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A quacultural Engineering 6 ( 198 7) 141 - 150

A p p r oac h to B i om ass P r od u c t ion o f t h e M ar i n eM i c r oa l ga T etr aselm is su eci ca (Kyl in) Bu tch U singC om m on G arden Fer tilizer and Soi l Ex trac t as Ch eapN utr ient Supp ly in Ba tch C ul turesJ a i m e F a b r e g a s , i L u i s a T o ri b i o , J u li o A b a l d e , B u e n a v e n t u r a

C a b e z a s a n d C o n c e p c i o n H e / ' r e roD e p a r t a m e n t o d e M i c r o b io l o g i a , F a c u l t a d d e F a r m a c i a , U n i v e r s id a d d e S a n t ia g o ,S a n ti a g o d e C o m p o s t e l a , S p a in

A B S T R A C TW e re p o r t th e p o s s i b i l it y o f a n e c o n o m i c c u l ti v a ti o n o f t h e m a r i n e m i c ro -alga Tetraselmis suecica, u s in g d if fe r en t m i x tu r e s o f a c o m m o n g a rd e nf ert il iz er , s o i l ex tract , m icro nu t r i en t s and v i tamins . M ax im um ce l lu lardens i t ie s were ob ta ined w i th a n i trogen concen t ra t ion o f 14 I~g /m l an dm a x i m u m p ro t e i n c o n c e n t ra t io n s w e re o b t a i n e d w i t h 2 8 l ug N / m l , i n a l lcases . Th e m ix ture o f e r t i li z e r + so i l ex t rac t + m icronu t r i en t s gave t he bes tr e su lt s f o r o b t a i n i n g m a x i m u m c e ll u la r d e n s it ie s a n d p ro t e i n c o n c e n tra -t io n s p e r m l o f c ultu re . A n e c o n o m i c e v alu a ti o n s h o w e d a m a x i m u m o f2 7 7 0 g o f m i c ro a l g a l p ro t e i n / d o l l a r a n d 2 1 0 x 1 0 12 m i c ro a lg a l c el ls ~ d o ll arw i th t he m ix ture o f t he f e r t il i z e r and so i l ex t rac t.

I N T R O D U C T I O NA g r e a t i n t e r e s t i n m a r i n e m i c r o a l g a l b i o m a s s p r o d u c t i o n h a s b e e np r ovoked by t he a r t i f i c i a l cu l t i v a t i on o f s he l l f i s h i n a ha t che r y env i r on -m e n t ( U k e l e s, 1 9 80 ). I n all t y p e s o f m o l l u s c a n a q u a c u l t u r e ( r e s e a r c hi nve s t i g a t i on s , p i l o t p l an t s o r com m er c i a l h a t che r i e s ) i t i s n e c e s s a r y t oh a v e a la r g e m i c r o a l g a l b i o m a s s a v a i l a b le a s a s o u r c e o f f o o d f o r n o r m a ld e v e l o p m e n t a n d g r o w t h o f t h e c u l t u r e d s p e ci es . I n f a c t, p r o d u c t i o n o ft h i s b i om as s i s f r equen t l y c i t ed a s c r i t i c a l t o t h e s ucce s s o f t h e p r o j e c ta n d a s b e i n g a s e r io u s i m p e d i m e n t t o th e f u t u r e d e v e l o p m e n t o f s h e ll fi sha q u a c u l t u r e ( D e P a u w e t a L , 1 9 83 ) . T h e m a s s c u l tu r e o f c e r t a i n s p e c ie st A u t h o r t o w h o m c o r r e sp o n d e n c e s h o u ld b e s en t.

141Aquacultural Engineering 0 1 4 4 - 8 6 0 9 / 8 7 / S 0 3 . 5 0 - - P u b l i s h e r s L t d . E n g l a n d . 1 9 8 7 . P r i n t e d i n G r e a t B r i ta i n E l s e v i e r A p p l i e d S c i e n c e

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1 4 2 J . Fabregas, L. Toribio , J. Ab alde , B . Cabezas, C. Herreroo f m i c r o a l g a e i s c o n s i d e r e d t h e m a j o r b o t t le n e c k i n t h e n u r s e r y c u l t u ri n go f m o l lu s c s ( P e r s o o n e a n d C l a u s , 1 9 8 0 ).

