Laing 1983 Aquacultural-Engineering

7/28/2019 Laing 1983 Aquacultural-Engineering

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Aquacu l tu ra l Eng ineer ing 2 (1983) 203-212

L a r g e - sc a l e T u r b i d o s t a t C u l t u r e o f M a r i n e M i c r o a l g a e

I . Laing and E. Jon es

Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research,Fisheries Expe r iment Stat ion , Benarth Roa d, Conwy , Gw yned d LL32 8UB,

W ales, UK

A B S T R A C TA s i m p l e a n d i n e x p e n s i v e d e v i c e is d e s c r i b e d w h i c h m a i n t a in s c u l t u r e s o fm a r i n e m i e ro a lg a e a t p r e d e t e r m i n e d c e l l d e n s i t ie s b y t h e a u t o m a t i c c o n t r o lo f h a r v e s ti n g a n d r e p l e n i s h m e n t o f t h e c u l t u r e v o l u m e w i t h f r e s h m e d i u m .Cu l tu re s w ere ma in ta ined a t a range o f c e l l dens i t i e s i n 80 - l i t re i n t e rna l l yi l l umina t ed ve s se l s and t he da i l y c e l l p ro duc t ion y i eM was eva lua ted .M a x i m u m y i e M s o f 0 . 8 - 1 . 0 x 1 01 2 c el ls d a y -1 o f Isochrysis a f f galbanaGreen a nd 0 .8 -1 .2 x 1011 ce l ls day -1 o f Tetraselmis suecica ( K v l i n ) B u t c h .were ob ta ined a t cu l t u re dens i t i e s o f 1 5 - 17 x 103 ce ll s ~ l i tr e -1 and 1 9 -21 x102 ce l ls p l i t re -1, respec t ive ly . Tu rbid os ta t cu l tur es o f Skeletonemacos t a tum ( G r e y .) C l e ve c o u l d n o t c o n s i s t e n t l y b e m a i n t a i n e d a b o v e 5 - 7 x10 3 ce lls talitre -1.

I N T R O D U C T I O NT h e s u c c e s s f u l c o m m e r c i a l p r o d u c t i o n o f m a r i n e b i va l ve m o l l u s c s e e din h a t c h e r i e s d e p e n d s o n t h e r e li ab l e p r o d u c t i o n o f s u f f ic i e n t q u a n t i t i e so f m i c r o a lg a l f o o d c e ll s ( H e l m a n d L a i ng , 1 9 8 1 ) . T h e t y p e o f c e llc u l t u r e d is i m p o r t a n t a s o n l y c e r ta i n s p e c i e s w i l l p r o m o t e g o o d g r o w t h( e .g . , W a l n e , 1 9 7 0 ) . T h r e e v a l u a b l e s p e c i e s w i d e l y u s e d a re t h e u n i-c e l l u l a r f l a g e l l a t e s I s o c h r y s i s a f t . g a l b a n a G r e e n a n d T e t r a s e l m i s s u e c i c a( K y l i n ) B u t c h . , a n d t h e d i a t o m S k e l e t o n e m a c o s t a t u r n ( G r e y . ) C l e v e .

203 H MS O 1983, reprodu ced b y permission o f the C ontrol ler of Her Majesty 'sStat ionery Off ice, London.


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204 L Laing, E. JonesA large-scale semi-continuous culture system for these and other

useful species has been developed which effic ient ly utilises the availablelight energy and produces the large volumes of algal culture requiredfor bivalve feeding (Laing and Helm, 1981). This paper reports thefurther development of the system for continuous culture operation, soas to reduce labour costs of operation and improve reliability andproductivity.

Several methods for the continuous culture of marine microalgaehave been described (see review by Ukeles, 1976). These mainlyemployed small-scale chemostat culture systems of 15 litres or less involume. Palmer et al. (1975) and Trotta (1981) have described largerscale continuous culture systems of 40 litres and 50 litres, respectively.

When culturing algae on a large scale in medium prepared fromnatural seawater, the variation in growth and division rate makes itdesirable to operate turbidostat rather than chemostat cultures. Thisprevents vigorous cultures entering the stationary phase or slow growingcultures being washed out.

