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N A S A C o n t r a c t o r NCC
2-501
C h a r a c t e r i z a t i o n
o f
Spirul ina Biomass
for CELSS D ie t
Potential
/ f l & - < Z
1.
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NASA Contractor NCC 2-501
Tit le: Character izat ion o f Spirulino Biomass
fo r CELSS Diet Potential
Pr ncipa
I I
nvest igator
Alabama A &M Uni ver si ty
Normal, A L 35762
Mahasin
G o
adros,
Ph-D-
Techni cal Monitor Robert D o MacElroy, PhoDo
NASA/AMES
MOffet Field, CA 94035
Period
Covered 11/01/87
-
10/31/88
NASA Cooperative Agreement CELSS Program
October 1988
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TABLE OF CONTENTS
Page
i
ACKNOWLEDGMENT
AND PERSONNEL ...........................
ABSTRACT
...............................................
ii
INTRODUCTION AND BACKGROUND ............................
1
Objectives ............................................. 3
Significance ........................................... 3
MATERJALS
AND METHODS .................................. 4
Culturing ..............................................
4
Growth Conditions
......................................
5
Analysis
...............................................
7
EXPERIMENTAL
DESIGN
.................................... 9
Protocol ...............................................
9
Growth Parameters Characterization ..................... 9
Physiological Characterization
of
Spirulina
in Batch Cultures ...................................
11
RESULTS
AND
DISCUSSIONS
................................
13
Temperature
and
Light
13
Aeration Rate .......................................... 13
Air Enrichment with Carbon Dioxide
17
pH Effect ..............................................
17
physiological Characterization
of
Spirulina
..................................
.....................
Nutrient Requirement ................................... 20
28
in
Batch Cultures ...................................
37
CONCLUSIONS ............................................
40
FuT[TRE RESEARCH
........................................
REFERENCES .............................................
42
APPENDIX
...............................................
44
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LIST OF FIGURES
Page
Figure
1.
Flow Diagram for Experimental Design
............. 10
Figure 2. Growth Rate and Yield
of
S. maxima(a)
and S. platensis (b) as a Function of
Temperature and Light Irradiance.. ................
14915
Figure 3. Growth Rate and Yield of S. maxima
and S. platensis as a Function of
Aeration Rate..................................... 16
Figure
4.
Growth Rate and Yield
of S.
maxima
and S. platensis as
a
Function of
Carbon Dioxide Concentration in Air .............. 18
Figure 5. Growth Rate and Yield of
S.
maxima
and
S.
platensis as
a
Function
of
pH
...............................................
19
Figure 6. Growth Rate and Yield of S. maxima
and S. platensis as
a
Function
of
21
itrogen Concentration ...........................
Figure
7.
Growth Rate and Yield of S. maxima
and S. platensis as a Function
of
Phosphate Concentration
..........................
22
Figure
8.
Growth Rate and Yield
of S.
maxima
and
S.
platensis as a Function of
Sodium Chloride Concentration
....................
24
Figure 9.
Growth Rate and Yield of
S.
maxima
and
S. platensis as a Function of
Iron Concentration
25
...............................
Figure
10.
Growth Rate and Yield of
S.
maxima (a)
and S. platensis
(b)
as a Function
of
Bicarbonate Concentration ........................ 26,27
Figure
11.
Optical Density versus Dry Weight
S.
maxima
(a), S. plantensis (b) ...................
29
Figure 12. Physiological Characteristics, under
Op tbum Growth Conditions
........................
31,32
Figure 13. Physiological Characterization
of
Cultures, under Stress Conditions
................
35,36
Figure 14. Cells
of S.
maxima grown under
Optimum Conditions (a) and (b) ...................
39
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LIST
OF
TABLES
Page
Table 1. Molecular Composition of Spirulina strains
.........
33
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ACKNOWLEDGEMENT
Appreciation is extended to N A S A - A M E S for their support
of th is proje ct.
As the NASA Technical Monitor Dr . Robert MacEIroy has
made invaluable guidance and assistance to the project.
him, we are especially gra tef ul.
To
PERSONNEL
The following personnel has been employed on this contract.
Mahasin G . Tadros, Ph.D.,
P . I .
Woodrow Smith, B. Sc ., M.Sc.
Peter Mabuthi, B.Sc.
Bever ly Joseph, B. Sc.
i
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ABSTRACT
Spi r ul i na sp. as a bi oregenerat i ve phot osynt het i c and an edi bl e al ga
f or space cr af t cr ew i n a CELSS, was char act er i zed f or gr owt h r at e
and bi omass yi el d i n bat ch cul t ur es, under var i ous envi r onment al
condi t i ons. The cel l char acter i st i cs wer e i dent i f i ed f or t wo st r ai ns
of Spi rul i na: S . maxi ma and S . pl at ensi s. Fast growt h r ate and hi gh
yi el d of bot h st r ai ns wer e obt ai ned under t he f ol l owi n condi t i ons:
t emper at ur e ( 30C- 350C) , l i ght i r r adi ance 60-100 uE ms s-l, ni t r ate
30mM, phosphat e 2mM, aer at i on300 m / m n, and pH 9-10. The par t i t i oni ng
of t he assi mal at or y pr oduct s ( pr ot ei ns, carbohydr ates, l i pi ds) wer e
mani pul at ed by var yi ng t he envi r onment al gr owt h condi t i ons. Our
exper i ment s wi t h Spi r ul i na have demonst r ated t hat under st r ess
condi t i ons ( i .e. hi gh l i ght
ni t r ogen or phosphat e l i m t at i on;
0.1
M sodi um chl or i de) car bohydr at e
i ncr eased at t he expense of pr ot ei n. I n ot her exper i ment s, wher e the
gr owt h medi a were suf f i ci ent i n nut r i ent s and i ncubated under opt i mum
gr owt h condi t i ons, t he t ot al pr ot ei ns wer e i ncr eased up
t o
al most 70%
of
t he or gani c wei ght .
120 uE m 2 s- l , t emperat ur e 38OC,
I n ot her wor ds t he nut r i t i onal qual i t y of t he al ga coul d be mani pul at ed
by gr owt h condi t i ons. These r esul t s suppor t t he f easi bi l i t y of con-
si deri ng Spi r ul i na as a subsyst em i n CELSS because of t he ease wi t h
whi ch i t s nut r i ent cont ent can be mani pul ated.
i i
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INTRODUCTION AND
BACKGROUND
Cert ai n basi c physi ol ogi cal needs must be met
i n or der f or human bei ngs
t o st ay al i ve. On ear t h, t hese needs ar e met by ot her l i f e f orms i n
conj unct i on wi t h geochemci al pr ocesses t hat ef f ect i vel y use human wast e
pr oduct s i n conj unct i on wi t h ener gy f r om t he sun t o produce f r esh
suppl i es of f ood, oxygen and cl ean wat er . I n t he ar t i f i ci al envi r onment
of a spacecraf t ,
t hese mat er i al s must be pr ovi ded, and human wast es r e-
moved, wi t hout r el yi ng on t he nat ur al r esour ces of t he ear t h' s bi ospher e.
Pur sui t of our nat i onal goal s i n space expl or at i on wi l l event ual l y
r equi r e man' s l ong- dur at i on t enancy of cel est i al vehi cl es and pl anet ar y
bases. Requi r ement s f or l i f e suppor t coul d be met t hrough expendi t ure
of st ored suppl i es and by r egenerat i on and r euse of t he wast e pr oduct s
of human metabol i sm The l ogi st i cs necessar y of r egenerat i on f or
ext ended space m ssi ons ar e wel l document ed.
The use of bi ol ogi cal component s Cont r ol l ed Ecol ogi cal Li f e Suppor t
Syst em ( CELSS) pr ogr am as subsyst ems f or t he revi t al i zat i on of ai r ,
wast e pr ocessi ng,
and f or t he pr oduct i on of f ood has been proposed
f or t he l ong t er m
-
space f l i ght ( MacEl r oy, Br edt , 1 9 8 5 ) . St udi es of
bi ogener at i ve l i f e suppor t syst ems f or use i n space i ndi cat ed t hat
t hey ar e sci ent i f i cal l y f easi bl e. Suppor t of a cr ew i n space, whet her
i n an orbi t er or on t he sur f ace of a pl anetar y body r equi r es that oxygen,
pot abl e wat er and f ood be suppl i ed and t hat wast e mat er i al be r emoved.
Empl oyment of phot osynt het i c or gani sms ( hi gher pl ant s, gr een al gae,
cyanobact eri a)
al l ows bi omass pr oduct i on f r om r el at i vel y si mpl e com
ponent s whi ch ar e r eadi l y r ecycl ed i n a CELSS sys t em namel y car bon
di oxi de, m ner al s ( NO3- , PO4- 3, K+ Na+, et c. ) and m cr onut r i ent s.
