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Culture of the brine shrimp, Artemia salina L., utilizing Dunaliella tertiolecta grown in swine waste-seawater.. Tiro, Leonardo B. 1981

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CULTURE OF THE BRINE SHRIMP, ARTEMIA SALINA L . UTILIZING DUNALIELLA TERTIOLECTA GROWN IN SWINE WASTE-SEAWATER MIXTURES AND IN DEFINED INORGANIC MEDIUM by LEONARDO B. TIRO, JR. B. Sc., X a v i e r U n i v e r s i t y , Cagayan de Oro C i t y P h i l i p p i n e s , 197^ A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f A g r i c u l t u r a l Mechanics)  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1980 0  Leonardo B. T i r o , J r . , I98O  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the I  Library shall  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  for  reference and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  make i t  I agree  this  representatives. thesis  It  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  not be allowed without my  written permission.  LEONARDO B. TIRO, J R .  Department of  Agricultural  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  -12-th January, 1981.  Mechanics  Columbia  ABSTRACT A c o m p a r a t i v e s t u d y was c o n d u c t e d o n t h e g r o w t h o f Dunaliella t e r t i o l e c t a using  defined  i n o r g a n i c medium a n d s w i n e  w a s t e - s e a w a t e r medium a s s o u r c e s o f n u t r i e n t s . was m a i n t a i n e d a t c a 1200  concentration both media.  T h e r e w e r e no s i g n i f i c a n t  measurements o f c e l l grown i n d e f i n e d  nitrogen -1  + 5° u g ~ a t N 1  for  differences i n the d a i l y  d e n s i t i e s a n d d r y w e i g h t s o f D.  i n o r g a n i c medium a n d s w i n e  medium i n e i t h e r h a t c h o r c o n t i n u o u s c u l t u r e The  The  tertiolecta  waste-seawater systems.  a l g a l biomass produced from continuous c u l t u r e  was u s e d a s f o o d f o r t h e b r i n e  shrimp, Artemia s a l i n a  system  L.  The b r i n e s h r i m p f e d w i t h D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e s e a w a t e r medium a n d t h o s e f e d w i t h _D. t e r t i o l e c t a defined their  i n o r g a n i c medium showed no s i g n i f i c a n t  daily  t o t a l length  A 57-58% A r t e m i a b i o m a s s  S t a t i s t i c a l a n a l y s i s f o r biomass no s i g n i f i c a n t  differences i n  and p e r c e n t a g e s u r v i v a l conversion efficiency  cultured i n  measurements.  was c a l c u l a t e d .  conversion efficiency  d i f f e r e n c e s f o r A r t e m i a f e d w i t h D.  tertiolecta  grown i n s w i n e w a s t e - s e a w a t e r medium a n d t h a t f e d w i t h D. t e r t i o l e c t a  cultured i n defined  inorganic  showed  medium.  iii TABLE OF CONTENTS Page ABSTRACT  ii iii v vi viii  TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS INTRODUCTION  1  LITERATURE REVIEW Algal Production  3 3 6  Algal-Waste R e c y c l i n g I n Aquaculture Use and P o t e n t i a l o f A r t e m i a F o r A q u a c u l t u r e I n R e l a t i o n To I t s L i f e C y c l e  8  N u t r i t i o n a l V a l u e o f A r t e m i a N a u p l i i As Food In Aquaculture  Hatcheries  Present C u l t u r a l P r a c t i c e s of Artemia MATERIALS AND METHODS >'iA 'Algae!; B r i n e Shrimp M e d i a and S a l t w a t e r P r e p a r a t i o n Swine Waste-Seawater M i x t u r e D e f i n e d I n o r g a n i c Medium Seawater P r e p a r a t i o n Description of Culture Units A l g a l Batch C u l t u r e U n i t I A l g a l Continuous C u l t u r e U n i t I I A l g a l Continuous C u l t u r e U n i t I I I B r i n e , Shrimp C u l t u r e U n i t C u l t u r e Methods A l g a l C u l t u r e Method B r i n e Shrimp C u l t u r e Method Chemical A n a l y s i s  9 11 lk lk lk 15 15 16 16 17 17 18 20 21 21 21 23 2k  Kjeldahl Nitrogen  2k  Ammonia / N i t r a t e + N i t r i t e  25  T o t a l Phosphate  25  iv Page Dry Weight A n a l y s i s  25  T o t a l Suspended S o l i d s  26 27  RESULTS Swine Waste A n a l y s i s  27  A l g a l B a t c h C u l t u r e System  27  A l g a l Continuous C u l t u r e System  31  B r i n e Shrimp  40  Feeding Experiments  DISCUSSION L i v e s t o c k Waste As A N u t r i e n t Source I n ^7  Algal Culture Growth C o n s t a n t , C e l l D e n s i t y  and D r y Weight ^9  o f D. t e r t i o l e c t a Continuous C u l t u r e System  I n Algal Production  52  Artemia Feeding Experiments T o t a l Body L e n g t h and P e r c e n t a g e S u r v i v a l  52 53  Biomass C o n v e r s i o n E f f i c i e n c y Comhined L i v e s t o c k W a s t e - A r t e m i a  51  C u l t u r e System  5^  SUMMARY AND CONCLUSIONS  57  REFERENCES  59  APPENDICES  70  V  LIST OF TABLES  Table 1  Title  Page  Swine waste a n a l y s i s o f TKN, NO^-N  + N0 -N, PO^-P  NH^-N  and T o t a l Suspended  2  S o l i d s (TSS) d u r i n g the course o f the experimental work 2,  N i t r o g e n (TKN,  28  NH^-N, Organic-N;,'f NO^-N  +  measured i n the a l g a l e f f l u e n t d u r i n g the continuous 3  c u l t u r e (Expt 5) i  n  A d u l t Artemia biomass produced 14-day c u l t u r e p e r i o d i n wet  Growth Constant  -  39  a f t e r the  and dry weight 46  (K^ ^ 0  hr  ) and Mean  G e n e r a t i o n Time ( t g h r s ) from p r e s e n t e a r l i e r s t u d i e s on D. 5  second  Mg at N 1  "basis 4  NOg)  and  tertiolecta  C a l c u l a t e d Biomass Conversion (BCE %) o f Artemia s a l i n a L.  5Q  Efficiency 55  vi L I S T OF FIGURES Figure  Title  Page  1  Generalized view of the a l g a l continuous c u l t u r e system  19  2  F i v e - d a y measurement o f t o t a l K j e l d a h l n i t r o g e n (TKN) o f s w i n e w a s t e s l u r r y and t h r e e d i f f e r e n t r a t i o s o f s w i n e w a s t e - s e a w a t e r m i x t u r e s ( l i 1 ; 2:1; 1:2) s t o r e d a t k°C  29  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c m e d i a i n b a t c h s y s t e m ( E x p t 1)  30  D a i l y c e i l d e n s i t y o f D. t e r t i o l e c t a g r o w n i n swine w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c media i n b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 2)  32  3  k  5  D a i l y m e a s u r e m e n t o f d r y wt o f D. t e r t i o l e c t a grown i n swine w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c media i n b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c c a r b o y s ( E x p t 2) 33  6  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c media i n continuous c u l t u r e s y s t e m u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 3)  3k  7  D a i l y m e a s u r e m e n t o f d r y wt o f D. t e r t i o l e c t a grown i n swine w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c media i n continuous c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 3) 35  8  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a g r o w n i n swine w a s t e - s e a w a t e r and d e f i n e d inorganic m e d i a i n c o n t i n u o u s c u l t u r e s y s t e m u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 5) 36  9  D a i l y m e a s u r e m e n t o f d r y w t o f D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e - s e a w a t e r and d e f i n e d i n o r g a n i c media i n continuous c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 5) 37  10  Growth i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. o v e r a 12-day c u l t u r e p e r i o d f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium  kl  vii  Figure  11  Title  Percentage s u r v i v a l o f Artemia d u r i n g the 12-day c u l t u r e p e r i o d f e d with D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium  Page  42  12  Growth i n t o t a l l e n g t h measurement o f Artemia s a l i n a L . over a l4-day c u l t u r e p e r i o d f e d w i t h swine waste and d e f i n e d i n o r g a n i c media grown D. t e r t i o l e c t a 43  13  Percentage s u r v i v a l o f Artemia s a l i n a L. over a 14-day c u l t u r e p e r i o d f e d w i t h swine waste and d e f i n e d i n o r g a n i c media grown D. t e r t i o l e c t a  44  viii ACKNOWLEDGEMENTS My s i n c e r e s t g r a t i t u d e t o D r . J . W. Z a h r a d n i k , my Department'.- o f B i o - R e s o u r c e E n g i n e e r i n g . t o a l l t h e members  o f my c o m m i t t e e :  I am d e e p l y  D r . N. J . A n t i a ,  E n v i r o n m e n t I n s t i t u t e , West V a n c o u v e r L a b o r a t o r y , the  during  t h e i n i t i a l phase o f t h e r e s e a r c h ;  Department o f Bio-Resource E n g i n e e r i n g ,  indebted Pacific  f o r providing  a l g a used i n t h e e x p e r i m e n t s and f o r h i s v a l u a b l e  advices  adviser,  h e l p and  D r . N-. R. B u l l y ,  f o r t h e u s e o f some  equipment and f o r h i s c o n s t r u c t i v e c r i t i c i s m s i n t h e p r e p a r a t i o n o f t h e m a n u s c r i p t ; D r . T. R. P a r s o n s , f o r h i s comments. My s i n c e r e s t t h a n k s t o t h e s e s c i e n t i s t s f o r t h e i r and  valuable  encouragements  comments.  I a l s o w i s h t o g r a t e f u l l y t h a n k D r . W. S. H o a r , D e p a r t m e n t of Zoology, f o r t h e use o f h i s l a b o r a t o r y d u r i n g pf the research; for  Dr. J . Marliave,  the f i r s t  phase  Vancouver P u b l i c Aquarium,  t h e A r t e m i a e g g s ; D r . D. H i g g s , P a c i f i c E n v i r o n m e n t I n s t i t u t e ,  West V a n c o u v e r L a b o r a t o r y ,  f o r the use o f t h e f i b e r g l a s s c u l t u r e  tanks. My s i n c e r e a p p r e c i a t i o n t o t h e f o l l o w i n g : D r . P. L i a o , Department o f Bio-Resource E n g i n e e r i n g , the  f o r c o n d u c t i n g some o f  c h e m i c a l a n a l y s i s ; J . P e h l k e a n d N. J a c k s o n , D e p a r t m e n t o f  Bio-Resource Engineering,  f o r the technical assistance.  M r . H. G r e w e l , G r a d u a t e S t u d e n t , F o r e s t r y D e p a r t m e n t , f o r h i s assistance use  i n the s t a t i s t i c a l  a n a l y s i s ; M r . R. P l a t o n , f o r t h e  o f h i s t y p e w r i t e r ; M r . N. C a s t i l l o , f o r h i s h e l p  setting-up  of the culture units.  i n the  ix  Ms. D e b b i e T u r n b u l l , I n t e r n a t i o n a l D e v e l o p m e n t C e n t r e o f Canada My  Research  (IDRC), Vancouver O f f i c e .  s i n c e r e g r a t i t u d e t o L i l l i a n , my w i f e , f o r h e r  d e d i c a t i o n i n t y p i n g t h e m a n u s c r i p t and f o r h e r u n d e r s t a n d i n g (when t h e g o i n g g e t s r o u g h ) . F i n a n c i a l s u p p o r t from the I n t e r n a t i o n a l Development Research  C e n t r e o f Canada  (IDRC) i s i v e r y s i n c e r e l y a c k n o w l e d g e d .  INTRODUCTION A r t e m i a s a l i n a L. a r e f i l t e r consume o n l y algae,  feeding  The s u i t a b i l i t y o f A r t e m i a f o r  a q u a c u l t u r e i s enhanced by t h e f a c t t h a t temperature, s a l i n i t y ,  same f o r b o t h t h e l a r v a l nursery  and  s m a l l p a r t i c u l a t e m a t t e r such as u n i c e l l u l a r  y e a s t and b a c t e r i a .  conditions:  herbivores  the p h y s i c a l c u l t u r a l  pH a n d 0^ t e n s i o n  s t a g e s and a d u l t s .  are the  T h e r e i s no s p e c i a l  environment-  Most o f t h e p h y t o p l a n k t e r s used as f o o d f o r A . h - s a l i n a have been c u l t u r e d Provasoli 1976)  seawater media (Croghan, 1 9 5 8 ;  e t a l . , 1 9 5 9 ; Mason, 1 9 6 3 ; Reeve, 1 9 6 3 a ;  o r media enriched  1973) * the  on d e f i n e d  with  and S i c k ,  commercial f e r t i l i z e r ( H e l f r i c h ,  A g r i c u l t u r a l w a s t e s have been used s u c c e s s f u l l y f o r  c u l t u r e o f m i c r o o r g a n i s m s such as b a c t e r i a and p h y t o p l a n k t o n  (Hephner, 1 9 6 2 * production 1974) .  S c h r o e d e r , 1 9 7 4 ) and have i n c r e a s e d t h e  of cultured  aquatic  animals  (Hephner and S c h r o e d e r ,  The u t i l i z a t i o n o f l i v e s t o c k w a s t e a s a n u t r i e n t  source  f o r p h y t o p l a n k t o n c u l t u r e a n d s u b s e q u e n t l y , a s f o o d f o r A. s a l i n a has  n o t been The  investigated.  general objective  of the present research  to e v a l u a t e the c u l t u r e p o t e n t i a l o f the brin e r  s a l i n a L. f e d w i t h seawater scale  w o r k was  shrimp,  Artemia  D u n a l i e l l a t e r t i o l e c t a grown i n s w i n e w a s t e -  as compared t o d e f i n e d  continuous culture  system.  inorganic  media i n a M a b o r a t o r y  The experiments were conducted i n two phases: Phase I was the c u l t u r e study between D. t e r t i o l e c t a grown i n swine waste-seawater mixture compared to that grown i n defined i n o r g a n i c medium;  and Phase I I was the comparative study on  the s u r v i v a l and growth of Artemia on two feeding regimes: i.  D. t e r t i o l e c t a grown i n swine waste-seawater mixture.  ii.  D.. t e r t i o l e c t a grown i n defined i n o r g a n i c medium.  