MASS C U L T U R E OF SPIRULINA USING LOW-COST N U T R I E N T S

C. V. S e s h a d r i * , and S e b a s t i a n T h o m a s
S h r i A. M . M . M u r u g a p p a C h e t t i a r R e s e a r c h C e n t r e , T h a r a m a n i ,
M a d r a s 600 042, India.

SUMMARY: Two s p e c i e s of S p i r u l i n a w e r e c u l t i v a t e d
in o u t d o o r ponds u s i n g l o w - c o s t s u b s t i t u t e s for s o m e
of t h e r e c o m m e n d e d n u t r i e n t s . In p a r t i c u l a r , b o n e -
m e a l and b i o g a s effluent w e r e found to be v e r y
e f f e c t i v e for the g r o w t h of t h e s e s p e c i e s .

INTRODUCTION: M o n o c u l t u r e of an a l g a l s p e c i e s is an e x p e n s i v e p r o c e s s if we
use conventional techniques. T h i s C e n t r e h a s b e e n ac tive in p r o m o t i n g the u s e
of l o w - c o s t m e t h o d s for the m a s s c u l t u r e of S p i r u l i n a in r u r a l h a b i t a t s ( S e s h a d r i
and T h o m a s , 1978). Some of the d e v e l o p m e n t s that have h e l p e d in l o w e r i n g the
cost were: 1) c h e a p m e t h o d s of pond c o n s t r u c t i o n , 2) c h e a p a g i t a t i o n p r o c e -
d u r e s , 3) h a r v e s t i n g e v e r y a l t e r n a t e day, and 4) s o l a r d r y i n g of the algal m a t .
H o w e v e r , t h e c o s t of the m e d i u m ( Z a r r o u k , 1966) r e m a i n s v e r y high. This
s t u d y r e p o r t s on the m a s s c u l t u r e of a s p e c i e s of S p t r u l i n a ( r e c e i v e d f r o m
I n d i a n A g r i c u l t u r a l R e s e a r c h I n s t i t u t e , New Delhi and c l a s s i f i e d as S p i r u l i n a
p l a t e n s i s ) and a l o c a l l y i s o l a t e d s p e c i e s of S p i r u l i n a r e f e r r e d to as S p i r u l i n a (j),
( J e e j i Bai, 1978), u s i n g l o w - c o s t s u b s t i t u t e s for the p h o s p h o r o u s , c a l c i u m
n i t r o g e n s a l t s in the t r a d i t i o n a l m e d i u m .

A. S p i r u l i n a IARI - E x p e r i m e n t a l Methods

1. B o n e - m e a l s u b s t i t u t e : S p t r u l i n a IARI w a s c u l t i v a t e d in open a l g a l ponds of

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1 m a r e a c o n t a i n i n g 100 1 m e d i u m at 10 c m depth. In pond A, 50% Z a r r o u k ' s
m e d i u m w a s u s e d as c o n t r o l and in pond B, 50% Z a r r o u k ' s m e d i u m o m i t t i n g t h e
p h o s p h a t e and c a l c i u m s a l t s but w i t h a d d e d b o n e - m e a l was u s e d . The t r a c e

e l e m e n t s in the m e d i u m w e r e r e s t r i c t e d to the A5M (50%)~ none of the B 6 M

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c o n s t i t u e n t s w e r e added. E D T A w a s not i n c l u d e d in e i t h e r of the m e d i a . (Normal
Z a r r o u k ' s m e d i u m w i t h o u t EDTA and B 6 m i c r o - n u t r i e n t s is r e f e r r e d to as m o d i -
fied Z a r r o u k ' s , M2~nn). ]3onemeal w a s e n c l o s e d in a fine m e s h nylon bag a n d
s u s p e n d e d in t h e m e d i u m , to avoid r e m o v a l of the p a r t i c l e s d u r i n g a l g a l har~cesting.
The s a m e c u l t u r a l c o n d i t i o n s w e r e p r o v i d e d in b o t h the p o n d s , s u c h as i n o c u l u m
q u a n t i t y , pH, r a t e of i n t e r m i t t e n t a g i t a t i o n b y hand p a d d l e s , light i n t e n s i t y , t e m p e r a -
ture, etc. I n i t i a l o p t i c a l d e n s i t y of the c u l t u r e w a s 0 . 0 5 - 0 . 1 5 at 480 n m . Initial
pH in b o t h c a s e s w a s 8 . 5 and r e a c h e d 1 0 . 5 w i t h i n five days. The average cultural
t e m p e r a t u r e v a r i e d b e t w e e n 26°C at 0800 h, 37°C at 1200 h and 33°C at 1600 h.
T h e lux r e a d i n g s w e r e a v e r a g e d at 2 2 , 0 0 0 / 0 8 . 0 0 h, 8 5 , 0 0 0 / 1 2 . 0 0 h and 3 4 , 0 0 0 /
16.00 h. Coconut t h a t c h c o v e r s f o r the p o n d s w e r e u s e d b e t w e e n 1100 h and
1500 h f o r the f i r s t w e e k , to p r e v e n t p h o t o o x i d a t i o n . H a r v e s t i n g w a s done on
a l t e r n a t e days (when the c u l t u r e o p t i c a l d e n s i t y r e a c h e d 0 . 7 - 0 . 8 at 480 nm) by
p o u r i n g the algal s l u r r y o v e r c o t t o n c l o t h f i l t e r s . Chemicals were replenished
periodically.