A simple and inexpensive electric sensing device is described foroperating turbidostat cultures of Tetraselmis suecica, Isochrysis galbanaand Skeletonema costatum in 80-1itre internally illuminated vessels.The yields obtained over a range of culture densities are evaluated.

MATERIALS AND METHODSThe apparatus used is shown in Fig. 1. Seawater was diluted to25-28%0 salinity (20%0 for Skeletonema) with freshwater and passedthrough a filter (> 2/2m particle retention) into the 200-1itre reservoir.Nutrient enrichment was carried out with the stock salt solutionsdescribed by Walne (1966). For Skeletonema, a filtrable silicate stocksolution (McLachlan, 1973) containing 40 g sodium metasilicate perlitre was added at 5 ml litre -1.

The culture vessel was cylindrical (150 cm high X 30 cm diameter)and made of borosilicate glass, although other suitable materials such asglass-reinforced plastic or polycarbonate could also be used. A 15 cmdiameter acrylic cylinder containing the lighting unit of six 80 W'daylight' fluorescent lamps was fitted internally. The external surfaceof the vessel was painted white to reflect light transmitted through the


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Large-scale turbidostat culture of marine microalgae 205

' l~ ' ~ 2OV

, . , . ,! U IArCOz

I

A ir

Fig. 1. Diagram of turb idos tat cul ture apparatus (not draw n to scale). 1, Seawaterm ed ium reservoir (200-1itre); 2, peristaltic pu m p; 3, resistance sensing relay (5 0-5000~2); 4, l ight dependent resistor (ORP 12); 5, cartr idge f i l ter (0.45/am); 6,cultu re vessel (80-1itre); 7, six 80 W fluore sce nt lamps; 8, collecting vessel (125-1itre).

c u l t u r e . C o n s t r u c t i o n d e t a i ls a n d m e t h o d s o f c l e a n i n g a nd s te r il is in g t h ev e s se l p r i o r t o c u l t u r e t ri al s w e r e a s d e s c r i b e d f o r 2 0 0 -1 i tr e v e s s e ls b yH e l m e t al. ( 1 9 7 9 ) .

T h e v e ss e l w a s f i ll ed b y p u m p i n g n u t r i e n t e n r i c h e d s e a w a t e r m e d i u mf r o m t h e r e s e r v o ir th r o u g h a n a u t o c l a v e d 0 . 4 5 ~ m p a r t i c le r e t e n t i o nc a r t r id g e f il te r . A f i x e d s p e e d p e r i s t a l t ic p u m p t u r n i n g a t 5 0 r p m w a su s e d t o d e l i ve r a b o u t 2 0 0 m l m e d i u m m i n -1 t h r o u g h 8 .5 m m b o r es i l i c o n r u b b e r t u b i n g .

I n o c u l a w e r e p r o v i d e d b y 2 -1 itr e c u l t u r e s g r o w n in E r d s c h r e i b e rm e d i u m f o r 7 - 1 4 d a y s o r in m e d i u m p r e p a r e d f r o m a u t o c l a v e d se a-w a t e r f o r 4 - 5 d a y s . I n it ia l c el l c o n c e n t r a t i o n s w e r e a b o u t 5 0 Tetra-selrnis ce l l s ~ l i t r e - 1 , 5 0 0 Isochrysis ce l l s l a l it r e -~ o r 2 5 0 Skeletonemac el ls ~ l i tr e -~ . C u l t u r e t e m p e r a t u r e w a s m a i n t a i n e d a t 2 0 + 2 C b yc o o l i n g w a t e r p a s s in g o v e r t h e s u r f a c e o f t h e v e s se l. A b o u t 1 5 l it rem i n -1 o f a 0 . 2 5 % v / v m i x t u r e o f c a r b o n d i o x i d e in a ir w a s s u c c e s s i v e l yf i l te r e d t h r o u g h c a r tr id g e u n i ts o f 8 . 0 , 2. 0 a n d 0 . 3 # m n o m i n a l p o r o s i t ya n d i n t r o d u c e d a t t w o p o r t s i n t h e b a s e o f t h e v e ss e l. T h e c a r b o nd i o x i d e e n r i c h m e n t w a s s u f f ic i e n t to m a i n t a i n c u l t u r e p H a t 7 . 6 - 7 . 8 .