3  LITERATURE REVIEW Algal  Production  E a r l y p u b l i c a t i o n s by A l l e n and N e l s o n E m e r s o n a n d L e w i s (1939) p r o v e d  (1910) a n d  t h a t a l g a l c u l t u r e was a s u b j e c t  o f much i n t e r e s t i n t h e e a r l y 1900's.  However, much o f t h e  e a r l y work d e a l t p r i m a r i l y w i t h t h e s t u d y o f p h o t o s y n t h e t i c pathways i n green a l g a l c e l l s Milner  (I96I) analysed  (Sargent,  the chemical  number o f m a r i n e a n d f r e s h w a t e r s p e c i e s . freshwater  species g e n e r a l l y ranked  than t h e marine s p e c i e s .  19^0; P r a t t , 19^3)* composition He f o u n d  of a  that  much h i g h e r i n e n e r g y  content  This d i f f e r e n c e r e s u l t e d from t h e  r e l a t i v e l y greater f a t content  of the freshwater  species.  P a r s o n s e t a l . (1961) observed  t h a t among m a r i n e a l g a l  species  c a r b o h y d r a t e , l l i p i d , p r o t e i n and a s h content v a r i e d w i d e l y . D u n a l i e l l a s a l i n a was f o u n d  t o c o n t a i n 57$  protein, the highest  v a l u e among t h e s p e c i e s a n a l y z e d , h o w e v e r , t h e l i p i d  content  was q u i t e l o w , S.k%. The cell  genus D u n a l i e l l a c o m p r i s e s  a group o f a l g a e w i t h e t h i n  e n v e l o p e whose a b i l i t y t o w i t h s t a n d c e r t a i n  environmental  c h a n g e s i s w e l l known (Ben-Amotz a n d A v r o n ,  However, a c e l l observed  extreme 1978).  coat o f D u n a l i e l l a t e r t i o l e c t a has been r e c e n t l y  w h i c h a p p e a r s ftofbe l a r g e l y composed, o f g l y c o p r o t e i n  containing neuraminic (Oliveira et a l . ,  acid residues i n i t s molecular structure  I98O).  F o r sometime, i t has been  t h a t heavy metal r e s i s t a n c e can develop  recognized  i n populations of  m i c r o o r g a n i s m s such a s f u n g i , y e a s t s and b a c t e r i a w h i c h have  4  "been s u b j e c t e d t o p r o l o n g e d concentrations al.,  1971)  but  exposure t o s u b - l e t h a l m e t a l  ( c . f . r.eviews b y A s h i d a , 7  I965 a n d A n t o n o v i c s e t  t h e r e appear t o be v e r y few examples o f organisms  whi:ch p o s s e s s a n i n n a t e r e s i s t a n c e t o m e t a l s . w i t h D. t e r t i o l e c t a .  T h i s i s t h e case  The s p e c i f i c g r o w t h r a t e o f D. t e r t i o l e c t a  was u n a f f e c t e d b y m e r c u r y I I c o n c e n t r a t i o n s o f a t l e a s t 2.03 J i g at 1  -1  A t 10 / i g - a t 1  .  -1  , t h e s p e c i f i c g r o w t h r a t e was e v e n t u a l l y  r e d u c e d b y Qk% b u t g r o w t h c o n t i n u e d ,  giving a final  m a t e r i a l o n l y 13% below t h a t i n a m e r c u r y - f r e e 1976).  level of cell  control  (Davies,  The a l g a h a s b e e n shown t o b e u n a f f e c t e d b y DDT a t 1 ppm  as m e a s u r e d b y c e l l of 43% a t 10  3  division  ppm ( L u a r d ,  (Bowes, 1971).  1973).  PCB g a v e a n i n h i b i t i o n  In a d d i t i o n , t h i s s p e c i e s  could  t o l e r a t e f l u o r i d e c o n c e n t r a t i o n f r o m 0 t o 100 ppm ( O l i v e i r a e t  1978).  al.,  By  the mid-1960's, the production o f algae on a semi-  c o m m e r c i a l b a s i s c r e a t e d much i n t e r e s t . Davis  e t a l . (1961),  (1963) a n d U k e l e s  Gaucher e t a l . (i960),  Casey e t a l . (1963), L o o s a n o f f  and Davis  (I965) i n v e s t i g a t e d t h e p o s s i b i l i t y o f c u l t u r i n g  a l g a e o n a mass s c a l e .  Ukeles  and Loosanoff  and Davis  were  primarily interested i nobtaining a reliable  feed source  and  and Davis  oyster larvae.  The e f f o r t s o f L o o s a n o f f  were d i r e c t e d t o w a r d c u l t u r i n g a l g a e resembling  i nlarge outdoor  f o r clam (1963)  vats  s m a l l ponds.  A n o t h e r t y p e o f mass c u l t u r e u n i t was d e v i s e d b y W i s e l y and P u r d a y ( I 9 6 I ) ,  the batch  c u l t u r e system.  o f l a r g e drums c o n t a i n i n g 200 l i t e r s cultures.  This unit consisted  o f I s o c h r y s i s galbana  I l l u m i n a t i o n showed l i g h t i n t e n s i t y t o be r e d u c e d  5 greatly  at.the  periphery  i n t e n s i t y may  and  the  have i n f l u e n c e d  culture.  o f the  A p r i m a r y drawback t o b a t c h  culture  lies  culture  dismantled,  such u n i t s .  Also,  cannot occur without  supplementation.  Furthermore,  Because of  Much o f  cultures  the  has  Herbert  condition  the  recent  been based  the  work done w i t h  on  in  nutrient  cultures  algae.  difficulties  associated  with  batch  continuous culture continuous  of  algal  b a c t e r i o l o g i c a l models p i o n e e r e d  (1958; 1961)  a l l o f whom c o n t r i b u t e d  m a t h e m a t i c a l models f o r c o n t i n u o u s The that  been s e t adjust  essential feature  at a constant value,  itself  to  the  two  ( N o v i c k and  Szilard,  principle  by  by  the  1950) are  and  regulates  amount o f  continuous Once t h e  (^1)  the  system w i l l (Herbert,  culture  of  lies  in  1958).  There  units,  'turbidostat'  the  has  1958).  basic differs.  of the  ' l i m i t i n g growth s u b s t r a t e '  are  'chemostat'  (Herbert,  the  i s controlled  growth r a t e  the  automatically  growth through c o n t r o l o f the  use  i n f l o w i n g medium  s i m i l a r i n operation,  chemostat r e g u l a t e s  the  the  to  cultures.  continuous culture  which growth r a t e  turbidostat  the  steady state  types of  Although these u n i t s  varying  o f the  i t i s self-regulating.  essentially  while  handling  s u c h u n i t s must be p e r i o d i c a l l y  supply of  inherent  light  ( 1 9 5 0 ) , N o v i c k and S z i l a r d (1950) and H e r b e r t e t a l . (1956)  Monod  The  i n the  state  additional  c u l t u r e , many e x p e r i m e n t e r s t u r n e d t o algae.  steady  in  culture.  scrubbed down,andc.reinoculated w i t h f r e s h  o r d e r to keep a h e a l t h y  fact  reduction  density  maintenance of  and  This  the  batch  in  of  cell cells  (Monod,  density by  1950).  6 A l g a l Waste-Recycling  System i n A q u a c u l t u r e  C u l t u r e o f m a r i n e a l g a e a t h i g h "biomass l e v e l s i n b o t h l a b o r a t o r y and l a r g e - s c a l e outdoor waste and seawater Menzel,  1971;  cultures using municipal  m i x t u r e s has been conducted  Goldman a n d S t a n l e y , 197*0 •  c u l t u r e t e c h n i q u e has been generated waste water  Interest i n this  by t h e d e m o n s t r a t i o n  n u t r i e n t s may be r e c y c l e d i n m a r i n e  systems designed and  (Dunstan and  f o r simultaneous  A s e r i e s o f papers suitability of liquid  aquaculture  t e r t i a r y treatment  food production (Ryther et a l . ,  1972;  i n the l i t e r a t u r e indicate the  o r g a n i c w a s t e s s u c h a s sewage a n d s l u r r y  1 9 7 6 ; Dugan e t a l . ,  ( G a r r e t and A l l e n ,  1976).  o f waste  1975).  as m e d i a f o r t h e c o n t r o l l e d c u l t i v a t i o n o f f r e s h w a t e r algae  that  1971;  micro-  Garret et a l . ,  The c o m p o s i t i o n a n d n u t r i t i o n a l p o t e n t i a l o f a l g a e  a l s o a t t r a c t e d i n t e r e s t f o r many y e a r s  (Burlew  have  ( e d ) , 1953)•  Of p a r t i c u l a r i n t e r e s t a r e r e p o r t s o n t h e n u t r i t i v e v a l u e s o f sewage g r o w n a l g a e f o r r a t s ,  c h i c k , p i g s , c a t t l e and sheep  (Cook e t a l , 1 9 6 3 ; H i n t z e t a l . ,  1966).  M o s t o f t h e i n v e s t i g a t i o n s i n t h e 1960's a n d m i d - 1 9 7 0 ' s were c o n c e r n e d  w i t h t h e c u l t u r e o f a l g a e o n m u n i c i p a l sewage  e. g., i n C a l i f o r n i a , U.SAA. ( O s w a l d (McGarry  et a l . ,  Studies,  1975)  1972),  Australia  and I s r a e l  et a l , 1959).  (Bureau  (Shelef et al., 5  i n Thailand  of Environmental 1972).  Initial  work  w i t h animal wastes d u r i n g t h i s p e r i o d i n c l u d e d p i g wastewater s t u d i e s i n t h e U."*S'. A. a n d i n t h e P h i l i p p i n e s . use  The l a t t e r made  o f r o o f s o f p i g houses as s h a l l o w ponds f o r a l g a l  (Eusebio,  1976).  culture  7 One algae  areas  f o r animal food  question in  of the  the  (either  substrate  d e r i v e d from the toxicants  and  may  animals.  or t e r r e s t r i a l ) i s  from f e e d r e s i d u e s ration,  of algae  toxic  could  D.  growth s t i m u l a n t s  these  are  that  s u c h as  small  t o be  from the pond e f f l u e n t  search  (Dodd, 1 9 7 9 ) '  could  by  f o r an  research  (1963).  Oswald  technological But  still  and  successful.  use of  nitrogen certain  which predominate i n  low  filtered  or  ©-glucosamine  screened employ  C o n s e q u e n t l y , o v e r many c o s t method o f  i n pond t e c h n o l o g y .  (1979)  high-rate  equipment w h i c h  q u a l i t y of a l g a l product  e x t e n s i v e l y by Dodd  harvesting has  Algal harvesting  G o l u e k e and  Oswald  gave a r e v i e w on  m a n a g e r i a l advancement o f a l g a l  t o d a t e no  as  important  hypoxanthine, urea,  effective,  paralleled  and  such  An  certains organic  conventional  decrease the  r e m o v a l were s t u d i e d  contain  tolerate concentration  satisfactorily  w h i c h does n o t the  foods  g e n e r a l l y more p r e d i c t a b l e  f a b r i c m a t e r i a l s u c h as m i c r o s t r a i n e r s . the  in  1980).  planktonic microalgae too  present  effects  i n the  t e r t i o l e c t a have b e e n known t o u t i l i z e  ( A n t i a e t a l . , 1975;  ponds a r e  and  utilize  n i t r o g e n sources  The  toxic residues  the  l i v e s t o c k w a s t e o r sewage i s t h e  of c e r t a i n species  sources.  as  sewage  consideration i n u t i l i z i n g  compounds o r  he  A l t h o u g h a n i m a l w a s t e s may  c o n t r o l l a b l e than with  years  o f waste-grown  have p o s s i b l e c o n c e n t r a t i n g  carryover  c o p p e r added t o t h e  organic  aquatic  use  o f t o x i c a n t s s u c h as h e a v y m e t a l s w h i c h may  p o n d o r g a n i s m s and  and  o f u n c e r t a i n t y i n the  (I965)  different harvesting.  e c o n o m i c a l method o f h a r v e s t i n g has  been  and  8  One  h i g h l y e f f i c i e n t method o f u t i l i z i n g t h e  biomass produced  i s to f e e d i t to a q u a t i c f i l t e r  a s some s p e c i e s o f c a r p , z o o p l a n k t o n and Use  to  f e e d e r s , such  Artemia.  and P o t e n t i a l o f A r t e m i a I n A q u a c u l t u r e In  The  cultured  R e l a t i o n To I t s L i f e  Cycle  b r i n e s h r i m p , A r t e m i a s a l i n a L. i s h i g h l y  an e x t r e m e l y w i d e r a n g e  o f s a l i n i t i e s and  The A r t e m i a i s a n o n - s e l e c t i v e p a r t i c l e  adaptable  temperatures.  filter-feeder.  C o n t r a r y t o many o t h e r c r u s t a c e a n s , i t s f o o d r e q u i r e m e n t s do change d u r i n g growth  1963a;  (Reeve,  1 9 6 3 b ) and i t h a s a  r e p r o d u c t i v e c y c l e which i s very w e l l - s u i t e d to a system  even i n h i g h d e n s i t i e s  Persoone,  1975;  (Helfrich,  Tobias et a l . ,  1973;  weeks),  ( o v e r 100  a high fecundity  reproduce  continuously throughout  life  (Gilchrist,  span The  i960;  cultural  Sorgeloos  (maximum o f  o f f s p r i n g day  t h e i r six-month  Bowen, 1 9 6 2 ;  s a l i n i t y , pH,  Og  s t a g e s and a d u l t s .  N i m u r a , 1967).  t h e r e i s no  as:  special  r a t e and t i m e t o a t t a i n m a t u r i t y  c i t e d i n the l i t e r a t u r e are probably a f u n c t i o n of: a l g a l food a v a i l a b l e ,  by  stages.  D i f f e r e n c e s i n growth  (3)  year  t e n s i o n a r e t h e same f o r "both t h e  That i s ,  nursery; environment/for a l l  and  ) and  s u i t a b i l i t y o f A r t e m i a f o r a q u a c u l t u r e i s enhanced  temperature,  of  and  about  t o one  the f a c t t h a t the p h y s i c a l c u l t u r a l c o n d i t i o n s such  larval  unique  1979).  A r t e m i a have a r a p i d g e n e r a t i o n time 3-4  not  (2)  the presence  (1)  o r absence o f  species bacteria  p h y s i o l o g i c a l s t a t e of the a l g a l food ( G i b o r , 1956a;  9 Mason, 1963;  Reeve, 1963a; S i c k , 1976).  I n a d d i t i o n to the  above environmental c o n d i t i o n s , g e o g r a p h i c a l s t r a i n s have been included ( G i l c h r i s t , Sorgeloos et a l . ,  1956;  1975).  B a i d , 1963;  D'Agostino,  1965;  However, r e c e n t s t u d i e s showed t h a t  Artemia can be r e a r e d and reproduced s u c c e s s f u l l y u s i n g inanimate food ( P e r s o n - l e Ruyet, al.,  1975;  Jacob, 1978;  Sorgeloos et  1980). T e c h n i c a l l y , the advantagesof Artemia f o r a q u a c u l t u r e i s "' u  t h a t i t s t a r t s as dry c y s t s .  These c y s t s a r e , i n f a c t ,  inactive  embryos and are commercially a v a i l a b l e , can be s t o r e d f o r years and o n l y have to be i n c u b a t e d f o r 24-48 hours i n seawater to produce  free-swimming n a u p l i i .  