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2. B i o g a s e f f l u e n t s u p p l e m e n t : S p i r u l i n a IARI w a s c u l t i v a t e d in 2 m ponds
c o n t a i n i n g 200 1 m e d i u m at 10 c m depth. T h r e e s e t s of n u t r i e n t m e d i a w e r e t r i e d :
a) Pond A: Zm as initial d o s e , b) P o n d B: 1 / 2 Zm + 5% v o l u m e / v o l u m e u n f i l t e r e d
b i o g a s e f f l u e n t as initial d o s e and c) Pond C: 1 / 3 Z m + 5% v o l u m e / v o l u m e b i o g a s
e f f l u e n t as initial d o s e . O t h e r c u l t u r a l c o n d i t i o n s w e r e s i m i l a r to the one w i t h
bonemeal substitute. The h a r v e s t i n g w a s on e v e r y a l t e r n a t e day. Small a m o u n t s
of b i c a r b o n a t e , p h o s p h a t e and n i t r a t e w e r e added to t h e p u r e s y n t h e t i c m e d i u m
c u l t u r e z.fter h a r v e s t , and a l s o o c c a s i o n a l l y to o t h e r c u l t u r e s (once in 2-3 w e e k s ,
d e p e n d i n g on t h e c u l t u r e p e r f o r m a n c e ) . B i o g a s effluent at 1% level w a s added to
pond ]3 and C a f t e r e v e r y h a r v e s t .

B. S p i r u l i n a (j): E x p e r i m e n t a l Methods

1. B i o g a s e f f l u e n t s u p p l e m e n t : S p i r u l i n a (j), a l o c a l l y i s o l a t e d s p e c i e s , w a s
2
g r o w n in ponds of 10 m a r e a c o n t a i n i n g 3000 1 m e d i u m at 30 c m depth. Two s e t s
of e x p e r i m e n t s w e r e c a r r i e d out. In pond A, 50% Zm m e d i u m w a s used; in pond B,

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50% Z m m e d i u m w a s s u p p l e m e n t e d w i t h 5% v o l u m e / v o l u m e b i o g a s e f f l u e n t as
initial d o s e . A g i t a t i o n w a s e s s e n t i a l l y by hand p a d d l e , and i n t e r m i t t e n t l y .
T h e ponds w e r e n e v e r c o v e r e d . The pH value r a n g e d f r o m 9 . 0 i n i t i a l l y to
1 0 . 5 and above, five days a f t e r i n o c u l a t i o n . L i g h t i n t e n s i t y (lux) v a r i e d
b e t w e e n 2 8 , 0 0 0 / 0 8 . 0 0 h, 7 8 , 0 0 0 / 1 2 . 0 0 h and 3 0 , 0 0 0 / 1 6 . 0 0 h. The a v e r a g e
c u l t u r e t e m p e r a t u r e s w e r e 31°C at 08.00 h, 37°C at 12.00 h and 35°C at
16.00 h. H a r v e s t i n g w a s done on a l t e r n a t e days. A special phenomenon
o b s e r v e d in t h e s e ponds w a s that the algae f l o a t e d as m a t s in the e a r l y h o u r s ,
f a c i l i t a t i n g e a s y h a r v e s t by s i m p l y s c o o p i n g the algal m a t with c o t t o n f i l t e r s ,
A b i c a r b o n a t e - p h o s p h a t e - n i t r a t e b o o s t w a s g i v e n to pond A a f t e r e a c h h a r v e s t
w h e r e a s in pond B only b i o g a s effluent w a s added, at 1% l e v e l a f t e r e a c h h a r v e s t .