The pr i mar y s our ce of al l man' s f ood and or gani c r aw mat er i al s i s
sol ar ener gy. Convent i onal f ood sour ces consi st of hi gher pl ant s
and ani mal s. Unconvent i onal f ood sour ces f or human consumpt i on ar e
phot osynt het i c al gae and bact er i a and non- phot osynt het i c bact er i a,
yeast s and f ungi . Convent i onal f ood sour ces are hi ghl y pal atabl e,
but r equi r e
a
l ong t i me t o pr oduce. The phot osynt het i c energy
ef f i c i ency of hi gher pl ant s i s l ess than 3 % . Al gae, on t he other
hand, gr ow r api dl y; t hei r met abol i sm can be cont r ol l ed; t hey pr oduce
a hi gh r at i o of edi bl e to nonedi bl e bi omass; and t hei r gas- exchange
char acter i s t i cs ar e
compat i bl e wi t h human requi r ement s.
The sem - m cr oscopi c bl ue- gr een al gae
( Cyanophyt a; Cyanobact er i a)
occupy a uni que t axonom c posi t i on, si nce t hey combi ne an aut or ophi c
mode of growt h that i s common t o eukaryot i c pl ant cel l s wi t h a
met abol i c syst em t hat i s gener al l y regar ded as bact er i al , r at her t han
pl ant - l i ke.
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Cyanobacter i a s i ngl e cel l pr ot ei n
(SCP)
has been used as a f ood source
i n var i ous par t s of t he wor l d (e.g. Mexi co, Chi na and Af r i ca) si nce
anci ent t i mes; i n f act , dri ed cyanobact er i a and cyanobact er i al t abl et s .
are now sol d i n heal t h f ood st ores i n J apan, Nort h Amer i ca and Eur ope
because t hey are r ecogni zed f or t hei r nut r i t i onal val ue. The nut r i -
t i onal qual i t y of al l cyanobact eri a whi ch have been t est ed ( See
Appendi x) appear s to be ver y hi gh. For exampl e, Spi r ul i na, i n
addi t i on t o bei ng t he
r i chest known sour ce of vi t am n Bi z, al so
cont ai ns s i gni f i cant amount s of vi t am ns
B1
and B2. Si m l arl y, one
gr am of Spi r ul i na cont ai ns one- hal f of t he adul t dai l y r equi r ement s
of v i t am n
A
( B- carot ene) . The t r ace el ement s and i odi ne f ound i n
cyanobact er i a ar e al so i mpor t ant when consi der i ng t he nut r i t i onal
qual i t y cyanobact er i a. The pr otei n of
S.
maxi ma and Anabaena
cyl i ndr i ca i s easi l y di gest i bl e and appr oxi mat el y 65 of t he pr ot ei n
i s assi m l at i bl e.
Changes i n t he suppl y of consumpt i on of met abol i t es may have con-
si derabl e ef f ect s on met abol i c pat t er ns. The accumul at i on of phot o-
synt het i c pr oduct s i n al gae can be i nduced by mani pul at i ng t he
envi r onment al condi t i ons under whi ch t he al gae are gr own ( Fogg, 1956) .
Physi ol ogi cal changes have been i ndi cat i ve
of
par t i cul ar changes i n
nut r i ent def i c i ency (Heal ey, 1975) . St udi es have shown t hat l i m t at i on
of ni t r ogen, phosphorus, and i r on i n cul t ur e medi a, af f ect s t he gr owt h
and physi ol ogy of cyanobact er i a. Agi t at i onof t he cul t ur e wi t h ai r
l eads to bi omass i ncr ease.
Enr i chment wi t h
5
C02 i n the bubbl i ng
ai r was
an ef f i ci ent way of obt ai ni ng a good pr oduct i vi t y
(DE
l a Noue
et . al . ,
1984 ) .
Packer , et . al . ,
( 1986 ) ,
have shown t hat wi t h proper
mani pul at i on of t he osmot i c envi r onment , macr omol ecul es of car bohydr at es
can be pr oduced by N2- f i xi ng cyanobact er i a.
The most di f f i cul t pr obl em i n usi ng al gae as f ood i s t he conver si on
of al gal bi omass i nt o pr oduct s t hat a space cr ew coul d act ual l y eat
over a l ong per i od of t i me. I f al gae are t o be consi dered as a pr i -
mar y f ood sourc e, i t wi l l be necessar y to det erm ne t hat t hey can
be
conver t ed i nt o a wi de enough range of a pal at abl e compl et e di et.
Theref ore, Spi r ul i na, an edi bl e al ga wi t h l ess nucl ei c aci ds and no
cel l wal l s , of f er s a gaod
pr ospect f or f ur t her st udi es by mani pul at i ng
growt h par amet er s.
I n or der t o eval uat e
the pot ent i al of Spi r ul i na f or a
CELSS
d i e t , i t
i s essent i al t o have backgr ound i nf ormat i on on t he envi r onment al
t ol er ances of t he speci es and event ual l y the responses of physi ol ogi cal
charact er i s t i cs.
speci es i n bat ch and cont i nuous cul t ur es.
Thi s backgr ound wi l l be obt ai ned f r om st udyi ng t he
The pur pose of t hi s pr oj ect was t o eval uat e t he gr owt h and chemci al
composi t i on of t wo st r ai ns of Spi r ul i na under di f f er ent gr owt h
condi t i ons .
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OBJECTIVES
To charact er i ze bat ch cul t ur es
of
Spi r ul i na: Gr owt h, Bi omass Yi el d,
and Chem cal Composi t on under var yi ng:
.
Temper at ur e
.
Li ght i nt ens i t y
.
Aer at i on r at e
. Nut r i ent concent r at i ons
SIGNIFICANCE:
. Devel opment of CELSS r el i es, i n par t , on the abi l i t y t o
mani pul at e and cont r ol t he or gani sms whi ch ar e a par t of t he
syst em
. Bi ol ogi cal r egener at i on of suppl i es consumed i n CELSS.
. Di r ect ut i l i zat i on of al gae i n space craf t crew di et .
Thi s pr oj ect st art ed i n November
1987.
The accompl i shment s i n t he
per i od November 1987 t o Oct ober 1988 ar e descri bed i n t hi s repor t .
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MATERIALS AND HETEODS
Culturing
Or gani sm
Two
st r ai ns of cyanobact er i a Spi r ul i na:
. S.
Maxi ma: ( UTEXLB 2342) was obt ai ned f r om Ut ex Al gal
Col l ec t i on
. S . Pl atensi s: was obt ai ned f r om Dr . Becker W Ger many
. S.
Maxi ma f i l ament s have t ur ns, whi l e
S.
Pl ant es i s f i l ament s
ar e str ai ght ones.
Gr owt h Medi um Zar r ouk medi um ( 1966) was used as f ol l ows:
NaHC03 16. 0g; K2HP04 0. 5g; NaN03 2. 5g; K2SO4 1. Og; NaCl 1. Og;
MgS04. 7H20 0- 2g ; CaC12 0. 4g; FeS04 0. 01g; EDTA 0. 08g; Sol ut i on
A5 1mL; Sol ut i on B6 1mL; i n 1L di st i l l ed wat er .
Sol ut i on A5 i n gr ams per l i t r e
of
di st i l l ed wat er : H3BO3, 2. 86;
MnC12. 4H20, 1. 81; ZnS04. 7H20, 0. 222; CuS04. 5H20, 0. 079; and
MoO3, 0. 015.
Sol ut i on B6, i n m l l i gr ams per l i t r e
of
di st i l l ed wat er : NH4V03,
22* 96; KCr
(s04)2. 12H20, 192. 0; Ni S04. 6H20,
44. 8;
Na2W04. 2H20,
17. 94; Ti OSOq. H2S04, 8H20, 61. 1; and Co(N03) 2. 6H20, 43. 98.
The medi um was aut ocl aved wi t hout t he bi carbonat e sal t s. The
bi car bonat e sol ut i ons wer e st er i l i zed by f i l t r at i on t hr ough 0. 2 mm
pore s i ze f i l t e r s.
The cul t ur e medi um was modi f i ed f or nut r i ent l i m t at i on st udi es:
. For N2 l i m t ed cul t ur es, NaN03 was r epl aced
by
KC1, and ni t r ate
ammoni a, and ur ea were t ested i n di f f er ent concent r at i ons as
ni t r ogen sour ces.
.
For P- l i m t ed medi um t he P was r epl aced
by
NaCl and
H3P04
was
used as P- sour ce, i n di f f er ent concent r at i ons.
. For sal i ni t y st udi es, NaCl was used i n di f f er ent concent r at i ons.
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.
FeSO4 was used i n di f f er ent concent r at i ons.
.
pH was mai nt ai ned i n al l cases at 9 wi t h 2N NaOH
Cont am nat i on: The st andard pl at e- count met hod was used t o det erm ne
t he number of bact eri a pr esent i n t he cul t ur e. Al i quot s wer e pl at ed
usi ng a bent gl ass r od on an agar medi um whi ch i s pr epared f r om
Zar r ouk (1966) medi um ( al gae medi umment i oned pr evi ousl y) enr i ched
wi t h t he f ol l owi ng i ngr edi ent s:
Trypt one gl ucose yeast agar : Trypt one, 5. 0g; Yeast ext r act , 2. 5g;
Gl ucose,
1. Og;
Agar, 15. 0g; i n 1L di st i l l ed wat er , pH 7 .
0.1 m f i l t r at e was spr ead on t he agar s ur f ace and i ncubat ed.
Col oni es were count ed, di l ut i on was made i f necessar y. Pl at es
wer e i ncubat ed at 30C and count ed af t er
48
hour s or l onger t o
det ect al l or gani sms.