M a s s p r o d u c t i o n o f m i c r o a l g a e h a s lo n g b e e n s u c c e ss fu l ly c a r r ie d o u te i t h e r b y u s i n g d o m e s t i c o r b i o - i n d u s t r i a l w a s t e s o r i n o r g a n i cc o m p o u n d s ( Sh e le f a n d S o e d e r , 1 9 8 0 ; F a b r e g a s et al . , 1 9 8 5 a ) . T h ec u l t u r e m e d i u m s h o u l d b e n o n t o x i c t o l a r v a e , s o t h a t t h e u s e i n l a r v a lr e a r i n g o f w a s t e - s u p p o r t e d m i c r o a lg a l b i o m a s s is o f t e n q u e s t io n a b l e .T h e r e f o r e , c l e a n n u t r i e n t s o u r c e s a r e o f t e n u s e d a n d t h e s e m e d i a a r eg e n e r a l l y l a b o r a t o r y p r e p a r a t i o n s ( St ein , 1 9 7 3 ; S o e d e r , 1 9 8 0 ; F a b r e g a set al . , 1 9 8 4 a , 1 9 8 5 a ). A g r i c u l tu r a l o r c o m m o n g a r d e n f e r ti li z er s c o n t a in -i ng n i t ro g e n a n d p h o s p h o r u s h a v e b e e n u s e d a s a n a lt e rn a t iv e s o u r c e o fn u t r i e n t s ( P e r s o o n e a n d C l a u s , 1 9 8 0 ; U k e l e s , 1 9 8 0 ; G o n z a l e z - R o d r i -g u e z a n d M a e s t r i n i , 1 9 8 4 ) . S u c h f e r t i l i z e r s a r e m a i n l y c o m p o s e d o fn i tr o g e n o u s a n d p h o s p h o r o u s s u b s t a n c e s a n d s o m e , if a n y , m i c r o -n u t ri e n ts s u c h a s i ro n , p o t a s s iu m o r s o d i u m ( G o n z a l e z -R o d r ig u e z a n dM aes t r i n i , 1984) , bu t , i n gene r a l , t h ey do no t con t a i n a l l t h e m i c r o -nu t r i en t s an d v i t am i ns ne ce s s a r y f o r m i cr oal lga l g r o w t h (S te in , 1973 ).T h e m a r i n e m i c r o a l g a T e t r a s e l m i s s u e c i c a is a t p r e s e n t w i d e l y u s e d i na q u a c u l t u r e ( W a l n e , 1 9 7 4 ; B a y n e , 1 9 7 6 ; L a i n g a n d U t t i n g , 1 9 8 0 ) a n d i th a s be en sugge s t ed a s a po t en t i a l s ou r ce o f S ing l e C e l l P r o t e i n (S C P)( F a b r e g a s a n d H e r r e r o , 1 9 85 ). T h i s m a r i n e m i c r o a l g a is a b l e to to l e r a t e aw i d e r a n g e o f n u t r i e n t c o n c e n t r a t i o n - s a l i n i t y c o n d i t io n s ( F a b r e g a s et al . ,1 9 8 4 a ). T h e m a s s p r o d u c t i o n o f th is m i c r o a lg a w i th o p t i m u m i n o rg a n icn u t r i e n t c o n c e n t r a t io n h a s a ls o b e e n a c h i e v e d ( F a b r e g a s et al . , 1985 a , b).I n th is p a p e r , w e r e p o r t t h e p o s si b il it y o f a m o r e e c o n o m i c c u l ti v a ti o n o ft h i s m i c r oa l ga , u s i ng a com m on ga r den f e r t i l i z e r , s o i l ex t r a c t , m i c r o -n u t r i e n ts o r v i t a m i n s , i n s t e a d o f i n o r g a n i c n u t r ie n t s o f a d e f in e d m e d i u m .

