effect of growth rate on the eicosapentaenoic acid

7/28/2019 Effect of Growth Rate on the Eicosapentaenoic Acid

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A p p l M i c r o b i o l B i o t e c h n o l ( 1 9 9 4 ) 4 1 : 2 3 - 2 7 AppliedM icrobiologyBiotechnology Spr inger -Ver lag 1994

E f f e c t o f g r o w t h r a t e o n th e e i c o s a p e n t a e n o i c a c ida n d d o c o s a h e x a e n o i c a c id c o n t e n t o f I so c h r y si s g a l b a n ai n c h e m o s t a t c u l t u r eE . M o l i n a G r i m a , J . A . S f i n c h e z P ~ r e z , F . G a r c i a C a m a c h o , J . M . F e r n d n d e z S e v i ll a , F . G . A c i ~ n F e r n d n d e zD e p a r t a m e n t o d e I n g e n i e r f a Q u i m i c a, F a c u l t a d d e C i e n c i a s E x p e r i m e n t a le s , U n i v e r s i d a d d e A l m e r f a,E - 0 4 0 71 A l m e r f a , S p a i nR e c e i v e d : 1 J u n e 1 9 9 3 / R e c e i v e d r e v is i o n : 2 7 S e p t e m b e r 1 9 9 3 / A c c ep t e d : 3 0 S e p t e m b e r 1 9 9 3

A b s t r a c t . A n i s o l a t e o f Isochrysis galbana r i ch in e ico -s a p e n t a e n o i c a c i d ( E P A ) a n d d o c o s a h e x a e n o i c a c i d( D H A ) h a s b e e n g r o w n a s a c h e m o s t a t c u l t u r e a t 2 0 Ca n d p H 8 . 0 0 c o n t r o l l e d b y C O 2 i n j e c t i o n . F r o m a l o wd i l u t i o n r a t e (D ) o f 0 .0 0 2 4 h -1 t o 0 . 0 3 7 7 h - l , c l o s e t om a x i m u m g r o w th , a d e c r e a se i n E P A c o n t e n t f ro m5 .2 1 % d ry w e i g h t ( d .w . ) t o 2 .8 0 % d .w . w a s o b s e r v e d ,a l t h o u g h t h e p e r c e n t a g e o f E P A i n t h e t o t a l f a tt y a c id si n c r e a s e d . L i p i d s w e r e f r a c t i o n a t e d , E P A b e i n g t h em a j o r f a t t y a c i d fo u n d i n t h e g l y c o l i p i d f r a c t i o n ,w h e r e a s i n t h e n e u t r a l l i p i d f r a c t i o n w e r e m a i n l y p a l -m i t i c a n d p a l m i t o l e i c a c i d s . A t t h e s a m e t i m e , t h e b i o -m a s s c o n c e n t r a t i o n a l s o d e c r e a s e d f r o m 1 0 15 m g - 1 - 1t o 2 02 m g . 1 - 1 o v e r t h e r a n g e o f D s m e n t i o n e d . N o n e -t h e l e s s , E P A p r o d u c t i v i t y h a d a m a x i m u m v a l u e o f1 5.2 6 m g ' l - l . d a y - 1 a t D = 0 .0 2 0 8 h - 1.

I n t r o d u c t i o nP o l y u n s a t u r a t e d f a t t y a c i d s ( P U F A s ) p l a y a n i m p o r -t a n t r o l e in h u m a n h e a l th , f r o m t h e p r e v e n t i o n o f h e a r ta n d c i r c u l a t o r y d i s e a s e s t o b r a i n d e v e l o p m e n t i n i n fa n -c y (K y l e 1 9 92 ) . S o m e s t r a i n s o f m i c ro a l g a e h a v e r e -c e n t l y b e e n p r o p o s e d a s a n a l t e r n a ti v e t o f is h o il a s as o u r c e o f t h e s e p r o d u c t s ( R a d w a n 1 9 9 1 ). T h e m a i n f a t -t y a c i d s s t u d i e d w e r e t h e n - 3 P U F A s , e i c o s a p e n t a e n o i ca c i d ( E P A ) a n d d o c o s a h e x a e n o i c a c i d ( D H A ) .I n th e p r o d u c ti o n o f E P A a n d D H A i n m i c ro a lg a e ,s e v e r a l f a c t o r s a f f e c t i n g t h e g r o w t h a n d b i o c h e m i c a lc o m p o s i t i o n o f th e c e l ls m u s t b e b a l a n c e d . C h o i c e o fs p e c ie s , g r o w t h c o n d i t i o n s a n d a g e o f t h e c u l t u r e d e t e r -m i n e t h e p r o d u c t i v i t y o f f a t t y a c i d s a s e x p r e s s e d i nt e r m s o f c u l t u r e v o l u m e a n d t i m e . I n t h is c o n t e x t , as t r a i n s e l e c t i o n p r o g r a m m e h a s b e e n c a r r i e d o u t w i t ht h e m a r i n e m i c r o a l g a Isochrysis galbana a n d g r o w t hc o n d i t i o n s t h a t e n h a n c e t h e E P A a n d D H A c o n t e n t s