Pur i f i cat i on
of
Spi r ul i na Cul t ure: The or i gi nal cul t ures of Spi r ul i na
were cont am nat ed wi t h bact eri a. Di f f erent pr ocedur es were used to
pur i f y t he cul t ur es. However , t he f ol l owi ng pr ocedur e was t he most
successf ul one:
Cel l s wer e col l ect ed, f i l t er ed wi t h 8mm f i l t er ( Gel man) , washed
wi t h basal medi um and homogeni zed. Cel l s wer e spr ead i n a pl at e,
exposed to UV 5 m n (20 W UV l amp,
30
cm di st ance) and i nocul ated
i n t est t ube cul t ur es cont ai ni ng t he basal medi um One dr op
si ze, i nocul um per tube. From 100 cul t ur e t ubes,
10
t ubes wer e
bacter i a f ree.
Growth Conditions:
Cul t ur e Room A smal l r oom ( 3m D x 2. 45m W2 x 2. l m H), was avai l abl e
f or t hi s proj ect . I t was provi ded wi t h shel ves, whi ch have been
illuminated with cool white fluorescent tubes. Light intensity
var i ed f r om
80-100
uE m 2
was kept at 25C.
on t he shel ves. The r oom t emperat ur e
Li ght Measurement s: Li ght i r r adi at i on measur ement s wer e made wi t h a
Li - Cor Model Li - 185 M ( Lambda I nst r ument s) Met er equi pped wi t h a
spher i cal quant um sensor .
Al l experi ment s wer e i ncubat ed i n cont i nuous l i ght .
Culturing Techniques:
a. Cul t ur i ng bot t l es: Smal l bot t l es (60 m capaci t y) cont ai ni ng
3 m gr owt h medi um wer e i nocul at ed f r om st ock cul t ur e i n t he
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exponent i al phase and bubbl ed wi t h ai r . These cul t ur es wer e
used f or eval uat i ng t he gr owt h par amet er s of t he al ga.
b. Roux bot t l es: Exper i ment al amount s of al gal cel l s wer e gr own i n
r oux bot t l es , cont ai ni ng 800 m st er i l e gr owt h medi um by i n-
ocul at i ng t hemwi t h
50
m of pr eadapt ed r api dl y gr owi ng cul t ur e
i n a
125
m er l enmeyer f l ask. Cul t ur es wer e i l l um nat ed con-
t i nuousl y by pl aci ng them i n f r ont of a bank of t wo cool whi t e
f l uorescent l amps ( 4 0 W. Li ght i r r adi at i on, measur ed at t he
sur f ace of cul t ur e bot t l es was
80
uE m 2 s l .
gr own i n a wat er bat h kept at 29- 30C by t he use of a heat er -
t her most at combi nat i on.
The cul t ures wer e
c. Aer at i on: The cul t ur es were aer at ed wi t h ai r (0.03%C02)
or
ai r
enr i ched wi t h car bon di oxi de. The ai r was del i ver ed by an oi l -
l ess compr essor . The ai r was passed f i r st t hr ough concent r at ed
sul f ur i c aci d, a sat ur at ed ZnC12 sol ut i on and t hen di s t i l l ed
wat er . The ai r was t hen passed t hr ough a cot t on f i l l ed er l enmeyer
f ol l owed by a gl ass wool f i l l ed er l enmeyer pr ef i l t er . F i nal l y ,
t he ai r was st er i l i zed by f l owi ng t hr ough st er i l e f i l t er 0. 22 um
(Gel man) . The ai r f l ow r at e was moni t or ed by a f l ow met er . The
sour ce of car bon di oxi de was f r oma pr essur i zed t ank (50 l b)
whi ch was provi ded wi t h a r egul at or and sol enoi d val ve t o shut
of f t he gas aut omat i cal l y t hr ough an el ect r i c ti mer . The car bon
di oxi de f l ow was moni t or ed by a f l ow met er and was s t er i l i zed by
passi ng t hr ough st er i l e 0. 22 um f i l t er (Gel man). M xtur es of ai r
( 0. 36% C02) and car bon di oxi de wer e obt ai ned by bl endi ng gases t o
a desi r ed m xtur e i n a t wo- gas pr opor t i oner . The f l ow r at e of
t he m xed gas del i ver ed t o t he cul t ur e was mai nt ai ned at 300 m / m n.
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Anal ysi s :
Gr owt h Rat e: Gr owt h was measured by moni t or i ng change i n absor bance
(O D. )
at
560
nm wi t h spect r ophot omet er ( Per ki n El mer Lambda
I )
and
expr essed as doubl i ngs dayl. The mean dai l y di vi si on r at e t , K , i s
cal cul at ed f rom
Wher e,
t
= days si nce i nocul at i on
ODt = opt i cal dens i t y af t er
t
days
OD, = opt i cal densi t y when
t
= 0.
Har vest i ng of Cel l s: Cel l s wer e col l ect ed
by
f i l t r at i on usi ng f i l t er
paper
10
mm
por e si ze (Gel man) . Cel l s were washed wi t h buf f er sol ut i on
( pH
8),
di l ut ed t o known vol ume and pr ocessed f or f ur t her anal ysi s.
Cul t ures wer e har vest ed at O. D.
0.1
uni t s , to avoi d l i ght l i m tat i on.
Tr i pl i cat e sampl es
of
t he al gal suspensi on wer e t aken f or each
det erm nat i on. The mean val ue of t hese t r i pl i cat es was r ecor ded.
The f ol l woi ng det er m nat i ons wer e car r i ed out :
Tot al Chl or ophyl l : An al i quot f r om t he cul t ur e was cent r i f uged f or
2
m n at 2000g. The pr eci pi t at e was suspended i n
5 ml
met hanol f or
5
m n i n a wat er bath at 7OoC, and t hereaf t er cent r i f uged.
opt i cal densi t y of t he supernat ant was det erm ned
655
nm.
The
Dr y Wei ght Measur ement s (DW: A vol ume f r om t he cul t ure was f i l t ered
t hrough a f i l t er 10 um pore s i ze, dr i ed i n pr evi ous l y dr i ed, pre-
wei ghed f i l t er paper f or
4
h at 80C, and t hen wei ghed af t er cool i ng
i n a desi ccat or .
Ash- Free Dr y Wei ght (AFDW: Af t er r ecor di ng t he dr ywei ght , t he dr i ed
cel l s wer e ashed at
500C
f or 2hr s. Then t he ash wt . was recor ded.
The di f f er ence bet ween dr y wei ght and ash wei ght gave t he organi c
wei ght of t he sampl e.
Tot al Carbohydr ates: The ant hr one sul phur i c aci d method was f ol l owed
( St r i ckl and, Pears ons, 1972). The pr i nci pl e of t hi s met hod i s t he
f ormat i on of a bl ue- gr een col or whi ch i s t he pr oduct of ant hr one and
t he f ur f ur al der i vat i ves pr oduced by aci d decomposi t i on of t he sugar .
The ant hr one r eagent consi st s of 0. 2g ant hr one,
8
m et hyl al cohol ,
10 m di s t i l l ed water .
one m of al gal suspensi on
( cont ai ni ng known wei ght of al ga)
and
heat ed i n a wat er bat h f or seven m nut es and cool ed.
Then
ml
of t he ant hrone reagent was added t o
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The bl ue- gr een col or was measur ed by a spect r ophot omet er at wave
l engt h of
620
nm The val ue of t he r eadi ng was cal cul at ed as
m cr ogr ams of gl ucose f r oma st andard cur ve f or gl ucose whi ch has
been pr epared by t he same met hod.
Tot al l i pi ds: Cel l ul ar l i pi ds wer e sol ubi l i zed by r epeat ed ext r act i on
wi t h met hanol and met hanol - chl or of or m ( l : l ) , t hen phase separ at ed
af t er adj ust ment of t he sol vent r at i os t o
10:10:9
( met hanol : chl oro-
f orm wat er, v/ v) ( Bl i gh and Dyer
1959).
The chl orof or m phase was
col l ec ted,
evapor at ed to dr yness under
N2,
and the wei ght of t he l i pi d
was det erm ned. Li pi d cont ent was cal cul at ed as t he wei ght of t he
l i pi d extr act di vi ded by t he ash f r ee dr y wei ght of t he or i gi nal
sampl e.
Tot al Ni t r ogen and Prot ei n ( Kj el dahl ) : One m of al gal suspensi on
cont ai ni ng
a gi ven wei ght was di gest ed i n a Kj el dahl di gest i on f l ask
cont ai ni ng 0.3, sel eni umm xtur e and one m sul f ur i c aci d.
cont ent s became col orl ess, t hey were t r ansf er r ed to t he Kj el dahl
appar at us wi t h
10
m of
50
sodi umhydr oxi de sol ut i on. A st r ong
current of st eamwas passed f or
7
m nut es dur i ng whi ch t he l i ber at ed
ammoni a was r ecei ved i n a 100 m f l ask cont ai ni ng
5
m of 2% bor i c
aci d sol ut i on and 4 drops
of
i ndi cat or . The i ndi cat or was composed
of 0. 016g met hyl r ed and 0. 83g br omocr esol gr een i n 100 m of alcohol.