N u t r i t i o n a l Value o f Artemia N a u p l i i As Food I n Aquaculture H a t c h e r i e s The e a r l i e s t s i g n i f i c a n t r e a l i z a t i o n o f Artemia as an e x c e l l e n t source of food f o r e a r l y stages of l a r v a l f i s h been documented ( S e a l e , 1933;  R o l l e f s e n , 1939).  has  Moreover, r e c e n t  s t u d i e s showed t h a t b r i n e shrimp are very w e l l accepted by l a r v a l f i s h e s and crustaceans (May,  1970;  1971;  Houde,  1972).  I t i s not e x a c t l y known whether t h i s can be a t t r i b u t e d i n t h e i r b i o c h e m i c a l composition ( B e n i j t s et a l . , 1978b), or t h e i r t h i n carapace (1 pm) a moving prey (Houde, 1972) factors.  Furthermore,  1975;  Watanabe et a l . ,  or the f a c t t h a t they are  or a combination of a l l these  s t u d i e s showed t h a t a d i e t of l i v e  Artemia g i v e s b e t t e r r e s u l t s than any p r e p a r a t i o n of dead Artemia ( S e r f l i n g e t a l . ,  1974;  Beck, 1979;  Schauer  and  10  and S i m p s o n , 1 9 7 9 ) '  R e c e n t f i n d i n g s showed t h a t w h i t e f i s h l a r v a e  metamorphosed e q u a l l y w e l l i f f e d A r t e m i a p r e v i o u s l y f r o z e n i n l i q u i d n i t r o g e n o r l i v e A r t e m i a , h u t n o t when f e d s l o w - f r o z e n nauplii.  R e c e n t l y , emphasis on l a r v a l r e a r i n g o f f i n f i s h e s  and  c r u s t a c e a n s h a s b e e n p l a c e d on t h e u s e o f g e o g r a p h i c a l s t r a i n s o f the b r i n e shrimp  (Reed,  1969;  O l e y n i k o v a and P l e s k a c h e u s k a y a ,  P r o v e n z a n o a n d Goy, 1979;  U c a l , 1979;  Watanabe,  From t h e s e s t u d i e s , iifcttwas c o n c l u d e d t h a t t h e A r t e m i a G r e a t S a l t L a k e (USA) other strains Shark  Bay  showed p o o r p e r f o r m a n c e  1976;  from  compared w i t h  such as t h o s e from Buenos A i r e s ( A r g e n t i n a ) ,  ( A u s t r a l i a ) , Macau a r e a ( B r a z i l ) , C h a p l i n Lake (Canada),  Lavalduc  (France), Gujarat area (India), Tientsin  (People's  R e p u b l i c o f C h i n a ) , B a r o t a c Nuevo ( P h i l i p p i n e s ) , C a d i z ( S p a i n ) , San F r a n c i s c o Bay  performance  o f t h e G r e a t S a l t L a k e (GSL)  to e x p l a i n the  p o s s i b l e t h a t GSL toxic alkaloid  Artemia (Slodbokin, 1968).  A r t e m i a might have developed  residual I t i s also  immunity a g a i n s t  s e c r e t e d by a l g a l ^ b l o b m s e i n t h e l a k e and  concentrated i n the Artemia c y s t s ( P r o v a s o l i , Another  poor  s t r a i n of Artemia.  o f t h e s e p l a c e s t h e b l a m e on a c c u m u l a t i o n o f  p e s t i c i d e s i n t h e GSL  area  (USA).  V a r i o u s t h e o r i e s have been suggested  One  con.  I969).  s u g g e s t i o n b l a m e s m i n e r a l d i f i c i e n c y . .>  S i n c e t h e use o f A r t e m i a i s p r e s e n t l y l i m i t e d i n most to f r e s h l y hatched n a u p l i i , suggested  t h e use o f a d u l t A r t e m i a has  for further studies.  l a r g e r and w e i g h 5 0 ° (Reeve,  1979).  1963c).  A d u l t Artemia are twenty  t i m e s more t h a n f r e s h l y h a t c h e d  cases  been times  nauplii  T h e i r n u t r i t i o n a l v a l u e changes c o n s i d e r a b l y  11 d u r i n g growth.  T h e i r f a t decreases  20$ t o l e s s t h a n 10$  a n d 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 f r o m 42$ t o  of t h e dry weight  o v e r 60$ ( H e l f r i c h , Whereas n a u p l i i  from  1973; B e n i j t s  et al.,  1975).  aredeficient i n histidine,  methionine,  p h e n y l a l a n i n e and t h r e o n i n e , a d u l t A r t e m i a are r i c h i n a l l e s s e n t i a l amino a c i d s ( G a l l a g h e r a n d B r o w n , 1975; W a t a n a b e e t al.,  1978a; C l a u s e t a l . ,  1979).  Present Cultural Practices of Artemia E x p o n e n t i a l l y i n c r e a s i n g demand f o r b r i n e s h r i m p world-wide  cysts  by aquarium h o b b y i s t s and aquaculture h a t c h e r i e s  e x c e e d e d t h e y e a r l y h a r v e s t o f a p p r o x i m a t e l y 3 ° t o 5° m e t r i c tons  ( S o r g e l o o s , 1976).  been impeded s e r i o u s l y .  As a r e s u l t ,  commercial  aquaculture has  Macrobrachium and Penaeids  dependent on A r t e m i a d i e t d u r i n g J h e i r l o n g l a r v a l (Bledsoe e t a l . ,  1978; G l u d e , e 197.8; S m i t h  I n a d d i t i o n , T h i r d World the v e r y expensive  et al.,  are entirely development  1978).  countries could h a r d l y a f f o r d to import  cysts.  N a t u r a l b r i n e shrimp  p o p u l a t i o n are s t i l l  the most  important source o f commercially a v a i l a b l e Artemia.  However,  they- a r e o o i a l y e x p l o i t e d i n a f e w a r e a s i n C a n a d a , F r a n c e USA  w i t h t h e t o t a l y e a r l y output from  metric tons. Artemia has,  and t h e  t h e s e c o u n t r i e s o f c a . 1000  The p o t e n t i a l f u t u r e h a r v e s t f r o m n a t u r e  where  t o d a t e , b e e n r e c o r d e d f r o m more t h a n 150 h a b i t a t s  c o u l d c o n s i d e r a b l y i n c r e a s e (Persoone  1980).  and S o r g e l o o s ,  A major i n n o v a t i o n i nthe technology o f Artemia c u l t u r i n g i s t h e a i r - w a t e r - l i f t raceway, o r i g i n a l l y  batch  developed  12 for  the i n t e n s i v e c u l t u r e o f p o s t - l a r v a l Penaeid shrimp  al.,  1973)  o u t m o d i f i e d f o r "brine s h r i m p  B o s s u y t and S o r g e l o o s , 1 9 8 0 ) . r i c e bran proved s u f f i c i e n t . in  Monodiet  (Mock e t  u s i n g whey-powder o r  However, i n v i e w o f t h e  differences  f a t t y a c i d composition, i t i s suggested that s t u d i e s  are  needed to e v a l u a t e the n u t r i t i o n a l v a l u e o f b r i n e shrimp on w a s t e p r o d u c t s f o r m v a r l d u s s c u l t u r e d o r g a n i s m s al.,  1980).  1977;  (Sorgeloos et a l . ,  raised  (Dobbeleir et  A s i d e from the a i r - w a t e r - l i f t raceway technology,  a much more i n t e n s i f i e d mass p r o d u c t i o n c a n be a c h i e v e d i n f l o w through systems  (Tobias et a l . ,  c a r r i e d out i n S t . C r o i x  1979)*  The  flow through  (US V i r g i n I s l a n d s ) w e r e r u n s w i t h t h e  e f f l u e n t o f the 2 a l g a l ponds o f t h e l o c a l A r t i f i c i a l Project  1976).  (Roels et a l . ,  test  Upwelling  T o b i a s e t a l . (1979)  calculated -1  3  t h a t i n a 1 nr c a n be p r o d u c e d  t a n k 25 k g a d u l t b i o m a s s ( o r 12000 a n i m a l s 1 w i t h i n 2 weeks f r o m a n i n i t i a l  the above system.  30  g-  cysts using  H o w e v e r , t h e maximum p r o d u c t i v i t y  has n o t b e e n a c h i e v e d due  to lowowater  temperature  )  potential  (22-25°C).  Recently, another i n t e r e s t i n g source of Artemia p r o d u c t i o n has  come i n t o p e r s p e c t i v e .  industrial  T e r t i a r y treatment p l a n t s f o r  e f f l u e n t s of high s a l i n i t y are capable of producing 1979).  s u b s t a n t i a l amount o f a d u l t A r t e m i a ( M i l l i g a n e t a l . , O t h e r s i m i l a r s t u d y was  conducted  i n Bombay, I n d i a .  The  initial  study proved s u c c e s s f u l i n producing g r a v i d females r e a r e d i n manured ponds ( p i g dung) w i t h s u p e r p h o s p h a t e fertilizer.  as  additional  P h y t o p l a n k t o n p o p u l a t i o n c o n s i s t i n g o f N a v i c u l a sp_.,  and A m p h i p h o r a s p . b l o o m e d u n d e r t h i s feed f o r the shrimp.  c o n d i t i o n and  served  However, ground n u t o i l cake and  as  yeast  13 were added as supplement (Dwivedl e t a l . ,  1979).  A s i d e from an improved p e r s p e c t i v e f o r t h e use o f A r t e m i a i n t h e a q u a c u l t u r e h a t c h e r i e s , i t has become obvious t h a t o t h e r a p p l i c a t i o n s show v e r y h i g h p o t e n t i a l even i n c l u d i n g d i r e c t use i n human n u t r i t i o n .  I n a t a s t e p a n e l l t e s t i n H a w a i i o f an  e x p e r i m e n t a l shrimp tempura p r e p a r e d from f r o z e n b r i n e shrimp the response was q u i t e favorable•. (Davidson,  197*0 •  I f n o t used  d i r e c t l y as human f o o d , A r t e m i a meal can be used as a r i c h source o f a n i m a l p r o t e i n i n l i v e s t o c k d i e t s (Anonymous, 1978)• I n t h i s c o n t e x t , b r i n e shrimp may be used as a v a l u a b l e a l t e r n a t i v e t o f i s h m e a l , e s p e c i a l l y i n c o u n t r i e s t h a t a r e e n t i r e l y dependent on f i s h meal i m p o r t s .  14 MATERIALS AND METHODS Algae Dunaliella tertiolecta,  a euryhaline a l g a l species obtained  1  f r o m D r . N. J . A n t i a ' s The  c o l l e c t i o n s was u s e d i n t h e e x p e r i m e n t s .  a l g a was m a i n t a i n e d u n d e r a x e n i c c o n d i t i o n s i n t w e n t y f i v e m l  E r l e n m e y e r f l a s k s i n a l t e r n a t i n g l i g h t a n d d a r k n e s s a t 18°C. The of  a l g a was t h e n e x p o s e d  t o a 12 L,: 12 D r e g i m e f o r a c o u p l e  d a y s "before t r a n s f e r r i n g t o f i f t y m l E r l e n m e y e r f l a s k s a n d  where t h e y w e r e m a i n t a i n e d u n d e r  a x e n i c c o n d i t i o n s as a  "starter"  the Erlenmeyer f l a s k  culture.  When n e e d e d ,  w e r e i n o c u l a t e d i n t o one l i t e r  cultures  o f seawater and a l l o w e d t o r e a c h  a density suitable f o r inoculating the larger experimental culture vessels.  T h i s a l g a was u s e d f o r t h e c o m p a r a t i v e  s t u d y u s i n g two d i f f e r e n t m e d i a , n a m e l y , and d e f i n e d i n o r g a n i c m e d i a .  growth  swine waste-seawaterr  L i k e w i s e , a l g a l "biomass  produced  f r o m e a c h c u l t u r e m e d i a d u r i n g c o n t i n u o u s c u l t u r e was u s e d f o r the  f e e d i n g e x p e r i m e n t o f t h e "brine s h r i m p . Brine  Shrimp  The "brine s h r i m p , A r t e m i a s a l i n a L . eggs w e r e o b t a i n e d 2 from D r . J . M a r l i e v e  .  T h i s s t r a i n o f A r t e m i a came f r o m S a n  F r a n c i s c o Bay, C a l i f o r n i a  (USA).  Research S c i e n t i s t , P a c i f i c Environment I n s t i t u t e 4 l 6 0 M a r i n e D r i v e , West V a n c o u v e r , B. C. >  Research S c i e n t i s t , Vancouver P u b l i c i n S t a n l e y P a r k P. 0 . B o x 3232 V a n c o u v e r , B. C. V6B 3X8  Aquarium  15 The The  b r i n e s h r i m p eggs w e r e s t o r e d a t 4°C p r i o r t o u s e .  eggs w e r e h a t c h e d i n a f u n n e l s h a p e d b o t t o m  c o n t a i n e r a t a d e n s i t y o f 10 g 1  .  The t e m p e r a t u r e a n d  s a l i n i t y w e r e m a i n t a i n e d a t 28 + 1°C a n d 31+1 i n the hatching funnels.  plexiglass  ppt respectively  The t e m p e r a t u r e was m a i n t a i n e d i n a  water bath heated by a g l a s s thermo-heater T h e r m o s t a t i c 100 w a t t s ) .  (Supreme  The n e w l y h a t c h e d n a u p l i i  Heatmaster were  s e p a r a t e d by a s e p a r a t o r d e s i g n e d a f t e r S o r g e l o o s and Persoone^  (1975) Media and S a l t w a t e r P r e p a r a t i o n Swine Waste-Seawater Swine waste  Mixture  samples were o b t a i n e d from t h e f i n i s h i n g h o g  in-housed barn f a c i l i t y  a t t h e UBC F a r m .  Wastes were t a k e n  f r o m t h e s u p e r n a t a n t p o r t i o n o f t h e manure p i t b y u s i n g a B i l g e pump (Whale  g u s h e r 8 pump MK I I I w i t h 3-81 cm i n l e t a n d  o u t l e t ) m a n u a l l y o p e r a t e d and t h e waste f i l t e r e d w i t h a n y l o n , s c r e e n mesh ( 0 . 3 - 0 . 5 c m ) a a t  the outlet.  The f i l t e r e d  swine  w a s t e s were t h e n s t o r e d i n a p l a s t i c c o n t a i n e r (20 1) and transported to the laboratory  (Bio-Resource Engineering  D e p a r t m e n t ) . . The s w i n e w a s t e s w e r e s t o r e d i n a 4°C w a l k - i n refrigerator. UBC  New s a m p l e s  barn every four The  o f swine waste were o b t a i n e d from t h e  days.  samples were a n a l y s e d f o r t o t a l K j e l d a h l  nitrogen,  NH^-N, N0^-N + N 0 - N a n d t o t a l p h o s p h a t e w i t h i n 24 h r s o f 2  collection. was  The r a t i o b e t w e e n s w i n e w a s t e  and seawater volume  v a r i e d d e p e n d i n g on t h e n i t r o g e n c o n c e n t r a t i o n , b u t on t h e  16  a v e r a g e c a 5-10%  w a s t e s and 90-95% s e a w a t e r .  The  nitrogen  -1 c o n c e n t r a t i o n was m a i n t a i n e d a t c a Defined  Inorganic  D.  1200  + 50 j i g - a t N  Medium  t e r t i o l e c t a has a c o n s p i c u o u s r e q u i r e m e n t f o r sodium  (Mclachlan,  i960).  (1975) c o n t a i n s  The medium, . ' f / 2 * d e v e l o p e d b y  sufficient  as a n u t r i e n t s o u r c e .  The  The (20  1)  T h e r e was  vitamins  compared  composition).  