Correspondence to : E . M o l i n a G r i m a

h a v e b e e n s t u d i e d , w i t h i m p r o v e d p r o d u c t i v i t y o f E P A(L6 p e z A l o n s o e t a l . 1 9 9 2 a , b ; M o l i n a G r i m a e t a l .1 9 9 3 ) . I n t h e p r e s e n t p a p e r , d a t a f r o m e x p e r i m e n t sa i m e d a t i n c r e a s i n g P U F A p r o d u c t i v i t y a r e g i v e n a n dt h e b i o c h e m i c a l c h a n g e s r e l a t e d t o g r o w t h r a t e a r e d is -c u s s e d .

M a t e r ia l s a n d m e t h o d sOrganism. T h e m i c r o a l g a u s e d w a s a n i s o l a t e ( l a b e l l e d I I - 4 ) s e -l e c t e d f r o m a m o n g 4 2 i s o l a t e s o f a s i n g l e s t r a i n o f L ga lbana i n ap h e n o t y p i c s e l e c ti o n p r o g r a m m e c a r r i e d o u t i n o u r l a b o r a t o r y t os e l e c t a n E P A - r i c h s t r a i n o f m i c r o a l g a e ( L 6 p e z A l o n s o e t a l .1992a , b ) .Growth cond i t ions . C u l t u r e s w e r e c a r r i e d o u t i n a 5 - 1 f e r m e n t o r( N e w B r u n s w i c k S c i e n t i f ic B i o f l o I I I ) t h a t w a s c o m p u t e r c o n -t r o l l e d . T h e c u l t u r e v e s s e l , h e a d p l a t e a n d c o m p o n e n t s o f t h eh e a d p l a t e c o u l d b e s t e r il i z e d b y a u t o c l a v in g .T h e c u l t u r e m e d i u m a n d s t e r i l iz a t i o n p r o c e s s e s w e r e a s d e -s c r i b e d b y M o l i n a e t a l. ( 1 9 91 ) . T h e c u l t u r e s w e r e c o n s t a n t l y i l lu -m i n a t e d w i t h f o u r O s r a m D u l u x E L ( 20 W ) f l u o r e s c en t l am p s a r -r a n g e d a r o u n d t h e c u l t u r e v e ss e l. T h e i n c i d e n t li g h t in t e n s i t y o nt h e c u l t u r e s u r f a c e w a s m e a s u r e d w i t h a M a v o l u x d i g i t a l m e t e r( G o s s e n ) i n W . m - 2 ( P h o t o s y n t h e t i c a l l y a c t i ve r a d i a t i o n1 W . m - 2 = 4 . 6 6 ~ E . m - 2 . s - 1 ) . C e ll c o n c e nt r a ti o n w a s d et e r-m i n e d b y a b s o r b a n c e a t 5 3 0 n m . E x p e r i m e n t s w e r e c a r r i e d o u t a ta te m p e r a t u r e o f 2 0 C , C O 2 - i n j e c t i o n - c o n t r o l l e d p H a t 8 . 00 , a i rs u p p l y a t 1 .5 1 . m i n - I s t e r i l i z e d w i t h 0 . 2 2- t ~ m M i l l i p o r e f i l t er s ,a n d 1 5 0 r p m a g i t a t io n .Analy t i ca l me thods . C h l o r o p h y l l s w e r e m e a s u r e d a c c o r d i n g t ot h e m e t h o d o f H a n s m a n n ( 1 9 73 ). C a r o t e n o i d d e t e r m i n a t i o n w a st h e s a m e a s t h a t u s e d b y W h y t e ( 1 9 8 7) . L i p i d s w e r e e x t r a c t e d b yt h e m e t h o d o f K o c h e r t ( 1 9 78 ) . F a t t y a c i d m e t h y l a t i o n w a s d o n eb y d i r e c t t r a n s e s t e r i f i c a ti o n w i t h a c e t y l c h l o r i d e : m e t h a n o l ( 1 : 2 0 )f o l l o w i n g t h e m e t h o d o f L e p a g e a n d R o y ( 1 98 4 ). T h e a n a l y s is o fm e t h y l e s t e r s w a s c a r r i e d o u t b y g a s c h r o m a t o g r a p h y u s i n g a 3 0 -m c a p i l l a r y c o l u m n o f f u s e d s i l i c a ( S P 2 3 3 0 , S u p e l c o , B e l l e f o n t e ,P a . , U S A ) , i n t e r n a l d i a m e t e r o f 0 .2 5 m m , 0 . 20 tx m s t a n d a r d f i l m ,s p l it r a t io 1 0 0 : 1 , a n d a f l a m e i o n i z a t i o n d e te c t o r . S i g m a L i p i dS t a n d a r d 1 8 9 - 15 , S u p e l c o r a p e s e e d o i l m i x t u r e a n d S u p e l c oP U F A - 1 p a t t e r n s w e r e u s e d f o r t h e d e t e r m i n a t i o n . N o n a d e c a n o i ca c i d w a s u s e d a s a n i n t e r n a l s t a n d a r d t o q u a n t i f y th e f a t t y a c idc o n t e n t i n b io m a s s d r y w e i g h t ( d . w .) . P r o t e i n s w e r e a s s a y e d a sd e s c r i b e d b y L o w r y e t a l . ( 1 9 5 1 ) .