M A T E R I A L S A N D M E T H O D ST h e m a r i n e m i c ro a l g a u se d w a s T e t r a s e l m i s s u e c i c a , i s o la t e d f r o m R i a d eA r o s a w a t e r s ( N W S p a i n ) . I t w a s c u l t u r e d i n s e a w a t e r f i l t e r e d t h r o u g h a0 - 4 5 / ~ m M i l l i p o r e f i l t e r , a u t o c l a v e d a t 1 2 0 C f o r 2 0 m i n a n d e n r i c h e dw i t h th e d i f f e r e n t n u t ri e n ts . W e u s e d f o u r t y p e s o f n u t ri e n ts : c o m m o ng a r d e n f e r ti li ze r , F e r t i c ro s 7 - 1 2 - 7 ( F ) ; c o m m o n g a r d e n f e r t i l i z e r + s o i le x t r a c t ( F + S E ) ; c o m m o n g a r d e n f e rt il iz e r + so il e x t r a c t + m i c r o n u t r i e n t( F + S E + M ) ; c o m m o n g a r d e n f e rt il iz e r + m i c r o n u t r i e n t v i ta m i n s( F + M + V ) . A s a b l a n k , w e u s e d a c u lt u re w i th th e in o r g a n ic m e d i u mc o m m o n l y u s e d i n o u r l a b o r a t o r y ( F a b r e g a s a n d H e r r e r o , 1 9 8 5 ;F a b r e g a s et al . , 1 9 8 4 a , 1 9 8 5 a ) . A l l t h e 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 i nt r ip l ica te .

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Biomass production of the marine microalgaT e t r a s e l m i s s u e c i c a 1 4 3T h e c o m p o s i t io n o f F e r t ic r o s 7 - 1 2 - 7 is : n i t r o g e n , 7 % ; p h o s p h o r i ca n h y d r i d e , 1 2 % ; p o t a s s i u m o x i d e , 7 % . T h i s f e r t i l i z e r c o n s t i t u t e d t h e

n i t r o g e n a n d p h o s p h o r u s s o u r c e f o r t h e m i c ro a l g a l c u l tu r e s. I t w a sd i s s o l v e d i n d i s ti ll e d w a t e r , f i lt e r e d t h r o u g h a p a p e r f i l te r a n d a u t o c l a v e da t 1 2 0 C f o r 1 5 m i n . W e u s e d s ix d i f f e r e n t c o n c e n t r a t i o n s : 0 . 0 5 , 0-1 , 0 -2 ,0 .4 , 0 "8 an d 1 .6 m g / m l , g i v i ng 3" 5 , 7 .0 , 14 .0 , 28 -0 , 56 -0 an d 112 .0 p g o fn i t r o g e n p e r m l o f c u l tu r e , r e sp e c t iv e l y .S o il e x t r a c t w a s p r e p a r e d b y m i x in g 1 v o l u m e o f s o il w i t h 2 v o l u m e so f d i s t il le d w a t e r , f il te r in g t h r o u g h a p a p e r f i l te r a n d a u t o c l a v i n g a t1 2 0 C f o r 2 0 m i n (S te in , 1 9 7 3 ). T h i s e x t r a c t w a s a d d e d t o a l l t h e c u l t u r e sa t a c o n c e n t r a t i o n o f 5 0 m l /l it re .T h e m i c r o n u t r i e n t s u s e d w e r e : Z n C 1 v 0 "1 3 6 m g / l it re ; M n C 12 . 4 H 2 0 ,0 -1 9 8 m g / l it re ; M o O n N a 2 . 2 H 2 O , 0 . 2 4 2 m g / l it re ; C O C 1 3, 0 - 01 6 5 m g /l i t r e; C u S O 4 . 5 ~ , 0 . 0 2 4 9 m g / li tr e ; f e rr ic c i t ra te , 6 .7 m g / li tr e ; E D T A ,9 . 3 8 m g / l i tr e .T h e v i t a m i n s u s e d w e r e : t h ia m i n e , 3 5 / z g / l i t r e ; b i o t in , 5 p g / l it r e ; B12,3 p g / l i t r e .T h e c o n c e n t r a t i o n s o f s o il ex t ra c t, m i c r o n u t r ie n t s a n d v i ta m i n s w e r ec o n s t a n t i n a l l t h e e x p e r i m e n t s . C u l t u r e s w e r e c a r r i e d o u t i n K i m a xs c r e w - c a p p e d t e s t t u b e s ( 15 x 2 .5 c m ) w i th 2 5 m l o f m e d i u m . A l l c u lt u r e sw e r e m a i n t a i n e d in a c o n t r o l l e d e n v i r o n m e n t a l i n c u b a t o r (N e w B r u n s -w i c k ) a t 1 5 C i n 3 9 0 0- 1u x l i g h t f r o m f l u o r e s c e n t la m p s ( P h i li p s T L2 0 W / 55 ). A 1 2 h : 1 2 h l ig h t : d a r k r e g im e w a s m a i n t a i n e d in o r d e r t oo b t a i n s y n c h r o n o u s c u l tu r e s. A n i n o c u l u m o f 1 x 104 l o g a r it h m i c - p h a s ec e l l s /m l w a s u s e d .T o o b t a i n t h e o p t i c a l d e n s i ty , t h e t r a n s m i t ta n c e T o f th e c u l tu r e s w a sd e t e r m i n e d b y u s i ng a C o l e m a n I I 6 / 2 0 s p e c t r o p h o t o m e t e r r e a d in g a t5 3 0 n m .C e l l u l a r d e n s i ty o f t h e c u l t u re s w a s d e t e r m i n e d b y c o u n t i n g c u l tu r ea l iq u o t s in a T h o m a c h a m b e r .P r o t e i n w a s m e a s u r e d i n t h e s t a ti o n a r y p h a s e b y t h e d y e - b i n d i n gm e t h o d ( B r a d f o rd , 1 97 6) .C h l o r o p h y l l s w e r e e x t ra c t e d i n a c e t o n e : m e t h a n o l ( 2: 1) a t 4 C f o r4 8 h . T h e e x t r a c ts w e r e f i l t e r e d ' t h r o u g h a F l u o r o p o r e M i ll ip o r e f il te r f o rc l a r i f i c a t i o n ( F a b r e g a s et a l . , 1 9 8 4 b ), a n d a b s o r b a n c e s o f t h e p i g m e n te x t ra c t a t s p e c if ic w a v e l e n g th s w e r e r e c o r d e d . T h e c o n c e n t r a t i o n o fc h l o r o p h y l l a w a s d e t e r m i n e d b y t h e f o r m u l a o f P a r s o n s a n d S t ri c k la n d(1965) .A f t e r th e s ix t h d a y o f c u l tu r i n g , C O , w a s a d d e d t o t h e c u l tu r e s ino r d e r t o m a i n t a i n t h e p H w i th i n th e o p t i m u m i n t e r v a l ( 7. 4 -8 .5 ) , a t a r a teb e t w e e n 0"2 a n d 2 m l p e r t u b e o f c u l tu r e .