R E S U L T S AND DISCUSSION.. H a r v e s t i n g w a s c a r r i e d out on a l t e r n a t e days;

only h a l f the c u l t u r e q u a n t i t y w a s h a r v e s t e d , so t h a t the r e m a i n i n g c u l t u r e
a c t e d as the i n o c u l u m to p r o v i d e a c o n t i n u o u s c u l t i v a t i o n s y s t e m . These
open pond s y t e m s have b e e n r u n n i n g c o n t i n u o u s l y f o r s i x to eight m o n t h s and
it is w o r t h w h i l e to add t h a t the s t a r t i n g i n o c u l u m for o t h e r e x p e r i m e n t s h a v e
b e e n t a k e n f r o m t h e s e ponds. In o t h e r w o r d s , the pond c u l t u r e s h a v e b e e n
c o n s i s t e n t l y h e a l t h y , though t h e r e w e r e i n f r e q u e n t c a s e s of c o n t a m i n a t i o n b y
d i a t o m s and f l a g e l l a t e s .

No s t r o n g c o r r e l a t i o n s could be m a d e b e t w e e n d e p t h of t h e c u l t u r e and
harvest. In open ponds of 10 c m d e p t h , the h a r v e s t i n g w a s done by p o u r i n g
t h e s l u r r y o v e r c o t t o n c l o t h f i l t e r s f o r b o t h S p i r u l i n a IARI and S p i r u l i n a (j),
w h e r e a s by i n c r e a s i n g the c u l t u r e d e p t h in t h e c a s e of S p i r u l i n a (j),
h a r v e s t i n g could be e a s i l y c a r r i e d out by s c o o p i n g t h e floating algal m a t .
H o w e v e r , the i n c r e a s e d depth a l s o n e c e s s i t a t e d i n c r e a s e d n u t r i e n t input as
the initial d o s e . We h a v e yet to r e a c h a c o m p r o m i s e w h e r e algal m a t

f o r m a t i o n c a n be c o n s i s t e n t l y a c h i e v e d w i t h a c u l t u r e d e p t h l e s s than 30 c m .

T h e b o n e m e a l s u b s t i t u t e d e x p e r i m e n t u s i n g S p i r u l i n a IARI gave as good

a h a r v e s t as the 50% Zm m e d i u m . T h e a l t e r n a t e day h n r v e s t in 50% Zm

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medium varied from 12 gm/m2/2--- days to 23 gm/m2/2- days with an a v e r a g e
h a r v e s t of 10.1 g m / m 2 day. In the bonemeal substituted m e d i u m the altern ate
day h a r v e s t varied from 14-20 g m / m 2 / 2 days with an av erag e h a r v e s t of
9.8 g m / m 2 day for a period of 60 days.

Spirulina IARI utilizing biogas effluent as supplement to 1/2 Zm m e d i u m

gave consistently b e t t e r h a r v e s t s c o m p a r e d with the utilization of full Z m .
medium. The average h a r v e s t , over a period of 45 days, was 8.86 g m / m 2
day in full Zm medium, 10.88 g m / m 2 day in 1/2 Zm medium supplemented
with biogas effluent and 9.41 g m / m 2 day in 1/3 Zm medium supplemented
with biogas effluent.

F o r Spirulina (j) the a ve r a ge h a r v e s t in the biogas effluent supplemented

50% Zm m e d i u m was 12.39 g m / m 2 day as c o m p a r e d to 10.3 g m / m 2 day
r e c o r d e d in 50% Zm medium.

It appears that the useful c h a r a c t e r i s t i c of algal mat formation found

with Spirulina (j) is favoured by: 1) the p r e s e n c e of gas vacuoles in the alga,
2) the g r e a t e r culture depth used, and 3) the reduction of light penetration
due to the addition of biogas effluent to the medium. Such algal mat formation
was not often observed in ponds of 10 cm depth.

The economics of Spirulina cultivation in a medium supplemented with

biogas effluent have been worked out (Seshadri, 1978). Pro tein analysis c a r r i e d
out at the National Dairy R e s e a r c h Institute, Bangalore, India showed 40.11%
crude protein in Spirulina IARI and 59% c r u d e protein in Spirulina (j).
Microbiological analysis of the p r o c e s s e d algae, and feeding t r i a l s , have been
s t a r ted.

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REFERENCES:

1, Seshadri, C. V., and Thomas, S. (1978). National Solar Energy Convention,

Bhavnagar, India. A7, 41-46,

2. Zarrouk, C. (1966). Contribution ~ It~tude dtune cyanophyces: influence de

divers facteurs physiques et chimiques sur la croissance et [a photosynth~se
de Spirulina maxima (Setchell et Gardner) Geitler; Thesis, Universit~ de Paris.

3. Seshadri, C. V. (1978). Conference on 'The State of the Art of Bioconversion

of Organic Residues for Rural Communities t, The United Nations University,
Guatemala.

4. Jeeji Bai, N. (1978). A local isolate, taxonomically classified by Dr. N. J e e j i Ba

of the University of Madras, as Spirulina geitleri.