The di s t i l l at e i n t he bor i c aci d sol ut i on was back t i t r at ed wi t h
0.1
N
sul phur i c ac i d, unt i l the col or of the i ndi cat or t ur ned pal e pi nk.
A bl ank sampl e was done f or each seri es of ni t r ogen est i mati on, usi ng
D .W . The val ue of t he readi ngs was cal cul at ed i n ug
N,
f r om a
st andard cur ve f or ni t r ogen sour ce as ammoni ums ul f at e, whi ch has
been t r eat ed by t he same met hod.
t ot al N x
6.25.
When t he
Tot al pr ot ei n was cal cul at ed f r om
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EXPERIMENTAL
DESIGN
Pr ot ocol : Fl ow di agr am f or t he exper i ment al desi gn was f ol l owed
(Fi g.
1 .
A
Gr ovt h par amet er s char act er i zat i on:
I . Temperat ur e, Li ght :
The al gal gr owt h was eval uated f or t emper at ure and l i ght
t ol erance on a gr adi ent pl at e. Temper at ure coul d be adj ust ed
i n r ange f r om 10C t o 50C. I l l um nat i on was pr ovi ded by
ei ght cool whi t e f l uor escent t ubes
( 4 0
W. Di f f erent l i ght
i nt ensi t i es were obt ai ned by var yi ng t he di st ance bet ween
t he cul t ur es and l i ght sour ce. The al gal speci es wer e
cul t ur ed i n smal l bot t l es (60 m capaci t y) cont ai ni ng 30 m
gr owt h medi um Tri pl i cate cul t ur es wer e pl aced on t he
gr adi ent pl at e, at t emperat ur es: 20C, 25 C, 35OC and 40C.
The cul t ur es wer e exposed t o t wo l i ght i nt ensi t i es and wer e
aer at ed wi t h ai r (0. 03%C02).
11.
pH
E f fec t :
The al ga was i ncubat ed i n smal l bot t l es as descr i bed i n
sect i on A(1) at 35C on a t emper at ur e gradi ent pl at e and
80 uE ml
s - ~
r r adi ance.
cul t ur i ng, except t he pH used f or cul t ur i ng was var i ed by
usi ng NaOH or HC1. The pH of cul t ures was adj ust ed dai l y t o
t he or i gi nal pH. The cul t ur es wer e aer at ed wi t h ai r
(0. 03%C02).
The ori gi nal medi umwas used f or
111. Aer at i on Rat e, Carbon Di oxi de Enr i chment , Bi carbonat e Con-
cent r at i on:
The alga was incubated in small bottles described in Section
A(1) at 35C on a t emper at ur e gr adi ent pl at e and 80 uE m l
s - ~
i r r adi ance. Thr ee set s of cul t ur es wer e t r eat ed di f f er ent l y:
a. Cul t ur es wer e aer at ed wi t h di f f er ent f l ow r ates
( ai r 0. 03% C02).
b. The f l ow r at e whi ch gave t he best gr owt h r at e, was
sel ected f r om ' la ' ' . The cul t ures wer e aer at ed wi t h
ai r enr i ched wi t h car bon di oxi de i n di f f erent
concent r at i ons 1% 32, 52,
10%
c. Cul t ur es wer e t r eat ed wi t h di f f er ent bi car bonat e
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rl
(d
u
l
B
a
rl
10
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concent r at i ons i n whi ch one set was aer at ed wi t h ai r
( 0 . 0 3 C 0 2 ) and ot her set was aer ated wi t h ai r
cont ai ni ng
1%
C02.
The pH of al l cul t ur es was adj ust ed t wi ce dai l y.
I V.
Nut r i ent Requi r ement s:
Cul t ures wer e i ncubated i n smal l bot t l es under t he same
condi t i ons as descr i bed i n Sect i on A ( 1 ) . The or i gi nal
growt h medi um was modi f i ed by changi ng t he concent r at i on of
one nut r i ent . Ni t r ogen, phosphor us, i r on, bi car bonat e and
sodi um chl or i de wer e studi ed i n suf f i c i ent and l i m t i ng
concent r at i ons. The bi carbonate ef f ect was st udi ed t oget her
wi t h the aer at i on ef f ect
111 .
I n al l exper i ment s t r i pl i cat e cul t ur e bot t l es were i nocul at ed
f r oms t ock cul t ur es i n t he exponent i al phase. Gr owt h r esponse
was measur ed as opt i cal densi t y and t he gr owt h r at e was expressed
as doubl i ngs per day. The yi el d of cul t ur es was expr essed as t he
t ot al dr y wei ght af t er 5 days of gr owt h. The t otal day wei ght
was det erm ned by har vest i ng t he cel l s and dr yi ng i t ( see Met hods).
B Physi ol ogi cal Char acter i zat i on of Spi r ul i na i n Bat ch Cul t ures:
For t hi s exper i ment , t he al ga was gr own i n bat ch cul t ur es ( Roux
bot t l es) as ment i oned i n Met hods . The cul t ures were mai nt ai ned
under opt i mum gr owt h condi t i ons and moni t or ed i n t he exponent i al
phase by t he absor pt i on measur ement ( see Met hods) .
I.
11.
O. D.
of Cel l Suspensi on ver sus
D. W
and Chl orophyl l : Both
speci es gr own i nt r i pl i cate Roux bot t l es under t he same
condi t i ons descr i bed bef or e ( see Met hods). Twent y m of
cul t ur e sampl es wer e t aken dai l y f or measur ement s of t heD. W ,
and chlorophyll. The experiment was continued for one week,
and
20
m of f r esh cul t ure medi um were added to t he Roux
bot t l es i mmedi atel y af t er each sampl i ng i n order to mai nt ai n
t he same vol ume of t he cul t ur e medi um dur i ng t he cul t i vat i on.
Under Opt i mum Gr owt h Condi t i ons:
Both speci es wer e gr own i n dupl i cat e Roux bot t l es under t he
same condi t i ons descr i bed bef or e ( see Methods). Cul t ur es
were anal yzed f or gr owt h paramet ers dur i ng t he ei ght days.
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111.
Var i ous St r ess Condi t i ons:
Li ght and Temper at ure:
Bat ch cul t ur es were i ncubat ed under hi gh l i ght i r r adi at i on
and ot her s at hi gh t emper at ure ( 38C) i n water bat h.
Nut r i ent s:
Bot h speci es wer e gr own i n Roux bot t l es under t he same
condi t i ons descr i bed pr evi ousl y. Bat ch cul t ur es wer e gr own
i n dupl i cat e unt i l t he exponent i al phase was r eached at 0.1
OD, t o avoi d l i ght l i m t at i on. One bat ch was anal yzed and
r epr esent ed t he cul t ur e suf f i ci ent i n nut r i ent s. The
exponent i al phase l ast ed t hr ee t o f i ve days. Bat ch cul t ur es
were concent r at ed and di l ut ed t o the or i gi nal bat ch vol ume
but wi t h a new medi um modi f i ed i n one el ement . The ni t r ogen
l i m t ed bat ch recei ved
0.05
mM
ni t r at e; t he phosphat e l i m t ed
batch r ecei ved 0. 01 mM phosphat e; t he bi car bonat e l i m t ed
bat ch r ecei ved 4g/ L bi car bonat e; and sodi um chl or i de was
added i n
t wo
concent r at i ons;
0.1
M and
0. 5
M When one
el ement was l i m t ed, t he ot her s wer e i n suf f i ci ent concen-
t r at i ons. The cul t ur es were i ncubat ed under st r essed condi -
t i ons f or t wo days and t en har vest ed f or anal ysi s.
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RESULTS
AND DI SCUSSI ON
Temper at ue
and
Light:
Fi gur e 2 (a and b) depi ct t he gr owt h and yi el d of bot h st r ai ns of
Spi r ul i na at t wo l i ght i r r adi at i ons and di f f er ent t emper at ur es rangi ng
f r om 20C t o 40C.
gr ow at 25C at ver y s l ow r at e.
al gal f ast est gr owt h r at e and hi ghest yi el d of cel l s.
t emperat ur e was r ai sed to 40 C, t he al gal cel l s t ur ned yel l ow and
Nei t her st r ai ns gr ew at 20C but t hey st ar t ed t o
Temperat ur es
30
and
35C
enabl ed the
When t he
gave a l ower yi el d. S. pl at ensi s, on t he ot her hand gave opt i mum
yi el d at l i ght i r r adi at i on- 80 uE m 2 s- l whi l e S. maxi ma t ol er at ed
l i ght i r r adi ance 120 uE mz s-l.
Aer at i on Rat e:
The ef f ect s
of
ai r agi t at i on r at e on the gr owt h r at e and cel l yi el d
aredepi ct ed i n Fi gur e
3 .
The gr owt h r at e of bot h Spi r ul i na st r ai ns
i ncreased wi t h i ncreasi ng t he f l ow r at e of ai r i n r ange of 150 m / m n
and 500 m / m n. When t he f l ow r at e of aer at i on was i ncr eased t o
2000 m / m n, t he gr owt h r at e st ar t ed to decl i ne and cel l s t ur ned
yel l ow. On t he ot her hand the cel l yi el d i n t er ms of dr y wei ght
was not af f ect ed. The pH of al l cul t ur es i ncreased t o 11. The cel l
yi el d of bot h st r ai ns showed par al l el f l uct uat i on t o t he gr owt h r at e
of t he agl a.