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2 4L i p i d s w e r e f r a c t i o n e d i n a c h r o m a t o g r a p h y c o l u m n u s i n g2 5 0 - 4 0 0 m e s h s i li c a g e l a n d e l u t e d w i t h c h l o r o f o r m , a c e t o n e a n dm e t h a n o l a t 1 0 , 4 0 a n d 1 0 t i m e s t h e v o l u m e o f b e d o f e a c h s o l -v e n t , r e s p e c t iv e l y ( K a t e s 1 98 6) . T h e v o l m n e o f e a c h s o l v e n t w a st h e n c o l l e c t e d a n d a n a l y s e d b y g a s c h r o m a t o g r a p h y a s in d i c a t e da b o v e .

R e s u l t s a n d d i s c u s s io nGrowth rateT h e d i l u ti o n r a t e ( D ) r a n g e d b e t w e e n 0 . 0 0 24 h - ~ a n d0 .03 7 7 h - ~ , g i v in g r i s e t o a d e c re a s e i n b i o m a s s c o n -c e n t r a t i o n f r o m 1 0 24 m g . 1 - a t o 2 0 2 m g - 1 - 1 . O n t h eo t h e r h a n d , b i o m a s s p r o d u c t i v i t y i n c r e a s e d w i t h D t o am a x i m u m p l a te a u o f 1 3 . 0 5 m g . l - ~ - h - 1 b e t w e e nD = 0 . 0 2 0 8 h - ~ a n d D = 0 . 0 2 9 h - a ( s e e F i g. 1 ). T h e s ev a r i a t io n s o f b i o m a s s a n d p r o d u c t i v i t y w i t h D a r e c h a r -a c t e r i s t i c o f li g h t - l im i t e d c o n t i n u o u s c u l t u r e s , wh e res t e a d y - s t a t e g r o w t h i s d e t e r m i n e d b y t h e a v a i l a b il i ty o fl i gh t , t h a t i s to s a y t h e a v e ra g e l i g h t i n t e n s i t y i n s i d e t h ec u l t u r e, w h i c h i s d e p e n d e n t o n t h e c e l l c o n c e n t r a t i o n( s e l f - s h a d i n g e f f e c t ) . T h u s , a s th e b i o m a s s c o n c e n t r a -t i o n i n c r e a s e s , t h e m e a n l i g h t i n t e n s i t y d e c r e a s e s a n ds o d o e s t h e g r o w t h r a t e .

Biochemical compositionT h e r e l a t io n s h i p o f b i o m a s s c o n c e n t r a t i o n , l i g h t a n dg r o w t h d e s c r i b e d a b o v e h a s a p a r a m o u n t i n f l u e n c e o nc e l l u l a r b i o c h e m i c a l c o m p o s i t i o n . A s c a n b e s e e n i nF i g. 2 a, t h e p i g m e n t c o n t e n t t e n d s t o d e c r e a s e a s D i n -c r e a s e s a n d t h e r e i s m o r e l ig h t a v a i l a b le f o r p h o t o s y n -t h e s i s , s o t h e n e e d f o r c h l o r o p h y l l a n d c a r o t e n o i d s i sl o w e r . T h i s is g e n e r a l l y o b s e r v e d i n c h e m o s t a t c u l t u r e so f p h o t o s y n t h e t i c m i c r o - o r g a n i s m s d u e t o t h e i n v e r s ev a r i a t i o n b e t w e e n p i g m e n t a n d l i g h t i n t e n s i t y ( H e r z i ga n d F a l k o w s k i 1 9 8 9 ). O v e r t h e d i l u t io n r a n g e a s s a y e d ,