s t e r i l e n u t r i e n t s were added  a n d f i l l e d w i t h 15  such  e f f e c t o f f / 2 medium and  s w i n e w a s t e - s e a w a t e r m i x t u r e on a l g a l g r o w t h was (see Appendix 1 f o r f / 2  Guillard  sodium, otherrmag.orrnutrients  as n i t r o g e n , p h o s p h o r u s w i t h t r a c e m e t a l i o n s and served  1  to n o n - s t e r i l i z e d  1 seawater before  carboys  algal inoculation.  no s t e r i l i z a t i o n a t t e m p t i n a n y o f t h e c u l t u r e  experiments. Seawater  Preparation  Since experimental laboratory . ;  the Zoology Department w o r k was  initially  has s e a w a t e r f a c i l i t i e s ,  c o n d u c t e d a t D r . W.  S.  Hoar's  H o w e v e r , a t t h e o n s e t o f s p r i n g I98O, t h e s e a w a t e r  h a d a h i g h c o n c e n t r a t i o n o f m i x e d a l g a l p o p u l a t i o n due temperature.  the 3  A preliminary.experiment  to high  showed t h a t t h e s y n t h e t i c  s e a w a t e r , F o r t y Fathoms ( B i o - c r y s t a l M a r i n e M i x ; A p p e n d i x when s u p p l e m e n t e d w i t h n u t r i e n t s c o u l d be u s e d t o c u l t u r e  P r o f e s s o r , Department o f Zoology U n i v e r s i t y o f B r i t i s h Columbia V a n c o u v e r , B.C. V6T 1W5  2),  17 D.  tertiolecta.  This  artificial  s e a w a t e r was u s e d  i n further  experiments. The from  seawater used  c u l t u r e was  transported  t h e Zoology Department t o t h e Bio-Resource  Engineering  Department.  f o r Artemia  The s e a w a t e r was a g e d f o ra week b e f o r e  i t was  used  Description Algal  of Culture  Batch  Culture  Algal  c u l t u r e u n i t s were  laboratory  Units  Unit  constructed  a t t h e Zoology Department and a t t h e  laboratory o f the Bio-Resource Engineering Batch Unit  I I .  carboys. in  a t D r . W.  c u l t u r e experiments were  Culture  containers  For Unit  f o rU n i t  Department.  culture unit.  flasks Glass  build-up the  glass The  24 + l ° c x  49  the culture flasks  A i r was f i l t e r e d with  tubing  absorbent  with  which  temperature  ports.  also prevented  tubing •  the aeration into the  i n s i d e a Nalgene  leakage  filter  f o ra i r  Rubber stoppers  grown  tubing  to glass  i t was i n t r o d u c e d  cottonc  were  Plastic  o u t l e t s were a l s o p r o v i d e d  and as sampling tubing?  before  borosilicate  tertiolecta  ( T y g o n R-3603 0.476 x O.635 x 0.14 cm) f i t t e d  system.  aquaculture  I I w e r e 20 1  I (Experiment I),,D.  cm o . d . ) c o n n e c t e d  Hoar's  conducted both i n U n i t I and  o n e - l i t e r wide mouth Erlenmeyer f l a s k s .  (0.5  S.  pressure  ( n o . 8)  and  supported  evaporation.  i n t h e c u l t u r e v e s s e l s was m a i n t a i n e d  b y p l a c i n g t h e v e s s e l s , i n t h e wooden w a t e r b a t h  cm x 9 cm) w i t h w a t e r f l o w i n g t h r o u g h a t 1 1 h r  .  at  (115  cm  18 Four 40-Watt*Cool  W h i t e " F l u o r e s c e n t Tubes ( F 2 4 7 1 2 ; H. 0. Lamps)  provided continuous i l l u m i n a t i o n .  Two t u b e s w e r e  installed  h o r i z o n t a l l y o n b o t h s i d e s a p p r o x i m a t e l y 15-20 cm away f r o m t h e culture flasks.  Fluoresecent fixtures  (BL-248,  98 W a t t s , 0.81  amps, 120 v o l t s ) p r o v i d e d p o w e r f o r t h e l i g h t s . were a t t a c h e d on H a n d y - a n g l e s u p p o r t f r a m e s . covered w i t h peg board  These  fixtures  The f r a m e was  ( 1 2 2 x 45 c m ) .  B a t c h c u l t u r e s u s i n g 20 1 b o r o s i l i c a t e  c a r b o y s was  conducted i n U n i t I I w i t h a set-up s i m i l a r t o continuous  culture  experiments. A l g a l Continuous Culture U n i t I I U n i t I I was u s e d f o r c o n t i n u o u s c u l t u r e e x p e r i m e n t s a s w e l l as f o r b a t c h c u l t u r e e x p e r i m e n t u s i n g 20 1 b o r o s i l i c a t e culture container. a n g l e frame tubes  (4 f t ,  T h i s U n i t was h o u s e d  (230 x 131 40 w a t t s  (Pyrex)  i n a p e g board-Handy  x 220 c m ) . A bank o f f o u r  fluorescent  " c o o l w h i t e " ) were a t t a c h e d on b o t h  of the culture containers.  sides  L i k e w i s e , two t u b e s w e r e a t t a c h e d  overhead. The c o n t i n u o u s c u l t u r e a p p a r a t u s c o n s i s t e d o f a n u t r i e n t r e s e r v o i r c o n n e c t e d t o t h r e e c a r b o y s a n d a common y i e l d aspirator bottles.  4-liter  The a l g a l e f f l u e n t was c o l l e c t e d a n d s t o r e d  i n 20 1 c a r b o y s ( F i g u r e 1 ) . The n u t r i e n t m i x t u r e s ( d e f i n e d i n o r g a n i c a n d s w i n e w a s t e - . seawater media) were s t o r e d s e p a r a t e l y i n p l a s t i c c o n t a i n e r s (20 1 ) o n a n e l e v a t e d p l a t f o r m .  A multi-channel p e r i s t a l t i c  c a s e t t e pump ( M a n o s t a t , New Y o r k , N.Y.) was u s e d t o c o n t r o l t h e  AER Figure!.  .Generalized view o f the a l g a l continuous c u l t u r e system. . A, a i r ; AE, a l g a l e f f l u e n t ; APP, a i r p r e s s u r e p o r t ; AS, a i r s t o n e ; AER, a l g a l e f f l u e n t r e s e r v o i r ; BC, b o r o s i l i c a t e c a r b o y s ; CYB, common y i e l d b o t t l e ; FL, f l u o r e s c e n t l i g h t s ; N I , n u t r i e n t inflow; NR, n u t r i e n t r e s e r v o i r ; P P , p e r i s t a l t i c pump; RS, r u b b e r s t o p p e r ; SP, s a m p l i n g p o r t .  i n f l o w of n u t r i e n t s to a l l culture containers. was  m a i n t a i n e d a t a n a v e r a g e o f 2.4  (I.  D.  1  day  .  The  flow rate  Tygon t u b i n g  0.4-76 cm) was u s e d t o c c o n v e y t h e n u t r i e n t s .  f i l t e r e d b e f o r e i t was through a Nalgene  A i r was  i n t r o d u c e d i n t o the c u l t u r e c o n t a i n e r  filter  u n i t with absorbent  cotton.  G l a s s t u b i n g o u t l e t s were p r o v i d e d f o r a i r - p r e s s u r e b u i l d - u p and a s a s a m p l i n g p o r t .  A r u b b e r s t o p p e r ( n o . 11)  g l a s s t u b i n g , p r e v e n t e d l e a k a g e and attempt to s t e r i l i z e glass tubings.  24 15.2  + 1°C cm)  T h e r e was  no  the c u l t u r e carboys, stoppers, tygon  and  However, c u l t u r e v e s s e l s were a c i d - w a s h e d  and  r i n s e d w i t h d i s t i l l e d w a t e r and oven The  evaporation.  supported the  dried.  t e m p e r a t u r e i n t h e c u l t u r e c o n t a i n e r was  maintained at  by p l a c i n g i n wooden w a t e r b a t h ( 2 4 4 cm x 122 with water f l o w i n g through a t 1 1  cm  x  hr  A l g a l Continuous Culture U n i t I I I The  s e a w a t e r a t t h e Z o o l o g y Department had a h i g h  c o n c e n t r a t i o n of contaminants which b a s i c a l l y c o n s i s t e d m i x t u r e s o f a l g a l f l a g e l l a t e s d u r i n g t h e summer t i m e .  of Also,  the  l a b o r a t o r y a t t h e Z o o l o g y D e p a r t m e n t was  t o be r e n o v a t e d b e f o r e  the  E x p e r i m e n t a l work  a n t i c i p a t e d c o m p l e t i o n o f the work.  moved t o t h e a q u a c u l t u r e l a b o r a t o r y o f t h e B i o - R e s o u r c e Engineering  ^-i^i^er  Department.  U n i t I I I se,±-up was the  was  w h o l e s e t - u p was  s i m i l a r to that of Unit I I .  However,  conducted i n a c o n t r o l l e d environmental  chamber ( C o n v i r o n M o d e l E8M,  Controlled Environmental L t d . ,  W i n n i p e g , M a n i t o b a , Canada.  Temperature  was  controlled  and  m a i n t a i n e d a t 24 C,  Continuous  6 f l u o r e s c e n t tubes  (Powertubes  i l l u m i n a t i o n was 48" VHO  S y l v a n i a , Canada) p l a c e d o v e r h e a d .  provided with  'Cool W h i t e  F48T1Z-CW,  1  I l l u m i n a t i o n was  by Quantum/Radiometer/Photometer s e n s o r s  measured  (Lambda L I - 1 8 5 ) • -1  The 0.05  c a 0.04  a v e r a g e . l i g h t i n t e n s i t y was -1 l y min  c a l min  -2 (0.04-  cm  ) measured i n s i d e the carboys w i t h o u t the  liquid  being present. B r i n e Shrimp C u l t u r e U n i t B r i n e shrimp were c u l t u r e d i n c y l i n d r i c a l of  40 l i t e r  capacity.  A PVC  (127 mm  w i t h n y l o n s c r e e n mesh ( 0 . 1 - 0 . 3 mm) v o l u m e a t 20 l i t e r s .  fiberglass  diamter) stand pipe  A handy a n g l e frame h e l d the was  c o l u m n was  order to resuspend  provided.  p r o v i d e oxygen f o r the b r i n e shrimps. Gilmont Flowmeter (F-1200). m a i n t a i n e d a t 27 Thermostatic  -1  c o n s t a n t l y are'rafte'd ( 6 0 0 - 7 0 0 m l  the algae throughout  + 1°C  The  the water  min  measured  i n the tanks  by g l a s s t h e r m o - h e a t e r s  ) in  c o l u m n and  A i r f l o w was  temperature  the  fiberglass  -1 water  fitted  a t the o u t l e t m a i n t a i n e d  t a n k s u p r i g h t and a wooden d r a i n a g e s y s t e m The  tanks  to by  was  (Supreme H e a t m a s t e r  100 w a t t s ) . C u l t u r e Methods  A l g a l C u l t u r e Methods A l t h o u g h none o f t h e c u l t u r e u n i t s r e m a i n e d c o n t a m i n a t i o n b y o t h e r a l g a e was  a v o i d e d by i n o c u l a t i n g w i t h  a x e n i c c u l t u r e and f i l t e r i n g t h e s e a w a t e r filter  unit  (AMF/CUNO F i l t e r M o d e l 1A1)  p r i m i n g pump ( J a b s c o M o d e l No.  bacteria-free, an  with a high pressure  attached to a  12310-0001).  self-  The  d e n s i t y o f t h e i n o c u l a n t was d e t e r m i n e d b e f o r e i t was  introduced . to the culture units.  The a l g a l c e l l s w e r e  counted  d a i l y u s i n g a h e m a c y t o m e t e r (New I m p r o v e d N e u b a u e r Chamber, Hausser S c i e n t i f i c ! ) .  To i m m o b i l i z e t h e m o t i l e D.  tertiolecta,  2 o r 3 d r o p s o f 5% f o r m a l i n were a d d e d t o t h e a l g a l before counting.  samples  The same f o u r s q u a r e s o n t h e g r i d o f e a c h  chamber w e r e c o u n t e d e a c h t i m e a n d t h e a v e r a g e  c o u n t was  4 m u l t i p l i e d b y 10 , ( c e l l s ml  T h u s , g i v e n t h e a v e r a g e Q, t h e d e n s i t y e,'  ) o f t h e s u s p e n s i o n i n t h e h e m a c y t o m e t e r was c a r a u l a t e d  from the e x p r e s s i o n , d = 10* x Q Three  r e p l i c a t e c o u n t s w e r e made a n d t h e mean v a l u e , c a l c u l a t e d . S a l i n i t y was m a i n t a i n e d a t 32 + 1 p p t a n d was m e a s u r e d  u s i n g a hydrometer  ( S p e c i f i c g r a v i t y f o r h e a v y l i q u i d s TP 60/60°F  range  Temperature  1000-1220).  culture units.  r e a d i n g s were t a k e n d a i l y i n a l l  P e r i o d i c c h e c k s o n pH change i n t h e c u l t u r e s  helped t o m o n i t o r a c i d i c and/or b a s i c c o n d i t i o n s w i t h i n t h e culture.  The pH was m e a s u r e d u s i n g a pH m e t e r ( F i s h e r A c c u m e t  M o d e l 420 D i g i t a l p H / i o n  meter).  Once t h e a l g a l g r o w t h p h a s e r e a c h e d t h e d e c r e a s i n g o r declining logarithmic p h a s e ( M y e r s , 19^2) a t a d e n s i t y o f 6 -1 a p p r o x i m a t e l y 4 x 10 c e l l m l , the nutrient inflow to the c u l t u r e v e s s e l was i n i t i a t e d .  F l o w r a t e was m a i n t a i n e d a t -1  a p p r o x i m a t e l y 2.33  - 2.50 l i t e r  day  -1 carboy  .  At this flow  rate, the c e l l  d e n s i t y reached steady s t a t e , as determined by  identical cell  c o u n t s o v e r a p e r i o d o f 2-3 d a y s .  d e n s i t i e s would  The  cell  drop t o a l o w e r c o n c e n t r a t i o n a t a h i g h e r f l o w  23 rate. B r i n e Shrimp C u l t u r e Method The  i n c u b a t i o n p e r i o d was  D e n s i t y d e t e r m i n a t i o n was  a b o u t 48r.hours a t 26  conducted  a f t e r the  n a u p l i i were s e p a r a t e d from unhatched Newly-hatched  d i a m e t e r , 250  cysts. plastic  mm  each were t a k e n from  l o n g g l a s s t u b i n g was  the  suspension.  used as  sampling  B o t h e n d s o f t h e t u b e w e r e o p e n e d and when s a m p l i n g ,  one  end was  was  sampled.  cylinders  c l o s e d by a thumb.  (50  Thus, the e n t i r e water  ml  cap).  Then t h e volume i n t h e c y l i n d e r s  s l o w l y to e v e n l y suspend each  column  Each o f t h e 5 ml samples were p l a c e d i n s e p a r a t e  made up t o 25 m l o f t h e same s a l i n i t y .  from  e g g s s a n d empty  a e r a t e d t o k e e p them e v e n l y s u s p e n d e d .  F i v e s e p a r a t e samples o f 5 ml  tube.  newly-hatched  A r t e m i a were p l a c e d i n a 4 - l i t e r  container, moderately  An 8 mm  + 1°C. .  the n a u p l i i .  was  T h i s v o l u m e was  aerated  A g a i n , 5 ml were  taken  c y l i n d e r and w e r e p l a c e d i n s m a l l g l a s s p e t r i d i s h e s .  S e v e r a l drops o f L u g o l ' s s o l u t i o n were added t o each p e t r i Then, the p e t r i  d i s h e s were p l a c e d under a d i s s e c t i n g  and  counted.  the n a u p l i i  c o n t a i n e r was T o t a l no.  Algal 5.0  cell  number o f n a u p l i i i n t h e  microscope original  o b t a i n e d by t h e f o l l o w i n g e q u a t i o n :  of nauplii *  . . Initial  The  dish.  =  T o t a l no. ~  of n a u p l i i -z  counted  volume o f - container -1  concentration m  c o n c e n t r a t i o n was  < -1 x 1CK m l i n each  tank.  e a c h t a n k was  2-3  nauplii  liter  maintained d a i l y at approximately  S u r v i v a l counts were conducted A 5 ° n i l "beaker was u s e d n a u p l i i were counted.  d a i l y i n each  t o samp He t h e w a t e r  column and t h e  The c u l t u r e was a g i t a t e d "by a r e l a t i v e l y  s t r o n g a e r a t i o n f o r a m i n u t e "before s a m p l i n g . 3 times f o r each  from  C o u n t i n g was done  tank.  To m e a s u r e g r o w t h , preseved  tank.  each  tank.  t e n a n i m a l s were sampled d a i l y and Preserved animals  ( i n a 5$ f o r m a l i n  s o l u t i o n ) v : w . e r e m e a s u r e d two weeks a f t e r t h e e x p e r i m e n t . The  t o t a l b o d y l e n g t h was m e a s u r e d f r o m  the a n t e r i o r margin o f  the head t o t h e base o f t h e c a u d a l f u r c a ( G i l c h r i s t ,  1956).  D u r i n g m e a s u r e m e n t , t h e a n i m a l s w e r e p l a c e d v e n t r a l s i d e down o n a mjr.c r o s e ope s l i d e . The  temperature  i n t h e c u l t u r e t a n k s was m a i n t a i n e d a t  27 + 1°C b y g l a s s t h e r m o s t a t i c h e a t e r s . was a t s a t u r a t i o n  Oxygen c o n c e n t r a t i o n  levels. Chemical A n a l y s i s  Kjeldahl Nitrogen The  m e t h o d f o l l o w e d was a n a d a p t a t i o n o f W.all e t a l . (197*0  and T e c h n i c o n A u t o A n a l y s e r I I ( 1 9 7 1 b ) .  F i v e m l o f t h e sample  ( s w i n e w a s t e o r a l g a l s u s p e n s i o n H ' w e r e i n t r o d u c e d i n t o a 50 m l d i g e s t i o n tube.  To e n h a n c e t h e o x i d a t i o n r e d u c t i o n , 0 . 5 g o f a  digestion catalyst  ( c o m p o s e d o f 9 9 6 0 g K^SO^, 3 5 g CuSO^, a n d 5 g  SeOg) w e r e a d d e d t o e a c h t u b e .  The t u b e s were t h e n p l a c e d i n a  d i g e s t o r r a c k a t 360°C f o r 6 - 1 2 h o u r s . chips prevented  The a d d i t i o n o f b o i l i n g  e x c e s s i v e bumping d u r i n g d i g e s t i o n and a s m a l l  g l a s s f u n n e l on t h e open end o f t h e t u b e s p r e v e n t e d  spillage  25 and e x c e s s i v e e v a p o r a t i o n .  A f t e r d i g e s t i o n , t h e t u b e s w e r e ei:.  r e m o v e d f r o m t h e d i g e s t o r and c o o l e d f o r a b o u t a n h o u r . w a t e r was  t h e n added  Distilled  t o e a c h o f t h e f l a s k s t o a v o l u m e o f 50  ml.  F u r t h e r d i l u t i o n o f t h e s a m p l e w i t h d i s t i l l e d w a t e r was n e c e s s a r y when t h e a u t o - a n a l y s i s g r a p h i c a l r e c o r d e r w e n t  off-scale.  A p p r o x i m a t e l y 10 m l o f e a c h d i l u t e d s a m p l e w e r e p l a c e d on a r o t a t i n g sampler o f the auto-analyser. Ammonia / N i t r a t e + N i t r i t e The  automated p r o c e d u r e f o r t h e d e t e r m i n a t i o n o f t h e above  n i t r o g e n o u s compounds was  II,  1969;  Total  adapted to that of Technicon AutoAnalyser  1971a.  Phosphate Samples  were d i g e s t e d b e f o r e d e t e r m i n i n g t o t a l  through the auto-analyser. to The  The  d i g e s t i o n p r o c e d u r e was  t h a t used f o r the d e t e r m i n a t i o n o f t o t a l K j e l d a h l 1  automated  phosphate similar  nitrogen.  procedure f o r the d e t e r m i n a t i o n o o f t o t a l  phosphate  w a s r a d a p t e d t o t h a t o f T e c h n i c o n A u t o A n a l y s e r , 1971c. Dry Weight  Analysis  An i n c r e a s e i n d r y w e i g h t o f c e l l s i s a w i d e l y u s e d m e t h o d of  growth measurement. F i f t y ml a l i q u o t samples from each c u l t u r e c o n t a i n e r were  filtered s i z e 4.25  through glass f i b e r f i l t e r  (Reeve A n g e l Grade  cm d i a m e t e r ) u s i n g vacuum s u c t i o n .  The  93^  filter  AH,  paper  was p l a c e d on t h e . c r u c i b l e and o v e n d r i e d a t lOO^C f o r a b o u t  an  26 h o u r and w e i g h e d b e f o r e u s i n g .  After filtration,  crucible  f i l t e r p a p e r were oven d r i e d t o a c o n s t a n t w e i g h t .  Three  r e p l i c a t e s were r u n e f o r each o f t h e c u l t u r e v e s s e l .  The  and  filter  p a p e r a n d c r u c i b l e w e r e w e i g h e d on a M e t t l e r b a l a n c e (Type dig  cap 160  g, M e t t l e r I n s t r u m e n t , H i g h s t o w n N. S.  The w e i g h t was r e c o r d e d t o f o u r d e c i m a l p l a c e s . b i o m a s s o f A r t e m i a was m e a s u r e d  as d e s c r i b e d  T o t a l Suspended  swine waste samples, t o t a l  U s i n g s u c t i o n , 100 2-5  ml d i s t i l l e d  (Whatman 7.0  cm n o .  The  concentration i n  suspended s o l i d s were a n a l y s e d . and w a s h e d w i t h  c r u c i b l e and f i l t e r  paper  h a r d e n e d ? , a s h l e s s ) were oven d r i e d a t  103-105°C f o r a n h o u r b e f o r e u s i n g . p a p e r was  Dry weight  Solids  ml samples were f i l t e r d  water.  Canada).  above.  To d e t e r m i n e t h e v a r i a b i l i t y o f t h e s o l i d the  The o v e n d r i e d  filter  c o o l e d i n a d e s i c c a t o r and t h e w e i g h t was  After filtration,  recorded.  t h e f i l t e r p a p e r and c r u c i b l e w e r e t h e n  p l a c e d i n t h e oven f o r an h o u r , c o o l e d i n a d e s i c c a t o r weighed  H6  (gross weight).  u n t i l l c o n s t a n t w e i g h t was  and  D r y i n g and w e i g h i n g w e r e c a r r i e d achieved  (normally three  out  times).  F i v e r e p l i c a t e s were r u n f o r each sample. Analysis for total  s u s p e n d e d s o l i d s was  'fresh'ssamples taken from the pit-manure. was  c a l c u l a t e d usiriglrfchersfollowing  TSS ppm  (mg 1 ) 1  =  T o t a l suspended  equation:  G r o s s wt - T a r e wt vol  conducted f o r  o f sample  (mg)  (ml)  x  10  6  solids  27 RESULTS Swine Waste A n a l y s i s During  the  course  nitrogen concentration n i t r o g e n ) and of the and  of the experiments, (TKN  the t o t a l K j e l d a h l  = o r g a n i c n i t r o g e n and  ammonia  o t h e r i n o r g a n i c n i t r o g e n (NOg-N + NO^-N) and  supernatant  p a r t of the swine waste v a r i e d o n l y  a l s o t h e r e was  suspended s o l i d s  n o t much c o n s i d e r a b l e (TSS)  as  changes i n  shown i n T a b l e 1 .  total  t o t a l K j e l d a h l n i t r o g e n o f t h e s w i n e w a s t e s a m p l e s and  1 : 2 ) s t o r e d a t 4°C if The  r e f r i g e r a t o r were c a r r i e d out  changes i n N d i s t r i b u t i o n might occur r e s u l t s showed no  considerable  n i t r o g e n c o n c e n t r a t i o n b e t w e e n day A l g a l Batch The D.  first  Culture  e x p e r i m e n t on t h e  during  f l a s k s i n batch  three  ( 1 : 1 , 2:1  to  and  change o f t h e t o t a l K j e l d a h l 1. and  day  5 (Figure 2 ) .  System comparative growth study and  of  swine waste-  c o n d u c t e d i n o n e - l i t e r w i d e mouth E r l e n m e y e r  c u l t u r e system.  Growth c o n s t a n t  t h e mean g e n e r a t i o n  d e f i n e d i n o r g a n i c and Another batch liter  and  determine  F i g u r e 3 shows d a i l y c e l l d e n s i t y d u r i n g t h e s e v e n culture period.  of  storage.  t e r t i o l e c t a g r o w n i n d e f i n e d inorganicir,medium  s e a w a t e r medium was  slightly  Measurement  d i f f e r e n t ratio:s;:- o f s w i n e w a s t e - s e a w a t e r m i x t u r e s  PO^-P  (K^Q^hr  day  ) w e r e 0 . 0 2 3 and  t i m e ( t g h r s ) w e r e 13.0  and  12.5  0.024  f°  r  swine waste-seawater media, r e s p e c t i v e l y .  c u l t u r e study  capacity Pyrex carboys.  (Expt  2 ) was  However, the  conducted i n 2 0  c u l t u r e volume  was  T a b l e 1.  T o t a l K j e l d a h l n i t r o g e n ( T K N ) , NH^-N, N O ^ - N ^ N 0 ~ N 2  Solids  f  PO^-P a n d T o t a l  (TSS) o f s w i n e w a s t e d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t a l  work.  Total Kjeldahl NH^-N N0~-N+N0 -N k~ N i t r o g e n (TKN) ^ -1 -1 -1 (jig-at N 1 ) (ug-at N 1 ) ( u g - a t N 1 ) (/ig-at E p o  Suspended  P  ?  Sampling Date  25 S e p t 28 O c t  1979*  Experiment No.  1 2  1  29 Nov 07 Dec 10 Dec  3 3 3  2.5 4.9 5.0 4.6 5.4  13 Dec 16  3 3  5.1 4.8  04 F e d ( E x p t 4,...was discontinued)  3.9  10 M a r c h  4.2  x  4 10*  1.0 4.0 3.5 3.0 3.6 3.6 3.4 2.8  18 J u n e  5  3.8  22  5  26  5  3.7 3.8  2.5 2.3 2.1 2.2  30  5 5  4.1 3.6  2.5 2.2  10  5  15  5  4.3 3.7  2.5 2.2  05  July  x  4 10^  3-57 5.00 6.43 3.57 3.57 2.14  3.2  x  l  10  2  _1 )  Total Suspended Solids., (mg 1 ) l  3-0 :  3.87 5.16 3.20 6.12 5.80  3.9 3.31  3.20  3.09  3.87 3.20  3-50  3.4 4.05  5-0  5-71  3.57 5-0 3.57 5.0 3.57  2.9 3.54  3-7 3.05 3.12  4.83 3.20  3.8  4.19 2.58  3.5  * T h i s p a r t i c u l a r s w i n e w a s t e s a m p l e was o b t a i n e d f r o m R a n d H Farm L a n g l e y , B. C. The r e s t o f t h e s a m p l e s were f r o m U. B. C. No. 1 R e s e a r c h Farm.  29 800  n  700'  600-  £ c  G.  C OJ  500^  o> o  .c o  5  400  300^  200^  10-0-1 0  F i g u r e 2,  -r  TIME (days)  2  o f swme waste-seawater m i x t u r e s ( 1  i  D  f o means  30  TIME ( d a y s ) F i g u r e 3-  D a i l y c e l l d e n s i t y o f ' D . t e r t i o l e c t a grown i n s w i n e w a s t e - s e a w a t e r ( O ) and d e f i n e d i n o r g a n i c . m e d i a (® ) i n b a t c h s y s t e m ( E x p t 1 ) .  31 m a i n t a i n e d o n l y up t o 15 w i t h the l a t t e r  I n t h i s e x p e r i m e n t and  weight, r e s p e c t i v e l y .  calculated.  also  ones, t h r e e r e p l i c a t e s were used f o r each  F i g u r e s 4 and 5 show d a i l y c e l l  treatment. dry  liters.  Statistical  d e n s i t y and  algal  A g a i n , t h e growth c o n s t a n t s were analysis using analysis of variance  (ANOVA) showed no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t r e a t m e n t s and r e p l i c a t e s f o r growth a n a l y s i s was  constant (Appendix 3).  done t h r o u g h t h e UBG  The  statistical  Computer System  using  a  G e n e r a l L e a s t S q u a r e s A n a l y s i s o f V a r i a n c e Programme known as GENLIN ( G r e i g and B j e r r i n g ,  1977).  A l g a l Continuous Culture  System  C o n t i n u o u s c u l t u r e s t u d i e s w e r e c o n d u c t e d i n 20 capacity Pyrex carboys. was  t e r m i n a t e d on d a y 16  The  f i r s t continuous culture  because  ( D r . W.  differences 4a,  4b, The  day 2 7 .  (P<0.05)  S. H o a r ' s  Statistical  with  laboratory,  F i g u r e s 6 a n d 7 show d a i l y c e l l  and a l g a l d r y w e i g h t , r r e s p e c t i v e l y . growth c o n s t a n t , c e l l  experiment  t h e r e w e r e some p r o b l e m s  seawater supply i n the l a b o r a t o r y Zoology Department).  liter  density  a n a l y s i s f o r the  d e n s i t y and d r y w e i g h t showed no  significant  b e t w e e n treatmentsr>,arid r e p l i c a t e s  (Appendices  4c). s e c o n d c o n t i n u o u s c u l t u r e e x p e r i m e n t was The  algae produced d a i l y from t h i s  i n the feeding studies of Artemia.  t e r m i n a t e d on  e x p e r i m e n t were used  F i g u r e s 8 and 9 show  cell  d e n s i t y and d r y w e i g h t , r e s p e c t i v e l y , measured d a i l y d u r i n g t h e culture period.  T h i s e x p e r i m e n t was  conducted i n a  e n v i r o n m e n t a l chamber a t t h e D e p a r t m e n t  controlled  of Bio-Resource  32  T I M E (days) F i g u r e 4.  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e - s e a w a t e r (o ) a n d d e f i n e d i n o r g a n i c m e d i a ( • ) i n b a t c h c u l t u r e s y s t e m u s i n g 20 1 b o r o s i l i c a t e carboys (Expt 2 ) , The a b o v e v a l u e s a r e r a n g e s a n d means o f r e p l i c a t e s p e r t r e a t m e n t .  33  0.20-,  i  o>  0.15-  t_ xi  o  0.10-  < 0.051  0 TlME(days) Figure  5-  D a i l y measurement o f d r y wt o f D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e - s e a w a t e r ( O ) and d e f i n e d i n o r g a n i c media ( • ) i n a b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 2 ) . The a b o v e v a l u e s a r e r a n g e s and means o f r e p l i c a t e s per treatment.  3k  F i g u r e 6.  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n s w i n e w a s t e - s e a w a t e r ( O ) and d e f i n e d i n o r g a n i c m e d i a ( # ) i n c o n t i n u o u s c u l t u r e s y s t e m u s i n g - 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 3 ) • The a b o v e v a l u e s a r e r a n g e s and means o f r e p l i c a t e s p e r t r e a t m e n t .  35  u  '  '  1  F i g u r e 7.  I  1  1  1  5  1  1  1  1  1  10 TIME (days)  1  1  1  1  r-—i 1 5  D a i l y m e a s u r e m e n t o f d r y wt Of D. t e r t i o l e c t a g r o w n i n s w i n e w a s t e - s e a w a t e r (o ) and d e f i n e d i n o r g a n i c m e d i a ( • ) i n c o n t i n u o u s c u l t u r e s y s t e m u s i n g 20 1 b o r o s i l i c a t e carboys (Expt 3 ) . Arrow i n d i c a t e s s t a r t of continuous culture. The a b o v e v a l u e s a r e r a n g e s a n d means o f r e p l i c a t e s p e r t r e a t m e n t .  