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I
u1
N
I
E
W
3
ea
d
I I I
rl
I
rn
si
I
E
w
1
00
N
00
rl
Q
In
m
m
Lo
N
N
e
Lo
e3
m
Lo
N
N
e
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m
W
a
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d
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N
I
E
w
1
hl
d
*
D
CJ rl
/
4
---?
I
I I I
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E
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s
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m
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t
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/
Y
/
Y
/
ea
d
I
0
0
0
W m
1 6
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Ai r Enr i chment wi t h Carbon Di oxi de:
Fi gur e
4
shows t he ef f ect of ai r enr i ched wi t h di f f er ent concent r at i ons
of carbon di oxi de on t he gr owt h r ate and yei l d of bot h Spi r ul i na
s t r ai ns
:
.
Cul t ur es aer at ed wi t h
10
C02
-
i n ai r , di d not gr ow and
t ur ned yel l ow. The pH dr opped r api dl y, wi t hi n
3
hour s , f r om
9 . 4
t o
7 .
. When t he C02
-
concent r at i on i n ai r was decr eased t o
5%
or
3%
t he cul t ur es st art ed t o gr ow. The pH of t he cul t ur es were
mai nt ai ned at
9 . 4
by t he addi t i on of s odi um hydr oxi de. How-
ever, t he pH of cul t ur es aerat ed wi t h 1 C02
-
enr i ched ai r
was mai nt ai ned st abl e.
.
Cul t ur es aer at ed wi t h ai r
( 0 . 3 %
C02) gr ew at mor e or l ess
t he same gr owt h r at e of t hose aerat ed wi t h
1
C02
-
enr i ched
ai r . The yei l d of cul t ur es t r eat ed wi t h di f f er ent C02
concent r at i ons, i n t erms of dr y wei ght , was equi val ent t o t he
gr owt h r ate.
The r esul t s of t hi s experi ment are i n agr eement wi t h t hose of Faucher
and Coupal
( 1 9 7 9 ) .
They r eport ed t hat spar gi ng
1%
C02
-
ai r i n
Spi r ul i na cul t ur es coul d mai nt ai n a const ant pH of t he cul t ur e
medi um and at t he same t i me generat e HCO3 i ons whi ch wer e used as
carbon sour ce f or
S
maxi ma. I n a si m l ar st udy wi t h gr een al gae,
Gol dman and Gr aham (1981) , r epor t ed t hat i n bat ch cul t ur es, maxi mum
gr owt h rat es wer e achi eved at t he C02 l evel s pr esent i n at mospher i c
ai r and at HCO?j concent r at i ons of
3
mM.
pE Effect:
The gr owt h r at e of
b o t h
Spi rul i na st ra i ns
i s
cl ear l y af f ect ed by t he
pH of t he gr owt h medi um as i n shown i n Fi gur e 5. Bot h st r ai ns
exhi bi t ed hi gher gr owt h rat e i n medi a of pH r ange of
9
t o
10.
The
growt h r at e decr eased wi t h i ncr easi ng pH above
10
and the cel l s
t ur ned yel l ow i n case of S. maxi ma whi l e i n S pl at ens i s t he cel l s
r emai ned i n bl ui sh gr een i n col or . The cel l concent r at i on i ncr eased
when i ncr easi ng t he pH of t he medi um f r om 8 t o
10
and t hen decr eased
above pH
1 0 .
1 7
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/
/
I
/
W
0
0
0
4
0 0 0
W
m
0
0
rl
0
m
0
m
rl
m
0
0
d
m
0
m
m
0
0
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R
f
2
c r
f
I
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\
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\
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L
v
co m
co
d
v
b
cp
e3
w
0
19
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Nut r i ent Requi r ement :
Ni t r ogen:
Ni t r ogen sour ces i n t he f or m of ni t r at e, ur ea and ammoni a were t est ed
i n
di f f er ent concent r at i ons i n or der t o det er m ne t hei r ef f ect i veness
as N- sour ces. Ammoni a i nhi bi t ed t he gr owt h of both st r ai ns and t here-
f ore the dat a were del et ed. The r esul t s of ni t r at e- N and ur ea- N are
r epr esent ed ( Fi gur e 6 ) . The gr owt h r at e of bot h Spi r ul i na st r ai ns
was i nhanced wi t h i ncr easi ng t he concent r at i on
of
ur ea- N and ni t r at e-
N. The urea- N at
20 mM
concent r at i on enhanced t he gr owt h r at e, whi l e
f ur t her i ncrease i n i t s concent r at i on l i m t ed t he gr owt h of bot h
st r ai ns. On t he ot her hand, ni t r at e- N at concent r at i on
30
mM, enabl ed
bot h st r ai ns t o reach f ast gr owt h r at e and hi gh yi el d
i n
t er ms of dry
wei ght .
t he gr owt h of bot h st r ai ns t o some ext ent , t he st r ai ns bl eached and
l ost t hei r pi gment s. Thi s experi ment demonst r at ed t hat t he l east
amount
of
ni t r at e- N necessar y t o mai nt ai n t he gr owt h
of
Spi ru l i na
i n
cul t ur e was
10 mM.
M cr oscopi cal l y, t he t r i chomes became shor t er
i n bot h st r ai ns and wi t h aver age 6 t ur ns/ t r i chome, i n medi a l i m t ed
i n ni t r ogen concent r at i on. I n agr eement wi t h our r esul t s, Faucher
et . al .
( 1 9 7 9 ) ,
r eport ed
t h a t
urea- N i n l ow concent r at i on coul d
suppor t t he gr owt h of
S.
maxi ma, at hi gh concent r at i on of ni t ate- N.
Al t hough l ower concent r at i on of ni t r at e- N
(10mM)
suppor t ed
Phosphat e:
I ncr easi ng t he phoshat e- P concent r at i on i n t he cul t ur i ng medi a t o
1 mM
and 5 mM, enhanced t he gr owt h rat e of bot h st r ai ns ( Fi gur e 7) . But as
t he concent r at i on i ncr eased t o
10 mM,
t he gr owt h r at e of bot h st r ai ns
decl i ned. The mass yi el d of bot h st r ai ns showed si m l ar r esponses
coi nci di ng wi t h t hei r gr owt h rate. M cr oscopi cal l y, the t r i chomes
became shor t er
i n
medi a of phosphat e- P concent r at i on bel ow
1 mM
and
wi t h f ew number of t urns i n case of S. maxi ma
5
t ur ns/ t r i chome) .
General l y, cyanobact eri a r equi r e smal l concent r ati ons of phosphat e- P
f or gr owt h. They can grow i nphosphor us- l i m t ed medi a ( Lang and Br own,
1981).
20
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w
c
I
\\\
\\\
\ \ \ \
c
n:
a
v
3
k
yO< \
\ \
w
b
e
w
a:
c
p:
5
w
N
W
d
3
1
*
m
el
d
w
m
hl
d
rb
a
rb
n
s Q
E 4
2 1
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2.25
1.5
0 . 7 5
0 . 0
6 0
30
0
-
-
-
-
-
2
I
I
I
S P I R U L I N A m a x i m a
.
0.0 2.0 5.0
10.0
S P I RU L I N A p l a t e n s i s
P,
I '
I
0
\
\
I
I
/
i
\
\
\
b
C O N C E N T R A T IO N
(mM)
PHOSPHATE
Figure
7.
Growth Rate
and
Yield
of
S.
maxima
and
S. platensis as a Function
of
Phosphate Con-
centration
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Sodi um Chl or i de:
Bot h Spi r ul i na st r ai ns gr ew i n medi a l acki ng sodi um chl or i de ( Fi gur e
8).
They showed r esponse i n gr owt h r ate as t he sodi um chl or i de
concent r at i on i ncr eased to
10
mM. Fur t her i ncrease i n sodi um chl or i de
concent r at i on (100 mM) af f ect ed t he gr owt h r at e of bot h st r ai ns and
r esul t ed i n l ower y i el d of cel l s . I n addi t i on, m croscopi c exam nat i on
of bot h st r ai ns i ndi cat ed t hat i n medi a t r eat ed wi t h a hi gh concent at i on
of sodi um chl or i de 100
mM,
t he t r i chomes were shor t and wi t h l ess t ur ns
i n case of
S.
maxi ma
(6
t ur ns/ t r i chome) . The r esul t s of t hi s exper i -
ment , i ndi cat e t hat Spi r ul i na t ol er at e i ncreases i n sodi um chl or i de
concent r at i on up t o
100 mM.
Spi r ul i na t ol er ance t o sal t had been
pr evi ousl y r epor t ed ( Faucher , et . al . , 1979).
I ron:
I r on concent r at i ons ( FeS04) i nf l uenced
t he
growt h and yi el d of both
st r ai ns ( Fi g. 9). Concent r at i on of 0. 05mMwas suf f i ci ent f or
t he
gr owt h of bot h st r ai ns, al t hough medi a def i ci ent i n i r on di d not show
any growt h r esponse. I ncr easi ng t he concent r at i on of i r on beyond
0.1
mM l ower ed t he yi el d
of t he
al ga and cel l s t ur ned yel l ow.