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Fig . 2 a - c . B i o c h e m i c a l c o m p o s i t i o n c h a n g e s a t d i f f e r e n t d i l u t i o nr a t e s , a P i g m e n t s : x , c h l o r o p h y l l a ; @ , c h l o r o p h y l l c ; 2 x, c a r o t e -n o i d s , b T o t a l l i p i d s a n d p r o t e i n s : A , l i p i d s ; O , p r o t e i n s , c M a i nf a t t y a c id s : A , 1 4 : 0 ; . , 1 6 : 0 ; , 1 6 : 1 ; x , 1 8 : 4 ; 7 ?, 2 0 : 5 ; ~ ,2 2 : 6

c h l o ro p h y l l a d e c re a s e d f ro m 1 .5 9 % d .w . t o 0 . 6 5 % d .w .a n d c h l o ro p h y l l c f ro m 0 .3 9 % d .w . t o 0 . 1 1 % d .w . C a ro -t e n o i d s a l s o d r o p p e d f r o m 1 . 07 % d .w . to 0 . 29 % d .w .I n t h e s y n t h e si s o f b i o c h e m i c a l c o m p o n e n t s , c e ll st h a t h a v e a s h o r t d u p l i c a t i o n p e r i o d c o n t a i n m o r e p r o -t e i n s a n d l e s s l i p i d s i n t h e b i o m a s s t h a n wh e n t h e d u -p l i c a t i o n p e r i o d i s l o n g e r . T h i s i s s h o wn i n F i g . 2 b , i nw h i c h t h e p r o t e i n c o n t e n t i n c r e a s e s m a r k e d l y w i t h Da c c o m p a n i e d b y a s i g n i f i c a n t d e c re a s e i n l i p i d c o n t e n t .T h i s v a r i a t i o n i s r e l a t e d t o t h e s t ru c t u ra l f u n c t i o n o fp r o t e i n s a s w e l l a s t o t h e r o l e p l a y e d b y s o m e k i n d s o f ,m a i n l y n e u t r a l , l i p i d s i n s t o r a g e (W h y t e 1 9 8 8 ) .


3/5

25A s o l d c e l l s s y n t h e s i z e t h r e e fo l d m o re l i p i d s t h a ny o u n g c e l ls , t h e l i pi d c o n t e n t m a y v a r y f r o m 3 3 % d .w .a t D = 0 . 0 0 2 4 h - l i t o 1 1 % d . w . a t D = 0 . 0 3 7 7 h - 1 . T h i sc h a n g e a l s o a f f e c t s t h e f a t t y a c i d c o m p o s i t i o n o f li p id s .T a k i n g i n to a c c o u n t o n l y t h e m a i n f a t t y a c id s f o u n d i nt h e t o t a l s a p o n i f i c a b l e f r a c t i o n o f l ip i d s , in F i g . 2 c s a t u -r a t e d a n d m o n o - u n s a t u r a t e d a c id s , p a l m i t ic ( 1 6 : 0 ) a n d