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1 4 4 J . Fabregas, L. Toribio , J . Ab ald e, B . C abezas, C HerreroR E S U L T S A N D D I SC U S S IO N

The cul tures were not cont inuous ly ae ra ted , a l though CO 2 was a d d e dda i ly a f te r the s ix th day a t a ra te of 0 .2-2 ml per cu l ture . This CO2supp ly p r even ted the ca rbon source f rom becoming l im i t ing and a t thesame t ime ma in ta ined the pH wi th in the op t imum range fo r T . s u e c i c agrowth; because of th is , the va lues obta ined in the cont ro l for ce l l ,p ro te in an d ch lo rophy l l a concen t r a tions a r e g r ea te r than those ob ta inedin ba tc h condi tions w i thout CO 2 supply (Fabregas et al . , 1984a).W e p lo t ted opt ica l d ens ity , expressed as (1 0 0 - T ), aga ins t time an daga inst n i t rog en co nce nt ra t ion for each typ e of nut rien t , ob ta in ing three-dim ensional f igures (Fig. 1). W e also plo t ted cel lular d ens i ty against t im eand aga ins t n i t rogen concent ra t ion for each type of nut r ien t , aga inobta ining three-dimensional f igures (Fig . 2).M axim um ce l lu la r dens i tie s w ere obta ine d w i th a n it rogen concent ra -t ion of 14/~g/ml of cu l ture medium, wi th a l l the d i f fe ren t mixturesa ssayed . Op t imum growth can be obse rved bo th by op t i c a l dens i tym easurem ents (F ig . 1) an d by ce l l count ing (F ig . 2). T he growth (cel ls /ml) was s igni f icant ly grea te r wi th 14/~g/ml of n i t rogen than wi th the

" , , s ~ " F " ~ . s 6 ~ - - - ' ~ ' ~ \ ." / " ~ T I ~ E ( ~ a y ~ ) T I M E ( d a y s )

F i g . 1 . G r o w t h o f c u l t u r e s o f T. suecica w i t h d if f e re n t m i x t u r e s a n d c o n c e n t r a t io n s o fn u t r i e n t s : A , f e r t il iz e r ; B , f e r t i l i z e r + s o i l e x t r a c t ; C , f e r t i l i z e r + s o i l e x t r a c t + m i c r o -

n u t r i e n t s ; D , f e r t il i z e r + m i c r o n u t r i e n t s + v i t a m i n s .