Bi car bonate Concent r at i on:
Fi gur e 10 a shows t hat S. maxi ma gr ows i n t he medi um even wi t hout
bi car bonat e sal t , pr ovi di ng t hat t he cul t ur e was aer at ed wi t h ai r
(0.03%CO2).
A s
t he bi car bonat e concent r at i on i ncr eased, t he gr owt h
r at e as wel l as pr oduct i vi t y i ncr eased. Fur t her i ncr ease i n bi car bonat e
concent r at i on above 16g/ L ( 190
mM)
di d not af f ect t he gr owt h r at e.
When t he car bon di oxi de concent r ati on i n t he ai r i ncr eased f r om 0. 03%
t o 1% as s hown i n Fi gur e
10
b , t he growt h r ate i ncr eased r emarkabl y
by decr easi ng t he bi car bonate concent r at i on i n t he medi um as l ow as
4g/ L (48
mM
i . e. one quar t er of t he concent r ati on i n t he Zarr ouk
medi um ( see Met hods) . Spi r ul i na pl at ensi s, does not show much var i at i on
i n i t s r esponse t o i ncreasi ng
CO2
concent r at i on i n ai r , when compar ed t o
S. maxi ma ( Fi gur e 10 b ). The cel l concent r at i on based on dry/ wei ght
measur ement was r el at ed t o t he gr owt h r ate i n bot h st r ai ns, i n al l
t r eat ment s. The r esul t s of t hi s exper i ment i ndi cat e t hat bot h st r ai ns
can ut i l i ze at mospher i c car bon di oxi de when t he medi a bi car bonat e
concent r at i on i s m ni mum i n t he cul t ur e medi um
was adj ust ed dai l y t o 9.4.
The pH
of
al l cul t ur es
2 3
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S P I R U L I N A maxima S P I RU L I NA platensis
0 . 0
0.01
0.1
0 . 5
I I
I
0 . 0 0.01
0.1 0 .5
CONCENTRATION M )
SODIUM CHLORIDE
Figure 8.
Growth Rate and Yield of S.
m a x i m a and
S.
platensis
as a
Function
of
Sodium Chloride
Concentration
2 4
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n
i2
n
Q
s
70
n
c
H
U
w
t.c
4
p:
3:
w
5
0
p:
c3
n
4
4
E
M
E
w
X
c3
w
5
*
p:
U
Y
n
2 . 2 5
1.50
0 .75
0.0
60
30
0
S P I R U L I N A maxima
S P I R U L I N A platensis
-
7
I
?--
\
I
I
\
- I
\
I
\
I \
i
-I
-
-
1 I I I
I
I
I
L
.0
0.05 0.1 0 .2
0.0 0 . 0 5
0.1
0 .2
CONCENTRATI ON (mM)
IRON
Figure 9.
Growth Rate and Yield of
S.
m a x i m a and
S. platensis as a Function of
Iron
Concentration
25
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?
i
i
\
\
t
n
0
ea
@a
d
CD m
m
m
In
d
In
m
d
aa
m
e
rn
ea
m
d
In
m
r
Q
In
*
26
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T
p:
2
i
4
\
t
1
I
1
I t I
i
c3
4
0
Q
- 0
m
*
0
n
0
u
do
m
0
0
I
00 00
0
m
- m
d
0
0
Q
0
m
*
a:
1 1 :
0
0
0
W
m
n
-
E
0
0
d
M
z
t c
c
p:
t c
z
W
u
z
u
W
27
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Physiological Characterization of Spirulina in Batch Cultures:
Batch Cultures:
I.
op
tical Density (O.D.)
of
Cell Suspension versus Dry Weight (D.W.)
and Chlorophyll:
Gr owt h can be expr essed as gr owt h r at e or as yi el d. Yi el d, as an
expr essi on of or gani c pr oduct i on, i s usual l y gi ven i n t erms of
dr y wei ght of t he organi c mass pr oduced over a per i od of t i me
per uni t vol ume. A r el at i onshi p bet ween opt i cal densi t y, dr y
wei ght and chl orophyl l was est abl i shed f or bot h st r ai ns of
Spi rul i na.
Resul t s ar e pr esent ed i n Fi gur e 11. For al l sampl es wi t hi n t he
f i r s t t hree days of cul t i vat i on, whi ch cont ai n r el at i vel y smal l
concent r at i ons of bi omass ( 4 0 0 mg DW L or l ess), r eadi ngs f el l
wi t hi n t he accur at e r ange of t he
O. D.
scal e and t hey coul d be
r ead di r ect l y f r om t he spect r ophot omet er wi t hout di l ut i on. How-
ever , f or al l sampl es dur i ng the l at er cul t i vat i on per i ods whi ch
cont ai ned hi gh concent r at i on
of
bi omass (500
mg
DW/L), di l ut i on
of t he sampl es wi t h di st i l l ed wat er was necessar y pr i or t o
OD
r eadi ngs. The gr aphs show l i near i t y bet ween
OD
and dr y wei ght .
Each
OD
uni t i s equi val ent t o a concent r at i on of
700
mg/ L
i n
t he
case of S. maxi ma and t o
750
mg/ L i n t he case of S. pl at ens i s .
I t i s obvi ous f r om t hi s exper i ment t hat ot her r el i abl e i ndi cat or s
of
est i mat i ng al gal pr oduct i vi t y can be comput ed f r om
OD
measur e-
ment s. Theref ore,
OD
measur ement s can be t r ansl at ed i nt o bi omass
yi el d i n t er ms of dr y wei ght or chl or ophyl l .
28
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1.5
1.0
E
c
(0
v
c,
a
0.5
0.0
0
250
400
550
700
850
n
0
1.
1.
10
n
d
+
bn
E
W
0.
0.
DRY
WEIGHT mg/l)
Figure 11.
Optical Density
versus
D r y Weight S. maxima (a)
and S. platensis
(b)
2 9
0
p:
0
bJ
3:
u
10
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11. Phys i ol ogi cal Char acter i s t i cs
of
Cul t ure, under Opt i mum Gr owt h
Condi t i ons:
Bot h speci es wer e grown i n dupl i cat e Roux bot t l es under t he same
condi t i ons descri bed bef ore ( see Met hods) . Cul t ur es were
assayed f or growt h paramet ers dur i ng
t he
ei ght days. ( Fi g.
12
a and
b ).
I ncr ement s of car bohydr at es, pr ot ei ns, dr y wei ght
and chl orophyl l ar e expr essed as ug/ m cul t ur e. The r esul t s
show t hat i ncr eases i n t he synt hesi s of chl or ophyl l , pr ot ei n
and yi el d of t he cul t ur e are cor r el ated. Gr owt h paramet er s of
cul t ur es anal yzed af t er 8 days st ar t ed to l evel of f , due the
nut r i ent exhaust i on and l i ght l i m t at i on caused by i ncreasi ng
cel l concent r at i on.
111.
Phys i ol ogi cal Char acter i zat i on of Cul t ur es, under St r ess
Condi t i ons:
The resul t s of anal ysi s were expr essed on t he basi s of organi c
wei ght ( Ash Fr ee Dr y Wei ght : AFDW and r epr esent ed i n Tabl e 1
( Fi gur e 13). Resul t s of cul t ur es gr own under opt i mum condi -
t i ons ( 11) were used as cont r ol f or al l exper i ment s i ncubated
under var i ous growt h condi t i ons.
L i ght I r r adi ance
and
Temper ature: I ncr easi ng
t he
l i ght
i r r adi ance to
120
uE
m-2 s-l,
l ed to an i ncrease i n t he t ot al
car bohydr at e cont ent and a decr ease i n t ot al pr ot ei n cont ent :
S.
maxi ma
19.58%,29.06%: S.
pl ant ensi s
15.222, 27.18%.
I n -
cr easi ng
t he
t emper at ur e of cul t ur e i ncubat i on t o 38 C, i n-
f l uenced t he composi t i on of bot h st r ai ns, i n a s i m l ar manner t o
t he l i ght i r r adi at i on exper i ment : S. maxi ma, 18.75%, 45.28%;
S.
pl at ens i s ,
13.12%, 35.32%;
f or pr ot ei n and carbohydr at es,
r espect i vel y. Bot h st r ai ns pr oduced a l ow per cent age of l i pi ds,
when gr own i n hi gh t emperat ure exper i ment s. Cel l s tur ned yel l ow
gr een i n col or . St udi es wi t h l i ght - l i m t ed cyanobact er i a
showed a hi gh l evel of pol ysacchar i de f or mat i on when t hey wer e
exposed to hi gh l i ght i nt ensi t i es ( Konopka, et . al . ,
1987).
Cohen et. al . , (1987) al so repor t ed a r educt i on i n f at cont ent
of
19
st r ai ns of Spi r ul i na by t emper at ur e and hi gh l i ght i nt ensi t y.
Nut r i ent Li m t at i on: Medi a l i m t ed i n ni t r at e- N and phosphat e- P,
f avored t he accumul at i on of carbohydr at e r at her t han pr otei n.