A u t o m a t i c h a r v e s ti n g o f t h e c u l t u r e w a s c o n t r o l l e d b y t h e f o l lo w i n gm e t h o d . A c a d m i u m s u lp h i d e p h o t o - c o n d u c t i v e c e ll ( O R P 12 L i gh tD e p e n d e n t R e s i s t o r ( R S C o m p o n e n t s L t d , L o n d o n ) ) w a s e n c l o se d in a


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206 L Laing, E. Jonesl i g h t - p r o o f h o u s i n g w i t h a ' w i n d o w ' a g a i ns t t h e o u t e r s u r f a c e o f th ev e s se l. C o n s t r u c t i o n o f th e s e n s o r h o u s i n g u n i t is d e s c r i b e d i n t h eA p p e n d i x . T h e h o u s i n g w a s p l a c e d a g a in s t a 4 c m 4 c m t r a n s p a r e n ta r ea a b o u t 5 0 c m f r o m t h e b a s e o f t h e v e s se l p o s i t i o n e d s o th a t t h es t re a m o f a i r b u b b l e s ris in g th r o u g h t h e c u l t u r e w o u l d n o t i n t e r f e rew i t h i ts o p e r a t i o n . T h e r e s is t a n c e o f t h e p h o t o - c o n d u c t i v e c e ll i n c re a s e da s t h e li g ht i n t e n s i t y r e a c h i n g i t f r o m t h e l a m p s f e ll , d u e t o g r o w t ha n d d i v i s i o n o f t h e a l g a l c e ll s i n t h e c u l t u r e . A c i r c u i t s w i t c h i n g t h ep e r i s ta l t i c p u m p w a s e n e r g is e d w h e n t h e r e s is t a n c e o f t h e c e ll b e c a m eg r e a t e r t h a n a p r e s e t v a l u e o n a r e l a y s e n s i ti v e t o i n p u t r e s i s ta n c e i nt h e ra n ge 5 0 - 5 0 0 0 1 2 ( R e s i s t a n c e S e n si ng R e l a y 3 4 9 - 8 2 2 , R S C o m -p o n e n t s L t d , L o n d o n ) . T h e 1 1 -p in r e la y w a s c o n n e c t e d t o th e lig h td e p e n d e n t r e s is t o r a n d p e r i s ta l t i c p u m p a s s h o w n i n F i g. 2 . N u t r i e n te n r i c h e d s e a w a t e r m e d i u m w a s t h e n p u m p e d t h r o u g h t h e f il te r i n to t h ev e ss e l a n d t h e v o l u m e m a i n t a i n e d b y a c o n s t a n t l ev e l d e v i ce , a lt h o u g ha s im p l e o v e r f l o w w o u l d b e j u s t a s e f f e c t i v e . T h e o u t f l o w o f a lg alc u l t u r e f r o m t h e v e s s e l w a s c o l l e c t e d i n a n a e r a t e d c o n t a i n e r . A s t h ec u l t u r e w a s d i l u t e d t h e d e c r e a s e in re s is t a n c e o f th e p h o t o - c o n d u c t i v ec e ll c a u s e d b y t h e h i g h e r l ig h t i n t e n s i t y n o w r e a c h i n g i t w a s s e n s e d b yt h e r e la y a n d t h e p u m p c i rc u i t s w i tc h e d o f f .

Fig. 2.

light dependentresistor ( '~ Resistance ensing

Peristalticump ~ ( ~

2z, vO.C

Circuit diagram f or resistance sen sing relay, light de pe nd en t resistor andperistaltic pump.


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Large-scale turbidostat culture of marine microalgae 207C u l t u r e s w e r e m a i n t a i n e d a t p r e d e t e r m i n e d l ev e ls , + 1 0 0 Tetraselmis