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Biom ass prod uct ion o f the mar ine microalga T e t r a s e l m i s s u e c i c a 1 4 5

~ 2

3 5

-~ % 3NS

3o x2

5 35'~'>'~o_~'%-% ~ X \ \ '~ > - ~ ~ ? / >7 \

TlvlEk:lays) TIME(days)F i g . 2 . C e l l u l a r d e n s i t i e s o f c u l t u r e s o f T . suecica w i t h d if f e re n t m i x t u re s a n d c o n c e n -t r a t i o n s o f n u t r i e n t s : A , f e r t i l i z e r ; B , f e r t i l i z e r + s o i l e x t r a c t ; C , f e r t i l i z e r + s o i le x t r a c t + m i c r o n u t r i e n t s ; D , fe r t il i z e r + m i c r o n u t r i e n t s + v i t a m i n s .

o t h e r n i t r o g e n c o n c e n t r a t i o n s i n a l l c a s e s . H o w e v e r , i n c u l t u r e s w i t hd i f f e r e n t c o n c e n t r a t i o n s o f t h e m e d i u m u s e d a s c o n t r o l s i n t h e s ee x p e r i m e n t s , o p t i m a l g r o w t h c o n d i t i o n s w e r e o b t a i n e d w i t h n i t r o g e nc o n c e n t r a t i o n s h ig h e r th a n 1 4 ~ g / m l , n o t o n l y f o r T. s u ec ica ( F a b r e g a set al. , 1 9 8 4 a ) b u t a l s o f o r o t h e r m a r i n e m i c r o a l g a e s u c h a s I s ochr ys i sg a l b a n a ( F a b r e g a s et al. , 1 9 8 5 b ) .

M a x i m u m c e l lu l a r d e n s i ty o b t a i n e d a t t h e s t a ti o n a r y p h a s e a t th isn i t r o g e n c o n c e n t r a t i o n w a s 3 x 106 c e U s /m l f o r t h e c u l t u r e w i thF + S E + M ( T a b l e 1 ). H o w e v e r , t h is g r o w t h w a s 5 4 % l es s t h a n t h a t o f th ec o n t r o l w i th t h e d e f i n e d m e d i u m , w h i c h r e a c h e d a f in a l b i o m a s s a t t h es t a t i on a r y ph a s e o f 5"5 x 106 c eU s / m l (Tab l e 1 ).F o r t h e s a m e n i t r o g e n c o n c e n t r a t i o n , t h e p a t t e r n o f m a x i m u m c e l l u la rd e n s i t y w a s a s f o l lo w s :

F + S E + M > F M + V > F + S E > FP r o t e i n c o n c e n t r a t io n is n o t p r o p o r t i o n a l t o c e l l u la r d e n si t y . M a x i -m u m p r o t e in c o n c e n t ra t io n s w e r e o b t a i n e d w i th 2 8 / z g / m l o f n i t r o g e n ina ll e x p e r i m e n t s ( T a b l e 1; F i g . 3 ), w i t h 6 4 . 7 / z g o f p r o t e i n / m l i n th e c u l t u r e

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146 J . Fabregas , L . Tor ib io , J . Ab ald e , B . C abezas , C . Herr eroT A B L E 1Growth and Protein and Chlorophyll a Concentrations of Cultures of T. suec ica withDifferent Mixtures and Concentrations of Nutrients

N u t r i e n t N i t r o g en O p t i c a l d e n si ty , l O p x c e l l s / m l P r o t e i n C h l o r o p h y l l am i x t u r e ~ ( ~ g / m l ) 1 0 0 - T ( ~ g / m l ) ( lt g/ m l )

F 3"5 52 1.34 9"58 0"097 63 1"26 17.77 0.4414 68 1"90 37.82 2.0328 67 1"85 55"73 1"5256 59 1"42 37"65 2.43112 48 1.60 26"99 1"20F+ SE 3"5 65 1.67 22.04 1"257 65 1"69 36"30 2-4814 73 1"99 38.49 2.6428 68 1.81 56.31 3.2056 63 1"74 38"60 3.27112 48 1"16 39-34 2"05F+ SE + M 3"5 71 2"66 31"73 1"117 75 2-50 33"50 1.4814 75 3"00 59.25 3"3028 62 1"72 64.71 4.4056 57 1-10 29-40 3"95112 48 1.17 39.91 2"58F +M +V 3"5 56 1.45 10.17 0"567 69 1"82 15.48 0"8414 78 2"25 32-77 2"1128 66 1.42 61"68 2.7956 57 1"26 44.15 2.53112 49 1"11 28.49 2.35Control 28 96 5"58 147.46 9.88

Abbreviations: E fertilizer; SE, soil extract; M, micronutrients; V, vitamins.with F+SE +M. Protein concentration in the control was 147 ktg/ml,228 % greater t han that of each of the other nutrients.