I n ni t r at e and phosphat e
l i m ted cul tures :
S.
maxi ma had 37.52%,
35.21% car bohydrat e and 21.562, 41.25% pr ot ei n, whi l e S. pl at ens i s
had
30.31%, 31.87%
car bohydr at e, and
32.81%, 34.683
pr otei n. When
t he cul t ur es wer e t r ansf er r ed t o medi a l i m t ed i n ni t r ogen and
phosphat e, cul t ur es changed i n col or f r om bl ue t o yel l ow- gr een.
N- l i m t ed cul t ur es of Anacyst i s ni dul ans ( Lehman and Wober,
1978),
Anabaena var i abi l i s ( Er nst and Boger,
1985)
and P- l i m t ed cul t ur es
30
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S P l R U L I N A maxima
B
C A R B O H Y D R A T E
P R O T E I N
8oo
t
w
c c
c
0 6 0 0 1
I
w 2
c c
2 400
*
X
0
200
eo
p:
4
u
n u
0
1400 c
DRY W E I GHT
HL OROP HYL L
I
d
bn 1050
E
v
*
CG
n
700
350
0
0
48
96
144 192
TIME hrs)
Figure 12a. Physiological Characteristics of
S. maxima under Optimum Growth
Conditions
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800
600
400
200
0
1600
1200
800
400
0
SPI RULI NA platensis
C A R B O H Y D R A T E
B
P R O T E I N
n
L
RY W E I GHT
A-A
C H L O R O P H Y L L
0 4 8 96
144 192
TIME (h r s )
Figure 12b. Physiological Characteristics of
S. platensis under Optimum Growth
Conditions
15
10
5
0
3 2
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Tabl e 1. Mol ecul ar Composi t i on of Spi r ul i na s t r ai ns
X Or gani c Ut. (AFDW)
Gr owt h
Condi t i ons
peci es
Pr ot ei n Car bohydr at e Li pi d
S. maxi ma *Suf f i ci ent
Nut r i ent s
69.75 11.5 4.68
Hi gh Li ght
(120
UE m-2
S-1)
Hi gh Temperat ur e
N- l i m t ed
P- I
m ed
Sodi um Chl or i de
(38C)
0.1M
0.5M
Bi car bonat e
4 g / L )
29.06 19.58 3.56
45.28
21.56
41.25
18.75
37.52
35.21
3.75
4.68
5.20
52.62
45.64
26.25
36.73
4.68
7.52
52.54 15.68
6.53
S.
pl at ensi s
*
Suf f i c i ent
Nut r i ent s
65.12
9.37 5.33
Hi gh Li ght
( 1 2 0 UE m-2
S-1
Hi gh Temper at ur e
(38C)
27.18 15.22
3.65
35.32 13.12 3.75
N- 1 m ed
32.81 30.31 5.43
P-limited 34.68 31.87
4. 68
Sodi um Chl or i de
0.1M 51.56 22.52 8.43
0.5M 37.50 32.10 10.31
Bi car bonate
(4g/L)
41.25 13.25 5.62
*Exper i ment al condi t i ons wer e:
t emper at ure 3 O o C; l i ght i r radi ance
80
uE I U - ~ s l;
ai r f l ow rate 300 m / m n;
The val ues shown are averages
of
f our i ndependent det er m nat i ons.
33
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of Osci l l at or i agar dhi i ( Ri egman et. al . , 1985 ) , showed el evated
l evel s
o f
pol ysacchari de st or age.
Depl et i on o f di ssol ved phosphat e- phosphor us f r om t he cul t ur e
medi um of Anabaena was accompani ed by a decl i ne
i n
chl or ophyl l
a , pr ot ei n, RNA and an i ncr ease i n car bohydr ate per uni t dr y
wei ght ( Heal ey, 1 9 7 3 ) .
1) I I
Sodium Chloride,
concentration influenced the storage
of
carbohydrates and proteins of both strains.
As
medi a ( Zarr ouk,
see Methods ) , wer e enr i ched wi t h
0.1 M
and 0 . 5 M NaC1, t he
carbohydrat e cont ent of t he cel l s i ncr eased, when compar ed t o
that
of
t he cont r ol ( Zar r ouk:
0.01
M NaCl ) ,
to 2 6 . 2 5 , 3 6 . 7 3
i n
S .
maxi ma and t o
2 2 . 5 2 , 3 2 . 1 0
i n S pl at ensi s. On t he
other hand t he tot al pr ot ei n decr eased r espect i vel y t o: 5 2 . 6 2
4 5 . 6 4
i n S maxi ma and
5 1 . 5 6 , 3 7 . 5 0 2 ,
i n S pl at ensi s. The
l i pi d per cent ages showed l i t t l e i ncr ease when compared t o t hose
of compl et e medi a (cont rol ) . Many cyanobact er i a ar e capabl e of
adapt i ng to a r ange of sal i ni t y i n t he envi r onment by syt hesi zi ng
i nt er nal osmot i c suppor t i n t he f or m of car bohydr ates ( Packer et .
al . 1986).
Bicarbonate: When bi car bonat e concent r at i on of Zar r ouch medi a
was reduced t o one quar t er (4. g/ L) , nei t her st r ai ns showed much
di f f erence i n t he chem cal composi t i on as compar ed wi t h the cont r ol
medi a except t hei r yi el d was s omewhat bel ow. t he cont r ol .
3 4
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w
n
H
hD
In
w
v
w
E
c
z
0
m
d
4
u
Y
m
w
d
X
u
E
n
n
H
j
0
u
dp
0
u
OQ
m
E
In
E
rl
z
E
0
d
a
E;
u
w
E
c
d
3.1
X
m
d
4
V
Q
n
CJ
z
z
W
c3
W
c3
;LHE)IBM 3 I N V E ) U O
d O
3 0 V & N 3 3 U 3 d
35
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I I
I I
0
0
W
m
W
m
z
w
t 1
p:
a
U
a
w
b
c
p:
*
x
m
p:
c
V
61
n
n
U
U
pc
I4
; LHE) I 3M 3I NVDHO o 3 3 V , L N 3 3 H 3 d
3 6
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CONCLUSIONS
I n an at t empt t o opt i m ze t he bi omass of cyanobact er i a
t he growt h par amet er s 0.f t wo st r ai ns were char act eri zed i n bat ch
cul t ur es
to gai n basi c i nf ormati on
t o
be consi dered i n a cont i nuous
cul t ur e syst em
Spi rul i na ,
Concl usi ons of
t he
f i r st year st udy ar e summar i zed as f ol l owi ng:
.
From t he envi r onment al var i abl es st udi ed, opt i mum gr owt h
t emper at ur e was cl ear l y speci es speci f i c. For S. maxi ma,
35C and f or S. pl atensi s, 3OOC.
. Opt i mum l i ght i r r adi at i on f or bot h st r ai ns was 80 uE m 2
s-1.
.
Bot h st r ai ns exhi bi t ed a t ol er ance f or a wi de r ange of pH, f r om
8 t o 11 wi t h opt i mum pH i n r ange of 9 t o
10.
.
When st udyi ng the
e f f e c t
of f l ow r ate of aer ati on and percen-
age of C02 present i n t he ai r on t he growt h r at e and yi el d
of t he al ga; i t was concl uded t hat t he
500
m / m n f or aerat i on
r at e and 1%C02 gave opt i mum condi t i ons of gr owt h.
I t was
concl uded al so that cul t ur es suppl i ed wi t h ai r
(0.03%
C02)
gave
t he same r esponsei n t erms of pr oduct i vi t y as wel l as
t hose suppl i ed wi t h 1% C02 enr i ched ai r , pr ovi di ng t hat t he
bi car bonat e concent r at i on pr esent i n t he medi um was r educed
t o 4 g/ L i nst ead of
16
g/ L ( Zarr ouk medi um .
. Var i at i ons i n suppl y of nut r i ent s: Ni t r ate- N, phosphat e- P,
sodi um chl or i de, bi car bonat e, af f ected the pr oduct i vi t y r at e
of t he al ga. They not onl y i nf l uenced t he pr oduct i on r at es,
but al so the qual i t y
of
t he produced bi omass as measur ed by
t he carbohydr at e and t ot al pr ot ei n. I n most of t he cases the
carbohydr at e cont ent i ncr eased when nut r i ent s were l i m t i ng or
i n excess as sodi um chl or i de concent r at i on of
0.1
M and
0. 5 M
( see Tabl e 1.
.
The l i pi d percent age, i n par t i cul ar, di d not show much i ncr ease
i n di f f er ent cul t ur e t r eat ment s. But , i ncreasi ng t he tern er at ur e
of cul t ur i ng to 38C or t he l i ght i r r adi ance t o 120 uE m$
s-l,
r educed
t he
t ot al l i pi ds drast i cal l y. However , i ncreas i ng
sodi um chl or i de t o
0.1
M i n t he cul t ur i ng medi a, t he l i pi ds
i ncr eased somewhat hi gher t han i n t he cont r ol medi a.
.
The abi l i t y of t he al ga to ut i l i ze macr oel ement s and m cr o-
el ement s, and t o conver t i t i nt o bi omass.
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.