ce l l s / a l it r e -1 and + 10 00 Isochrysis o r Skeletonema ce l l s t a l i t r e - ' f o r7 - 1 4 d a y s b y a d j u s t m e n t o f th e p r e s e t v a l u e o n t h e r e si s ta n c e s e n si ngr e la y . T h e y i e ld ( n u m b e r o f c e ll s p r o d u c e d d a y -1 ) w a s d e t e r m i n e d f r o md a i ly o b s e r v a t i o n s o f t h e c e ll d e n s i t y a n d v o l u m e o f c u l t u r e in t h ec o l l e c t i n g v e s s e l. A r a n g e o f c u l t u r e d e n s i t i e s a t i n t e r v a l s o f 2 0 0 c e ll s~ l i t r e - 1 (Tetraselmis) a n d 2 0 0 0 c e l ls ~ l it r e -1 (Isochrysis, Skeletonema)w a s i n v e s t i g a t e d d u r i n g 4 0 - to 5 0 - d a y t r ia l p e r i o d s , t h e l e n g t h o f t i m ed u r in g w h i c h a u t o m a t i c h a r v e st in g c o u l d b e c a rr ie d o u t b e f o r e t h ec u l t u r e c o l l a p s e d . A t t h e l o w e s t c el l d e n i s i ti e s o f Tetraselmis a n dIsochrysis w h i c h w e r e i n v e s t ig a t e d , a c e t a t e s h e e t c o l o u r fi lt e rs ( R e d 1 5a n d G r e e n 1 6, r e s p e c ti v e l y , B r it is h C e la n e s e L t d , L o n d o n ) w e r e p u ti m m e d i a t e l y in f r o n t o f th e p h o t o - c o n d u c t i v e c e ll in o r d e r t o b r in g i tsr e s i s ta n c e w i t h i n t h e r a n g e o f th e r e l a y .

T h e a s h - f re e d r y w e i g h t , e q u i v a l e n t t o t h e o r g a n i c w e i g h t , o f t h e a l ga lc e lls p r o d u c e d w a s d e t e r m i n e d u s in g th e m e t h o d d e s c r i b e d b y L a in ga n d H e l m ( 1 9 8 1 ) .

R E S U L T S A N D D I S C U S S I O NT h e y i e l d s o b t a i n e d w h e n c u l tu r e s o f Isochrysis a n d Tetraselmis w e r em a i n t a i n e d a t a r a ng e o f p r e d e t e r m i n e d c e ll d e n s i ti e s a re s h o w n inF i g . 3 . M a x i m u m y i e l d s of lsochrysis ( 0 . 8 - 1 . 0 X 1012 c e l ls d a y - 1) w e r eo b t a i n e d a t c u l t u r e d e n s i t i e s o f 1 5 - 1 7 l 0 s c e ll s ~ l i tr e -1 . T h e o r g a n i cw e i g h t s a t t h e s e d e n s i ti e s w e r e a b o u t 1 3 - 1 5 ~ g p e r m i l li o n c e lls ,e q u i v a l e n t t o a p r o d u c t i o n r a te o f 1 0 - 4 t o 1 5 . 0 g d a y -1. Tetraselmisc u l t u r e s m a i n t a i n e d a t 1 9 - 2 1 X 1 02 c e l ls ~ l i t re -1 y i e l d e d 0 . 8 - 1 . 2 X 1 01 1c e ll s d a y -1 , e q u i v a l e n t t o 1 6 . 0 - 2 6 . 4 g d a y -1 o r g a n i c w e i g h t ( a t 2 0 0 - 2 2 0/ag p e r m i l l io n c e ll s ). W i t h b o t h a lg a l s p e c i e s h i g h e r c e l l d e n s i t i e s g a v eg r e a t e r v a r i a b i l i t y i n y i e l d .

P r o d u c t i o n o f Skeletonema w a s a b o u t 2 - 5 - 4 X 1 0 1 1 c e ll s d a y -1 a t5 - 7 X 1 03 c e l ls / al it re -1 . T h e r e w a s n o i m p r o v e m e n t i n y i e l d a t h i g h e rc ell d e n s it ie s w h i c h c o u l d o n l y b e m a i n t a i n e d f o r o n e o r t w o d a y sb e f o r e d e c r e a s i n g s p o n t a n e o u s l y . U n o ( 1 9 7 1 ) a ls o n o t e d a d e c l in e ing r o w t h r a t e i n s m a l l- s ca l e t u r b i d o s t a t c u l t u r e s o f Skeletonerna costatum.H o w e v e r , i n a s e m i - c o n t i n u o u s c u l t u re s y s t e m y i e ld s o f 7 .5 X 1011Skeletonema c e ll s d a y -1 o v e r 5 0 - t o 6 0 - d a y p e r i o d s h a v e b e e n o b t a i n e db y h a r v es t in g 9 0 % o f t h e v o l u m e t h re e t im e s e a c h w e e k f ro m 2 0 0 -