The chlorophyl l a content presented more variation depending on thedifferent concentrations and types of nutrients. In general , maximumvalues were obtained with nitrogen concentrations of 14, 28 and 56/~g/ml (Table 1; Fig. 4). The maximum value of chlorophyll a was obtainedin the cultures with F+ SE + M, with 4.4/~g/ml. These cultures were theones with better growth, too. However, this value is 45% less than thatobtai ned with the control .

Excepti ng the control , the cultures with F+ SE + M show ed bet tergrowth than the other cultures, and the major protein and chlorophyll acontent.

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Biom ass p roduc t ion o f t he m ar ine m icroa lga T e t r a s e l m i s s u e c i c a 1 4 71 6 0 -

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F i g . 3 . P r o t e i n c o n c e n t r a t i o n s i n c u l t u r e s o f T. suecica w i t h d i f f e r e n t m i x t u r e s a n dco n c en t r a t i o n s o f n u t r i en t s : A , f e r t il i z e r ; B , f e r t i l i z e r + so i l ex t r a c t ; C , f e r t i li z e r + so i le x t r a c t + m i c r o n u t r i e n t s ; D , f e r ti l iz e r + m i c r o n u t r i e n t s + v i t a m i n s ; E , c o n t r o l . N i t r o g e nco n te n t s (~g ) pe r m l o f cu l tu re w ere : 1 , 3 -5 ; 2 , 7 ; 3 , 14; 4 , 28 ; 5 , 56 ; 6 , 112 .

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F i g . 4 . C h l o r o p h y l l a c o n c e n t r a t i o n s in c u l t u r e s o f T. suecica w i t h d i f f e r e n t m i x t u r e san d co n c en t r a t i o n s o f n u t r i en t s : A , f e r t il i z e r ; B , f e r t il i z e r + so i l ex t r a c t ; C , f e r t i l iz e r + so i le x t r a c t + m i c r o n u t r i e n t s ; D , f e r ti li z e r + m i c r o n u t r i e n t s + v i ta m i n s ; E , c o n t r o l . N i t r o g e n

co n te n t s ( /~g ) pe r m l o f cu l tu r e w ere : 1 , 3 .5 ; 2 , 7 ; 3 , 14 ; 4 , 28 ; 5 , 56 ; 6 , 112 .

T h e f e r t il iz e r F e r ti c ro s d o e s n o t c o n t a i n a ll t h e e l e m e n t s n e c e s s a r y f o rm a x i m u m m i c ro a l g a l g r o w t h , a l th o u g h i t c a n b e u s e d a s a s o u r c e o fn i tr o g e n a n d p h o s p h o r u s .T h e u s e o f s o i l e x t r a c t f o r m a r i n e m i c r o a l g a l g r o w t h i n s e a w a t e r i sv e r y c o m m o n ( P ra k a s h a n d R a s h i d , 1 9 68 ; P r a k a s h et a l . , 1 9 7 3 ) . T h i s s o i le x t r ac t c a n s u p p l y v i ta m i n s , m i c r o n u t r i e n t s a n d , p e r h a p s , c e r t a in o t h e rg r o w t h f a c t o rs , a c t in g b e s i d e s a s a c h e l a t i n g a g e n t (H o e f f n e r a n dM a n a h a n , 1 9 8 0 ) b e c a u s e h u m i c s u b s t a n c e s c h e l a te io n s o f F e , C a , A 1,C u a n d C o , s o t h a t t h e s e i o n s a r e p r e v e n t e d f r o m p r e c i p i ta t in g e s s e n ti a lm i c r o a l g a l n u t r i e n t s s u c h a s p h o s p h a t e ( P r a k a s h et a l . , 1 9 7 3 ) ; o n t h e

