tot al 'lipids, J. exp. mar. Biol . Ecol. , 12, 103-18.Flaak, A. R . & Epifanio, C. E. (1978). D ietary protein levels and grow th of theoys te r Crassostrea virginica, Mar. B iol ., 45, 157-63.


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In f luence o f n i t rogen avai labi l i t y on b iochemis t ry o f mar ine a lgae 189F o g g , G . E . ( 1 9 5 6 ) . P h o t o s y n t h e s i s an d f o r m a t i o n o f f a ts in a d i a to m . A n n . B o t .

( N . S . ) , 2 0 , 2 6 5 - 8 5 .F~ yn , B . ( 1934 ) . Lebe nszyk l us , Cy t o l og i e und Sexual it~ 't de r Ch l o r op hyc ee

Cladophora suhriana Kfi tz ing , Arch . Pro t i s t enk . , 8 3 , 1 - 5 6 .He lm, M. M. & Laing , I . (1981 ) . C os t -e f fec t ive cul ture of m ar ine unice l lu la r a lgae.

In : Energy Conserva ti on an d U se o f Ren ewa b l e Energi e s i n t he B i o - indus t r ie s ,ed . F . V og t , Pe r gam on Pr e s s , O xf o r d and N ew Y or k , pp . 247 - 59 .

He lm, M . M. , La ing , I . & Jo nes , E. (1979 ) . C ul ture o f a lgae for l arval f i sh and shel l-f i sh rear ing , P ar t 1 , Th e de ve lo pm ent o f a 20 0 l i t re a lga l cu l ture vesse l a t Co nwy ,Fish. Res . Tech . Rep . , M A F F D i rect. F ish. Res . Lo w es t o f t , 53 , 1 - 7 .

H o l l and , D . L . & H ann an t , P . J . ( 1973) . A dden dum t o a m i c r oan a l y t i c a l s chem e f o rthe b iochemica l ana lys i s of mar ine inver tebra te l a rvae , J . M ar. Bio l . Assoc . UK ,5 3 , 8 3 3 - 8 .

Laing , I . (197 9) . Cu l ture o f a lgae for l arval f i sh and she l l fi sh rear ing , Par t 2 , Rec om -m e n d e d p r o c e d u r e s f o r t h e c u l t u r e o f Chaetoceros calcitrans, Fish. Res. Tech.Rep . , M A F F D i rect. F ish. Res . Lo w es t o f t , 5 3 , 8 - 1 2 .

Lew i n , R . A . ( 1962) . P h y s i o lo g y a n d B i o c h e m i s tr y o f t h e A l g a e , Academic Press ,N ew Y or k .

Mar ke r , A . F . H . ( 1965 ) . Ex t r ace l l u l a r c a r boh ydr a t e l i be r a t ion in t he fl agel la t el sochrysis galbana an d Prym nes i um parvum , J. M ar . B i o l. As soc . U K, 4 5 , 7 5 5 - 7 2 .

Marsh, J . B . & Wei ns te i n , D . B . ( 1966 ) . 'N o t e s on m e t ho do l o gy ' . S im p l e cha r ri ngm e t ho d f o r de t e r m i na t i on o f li p id s , J . L i p i d R e s . , 7 , 5 7 4 - 6 .Mor r i s , I . ( 1981) . Pho t osyn t he t i c p r oduc t s , phys i o l og i ca l s t a t e and phy t op l ank t ongr ow t h . I n : 'Phys i o l og i ca l ba ses o f ph y t o p l an k t o n ec o l ogy ' ( ed . T r ev or P l a t O ,Can. Bul l . Fi sh . Aq ua t . Sc i . , 2 1 0 , 8 3 - 1 0 2 .

M or t , C . L . de ( 1970) . The cu l t u r e and b i ochem i ca l ana lys is o f som e e s t ua r i nephytoplankton spec ies , PhD thes i s , Oregon Sta te Univers i ty , Corva l l i s .

M y k l e s ta d , S . & H a u g , A . ( 1 9 7 2 ) . P r o d u c t i o n o f c a r b o h y d r a t e s b y t h e m a r in ed i a t om Chaetoceros a f f in i s vat . wi l le i ( G r an ) H us t ed t . I . E f f ec t o f t he concen -t r a t i on o f nu t r i en t s i n t he cu l t u r e m ed i um , J . exp. ma r . Biol . Eco l . , 9 , 1 2 5 - 3 6 .

Shi f r in , N . S . & Chisholm, S . W. (19 81) . P hy top lan kto n l ip ids : in te rspec i f i c d i f fe r -ences and e f fec t s of n i t ra te , s i l i ca te and l ight -dark cyc les , , / . P h y c o l . , 1 7 , 3 7 4 - 8 4 .

Steem an-N ie l sen , N . & J~brgensen , E. G . (196 8) . Th e ad apta t ion of p lankto n a lgae.1 . Ge nera l par t , Phys i o l . P l an t . , 2 1 , 4 0 1 - 1 3 .

S t ew ar t , W. D . P . ( 1974) . Alg al Physiology . a nd Bioc hem is t ry , Blackwel l Sc ient i f i cPub l i ca t i ons , O xf o r d .

S t r i ck l and , J . D . H . & Pa r sons , T . R . ( 19 68) . A p r ac t ic a l han dbo ok o f s ea w a t e ranalysis , Bull . Fish. Res. Bd. Can. , 1 6 7 , 2 2 7 - 3 0 .

U ke l e s , R . ( 196 1) . Th e e f f ec t o f t e m p e r a t u r e on t he g r ow t h and sur viva l o f seve ra lmarine algal species , Biol . bull . mar. biol . Lab. , W ood s H ole , 1 2 0 , 2 5 5 - 6 4 .

Walne , P . R. (1974) . Cul t ure o f B i va lve M oll uscs . 50 Year s ' Exper i ence a t Co nw y ,Fi sh ing N ew s ( Boo ks ) L t d , Whi t e f ri a rs P re ss L t d , L ond on .


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190 S. D. UttingW er ne r, D . ( 1970 ) . P r odu c t i v i t y s t ud ie s on d i a t om cu l t u r e s, Helgolfinder wiss.

Meeresunters., 2 0 , 9 7 - 1 0 3 .Wi lson , J . H . ( 1 979 ) . O bse r va t i ons on t he g r az i ng r a t es and g r ow t h o f Ostrea edulis

L. l a rvae when fed a lga l cu l tures of d i f fe rent ages . J . exp. mar. Biol. Ecol., 3 8 ,1 8 7 - 9 9 .

utting 1985 aquacultural-engineering

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