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208 LLaing, . o n e s121C

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O ( ~ 5 7 1 1 1 3 1 5 1 7t~ C e l t d e n s , t y ( c e l l s u l '~ x 10 "3 ]

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19 211

3

13Fig. 3. Dai ly yield ( N o. of cells x 10 -xl ) for a range of cell densit ies o f I sochrys i s ,

(A) and Tet rase lm is (B ) in 80-1itre internally i lluminated turbidostat cultures.

l it r e c u l t u r e s i n i n t e r n a l l y i l l u m i n a t e d v e s s e l s . It i s p o s s i b l e t h a t e x t r a -c e l l u l a r m e t a b o l i t e s o f S k e l e t o n e m a c e l ls o r b a c t e r ia g r o w i n g i na s s o c i a t i o n w i t h t h e d i a t o m a re i n h i b i ti n g g r o w t h a n d d i v i s io n inc o n t i n u o u s c u l tu r e , w h e r e a s m o s t o f t h e se in h i b i to r y f a c t or s a rer e g u l a r l y r e m o v e d i n s e m i - c o n t i n u o u s c u l t u r e .

Y i e l d s o f T e t r a s e l m i s w e r e s i m i la r t o t h o s e o b t a i n e d f r o m s e m i -c o n t i n u o u s c u l t u r e s in 2 0 0 -1 i tr e v e s se l s ( L a i n g a n d H e l m , 1 9 8 1 ) ,


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Large-scale turbidostat culture o f marine microalgae 209a l t h o u g h t h e c u l t u r e v o l u m e u s e d i n t h e s e tr ia l s w a s o n l y 8 0 l it re s .T u r b i d o s t a t c u l tu r e s o f Isochrysis h a v e p r o v e d m o r e p r o d u c t i v e a n dm o r e r e l i a b l e t h a n 2 0 0 - 1 i t r e s e m i - c o n t i n u o u s c u l t u r e s a t t h i s l a b o r a t o r y .

O r g a n i c w e i g h t p r o d u c t i o n o f a b o u t 1 2 g d a y -1 o f Isochrysis a n d2 0 g d a y - I o f Tetraselmis c o m p a r e s f a v o u r a b l y w i t h r e c e n t l y q u o t e dp r o d u c t i o n r a t es . P a l m e r et al. ( 1 9 7 5 ) o b t a i n e d 5 g d a y -1 d r y w e i g h t o fMonochrysis f r o m 4 0 - 1i tr e c o n t i n u o u s ( c h e m o s t a t ) c u l t u r e s a n d T r o t t a( 1 9 8 1 ) q u o t e s y i e l d s o f 2 0 - 3 0 g d a y -~ w e t w e i g h t o f Tetraselmis f r o m5 0 -1 itr e p o l y e t h y l e n e b a g c u l tu r e s o p e r a t e d a s t u r b i d o s t a t s . O r g a n icw e i g h t p r o d u c t i o n is a b o u t 8 0 - 9 0 % o f th e d r y w e i g h t a n d 2 0 - 3 0 %o f t h e w e t w e i g h t v a lu e .

A C K N O W L E D G E M E N TT h e d e s ig n o f t h e m e c h a n i s m f o r a u t o m a t i c c o n t r o l o f h a r v e st in g o f t h ec u l t u r e s w a s o r i g i n a l l y s u g g e s t e d b y M r B . T . H e p p e r .

R E F E R E N C E SHelm, M . M. & Laing , I . (1981 ). Cost effect ive cul ture o f marine unicellular algae.

In : Energy conservation and use of renewable energies in the bio-industries,ed. F. Vogt , O xford and N ew York, Pergamon Press , pp . 24 7-59 .

Helm, M. M., Laing, I. & Jones , E . (1979) . The dev elopm ent o f a 200 1 algal cul turevessel at Conwy. Fish. Res. Tech. Rep., MAFF Direct. Fish. Res., Lowestoft,No. 53, Part 1, pp. 1-7 .