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Biom ass prod uction o f the m arine microalgaTetraseimis suecica 149o t h e r h a n d , t h e i n v o l v e m e n t o f h u m i c s u b s t a n c e s i n c e l l u l a r m e t a b o l i cp r o c e s s e s i s a l s o a p o s s i b i l i ty ( P r a k a s h a n d R a s h i d , 1 9 6 8 ).W e e s t a b l i s h e d t h e e f f i c ie n c y a s th e r a t i o b e t w e e n t h e n i t r o g e n a d d e dt o t h e c u l t u r e m e d i u m a n d t h e p r o t e i n n i t r o g e n p r o d u c e d p e r c u l t u r e .M a x i m u m e f f i c ie n c y w a s o b t a i n e d i n t h e c u l tu r e s w i th th e m i x tu r e o fF + S E ( T a b l e 2 ).W e c a r r i e d o u t a n e c o n o m i c e v a l u a t io n , b u t o n l y r e f e r re d t o th e c o s to f th e d i f f e re n t c h e m i c a l c o m p o u n d s o f th e d i f f e re n t m i x tu r e s.

M a x i m u m c o n c e n t r a t io n o f m i c ro a l g a l p r o t e in , e x p r e s s e d a s g /d o l la r ,w a s o b t a i n e d w i t h F + S E , w i t h 2 7 7 0 g o f p r o t e in / d o l l a r , a t a n i t r o g e nc o n c e n t r a t i o n o f 3 "5 / z g /m l . M a x i m u m c e l l u l a r d e n s i t y o f 2 1 0 x 1012c e l l s /d o l l a r w a s o b t a i n e d w i th th e s a m e m i x t u r e o f F + S E a n d a t th es a m e n i t r o g e n c o n c e n t r a t i o n ( T a b le 2). C o m p a r i n g t h e s e d a t a w i th t h ec o n t r o l , w e o b t a i n e d b e t t e r e c o n o m i c r e s u l t s w i t h F + S E t h a n w i th th ec o n t r o l , th e d i f fe r e n c e s b e i n g u p t o 4 0 4 - f o l d i n t e r m s o f p r o t e i n / d o l l a ra n d 8 1 0 - f o l d i n t e r m s o f c e l ls / d o l la r .

A C K N O W I .E D G E M E N T SW e t h a n k P r o f e s s o r D r B . R e g u e i r o V a r e l a f o r h i s i n te r e s t i n g c o m m e n t sa n d C r o s S A f o r t h e ir c o o p e r a t i o n . T h i s w o r k w a s s u p p o r t e d b y a g r a n tf r o m D i r e c c i o n G e n e r a l d e O r d e n a c i o n P e s q u e r a , M i n i s t e r i o d e A g r i -c u l t u r a , P e s c a y A l i m e n t a c i o n , M a d r i d , S p a i n .

R E F E R E N C E SBayn e, B . L. (1976) . T he b io logy o f the m usse l la rvae . In : M arine M ussels: T heirEco logy and Phys io logy , ed. B . L. Bayne, Cam bridge Un ivers i ty Press ,Cam br idge , pp . 81-1 20 .Bradford , M. (1976) . A rapid and sens i t ive method for the quant i ta t ion ofm icrogram quant i ties of prote in u t il iz ing the p r inc ip le of prote in dye-binding.A n a l B i o c h e m . , 72 , 248 -54 .D e P auw , N. , V erboven , J . & C laus, Co (1983). Larg e-scale m icroalgae produ c-t ion for nursery rear ing of mar ine b iva lves . Aquacul tural Engineering, 2 ,2 7-47 .Fabregas , J . & Herrero , C . (1985) , Mar ine microalgae as a potent ia l source ofSingle Cel l Prote in (SCP). Appl. Microbiol . Technol. , 23, 110-13.Fab regas, J ., A balde, J ., H erre ro, C. , C abezas, B. & V eiga, M. (1984a). G row thof the m ar ine m icroalga Tetraselmis suecica in ba tch cul tures w i th d i f ferentsa lin it ies a nd nu t r ient concent ra tions . Aquacul ture , 42 , 207-1 5 .Fab regas, J ., H erre ro, C . & Ve iga, M. (1984b). Effect of oi l an d dispersant ongrowth and ch lo rophy l l a con ten t o f the mar ine m ic roa lga Tetraselmissuecica. Ap pl. Environ. M icrobiol . , 47(2) , 44 5-7 .