p a l m i t o l e ic ( 1 6 : 1 ) , ( a b o v e 7 % d . w .) a r e o b s e r v e d t o b et h e m a j o r c o m p o n e n t s o f l i pi d s in c e ll s g r o w n a t l o wD s . T h e p o l y u n s a t u r a t e d 2 0 : 5 c o n t e n t W a s l o w e r ,a r o u n d 5 % d . w . H o w e v e r , a s D i n c r e a s e d , t h i s f a t t ya c i d p r o f i le c h a n g e d , a n d a t D = 0 . 0 2 0 8 h - 1 t h e m a j o rf a t t y a c i d w a s E P A ( 5 % ) , w h i le th e 1 6 : 0 a n d 1 6 : 1 c o n -t e n t s d r o p p e d t o 2 . 7 4 % a n d 3 . 3 6 % , r e s p e c t i v e l y . A th i g h e r D s , a l l t h e f a t t y a c i d s d e c r e a s e d , E P A r e m a i n -i n g t h e m a j o r f a t t y a c i d .A t t h e l o w e s t D t e s t e d , 1 6 : 0 a n d 1 6 : 1 r e p r e s e n t e d2 1 .6 7 % a n d 2 2 .1 7 % o f t h e t o t a l f a t t y a c i d s, r e s p e c t i v e -ly , E P A 1 5 .7 6 % a n d D H A 7 . 82 % . A t D = 0 . 0 04 8 h - l ,c e l l g ro w t h w a s a l s o s t r o n g l y l i m i t e d b y l i g h t , a n d as i m i l a r f a t t y a c i d p ro f i l e w a s fo u n d , w i t h a s l i g h t i n -c r e a s e i n 1 6 : 0 , 1 6 : 1 a n d E P A , a l t h o u g h t h e d r y w e i g h to f to t a l f a t t y a c i d d e c r e a s e d . T h e m o s t i n t e r e s t in g f e a -t u r e a p p e a r s w h e n t h e f a t t y a c i d p r o f i l e a tD = 0 . 0 0 4 8 h - 1 i s c o m p a r e d t o D = 0 . 0 2 0 8 h - 1. T h ep e r c e n t a g e o f 1 4 : 0 i n c r e a s e d s l i gh t ly , b u t t h e 1 6 : 0 a n d1 6 : 1 p e r c e n t a g e s d r o p p e d t o 1 5 . 4 9 % a n d 1 9 . 0 4 % , r e -s p e c t i v e l y , a c c o rd i n g t o t h e d e c re a s e o b s e rv e d i n t h e i rd r y w e i g h ts . O n t h e o t h e r h a n d , E P A i n c r e a s e d t o2 7 . 9% o f th e t o t a l f a t t y a d d s , a s it s d r y w e i g h t c o n t e n tr e m a i n e d a p p r o x i m a t e l y c o n s t a n t . A t t h e h i g h e r D s o f0 .0 29 h -1 a n d 0 .0 37 7 h -1 t h e f a t t y a c i d p ro f i l e w a sf o u n d t o b e t h e s a m e i n s p it e o f t h e l o w e r t o t a l l i pi dc o n t e n t o f th e b i o m a s s .T h e v a r i a t i o n d e s c r i b e d i s r e l a t e d t o t h e a b o v e -m e n t i o n e d c h a n g e i n p ro t e i n s. A t l o w g r o w t h r a t es ,s t o r a g e l i p i d s (1 6 : 0 , 1 6 : 1 ) s e rv e d a s a s i n k fo r p h o t o -s y n t h e t i c a l l y f i x e d c a rb o n a s t h e n e e d fo r s t r u c t u r a lb i o m o l e c u l e s w a s r e d u c e d . T h i s w a s r e f le c t e d i n t h el o w a m o u n t o f p r o t e i n s s y n t h e s i ze d . N o n e t h e l e s s , a sg ro w t h w a s l i g h t - l i m i t e d t h e c e l l s h a d t o m a i n t a i n as u b s t a n t i a l p h o t o s y n t h e t i c a p p a r a t u s ( c h l o r o p l a s t s ) i no rd e r t o c o l l e c t t h e s c a r c e l i g h t a v a i l a b l e . Th u s , s t r u c -t u r a l l i p i d s , m a i n l y t h o s e a l l o c a t e d t o c h l o ro p l a s t m e m -b r a n e s , w h i c h a r e h i g h l y u n s a t u r a t e d ( K a t e s a n d V o l -c a n i 1 9 6 6 ) , w e re s t i l l f o u n d i n h i g h p ro p o r t i o n . Th i sc o u l d b e t h e r e a s o n w h y t h e E P A c o n t e n t w a s a p pr o x -i m a t e l y c o n s t a n t f r o m D = 0 .0 0 2 4 h -1 t o 0 . 0 2 0 8 h -1T h e t o t a l l i p i d s i n t h e b i o m a s s o b t a i n e d a tD = 0 . 0 0 4 8 h - 1 a n d D = 0 . 0 2 9 h - 1 w e r e s e p a r a t e d i n t ot h r e e m a i n f r a c t i o n s : n e u t r a l l i p i d s , g l y c o l i p i d s a n dp h o s p h o l i p i d s . I n F i g . 3 , t h e v a r i a t i o n s i n c l a s se s o f l ip -i d s a re p r e s e n t e d i n p e r c e n t a g e s o f t o t a l s a p o n if i a b l el ip i ds a n d p e r c e n t a g e s o f d r y w e i g h t. T h e p r o p o r t i o n o fn e u t r a l l ip i ds i n b i o m a s s g r o w n a t 0 . 0 0 4 8 h - 1 w a s6 7 .5 % , w h i c h c o r r e s p o n d s t o 1 9 .1 % d .w . Th i s i s a p -p ro x i m a t e l y t h r e e t i m e s h i g h e r t h a n g l y c o l i p i d f r a c t i o na n d f i v e t i m e s h i g h er t h a n p h o s p h o l ip i d s . O n t h e o t h e rh a n d , i n b i o m a s s g r o w n a t 0 .0 2 9 h - l , t h e n e u t r a l l i p idp r o p o r t i o n d e c r e a s e d d o w n t o h a l f o f t h a t o b t a i n e d a t0 .00 4 8 h -1 , w i t h a c o n s e q u e n t i n c r e a s e i n g l y c o l i p id s

