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
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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
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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 ) .
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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 .
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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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