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150 J. Fabregas, L. Toribio, J. Abald e, B . C abezas, C. HerreroFabregas, J ., H e r re ro , C . , Cab ezas , B . & A ba ld e , J . ( 1985a) . Mass cu l tu re an db i o c h e m i c a l v a r ia b i li ty o f t h e m a r i n e m i c r o a lg a Tetraselmis suecica Kyl in( B u t c h ) w i th h i g h n u t r i e n t c o n c e n t r a ti o n s . Aquacul ture , 4 9 , 2 3 1 - 4 4 .Fab regas , J . , He r re ro , C . , Aba lde , J . & Cabezas , B . ( 1985b) . Growth , ch l o ro -p h y l l a a n d p r o t e i n o f t h e m a r i n e m i c r o a l g a Isochrysis galbana i n b a t c hcu l t u re s w i t h d i f f e r en t s a lin iti es an d h i gh nu t r i en t conce n t r a t i ons . Aquacu l -ture, 50 , 1 -11 .G on za l ez -Ro dr i gu ez , E . & M aes t f i n i , S . Y . ( 1984) . T h e u se o f som e ag ricu ltu ralf e r ti li z e rs fo r t h e m a s s p r o d u c t i o n o f m a r i n e a l ga e. Aquacul ture , 3 6 , 2 4 5 - 5 6 .H oe f fn e r , S . L . & M anah an , S . E . ( 1980). In f l uen ce o f coa l hum ic ac i d on t heg r o w t h o f C hloreUa vulgaris algae. J. Env. Sci. Health, Part A, 15(2 ) , 149-61 .La ing , I. & U t ti ng , S . D . ( 1980). Th e i n f l uenc e o f s a li nity o n t h e p rod uc t i on o f

t w o c o m m e r c i a ll y i m p o r t a n t u n i c e l lu l a r m a r i n e . al g a e. Aquacul ture , 21,7 9 - 8 6 .Parsons , T . R . & St r i ck land , J . D. H . (1965) . Par t i cu la t e o rgan ic mat t e r . I I I . I .P i gmen t ana l y s i s . I I I . I I . De t e rm ina t i on o f phy top l ank ton p i gmen t s . J . Fish.Res . Bd. Can. , 18 , 117-27 .Pe r so one , G . & C l aus , C . ( 1980). Mass cu l t u re o f algae : a bo t t l enec k i n thenu r se ry cu l t u r i ng o f mo l lu sc s . In : Algae B iom ass : P roduc t ion and U se , ed . G .S h e l e f a n d C . J . S o e d e r , E l s e v i e r / N o r t h H o l l a n d B i o m e d i c a l P re ss , A m s t e r -d a m , p p . 2 6 5 - 8 5 .P r a k a s h , A . & R a s h i d , M . A . ( 1 9 6 8 ). I n f l u e n c e o f h u m i c su b s t a n c e s o n t h eg r o w t h o f t h e m a r i n e p h y t o p l a n k t o n : d i n o fl a g e ll a te s . Limnol . Oceanogr . , 13,5 9 8 - 6 0 6 .P r a k a s h , A . , R a s h i d , M . A . , J e n s e n , A . & R a o , D . ( 1 97 3 ) . .I n f l u e n c e o f h u m i cs u b s t a n c e s o n t h e g r o w t h o f m a r i n e p h y t o p l a n k t o n : d i a t o m s . L i m n o l .Oceanogr., 1 8 , 5 1 6 - 2 4 .She le f , G. & Soeder , C . J . ( eds) (1980) . Alga e Biom ass: Product ion an d Use.E l s e v i e r /N o r t h H o l l a n d B i o m e d i c a l P re ss , A m s t e r d a m .Soe de r , C . J . (1980) . Mass i ve cu l ti va t ion o f m i c roa lgae : r e su l ts a nd p ro spec t s .Hydrobiologia, 7 2 , 1 9 7 - 2 0 9 .Stein, J. R. (ed.) (1973). H an db oo k o f Phyco logical Methods: Cul ture M ethod sand Grow th Measurements . C a m b r i d g e U n i v e r s i ty P r es s, C a m b r i d g e .U k e l e s , S . R . ( 1 9 80 ). A m e r i c a n e x p e r i e n c e i n th e m a s s c u l tu r e o f m i c r o a l g a e f o rf e e d i n g l a r v a e o f t h e A m e r i c a n o y s t e r , Crassostrea virginica. In : AlgaeBiomass: Product ion and Use, ed . G . She l e f and C . J. Soede r , E l sev i e r /N or t hH o l l a n d B i o m e d i c a l P re ss , A m s t e r d a m , pp . 2 8 7 - 3 0 6 .W alne , E R . (1974) . Culture o f Bivalve M ollusc . 50 Years ' Exp erience at Conw y.F i s h i n g N e w s ( B oo k s), F a r n h a m .