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D i l u t i o n r a t e , h 1Fig. 3. Distribution of lipid classes of cells grown at low and highdilution rate . Conten ts are give n as percentages of total fattyacids and as pe rcentage s of dry weigh t: [],n eut ral lipids; rN, gly-colipids; B, phospholipids

a n d p h o s p h o l i p i d s . T h e r e m a r k a b l e f a c t i s t h a t t h e d e -c r e a s e i n t o t a l l i p i d s s h o w n i n F i g . 2 b w a s m a i n l y d u et o t h e r e d u c t i o n i n n e u t r a l l i p i d s , a s t h e g l y c o l i p i d s r e -m a i n e d c o n s t a n t, a l t h o u g h t h e p h o s p h o l i p i d s a l s o d e -c r e a s e d , b u t t o a m u c h l o w e r e x t e n t .T h e f a t t y a c id c o m p o s i t i o n o f t h e s e p a r a t e d l ip i df r a c t i o n s i s g i v e n i n F i g . 4 . Th e m a i n f a t t y a c i d s fo u n di n t h e n e u t r a l l i pi d s w e re 1 6 : 0 a n d 1 6 : 1 . P o l y u n s a t u -r a t e d f a t t y a c i d s w e r e a l s o p r e s e n t a n d E P A w a s t h et h ir d , i n o r d e r o f p e r c e n t a g e . A t 0 .0 2 9 h - l , u n s a t u r a -t i o n w a s s o m e w h a t h i g h e r t h a n a t 0 . 00 4 8 h -1 w i t h as ig n i fi c an t in c r e a s e in D H A c o n t e n t ( f r o m 3 % t o1 0 % ) . E P A c o n s t i t u t e d m o r e t h a n 3 0 % o f t h e g ly c o li -p i ds , f o ll o w e d b y 1 6 : 0 , 1 6 : 1 a n d 1 8 : 4 i n m u c h l o w e rp r o p o r t i o n s . T h e o v e r a l l p r o f i l e d i d n o t c h a n g e b e -t w e e n t h e t w o D s . O n t h e c o n t r a r y , t h e p h o s ' p h o l i p i d sb e c a m e m o r e s a t u r a t e d i n f a s t - g r o w i n g c e l l s , 1 6 : 0 b e -i n g t h e m a j o r c o n s t i t u e n t . I n g e n e ra l , 1 4 : 0 w a s fo u n di n t h e t h r e e f r a c t i o n s a s a m i n o r c o m p o n e n t , i n p e r -c e n t a g e s r a n g i n g f r o m 5 % t o 1 2 % . B o t h 1 6 : 0 a n d E P Aw e re t h e p r i n c i p a l f a t t y a c i d s i n t h e p h o s p h o l i p i d f r a c -t i o n . A t t h e s a m e t i m e , E P A w a s f o u n d m a i n l y i n g l y -c o l i p i d s a n d ]6 : 1 i n n e u t r a l l i p id s .F r o m t h e a b o v e d a t a , i t m a y b e c o n c l u d e d t h a tw h e n t h e t a r g e t u s i n g a m i c ro a l g a l c u l t u r e s y s t e m is t oo b t a i n P U F A s , i t i s n o t w o r t h g r o w i n g c e l l s u n d e rs t ro n g l y l i m i t i n g c o n d i t i o n s fo r h i g h l i p i d c o n t e n t i nb i o m a s s , b e c a u s e i n c r e a s e d l i p i d c o m e s f r o m t h e s y n -t h e s i s o f s t o r a g e l i p i d s, w h i c h a r e s a t u r a t e d f a t s . O n t h eo t h e r h a n d , t h e r e i s a n i n v e r s e r e l a t i o n s h i p b e t w e e nl i p i d c o n t e n t a n d g ro w t h (F i g . 2 b a n d c ) , t h e b e s tg r o w t h r a t e n o t b e i n g t h e h i g h e s t . T h u s , a b a l a n c e dg r o w t h m u s t b e o b t a i n e d .W h e n t h e f a t t y a c i d c on t e n t s p r e s e n t e d h e r e a r ec o m p a re d w i t h t h o s e c o r r e s p o n d i n g t o t h e s a m e s t r a i na n d c u l tu r e s y s t e m d e s c r i b e d p r e v i o u s ly ( M o l i n a G r i m aet a l . 1993), an inv erse var ia t ion o f fa t ty ac id p ro f i l e ve t -