Laing, I . & Helm, M. M. (1981). Factors affect ing the semi-continuous productiono f Tetraselmis suecica (Ky lin) Butch. in 200 1 vessels. Aquaculture, 22 , 137-48 .

McL achlan, J. (197 3). Gro w th media-marine. In: Handbook of phycologicalmethods, culture methods and growth measurements, ed. J. R. Stein, Cambridge,Cambridge Universi ty Press, pp. 25-53.

Palmer, F. E., Bal lard, K. A. & Taub, F. B. (1975). A continuous cul ture apparatusfor the m ass prod uct ion of algae. Aquaculture, 6 , 319-31 .Trot ta , P . (1981 ) . A s imple and inexpensive system for co nt inuous m onox enic m ass

cul ture of m ar ine microalgae. Aquaculture, 22 , 383-7 .Ukeles, R. (19 76) . Cult ivat ion of plants. In: Marine ecology, Vol. IlL Odtivation,Part 1, ed. O. Kinne, New York, John Wiley and Sons, pp . 36 7-4 66 .Uno , S . (1971) . Turb idomet r i c con t inuous cu l tu re o f phy top lank ton . Bull, Plankt.Soc. Japan, 18, 14-27.


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210 L Laing, E. JonesWalne, P . R. (196 6) . Ex per im ents in the la rge-scale c u l ture of the l a rvae of Ostreaedulis L. Fishery Invest., Lond., Ser. 2, 25 ( 4 ) , 53 pp .Wa lne, P . R . ( 1 970 ) . S t ud i e s on t he f oo d va lue o f n i ne t een gene r a o f algae t o

j uven i l e b i va l ve s o f t he gene r a Ostrea, Crassostea, Mercenar~a an d Mytilus.Fishery Invest. Lond., Set. 2, 26 ( 5 ) , 62 pp .T h e r e f e r e n c e t o p r o p r i e t a r y p r o d u c t s i n t h i s r e p o r t s h o u l d n o t b e c o n s tr u e d a s a no f f i c ia l end or s em en t o f t he s e p r o duc t s no r is any c r i ti c is m i m p l i ed o f si m i la rp r o d u c t s w h i c h h a v e n o t b e e n m e n t i o n e d .

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Ar r angem en t o f s ens o r hous i ng he l d i n pos i t i on o n c u l t u r e ve ss el.


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3 8 r a m x 2 B A3 m m / b r a s s s c r e w

/

3 8 r a m

L ig h t d ep en aen tr e s i s t o r 0 R P 1 2

Fig. A2. Sensor housing.

APPENDIX: CONSTRUCTION OF SENSOR HOUSING UNITThe uni t (Figs A1 and A2) was const ruc ted fro m a 38 mm squaredural block 38 mm long. The front end was milled to suit the radius ofthe glass culture vessel. The block was then mounted concentricallyin a 4-jaw chuck on the lathe and the milled face was bored outcentrall y to 11 mm diame ter to a dept h of 32 ram. To ac com mod atethe light de pen den t resistor the 11 mm bore was cou nte rbo re d toapproximately 13 mm diameter for a length of 18 mm to form a snugfit for the sensor. The front of the block was then opened out to25-4 mm diamet er for 10 mm length and threaded internally (Whit-worth form).

To make the unit wat erp roof a groove was machined to take a Viton'O' ring against which a 25 mm diameter transparent perspex windowwas tighte ned using a stainless steel ring thr ead ed ext er nal ly to suit thethrea ded 25.4 mm diame ter bore. The ring was machi ned with a 30 angled apert ure to ensure that ma ximu m am oun t of light could ente rthe unit, and with two slots across the oute rfa ce to take a suitablespanner. The electrical connections between the sensor and the control


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212 L La i ng , E . J on e sunit were made by drilling two 1 mm diameter holes into the cavitybehind the sensor and the wires sealed with 'Araldite'.

The sensor housing unit was held to the surface of the vessel using12.7 mm wide nylon power belting and a grip clamp supplied by themanufacturer (FA Power Ltd, Birmingham) of the belting. A groovewas milled 12.7 mm wide and 0-5 mm deep across the back of the unitto accommodate the belt and a dural securing plate 3 mm thick held inposition by two 2 BA brass screws.

Laing 1983 Aquacultural-Engineering

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