36  1  F i g u r e 8.  5  10  15 TIME (days)  20  25  D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n s w i n e waste-seawater ( O ) and d e f i n e d i n o r g a n i c media ( • ) i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e carboys (Expt 5 ) ' Arrow i n d i c a t e s s t a r t o f continuous culture. The above v a l u e s a r e r a n g e s and means o f r e p l i c a t e s p e r t r e a t m e n t .  30  37 0.35n  0.30H  0.05H  u  '—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—|—i—i  1  F i g u r e 9.  5  10  15 TIME (days)  20  25  D a i l y m e a s u r e m e n t o f d r y wt o f D.. t e r t i o l e c t a grown i n • s w i n e - w a s t e — s e a w a t e r (o) and d e f i n e d i n o r g a n i c m e d i a ( • ) i n c o n t i n u o u s c u l t u r e s y s t e m u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 5)• A r r o w i n d i c a t e s s t a r t o f continuous culture. The a b o v e v a l u e s a r e r a n g e s a n d means o f r e p l i c a t e s p e r t r e a t m e n t .  30  38  E n g i n e e r i n g Department A q u a c u l t u r e L a b o r a t o r y . a n a l y s i s showed a h i g h l y  Statistical (P<0.01)  significant difference  b e t w e e n t r e a t m e n t s b u t i t showed no s i g n i f i c a n t d i f f e r e n c e s f o r replicates,(Appendix 5a). (onset o f n u t r i e n t were d i f f e r e n t 5c,  5d)«  The d a t a a n a l y s e d w e r e f r o m d a y 8  i n f l o w ) up t o d a y 2 7 .  However,  when d a t a w e r e a n a l y s e d i n s e g m e n t s  Thus, t h e f o l l o w i n g  the results ( A p p e n d i c e s 5"b,  were o b t a i n e d :  Days  F-Ration  8-14  treatment replicate  highly significant not significant  15-22  treatment replicate  both n o t s i g n i f i c a n t  23-27  treatment replicate  both n o t s i g n i f i c a n t  The v a r i a b i l i t y  o f t h e above r e s u l t s  t h e v e r y dynamic system.  Statistical  (P<0.01)  was p r o b a b l y due t o  analysis  f o r growth  constant  showed no s i g n i f i c a n t d i f f e r e n c e s f o r t r e a t m e n t s a n d r e p l i c a t e s (Appendix  6).  However,  f o r a l g a l d r y weight,  differences f o r treatment were o b s e r v e d  (Appendix  (P<0.01)  significant (P<0.05)  and f o r r e p l i c a t e s  7).  N u t r i e n t s i n t h e form o f n i t r o g e n such as t o t a l  Kjeldahl  n i t r o g e n , NH^-N, 0 r g a n i c - N i a n d NO^-N + NOg-N w e r e m e a s u r e d i n t h e algal to  effluent  d u r i n g t h e second  continuous culture  determine whether these n u t r i e n t s  (Expt 5)  were f u l l y u t i l i z e d  (Table22).  1  39 Table  2".  Nitrogen  (TKN, NH^-N, ©rganic-N",- NO^-N + N 0 )  measured  i n t h e a l g a l e f f l u e n t d u r i n g t h e second  2  continuous waste  c u l t u r e ( E x p t 5) i n u g - a t N 1  s e a w a t e r medium; DM, d e f i n e d i n o r g a n i c mediumO  Date  Sample No.  26 J u n e (d9)  SW 1  3.8  2  3.6  3  3.4  3-5 2.8  DM 1  2.0  1.4  2  1.7  1.4  3  2.1  1.8  SW 1  3.2  2.1  2  2.8  1.4  3 DM 1  2.9  2.1 3.8  2  1.3 1.6  2.1  1.4  3  1.4  1.8  1.2  SW 1  1.8  2  2.5 2.1  3  1.8  1.1  . DM 1  7.1  X  2  1.4  X  3 SW 1  1.4  30 J u n e (dl3)  06 J u l y (dl9)  10 J u l y (d23)  TKN  X  !0  NH^-N  2  3.2  io  1  2 10  3.6  X  3 DM 1  1.9  8.9  9.3  2  3.6 X  10  2  1  X  IO  undetectable  1  II  6.0  it  1.9  X  1.6  2 10  X  10  l.l  X  X  X  10  1  2 10  X  X  10  1  2 10  X  2 10 IO  it  1  1.07 x i o X  io  2  X  io  1  1.28 undetectable  8.0  it  7.0  If  1.42 x 1 0 X  2 10  io  1  1.0  X  1.9  7.4  X  2 10^ IO  1.9 1.8  2.0 X  io  1.1  1.64 undetectable  1.3  1  1  it  II  1.28 x 1 0 1.50  2  1.78  S a m p l e n o s 1, 2 a n d 3 w e r e o b t a i n e d f r o m e a c h o f t h e culture vessels. ^Organic  N i t r o g e n * (TKN)  (NH--N)  2  1.14  X  io  2  1.14  9.0  X  undetectable II  9.2  7.0  2  1.28  1.4 8.0  1.78 x 1 0 1.85  1.9 2  NO^-N+NOg-N  1.0  1.2  1.4 1  10  6.0  2.1  2.3  io  2 10^  2.6  2  X  Organic Nitrogen  3-5 1.1  1.4  1.3  X  1.3  2.7  3  .(SW, s w i n e  X  2  40 B r i n e Shrimp Feeding Experiment s F i g u r e 10 shows the growth i n t o t a l l e n g t h o f Artemia ( f r e s h l y measured and p r e s e r v e d samples) d u r i n g the 12-day culture period.  I n t h i s p r e l i m i n a r y experiment the a l g a l  d e n s i t y was maintained d a i l y a t the range 3«0 to 5*0 x 10-^ c e l l s ml  .  F i g u r e 11 shows the percentage s u r v i v a l f o r t h i s  experiment. F i g u r e s 12 and 13 show the t o t a l l e n g t h measurement and percentage s u r v i v a l o f the s h r i m p , r e s p e c t i v e l y d u r i n g the 14-day (  culture period.  I n t h i s p a r t i c u l a r experiment f o u r r e p l i c a t e s  were used f o r each treatment. S t a t i s t i c a l a n a l y s i s f o r t o t a l l e n g t h measurement showed no s i g n i f i c a n t d i f f e r e n c e s f o r treatments and r e p l i c a t e s (Appendix 8 ) .  However, s t a t i s t i c a l a n a l y s i s f o r percentage  s u r v i v a l showed h i g h l y s i g n i f i c a n t d i f f e r e n c e s (P<0.01) f o r treatments and r e p l i c a t e s .  The o v e r a l l l m e a n l e n g t h o f Artemia  i n treatment 2 (Artemia f e d w i t h swine waste grown a l g a e ) was h i g h e r than tr.eatmenteibifcArtemia f e d with d e f i n e d i n o r g a n i c medium grown a l g a e ) .  Furthermore,  s t a t i s t i c a l a n a l y s i s showed  d i f f e r e n t r e s u l t s when percentage s u r v i v a l d a t a were a n a l y s e d i n segments (days 1-7 and 8-14).  The a n a l y s i s f o r the f i r s t  seven  days showed h i g h l y s i g n i f i c a n t d i f f e r e n c e s (P<0.01) i n percentage s u r v i v a l y f o r treatments and r e p l i c a t e s . last  seven days the percentage s u r v i v a l showed no s i g n i f i c a n t  d i f f e r e n c e s f o r treatments and r e p l i c a t e s 9c).  However, d u r i n g the  (Appendices 9a, 9t>,  U"  1  1  1  1  1  1  5  1  1  .  1  1  1  • 10  1  1  1  !  1  15  TIME (days) Figure.10.  Growth i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. over a 12-day c u l t u r e p e r i o d f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium. The above v a l u e s a r e average measurements o f a n e s t h e s i z e d samples ( • ) and 2-weeks o l d p r e s e r v e d s a m p l e s (o).  42  F i g u r e 11.  P e r c e n t a g e s u r v i v a l o f A r t e m i a s a l i n a L. f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium. The above v a l u e s a r e r a n g e s a n d means o f r e p l i c a t e s .  43  Figure  12.  G r o w t h i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. o v e r a 14-day c u l t u r e p e r i o d f e d w i t h s w i n e w a s t e - s e a w a t e r ( O ) and d e f i n e d i n o r g a n i c ( # ) m e d i a grown D. t e r t i o l e c t a . The a b o v e v a l u e s a r e r a n g e s and means o f f o u r r e p l i c a t e s p e r treatment.  T  5  gure 1 3 .  10 TIME (days)  Percentage s u r v i v a l o f A r t e m i a s a l i n a L. over a • 1'4-day c u l t u r e p e r i o d f e d w i t h s w i n e w a s t e seawater (o ) a n d d e f i n e d i n o r g a n i c m e d i a ( • ) grown D. t e r t i o l e c t a . T h e above v a l u e s a r e r a n g e s a n d means o f f o u r r e p l i c a t e s .  15  A d u l t Artemia produced i n the system a f t e r the 14-day c u l t u r e p e r i o d were measured i n wet and dry weight (Table 3 ) . S t a t i s t i c a l  basis  a n a l y s i s f o r wet and dry weight o f  Artemia showed no s i g n i f i c a n t d i f f e r e n c e s between treatments and r e p l i c a t e s (Appendices 10a and 10b, r e s p e c t i v e l y ) .  46  T a b l e 3-  A d u l t A r t e m i a biomass produced a f t e r t h e l4-day c u l t u r e p e r i o d i n wet a n d d r y w e i g h t b a s i s .  Artemia Fed With Swine Waste-Seawater Grown A l g a e  1 2 3 4  Wet W e i g h t (g) —  —-  Dry Weight (g) 1.82  18.82  19.^6 19.43 20.47 19.55 + 0.68  2.13 2.06 2.40 2.10 + 0.24  1  16.00  1.50  2....*  18.06  1.91  3 4  19.08  2.01  18.05  1.87  X Artemia Fed With Defined Inorganic Medium Grown A l g a e  X  17.80  + 1.29  1/82  +0.22  47 DISCUSSION L i v e s t o c k Waste As A N u t r i e n t Source In A l g a l C u l t u r e A n a l y s i s of t o t a l K j e l d a h l n i t r o g e n (TKN), -NH^-N, NO^-N NOg-N PO^-P  and  t o t a l suspended s o l i d s  samples c o l l e c t e d d u r i n g the course t h a t the n u t r i e n t content The  (TSS)  +  o f the swine waste  of the experiments showed  o f the waste v a r i e d o n l y  slightly.  constancy o f the manure n u t r i e n t s c o n c e n t r a t i o n found i n these  experiments i s s i g n i f i c a n t  (Table 1 ) .  It  i n d i c a t e s that f o r a  p a r t i c u l a r farm i t m.aybe .unnecessary t'ooanalyse waste and to p r o v i d e  each batch  t h a t a n a l y s i n g the waste p e r i o d i c a l l y may the necessary  nutrient loading rates.  of  be adequate  i n f o r m a t i o n f o r c a l c u l a t i n g the r e q u i r e d In t h i s case,  the samples were taken  from the same batches of f i n i s h i n g hogs,and j u s t p r i o r to p i t f l u s h i n g which may  have i n c r e a s e d the u n i f o r m i t y of the manure  samples. In the p r e s e n t  study,  found i n l i v e s t o c k waste was  the h i g h e s t percentage of n i t r o g e n i n the form o f ammonia n i t r o g e n which  i s in,;.- agreement with the r e s u l t s of Dunstan and Menzel (1971) and Thomas et a l . (1974) f o r the sewage.  T h i s form o f i n o r g a n i c  n i t r o g e n i s r e a d i l y u t i l i z e d by phytoplankton l e v e l s are not a t t a i n e d . mixtures,  as l o n g as t o x i c  Although i n the swine waste-seawater  ammonia n i t r o g e n c o n c e n t r a t i o n was  about 5°0 ug-at  -1 N 1^  and- 5 ° % h i g h e r than t h a t of d e f i n e d i n o r g a n i c medium, t h i s  NH^-N  l e v e l d i d not appear to e x h i b i t t o x i c e f f e c t s on the  algae.  48 Certain species of phytoplankton Dunaliella tertiolecta) nitrogen  can a l s o f u l l y  (Hemiselmis utilize  v i r e s c e n s and  organic sources o f  ( A n t i a a n d C h a r n e y , 1968; A n t i a e t a l . ,  1975).  I f t h e s e p h y t o p l a n k t o n w e r e grown o n l i v e s t o c k w a s t e o r s e w a g e , t h e y c o u l d have a g r e a t advantage i n terms o f n i t r o g e n u t i l i z a t i o n and  c o n v e r s i o n e f f i c i e n c y s i n c e l a r g e amounts o f o r g a n i c n i t r o g e n  are found i n these wastes.  I t s h o u l d he n o t e d , h o w e v e r , t h a t i n  the present study very l i t t l e medium due'Lto  organic nitrogen i spresent i n the  prefiltration.  A l t h o u g h a n i m a l w a s t e s may c o n t a i n t o x i c a n t s f r o m r e s i d u e s and growth  s t i m u l a n t s such as copper  feed  added t o t h e  r a t i o n , t h e s e a r e g e n e r a l l y more p r e d i c t a b l e a n d c o n t r o l l a b l e t h a n i n t h e c a s e o f sewage (Dodd, 1 9 7 9 ) .  Variability of  n u t r i e n t s f o u n d i n sewage c o u l d a l s o a f f e c t t h e s p e c i e s composition o f phytoplankton i n culture 1972)  g r o w n o n t h i s medium.  (Dunstan  and Tenore,  Moreover, the problem  of toxicants  i n l i v e s t o c k waste i s c o n s i d e r a b l y l e s s i n d e v e l o p i n g c o u n t r i e s than i n h i g h l y i n d u s t r i a l i z e d  countries.  h i g h l y i n t e n s i v e l i v e s t o c k c u l t u r e system  The l a t t e r u s e s  involving greater  amounts o f s y n t h e t i c s t i m u l a n t s i n t h e i r f o o d r a t i o n . present study, the presence probably i n s i g n i f i c a n t . conducted  a  I n the  o f t o x i c a n t s i n t h e swine i s  H o w e v e r , f u r t h e r s t u d i e s s h o u l d be  on t r a c e m e t a l s a c c u m u l a t i o n n o t o n l y on t h e a l g a e b u t  a l s o on t h e n e x t t r o p h i c  level.  49  Growth Constant,  C e l l Density  D r y W e i g h t o f D. I n "both h a t c h growth constant obtained  and  tertiolecta  continuous  (K^ ^  hr  0  And  ) and  algal  c u l t u r e systems,  mean g e n e r a t i o n  i n a l l e x p e r i m e n t s have s i m i l a r v a l u e s  i n o r g a n i c or swine waste-seawater media. (Table 1965;  4)  J i t t s . e t a l . , 1964)  either i n defined  values  obtained  t o be  Kalmakoff,  i n which the values , r e p o r t e d  are  1  c l o s e to o p t i m a l .  These r e s u l t s i n d i c a t e t h a t  c u l t u r e c o n d i t i o n s i n the present  c a s e s and  time ( t g hrs)  w e r e s i m i l a r t o e a r l i e r s t u d i e s ( A n t i a and  considered the  The  the  s t u d y were o p t i m a l  t h a t n e i t h e r t h e h i g h l e v e l s o f NH^-N  t o x i c a n t s i n t h e manure w e r e h a v i n g  nor  i n most  possible  a d e t r i m e n t a l e f f e c t on  algal  growth r a t e . Algal cell  d e n s i t i e s attained*..: a f t e r 7 - 8 6  batch  c u l t u r e w e r e 5 t o 7 x 10  days growth  -1  c e l l s ml  f o r both media.  S i m i l a r v a l u e s were a t t a i n e d d u r i n g c o n t i n u o u s maintained  f o r the d u r a t i o n of the  c u l t u r e s and  experiments.  