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P h o s p h o l i p i d s

whereas in the present experiments, carbon was sup-plied by CO2 injection into the medium to keep the pHconstant at 8.00. In practice, in the first set of experi-ments, the cells were CO2-1imited. This was evident inthe increase in pH up to 8.65, and in the l atter this lim-itation was prevented. Therefore, at low Ds, non-car-bon-limited cells are capable of synthesizing higheramounts of storage lipids than carbon-limited cells.Furthermore, the EPA content under both growingconditions at low Ds are analogous, around 5% d.w.,due to the effect of light on PUF A synthesis.Finally, the aim was to obtain high fatty acid pro-ductivity. EPA productivity varied with D, reaching amaximum of 15.26 mg .l - l . day -1 at D =0.0208 h-~.At this rate, neither EP A content (4.93% d.w.) norbiomass concentration (620 mg.1-1) were the highestobtained, but as the time factor is considered in pro-ductivity, the net EPA synthesis rate was maximal.Nevertheless, at D = 0.029 h-1, the biomass productivi-ty obta ined was the same as at D=0.0 208 h -~, al-though the biomass concentration was lower(450mg- 1-1) as was the EP A cont ent (3.19% d.w.).Thus, at D = 0.029 h - ~ effluen t from the culture vesselwas less concentrated, with a lower fatty acid content,making this D unfavorable in comparison toD =0.0208 h -1. This is a clear example of how condi-tions optimizing a bioproduct are generally differentfrom those that optimize: bi0mass productivity.Acknowledgement. T h i s r e s e a r c h w a s s u p p o r t e d b y a g r a n t f r o mt h e C o m i s i 6 n I n t e r m i n i s t e r i a l d e C i e n c i a y T e c n o l o g f a ( C I C Y T ) ,(BIO 91-0652) .

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F i g . 4 a - c . F a t t y a c i d p r o f i l e s o f e a c h l i p i d f r a c t i o n : I1 , 14:0 ; [ ] ,16 :0 ; [ ] , 16 :1 ; N , 18 :4 ; N , 20 :5 ; Nil , 22 :6 . a Ne u t ra l l ip ids , b G ly-c o l i p id s , c P h o s p h o l i p i d s

sus growth rate is found. It was reported that the per-centage of 16:0 and 16:1 over total fa tty acids increasedwith D, from 10.12% and 10.65% at D=0.006h -1 to17.64% and 16.07% at D=0 .0 37 7h -1, respectively,and the percentage of EPA decreased from 25.10% to22.87% for the same Ds. Wh at makes these sets of ex-periments different is that in Molina Grima et al.(1993), the culture received CO2 only by air bubbling,