t h e s w i n e w a s t e - s e a w a t e r medium h a s v . n e i t h e r  This  i n h i b i t o r y nor  t e r t i o l e c t a when  d e f i n e d i n o r g a n i c medium.  I n a s i m i l a r , s t u d y , u s i n g 15  sewage e f f l u e n t i s an populations The  I t was  toxic  compared w i t h  the  liter  demonstrated that secondary t r e a t e d  e x c e l l e n t medium f o r t h e g r o w t h o f m i x e d  of marine phytoplankton  ( D u n s t a n and M e n z e l ,  1971).  a l g a l dry w e i g h t measured d a i l y f o r swine waste-  s e a w a t e r and ( F i g u r e 2)  were  showed t h a t  e f f e c t on t h e g r o w t h o f D.  v o l u m e c u l t u r e s , i t was  during  d e f i n e d i n o r g a n i c media d u r i n g continuous  was  not  constant.  observed that although  I t ranged from 0.12 the  culture  to 0.25  c o n c e n t r a t i o n of c e l l s  g  1  remained  50  Table 4 .  Growth Constant ( K ^ ) ) h r - ) and Mean G e n e r a t i o n 1  1 0  Time ( t g h r s ) from p r e s e n t and e a r l i e r  studies  on D. t e r t i o l e c t a .  Defined Inorganic Medium  Swine Waste-Seawater Mixtures  (K  (K  ( i p )  hr  _ 1  )  P r e s e n t Study Expt 1 0.023  ( t g hrs)  ( 1 0 )  hr  _ 1  )  (tg hrs)  13.0  0.024  12.5  12.92+0.0  0.023 + 0.001  12.95+0^34  Expt 2  0.023 + 0 . 0  Expt 3  0.023 ± 0.002 13.92 + 0.36 0.0231 + 0.001  13.02+0.05  Expt 5  0.023 + 0.002 12.85 + 0.49 0.024 + 0.004  12.71+0.67  A n t i a and r Kalmakoff (19''; ) (1965) 0.025 Jitts'et a l . (1964)''  O.030  12.0  51 f a i r l y constant, the algal ( F i g u r e s 8 and 9 ) .  "biomass decreased a t d i f f e r e n t  times  I t was observed t h a t the a l g a l "biomass tended  to i n c r e a s e s h o r t l y a f t e r the n u t r i e n t media "being added to the c u l t u r e was r e p l e n i s h e d from the stock s o l u t i o n .  Moreover, the  h i g h e r v a r i a b i l i t y was observed i n swine waste-seawater due to c o n s i d e r a b l e amount o f suspended  medium  s o l i d s i n the swine waste-  seawater mixture as compared to d e f i n e d i n o r g a n i c medium. S i g n i f i c a n t d i f f e r e n c e was found i n the a l g a l d r y weight d a i l y f o r both media.  produced  F u r t h e r r e s e a r c h should be c a r r i e d out to  determine p o s s i b l e reasons why a t the time o f r e p l e n i s h i n g the n u t r i e n t s - s o l u t i o n an i n c r e a s e i n a l g a l biomass was observed. Continuous C u l t u r e System I n A l g a l P r o d u c t i o n One o f the major v a l u e s o f the present work was the demonstration t h a t the continuous c u l t u r e system more a l g a l biomass than the batch system.  can produce f a r  This culture  system  when compared to a b a t c h system i s f a r more conducive f o r o p t i m a l p r o d u c t i v i t y o f the a l g a e . o f p r o d u c i n g 0.015  Although the batch system was capable  g dry wt 1  -1 day -1 (0.10  dry wt 1  -1 i n . :  6 days), .thercontinuous c u l t u r e system was capable o f p r o d u c i n g 0.2 •C  to 0.25  g dry wt l  -  1  day . - 1  I t should be p o i n t e d out that the n u t r i e n t s i n both media  were i n excess and thereby the n u t r i e n t s were not f u l l y by the algae d u r i n g continuous c u l t u r e  (Table 2).  utilized  I t i s suggested  t h a t a d d i t i o n a l c u l t u r e v e s s e l s should be added i n s e r i e s to f u l l y u t i l i z e the n u t r i e n t s , thus r e s u l t i n g to a h i g h e r  biomass.  52 McAllister  e t a l (1964) c o n c l u d e d  photosynthetic efficiency at  0.1 l y m i n  limiting. was will  .  (P  m a x  )  ?  o  r  t h a t t h e maximum  D. t e r t i o l e c t a was a c h i e v e d  I n t h e p r e s e n t s e t - u p , l i g h t was p r o b a b l y  S i n c e t h e l i g h t i n t e n s i t y from t h e f l u o r e s c e n t tubes  b e t w e e n 0.04 t o 0.05 l y m i n  , i n c r e a s i n g the l i g h t  intensity  i n c r e a s e the p h o t o s y n t h e t i c p r o d u c t i o n . Furthermore,  using continuous  p r o v i d e a more u n i f o r m p r o d u c t  cultures, i t i s possible to  d a i l y by s e l e c t i n g  growth  c o n d i t i o n s t o o b t a i n t h e d e s i r e d composition o f the F o r example, by s e l e c t i n g the f l o w r a t e , a p r o d u c t low p r o t e i n c e l l s  c o u l d be p r o d u c e d  consistently  1975; L a m p e r t , 1 9 7 6 ) .  Palmer e t a l . ,  product. o f high o r  (Ballard,  1972;  On t h e o t h e r h a n d , i t i s a  c h a r a c t e r i s t i c o f batch cultures that t h e i r composition w i l l c o n t i n u o u s l y a s t h e y g e t o l d e r (Taub and D o l l a r , Artemia Feeding T o t a l Body L e n g t h  1965).  Experiments  and Percentage  Survival  T o t a l body l e n g t h and p e r c e n t a g e the b r i n e shrimp  vary  s u r v i v a l measurements o f  d u r i n g t h e 14-day c u l t u r e p e r i o d showed no  s i g n i f i c a n t d i f f e r e n c e s when f e d s w i n e w a s t e g r o w n a l g a e w i t h t h o s e f e d d e f i n e d i n o r g a n i c medium grown a l g a e .  Daily  observations  on t h e i r s w i m m i n g o r f e e d i n g b e h a v i o r gave no a p p a r e n t d i f f e r e n c e s b e t w e e n t h e two t r e a t m e n t s . The  r a p i d growth o f Artemia  p r e s e n t s t u d y was r e m a r k a b l e . growth p a t t e r n .  b e t w e e n 2 t o 7 mm s t a g e i n t h e  M a s o n (1963) o b s e r v e d  These l e n g t h s c o r r e s p o n d  a similar  t o t h e p e r i o d s when  growth r a t e i s most r a p i d and h i g h l y dependent upon f o o d  level  53 and w i t h the time when food f i r s t  4-6  shows i t s e f f e c t , days  ( P r o v a s o l i et a l , 1 9 5 9 ) . Although the same number of algae are b e i n g f e d and 25$  algae c o n t a i n about  l e s s o r g a n i c n i t r o g e n (Table 2 ) ,  d i d not a f f e c t the subsequent The algae are s t i l l  those this  growth r a t e of the Artemia.  able to grow and reproduce w h i l e the Artemia  are f e e d i n g on them. S c o t t and M i d d l e t o n ( 1 9 7 9 ) have shown t h a t t u r b o t l a r v a e v with D. t e r t i o l e c t a g i v e n d u r i n g the r o t i f e r f e e d i n g stage showed poor animal growth and s u r v i v a l  compared to l a r v a e i n tanks with  e i t h e r I s o c h r y s i s galbana, Phaedactylum tricornoturn or P a v l o v a l u t h e r i added.  S i n c e D. t e r t i o l e c t a i s known to be not  toxic,  I t s e f f e c t on the t u r b o t l a r v a e I s probably due to "a d i e t a r y efficiency.  I t has been p o i n t e d out that the n u t r i t i o n a l  state  of the a l g a l food i s important f o r the growth of h e r b i v o r e s (Parsons et a l . , 1 9 6 1 ) . survival  T h i s i s probably the cause of low Tobias et a l . (1979)  of Artemia i n the p r e s e n t study.  achieved 80$ s u r v i v a l  o f Artemia f e d with diatom  curvisetus (clone STX-I67) Biomass Conversion  Chaetoceros  i n a flow-through raceway  system.  Efficiency  The biomass c o n v e r s i o n e f f i c i e n c y  ($) i s d e f i n e d here as a  measure of the amount o f biomass of Artemia produced  by a  g i v e n q u a n t i t y o f algae f e d d u r i n g the c u l t u r e p e r i o d .  The  i s g i v e n as: dry weigth of Artemia B. C. E .  $  =  _ _ — _ —  produced  _ _  x  dry weight o f algae f e d i s biomass c o n v e r s i o n e f f i c i e n c y x  where B. C. E .  inn 1 U U  formula  The  biomass c o n v e r s i o n  e f f i c i e n c i e s f o r Artemia f e d with  swine waste-seawater grown algae algae were 57$ algal-Artemia  and  and  58$,respectively  conversion  d e f i n e d i n o r g a n i c grown 5)-  (Table  The  e f f i c i e n c i e s showed no  calculated  statistically  s i g n i f i c a n t d i f f e r e n c e s between treatments and r e p l i c a t e s (Appendix  11).  F i f t y percent D.  protein-N  conversion  e f f i c i e n c y from  t e r t i o l e c t a to Artemia c u l t u r e d l i n the l a b o r a t o r y was  by Reeve ( 1 9 6 3 c ) .  H e l f r i c h ( 1 9 6 3 ) i n d i c a t e d a 50$  e f f i c i e n c y .(ash-free dry wt  ) was  attained i n a  reported  conversion  feasibility  study conducted on Christmas I s l a n d f o r a p r o d u c t i o n  scheme of  Artemia eggs and  directly  adult.  The  present  study cannot be  compared with the above s t u d i e s s i n c e the u n i t s used were i n g dry wt.  The  present  r e s u l t s , however, may  reached a comparable c o n v e r s i o n  be  s a i d to have  efficiency.  Reports on e f f i c i e n c y of growth of Artemia are q u i t e v a r i e d i n terms o f u n i t s used such as wet dry weight, p r o t e i n - N Helfrich,  1973;  and mg  S i c k , 1976  weight, dry weight,  C (Gibor,  1963;  Mason,  and Bossuyt and S o r g e l o o s ,  I t i s suggested t h a t the use  of such u n i t s should  parameters such t h a t r e s u l t s w i l l be a p p l i c a b l e to a q u a c u l t u r a l  1957,  ash-free  198O).  have common  comparable and  also  studies.  Combined L i v e s t o c k Waste-Artemia C u l t u r e System The pres.ent study shows that the use  of swine waste as  a  source of n u t r i e n t s i n a l g a l c u l t u r e i s h i g h l y comparable to d e f i n e d i n o r g a n i c media.  The  study a l s o shows t h a t Artemia f e d  55 Table 5*  C a l c u l a t e d Biomass Conversion E f f i c i e n c y o f Artemia s a l i n a L. Fed With Swine Waste Grown D. t e r t i o l e c t a  Parameters Measured  (BCE %)  Fed With D e f i n e d I n o r g a n i c Medium Grown D. t e r t i o l e c t a  A l g a l Production 0.17  (X = g dry wt d a y ) - 1  +  0.15 ±0.61  0.72  Corresponding A l g a l C e l l Density (X = c e l l  ml  - 1  )  5.78 + 1.65 x 10°  5.31 + 1.62 x ier  2.99  2.78  C e l l Weight (X = g dry wt c e l l " ) 1  + 1.01 x I O  Algal C e l l Censity M a i n t a i n e d as Feed F o r Artemia day ~1 (X = c e l l ml  1  5.0 + 0.50 x 10 5  )  - 1 1  + 0.9  x  IO  5.0 + 0.50 x 10 5  No. of A l g a l C e l l s Added As Feed For Artemia day-1 (X = c e l l s  day  8.67 + 1.50 x 10  - 1  y  9.97 + 1.0 x 10'  A l g a l Dry Weight Added As Feed I n 14-Day C u l t u r e P e r i o d  3.63 + 1.40  (X = g dry wt)  3.10 + 1.09  Artemia Biomass Produced A f t e r 14 Days  2.10 + 0.42  (X = g dry wt) Biomass  Conversion  Efficiency  (fo)  57-93 + 5-84  1.82 +  0.38  58.79 + 7.19  - 1 1  56 O H algae grown i n swine waste-seawater mixture  grow as w e l l  as  those f e d with algae grown i n d e f i n e d i n o r g a n i c medium. T h i s i s an important  c o n s i d e r a t i o n , e s p e c i a l l y f o r an  a g r i c u l t u r e - a q u a c u l t u r e i n t e g r a t e d system, and i t shows,that Artemia has a h i g h p o t e n t i a l i n c o n v e r t i n g a l g a l biomass grown i n a g r i c u l t u r a l wastewater i n t o much needed p r o t e i n , p a r t i c u l a r l y i n T h i r d World c o u n t r i e s .  For example, Artemia  s u b s t i t u t e f o r f i s h meal.  can be used as a  I n t h i s regard, i n t e g r a t e d l i v e s t o c k  waste-Artemia p r o d u c t i o n system can be most economically in Third  World c o u n t r i e s .  feasible  I t should be p o i n t e d out, however,  t h a t there are s t i l l numerous areas o f u n c e r t a i n t i e s i n the system, such as b i o l o g i c a l , I t i s suggested outdoors  e n g i n e e r i n g and  said  economic problems.  that f u t u r e s t u d i e s should be conducted  on  f o r a l g a l - A r t e m i a p r o d u c t i o n scheme so t h a t data,  e s p e c i a l l y on e n g i n e e r i n g and economic aspect can be a p p l i e d to aquacultural production  ventures.  57 SUMMARY AND CONCLUSIONS A n a l y s i s o f t o t a l K j e l d a h l n i t r o g e n ( T K N ( ) , NH^-N, NO^-N + Nbg-N, PO^-P a n d t o t a l  suspended  s o l i d s ( T S S ) o f t h e swine  waste  s a m p l e s showed o n l y s l i g h t v a r i a t i o n d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t a l work.  I n b o t h "batch a n d c o n t i n u o u s  systems.,, t h e a l g a l g r o w t h  culture  constants obtained i n a l l experiments  were f o u n d t o have s i m i l a r v a l u e s f o r b o t h d e f i n e d i n o r g a n i c and s w i n e w a s t e - s e a w a t e r  media.  The p r o d u c t i o n p a r a m e t e r s  m a i n t a i n e d d u r i n g a l g a l c u l t u r e i n both b a t c h and c o n t i n u o u s c u l t u r e systems salinity, min  -1-2  cm  32+1  were * as f o l l o w s :  temperature,  2h + 1°C;  ppt; fluorescent l i g h t i n t e n s i t y ,  -1 ( o r 0.04 l y min" ) .  a f t e r 7-8 d a y s g r o w t h  The a l g a l c e l l  &a 0.04 c a l  density  attained  f o r b a t c h c u l t u r e was 5 t o 7 x 10  cells  _ i  ml  i n both media.  S i m i l a r v a l u e s were a t t a i n e d d u r i n g  c o n t i n u o u s c u l t u r e s and s u c c e s s f u l l y m a i n t a i n e d f o r t h e d u r a t i o n of the experiments. The  r e s u l t s show t h a t t h e u s e o f s w i n e  m i x t u r e s as a n u t r i e n t source i n a l g a l d e f i n e d i n o r g a n i c medium.  waste-seawater  c u l t u r e i s comparable t o  One a d v a n t a g e  of using livestock  waste as a n u t r i e n t source f o r a l g a l c u l t u r e i s t h a t l o a d i n g such as n i t r o g e n and phosphorus I t i salso The  nutrient  c a n be p r e d i c t e d .  c h e a p a n d a v a i l a b l e i n many a r e a s . t o t a l body l e n g t h and p e r c e n t a g e  survivalinmeasurements  o f A r t e m i a d u r i n g t h e 1 4 - d a y c u l t u r e p e r i o d showed no s i g n i f i c a n t d i f f e r e n c e s between A r t e m i a f e d swine waste-seawater  grown  D. t e r t i o l e e t a a n d t h o s e f e d d e f i n e d i n o r g a n i c medium g r o w n  58 D. t e r t i o l e c t a .  The c a l c u l a t e d biomass c o n v e r s i o n e f f i c i e n c i e s  f o r Artemia f e d w i t h swine waste-seawater grown algae and d e f i n e d i n o r g a n i c medium grown algae were 57% and 58%, respectively.  The a l g a l - A r t e m i a c o n v e r s i o n e f f i c i e n c i e s showed  no s t a t i s t i c a l l y  s i g n i f i c a n t d i f f e r e n c e s between treatments and  replicates. The  study shows t h a t Artemia f e d on algae grown i n swine  waste-seawater mixture  grow as w e l l as those f e d with algae  grown i n d e f i n e d i n o r g a n i c medium.  59 REFERENCES A l l e n , E. J . and E. W. N e l s o n . 1910. On t h e a r t i f i c i a l c u l t u r e o f marine p l a n k t o n organisms. J . Mar. B i o l . A s s o c . U. K. 8: 421-74. Anon. 1978. A q u a c u l t u r e development f o r H a w a i i . 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F i s h . 44(9): 979-84. Wickins, S. F. 1972. The food v a l u e s o f the b r i n e shrimp, Artemia s a l i n a L. to l a r v a e o f the prawn, Palaemon s e r r a t u s Pennat. J . Exp. Mar. B i o l . Z o o l . 10: 151-70. W i s e l y , B. and C. Purday. 1961. An a l g a l mass c u l t u r e u n i t f o r f e e d i n g marine i n v e r t e b r a t e l a r v a e . 1-B D i v . F i s h Ocenogr. Tech. Paper No. 12 C. S. I . RQ 0. Melbourne, A u s t r a l i a .  APPENDICES  71 Appendix 1  Composition o f Enrichment " f / 2 " Major N u t r i e n t s NaNO^  7 5 mg ( 8 8 3 uM)  NaH P0^'H 0 2  2  5 mg ( 3 6 . 3 uM)  Trace M e t a l s Na EDTA  +  4 . 3 6 mg ( ca  2  FeCly6H 0 2  +  11.7  uM)  3 . 1 5 mg ( O . 6 5 mg Fe or ca 1 1 . 7 uM  CuSO^•5H o  0.01  ZnS0^'7H 0  0 . 0 2 2 mg ( 5 ug Zn o r ca 0 . 0 8 uM)  CoCl *6H 0  0.01  MnCl «4H 0  0.18 mg ( 0 . 0 5 mg Mn o r ca 0 . 9 uM)  2  2  2  2  2  2  Na MoO^«2H 0 2  2  mg ( 2 . 5 ug Cu o r ca 0 . 0 4 uM)  mg ( 2 . 5 ug Co o r ca 0 . 0 5 uM)  0 . 0 0 6 mg ( 2 . 5 ug Mo o r ca 0 . 0 3 uM  Vitamins Thiamin-HCl  0.1  Biotin  0 . 5 ug  B  12  Seawater  mg  0 . 5 ug to one l i t e r  Appendix 2 Composition o f A r t i f i c i a l  Seawater  FORTY FATHOMS BIO-CRYSTALS MARINEMIX Assay Chart Average s o l u t i o n o f F o r t y Fathoms Marinemix hydrated to a d e n s i t y o f 1.025 u s i n g d i s t i l l e d water.. F i g u r e s c i t e d from a c t u a l independent l a b o r a t o r y a n a l y s i s . Concentration Aluminum Antimony Argon Arsenum Barium Bicarbonate Beryllium Bismuth Boron Bromide Cadmium Calcium Carbonate Cerium Cesium Chromium Chloride Copper Cobalt Dysprosium Erbium Europium Fluoride Gadolinium Gallium Germanium Gold Hafnium Helium Holmium Indium Iodine Iron Krypton Lanthanum Lead Lithium Lutetium Magnesium Manganese  0.06 0.0005  trace  0.01 0.12 174.0 0.0002 trace  2.1 62.0 0.009 410.0 10.0 0.0007 trace  0.02 18600.0 0.007 0.0025 trace trace trace  1.9  trace  0.0004 0.00005  trace trace trace trace trace  0.03 0.03  trace trace trace  0.24  trace  1290.0  0.008  (ppm)  C o m p o s i t i o n (ppm) Mercury Molybdenum Neodynium Neon Nickel Niobium Nitrogen Palladium Phosphorus Potassium Praeseodymium Protactinium Radium Radon Rubidium Ruthenium Samarium Scandium Selenium Silicon Sodium Strontium S u l f u r (as SO^) Tantalium Tellerium Terbium Thalium Thulium Tin Titanium Tungsten Uranium Vanadium Xenon Ytterbium Yttrium Zinc Zirconium  0.0007 0.005 trace trace 0.009 trace 0.85 trace 0.04 380.trace trace trace trace 0.06 trace trace trace trace 4.5 110400.0 12.4 2600.0 trace trace trace 0.00007 trace 0.006 0.004 0.004 0.00005 0.0009 trace trace trace 0.24 trace  Appendix 3 ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 2A) SOURCE  SUM OF SQUARES  DF  MEAN SQUARE  F-RATIO  P R O B A B I L I T Y TEST TERMTREATMENT RFP  RESIDUAL RESIDUAL  0.16666E-08  1.  0.16666E-08  0.76920E-01  0.80755  0.43333E-07  2.  0.21667E-07  1.0000  0.50000  2.  0.21667E-07  RESIDUAL  0.43333E-07  TOTAL  0.88333E-07 OVERALL MEAN  GROWTH  0.23283E-01  5OVERALL STANDARD DEVIATION 0.13292E-03  A p p e n d i x 4a  ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 3A) SUM OF SQUARES  DF  TREAT  0.20167E-06  1.  0.20167E-06  REP  0.13334E-07  2.  0.66668E-08  RESIDUAL  0.53334E-07  2.  0.26667E-07  TOTAL  0.26833E-06  5-  SOURCE  OVERALL MEAN GROWTH  0.22917E-01  MEAN SQUARE  F-RATIO  P R O B A B I L I T Y TEST TERM  7.5624  O.IIO7O  RESIDUAL  0.25000  0.80000  RESIDUAL  OVERALL STANDARD DEVIATION 0.23166E-03  -N3  A p p e n d i x 4b ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY SOURCE P R O B A B I L I T Y TEST TERM  SUM OF SQUARES  DF  MEAN SQUARE  TREAT  0.76163  1.  O.76163  4.2740  2.  2.1370  RESIDUAL REP DAY  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  202.71  22.524  3.9737 11.149  117.51  0.06160 0.00071 0.00000  3.9576  2.  5.6726  18.  0.31514  1.6442  0.15031  2.1127  9.  0 .23475  1.2248  O.33986  3.4500  18.  0.19167  222.94 OVERALL MEAN  DENSITY  9.  F-RATIO  3-5733  1.9788  10.324  59. OVERALL STANDARD DEVIATION  1.9439  0.00103  A p p e n d i x 4c ANALYSIS OF VARIANCE TABLE FOR ALGAL DRY WEIGHT (EXPT 3 C ) SOURCE PROBABILITY TEST TERM TREAT REP DAY  SUM OF SQUARES  DF  0.47538E-03  1.  0147538E - 0 3  0.79947E -01  0.78060  0.20040E-01  2.  0.10020E -01  1.6851  0.21339  0.59008  9.  O.65565E -01  0.16076E-01  2.  0.80382E -02  1.3518  0.28381  18.  0.59387E-02  0.99874  0.50105  9.  0.70743E -02  1.1897  O.35845  0.59462E'-02  RESIDUAL RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL  0.10703  18.  TOTAL  0.90240  59.  0.10690 O.63669E-OI  OVERALL MEAN DRY WEIGHT  0.18375  MEAN SQUARE  F- RATIO  11.026  OVERALL STANDARD DEVIATION 0.12367  0.00001  A p p e n d i x 5a ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY (EXPT 5 B ) SOURCE PROBABILITY TEST TERM TREAT REP DAY  RESIDUAL RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY>I RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL-  SUM OF SQUARES  MEAN SQUARE  6.6082  1.  6.6082  0.93253  2.  0.46627  294.11  19.  15.479  F-RATIO 45.442  3.2063 106.45  0.00000 0.05167 0.00000  2.  0.31293  2.1519  0.13023  5-5737  38.  0.14668  1.0086  0.48950  8.1307  19.  0.42793  2.9427  0.00227  5.5260  38.  0.14542  O.62585  321.51 OVERALL MEAN  DENSITY  DF  5.5438  119. OVERALL STANDARD DEVIATION 1.6437  Appendix ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY SOURCE P R O B A B I L I T Y TEST TERM TREAT REP DAY  RESIDUAL RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  (EXPT 5B1)  SUM OF SQUARES  DF  MEAN SQUARE  8.1420  1.  8.1420  0.63622  2.  0.31811  6.  5.2340  2.  0.31007  2.3576  0.14057  2.3135  12.  0.19279  1.4658  0.26708  2.2230  6.  0.37050  2.8170  0.06512  1.4467  11.  0.13152  31.404 0.62014  47.840 OVERALL MEAN  DENSITY  $b  4.9885  F-RATIO  61.907 2.4187 39.796  40. OVERALL STANDARD DEVIATION  1.0936  0.00001 0.13471 0 .00000  A p p e n d i x 5c ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY (EXPT 5B2) SOURCE P R O B A B I L I T Y TEST TERM  SUM OF SQUARES  DF  MEAN SQUARE  TREAT  1.1959  1.  1.1959  4.4383  0.05513  0.32929  2.  0.16464  0.61102  0.55767  5.0913  6.  0.848 54  3.1491  0.03924  0.76389  2.  0.38194  1.4175  0.27741  1.3014  11.  0.11831  0.43908  0.91017  1.4670  6.  0.24450  0.90740  0.51899  3.5029  13.  0.26946  REP DAY  RESIDUAL RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  12.791 OVERALL MEAN  DENSITY  6.6921  F-RATIO  41 OVERALL STANDARD DEVIATION  0-55855  00 o  Appendix ANALYSIS OF VARIANCE FOR CELL DENSITY (EXPT SOURCE P R O B A B I L I T Y TEST TERM TREAT RESIDUAL REP DAY  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL . REP*DAY RESIDUAL TREAT*DAY ^.£I>!RESIDUAL RESIDUAL TOTAL  5B3)  SUM OF SQUARES  DF  0.14963E-01  1.  0.14963E-01  0.15520  0.16247E-01  2.  0.81233E-02  0 . 8 4 2 5 5 E - 0 1 0.92000  0.15365  4.  0.38412E-01  0.39841  0.80482  0.40613  2.  0.20306  2.1062  0.18415  0.94345  8.  0.11793  1.2232  0.39130  0.22895  4.  0.57238E-01  0.59368  0.67726  0.77131  8.  0.96413E-01  2.5347 OVERALL MEAN  DENSITY  5d  5-8537  MEAN SQUARE  F-RATIO  29. OVERALL STANDARD DEVIATION  0.29564  0.70391  Appendix 6 ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 5A) SOURCE PROBABILITY TEST TERM  SUM OF SQUARES  DF  MEAN SQUARE  TREAT. RESIDUAL REP RESIDUAL RESIDUAL  0.10667E-06  1.  0.10667E-06  0.26556  O.65763  0.32333E-06  2.  0.16167E-06  0.40249  0.71302  0.80333E-06  2.  0.40167E-06  TOTAL  0.12333E-05  5.  OVERALL MEAN GROWTH  0.23533E-01  F-RATIO  OVERALL STANDARD DEVIATION 0.49666E-03  co ro  Appendix 7 ANALYSIS OF VARIANCE TABLE FOR ALGAL DRY WEIGHT ( EXPT 5C) SOURCE PROBABILITY TEST TERM TREAT REP DAY  RESIDUAL  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREATEDAY RESIDUAL RESIDUAL TOTAL  SUM OF SQUARES  DF  0.18800E-01  1.  0.18800E-01  0.84485E-03  2.  0.42243E-03  19.  0.24426E-01  0.22687E-02  2.  0.11344E-02  2.1867  0.12621  0.11038E-01  38.  0.29048E-03  0.55996  0.96109  0.24594E-01  19.  0.12944E-02  2.4953  0.00808  0.19712E-01  38.  0.51874E-03  0.46410  0.54135 OVERALL MEAN  DRY WEIGHT  0.16005  MEAN SQUARE  119. OVERALL STANDARD DEVIATION 0.67448E-01  F-RATIO 36.241 0.81432 47.087  0.00000 0.45052 0.00000  Appendix 8 ANALYSIS OF VARIANCE TABLE FOR TOTAL LENGTH OF ARTEMIA SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  SUM OF SQUARES  DF  0.36363E-03  1.  0.36363E-03  0.74867E-01  3.  0.249£6E-01  .2.3 • 0.12452  64.094  F-RATIO 0.14954E-01 1.0263  2635.8  0.90332 0.39183 0.00000  3.  0141508E-01  1.7070  0.18189  1.3383  39.  0.34315E-01  1.4111  0.14532  0.68787  13.  0.52913E-01  2.1760  0.03137  0.92405  38.  0.24317E-01  836.75 OVERALL MEAN  GROWTH  13.  MEAN SQUARE  2.8957  110. OVERALL STANDARD DEVIATION 2.7580  Appendix 9 a  ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OF ARTEMIA SOURCE PROBABILITY TEST TERM TREAT REP DAY  RESIDUAL RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  SUM OF SQUARES  DF  331.95  1.  242.75  3.  41120.  331.95 80.917 3163.1  39.365 9-5958 375.10  0.00000 0.00007 0.00000  105.10  3.  35.035  4.1547  0.01200  395.58  39.  10.143  1.2028  0.28346  591.09  13.  45.468  5.3920  0.00002  328.87  39.  43369. OVERALL MEAN  SURVIVAL  13.  F-RATIO  MEAN SQUARE  61.393  8.4326  111. OVERALL STANDARD DEVIATION 19.766  Appendix 9 b  ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OR ARTEMIA SOURCE PROBABILITY TEST TERM TREAT REP DAY  RESIDUAL  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL  SUM OF SQUARES  DF  MEAN SQUARE  631.86  1.  631.86  134.34  3-  10628.  1771.4  78.730 5.5798 220.72  0.00000 0.00600 0.00000  115.01  3.  38.338  4.7769  0.01141  229.41  18.  12.745  1.5880  0.15837  330.71  6.  55.119  6.8678  0.00045  160.51  20.  12311. OVERALL MEAN  SURVIVAL  6.  44.782  F-RATIO  77.862  8.0257  57OVERALL STANDARD DEVIATION 14.696  Appendix 9c  ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OF ARTEMIA F-RATIO  MEAN SQUARE  SOURCE PROBABILITY TEST TERM  SUM OF SQUARES  DF  TREAT'I' RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL  12.532  1.  12.532  3.  43.320  10.136  0.00033  6.  211.246  49.426  0.00000  66.274  3.  22.091  74.818  18.  20.097 81.204  TOTAL  129.96 1267.5  1653.3 OVERALL MEAN  SURVIVAL  45.368  2.9323  0.10310  5.1689  0.00883  4.1566  0.97255  0.52185  6.  3.3496  0.78373  0.59317  19.  4.2739  56. OVERALL STANDARD DEVIATION 5.4335  Appendix 10a  ANALYSIS OF VARIANCE TABLE FOR ARTEMIA WET WEIGHT SOURCE  SUM OF SQUARES  DF  6.1075  1.  6.1075  0.0  1.00000  RESIDUAL  4.5720  3-  1.5240  0.0  1.00000  RESIDUAL TREAT*REP RESIDUAL RESIDUAL TOTAL  1.8382  3.  0.61272  0.0  1.00000  PROBABILITY TEST TERM TREAT REP  0.0 12.518 OVERALL MEAN  WET WEIGHT  18.671  -0.  MEAN SQUARE  F-RATIO  0.0  7. OVERALL STANDARD DEVIATION 1.3373  Appendix 10b  ANALYSIS OF VARIANCE TABLE FOR ARTEMIA DRY WEIGHT SOURCE PROBABILITY TEST TERM  SUM OF SQUARES  DF  TREAT  0.15680  1,.'  0.25965 0.60300E-01  REP  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL RESIDUAL  0.0  TOTAL  0.47675 OVERALL MEAN  DRY WEIGHT  1.9625  MEAN SQUARE  F-RATIO  0.15680  0.0  1.00000  3.  0.86550E-01  0.0  1.00000  3.  0.20100E-01  0.0  1.00000  -0.  0.0  7. OVERALL STANDARD DEVIATION  0.26097  Appendix 11  ANALYSIS OF VARIANCE TABLE FOR ARTEMIA BIOMASS CONVERSION EFFICIENCY (BCE) SOURCE PROBABILITY TEST TERM TREAT REP  RESIDUAL  RESIDUAL TREAT*REP RESIDUAL RESIDUAL TOTAL  SUM OF SQUARES  DF  MEAN SQUARE  1.5051  1.  1.5051  230.91  0.0  1.00000  3.  76.969  0.0  1.00000  3.  17.954  0.0  1.00000  0.0  -0.  0.0  286.28  7.  53.863  OVERALL MEAN BCE  F-RATIO  58.356  OVERALL STANDARD DEVIATION  6.3950  

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