R e f e r e n c e s

H a n s m a n n E ( 19 7 3) P i g m e n t a n a l y si s . I n : S t e i n J R ( e d ) H a n d -b o o k o f p h y c o l o g ic a l m e t h o d s , c u l t u r e m e t h o d s a n d g r o w t hm e a s u r e m e n t s . C a m b r i d g e U n i v e r s it y P r es s , C a m b r i d g e ,p 359H e r z i g R , F a l k o w s k i P G ( 19 8 9) N i t r o g e n l i m i t a t i o n i n Isochrysisgalbana ( H a p t o p h y c e a e ) . I . P h o t o s y n t h e t i c e n e r g y c o n v e r s i o na n d g r o w t h e f f ic i e n c ie s . J P h y c o l 2 5 : 4 6 2 - 4 7 1K a t e s M ( 19 8 6) T e c h n i q u e s o f l i p i d o l o g y : i s o l a t io n , a n a l y s is a n di d e n t i f i c a t i o n o f l ip i d s . 2 n d r e v i s e d e d n . I n : B u r d o n R H ,K n i p p e n b e r g P H v a n ( e d s ) L a b o r a t o r y te c h n i q ue s i n b i o -c h e m i s t r y a n d m o l e c u l a r b i o l o g y , v o l 3 , p a r t 2 . E l s e v i e r, A m -s t e r d a m , p p 1 9 0 - 1 95K a t e s M , V o l c a n i B E ( 19 6 6) L i p i d c o m p o n e n t s o f d i a t o m s . B i o -c h e m B i o p h y s A c t a 1 1 6 :2 6 4 -2 7 8K y l e D ( 19 9 2) P r o d u c t i o n a n d u s e o f li p i d s f r o m m i c r o a l g a e . L i p -i d T e c h n o l M a y - J u n e : 5 9 - 6 4K o c h e r t G ( 1 97 8 ) Q u a n t i t a t i o n o f t h e m a c r o m o l e c u l a r c o m p o -n e n t s o f m i c r o a l g a e . I n : H e l l e b u s t J , C r a g i e S (e d s ) H a n d b o o ko f p h y c o l o g i c a l m e t h o d s . P h y s i o l o g i c a l a n d b i o c h e m i c a l m e t h -o d s . C a m b r i d g e U n i v e r s i t y P r e s s , C a m b r i d g e , p 1 8 9L e p a g e G , R o y C ( 19 8 4) I m p r o v e d r e c o v e r y o f f a t t y a c i d t h r o u g hd i r e c t t r a n s e s t e r i f i c a t io n w i t h o u t p r i o r e x t r a c t i o n o r p u r i f i c a -t i o n . J L i p i d R e s 2 5 : 1 3 9 1 - 1 3 96L d p e z A l o n s o D , M o l i n a G r i m a E , S ~ i n c h e z P 6 r e z J A , G a r c f aS 4 n c h e z J L , O a r c i a C a m a c h o F ( 1 9 9 2a ) I s o l a t i o n o f c l o n e s o fIsochrysis galbana r i c h in e i c o s a p e n t a e n o i c a c i d . A q u a c u l t u r e102 : 363-371L d p e z A l o n s o D , M o l i n a G r i m a E , S 4 n c h e z P 6 r e z J A , O a r c f aS 4 n c h e z J L , G a r c f a C a m a c h o F ( 1 9 92 b ) F a t t y a c i d v a r i a t i o n


5/5

27a m o n g d i f f e r e n t i s o l a t e s o f a s i n g le s t r a i n o f I s o c h r y s i s g a l b a -n a . Phy toche mis t ry 31 : 3901-3904L o w r y O H , R o s e b r o u g h N J , F a r r A L , R a n d a l l R J ( 19 51 ) P r o t e i nm e a s u r e m e n t w i t h t h e F o l i n p h e n o l r e a g e n t . J B i o l C h e m193: 265-27 5M o l i n a G r i m a E , M a r t f n e z M E , S ~ i n c h e z S , G a r c f a F , C o n t r e r a sA ( 19 9 1) G r o w t h a n d b i o c h e m i c a l c o m p o s i t i o n w i t h a m p h a s i so n t h e f a t t y a c i d s o f T e t r a s e l m i s s p . A p p l M i c r o b i o l B i o t e c h -no l 36 : 21-25M o l i n a G r i m a E , S f i n c h e z P 4 r e z J A , G a r c f a C a m a c h o F , G a r c ~ a

S ~ in c he z J L , L 6 p e z A l o n s o D ( 19 9 3) n - 3 P U F A p r o d u c t i v i t yi n c h e m o s t a t c u l t u r e s o f m i c r o a l g a e . A p p l M i c r o b i o l B i o t e c h -no l 38 : 599-605R a d w a n S S ( 19 9 1) S o u r c e s o f C 2 0 - p o l y u n s a t u r a t e d f a t t y a c id s f o rb i o t e c h n o l o g i c a l u s e . A p p l M i c r o b i o l B i o t e c h n o l 3 5 :4 2 1- -4 3 0W h y t e J N ( 1 9 87 ) B i o c h e m i c a l c o m p o s i t i o n a n d e n e r g y c o n t e n t o fs i x s p e c i e s o f p h y t o p l a n k t o n u s e d i n m a r i c u l t u r e o f b i v al u e s .A q u a c u l t u r e 7 5 : 1 93 - 2 0 3W h y t e J N ( 19 8 8) F a t t y a c i d p r o f i l e s f r o m d i r e c t m e t h a n o l y s i s o fl i p id s i n t i s su e o f c u l t u r e d s p e c i e s . A q u a c u l t u r e 7 5 : 1 9 3 - 2 0 3

effect of growth rate on the eicosapentaenoic acid

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