Vari at i on of si ngl e envi r onment al r egul ant s such as l i ght
i nt ensi t i es or t emper at ur e, dur i ng t he pr esent st udy al so
r eveal ed t hei r det r i ment al ef f ect even when t he cul t ur es
cont ai n suf f i ci ent nut r i ent s. Cul t ur es whose gr owt h r at es
and pr oduct i vi t i es wer e r educed by any f act or , became pr o-
gr ess i vel y mor e yel l ow ( l i ght , t emper at ur e, nut r i ent l i m t -
at i on, pH) and changed i n mor phol ogy ( Fi g. 1 4 ) .
. A sl i ght i nver se r el at i onshi p was obser ved bet ween the pr ot ei n
cont ent and car bohydrat es whi ch means t hat one i ncreased at
t he expenses of t he ot her . Thi s suggest s t hat qual i t y of
bi omass may be mani pul at ed f or di et ary pur poses.
An
edequat e
suppl y of nut r i ent s i s t her ef or e a pr e- r equi s i t e f or pr oduci ng
a uni f orm qual i t y of bi omass, whi ch i n t ur n coul d t hen be used
i n t he f or mul at i on of di et s. ( see Suf f i c i ent Nut r i ent s). The
possi bi l i t y of mani pul at i ng the qual i t y of t he bi omass coul d
have pot ent i al f or t he NASA/ CELSS Pr ogr am when speci f i c di et
f or muat i on i s needed (e. g. l ow pr ot ei n cont ent ) .
. Over al l al gal pr oduct i vi t y and qual i t y coul d be mani pul at ed by
means of var yi ng nut r i ent concent r at i ons or t emper at ur e and
l i ght i r r adi ance.
Fur t her wor k i s needed t o character i ze t he ef f i ci ency of t he al gal
cel l s under such envi r onment al condi t i ons i n t erms of gas exchange
and ener gy l oss or gai n i n st eady st at e.
I t can be concl uded t hat t hr ough mani pul at i ng envi r onment al condi t i ons
o f
t he al gal gr owt h, one can modi f y t he phot osynt het i c pr oduct s. Thus,
Spi r ul i na can be, t hr ough mani pul at i ng gr owt h f act ors , used as pal at abl e
di et compar abl e t o hi gher pl ant s ( see Appendi x) .
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Figure 14a.
Cells
of
S.
maxima grown under
Optimum Conditions. Scale:
1
cm = 25 u
Figure
14b.
Cells
of S.
m a x i m a grown under
Stress Conditions. Scale:
1 cm = 40 u
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FUTURE
RESEARCH ( 1 9 8 8 / 1 9 8 9 )
We pl an to i nvest i gat e t he ef f ect s of : l i ght i r r adi at i on, t emper at ur e,
car bon di oxi de on par t i t i oni ng of macr omol ecul es ( pr ot ei n, car bohydr ates,
l i pi ds) and on el ement al composi t i on ( carbon, hydr ogen, oxygen) of
Spi r ul i na maxi ma. The mani pul at i on of composi t i on
of
Spi r ul i na maxi ma
by t he envi r onment al f act or s wi l l be i nvest i gat ed at st eady stat e.
Tasks
:
I.
Bi omass ( Dr y Wei ght and Chl orophyl l ) and Cul t i vat i on Ti me
11. Di l ut i on Rat e and Dr y Wei ght
111. Product i vi t y and Dr y Wei ght
IV. Ef f ec t of L i ght I nt ens i t y:
Thi s exper i ment wi l l be per f or med usi ng t he best t emper at ur e
and aer at i on obt ai ned f r ombat ch cul t ur i ng. The gr owt h r at e
as a f unct i on of l i ght i nt ens i t y wi l l be det er m ned at :
3 0 ,
60, 100
uE m 2
s-l
and as wel l as 35C t emper at ur es i n
r el at i on t o t he pr oduct i vi t y and ef f i c i ency o f t he al ga.
V.
Aerat i on and Car bon Di oxi de Concent r at i ons
The f ol l owi ng experi ment s wi l l be perf ormed at st eady stat e:
. Bubbl i ng wi t h ai r ( 0. 03% C02)
.
Bubbl i ng wi t h ai r
(0.1%
C02)
.
Bubbl i ng wi t h ai r
(1%
C02)
.
Bubbl i ng wi t h ai r
(5%
C02)
Exper i ment s IV and
V
wi l l be anal yzed f or t he f ol l owi ng:
. Dr y Wei ght
.
Chl or ophyl l
Pr oduct i vi t y
.
Li ght ef f eci ency
. Par t i cul at e car bon
. Par t i cul at e ni t r ogen
, I nor gani c ni t r ogen and car bon
.
Tot al phosphorus
.
Pr ot ei ns
.
Car bohydr at es
.
02 - evol ut i on
. C02 measur ed i n f l ux i n and out of cul t ur e
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EXPECTED OUTCOMES
. Rel at i onhsi p between di l ut i on r at e ( gr owt h r at e) and dr y wei ght
.
Gr owt h r at e as f unct i on of l i ght i nt ensi t y e. g. 30 60, 100 uE m2 s- l
. Pr oduct i vi t y and chem cal anal ysi s i n r el at i on to three l i ght
i nt ensi t i es wi l l be det er m ned i n or der t o det er m ne ef f i ci ency of
t he al ga
. Rel at i onshi p bet ween C02 mass f l ux i n ( mg C. dayl) and al gal car bon
mass f l ux out (mg C. dayl) at f i xed di l ut i on r ate.
. Rel ati onshi p bet wen C02 mass f l ux ( i n- out) at di f f er ent concent r at i ons
and al gal car bon f l ux out (mg C. dayl) at f i xed di l ut i on r at e
. Rel at i onshi p bet ween C02 mass f l ux i n ( mg C. dayl) and al gal carbon:
ni t r ogen mass f l ux out ( mg C. dayl)
. Ral at i onshi p bet ween C02 mass f l ux i n ( mg. C. dayl) and al gal com
posi t i on ( car bohydr at e, pr ot ei n, l i pi d)
. Rel at i onshi p bet ween 02 evol ved by t he cul t ure and C02 f l ux
. Ef f i ci ency eval uat i on of Spi r ul i na bi omass f or CELSS, i n t er ms of
l i ght , C02 and nut r i ent par amet er s.
4 1
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FERERENCES
Cohen, Z., Vonshak,
A,
Richard, A.: 1987. Fatty acids Composition
of Spirulina strains grown under various environmental conditions.
Phytochen. 26: 2255-2258.
De la Noue,
J.,
Cloutier-Plantha, L., Walsh, P., Picard, G.: 1984.
Influence of agitation
and
aeration modes on biomass production by
Oocystis. Sp. grown on wastewaters. Biomass 4: 43-58.
Ernst, A., Boger, P.: 1985. glycogen accumulation and the induction
of nitrogenase activity in the heterocystforming cyanobacterium
Anabaena variabilis.
J.
of Gen. Hicrobio. 131: 3147-3153.
Faucher, O., Coupal, B., Ledny, A.: 1979. Utlization of Seawater -
urea as a culture medium for Spirulina maxima. Can. J. Microbiol.
25: 752-759.
Fogg, G.
E.:
1956. Phytosynthesis and formation of fats in a diatom.
Ann.
Bot (Loud.) 20: 265-285.
Goldman J. C., Graham,
S.
J.: 1981. Inorganic carbon limitation and
chemical composition of tvo freshwater green microalgae. Appl. Environ.
Microbiol. 41: 60-70.
Healey, F. P.: 1973. Characteristics of phosphorus deficiency in
Anabaena. J. Phycol. 9: 383-394.
Healey, F. P.: 1975. Physiological indicators of nutrient deficiency
in
algae. Can. Fish. Xar. Serv. Res. Dev. Tech. Rep. 585: 30.
Konopka, A. Kromkamp, J., Mur, L. R.:
vesicle content and buoyancy in light or phosphate-limited cultures
of Aphanizomenon flos-aquae (Cyanophyceae).
J.
Phycol. 23: 70-78.
Lang, D. S., Brown, E. S.: 1981. Phosphorus-limited growth of a
green alga and a bluejgreen alga.
1987. Regulation of gas
Appl. Environ. Hicrobiol. 42:
1002-1009.
Le-, M., Wober,
G.:
1976. Accumulation, mobilization
and
turn-
over of glycogen in the blue-green bacterium Anacystis nidulans.
Archiv
of
Microbiol. 111: 93-97.
IkcElroy, B. D., Bredt, J.: 1985. Current concepts
and
future
directions of CELSS, pp. 1-9, In: Controlled Ecological Life
Support System, NASA CP-2378,
Xgp
COSPAR meeting, Graz, Austria, July
1984 (MacElroy, B. C., Smeroff, D. T., Klein, H. P., eds.).
42
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Packer, L.,
F r y , I.,
Belkin, S.: 1986. Application of photosynthetic
N2-fixing cyanobacteria to the CELSS program p.
339-352, In:
Controlled
Ecological Life Support Syste: CELSS 1985. Workshop NASA TM 88215.
Riegman, R., Rutgers,
M.,
Mur,
L.
R.: 1985. Effects
of
photoperiodicity
and light irradiance
on
phosphate-limited Oscillatoria agardhii in
chemostat cultures.
I.
Photosynthesis and carbohydrrate storage.
Archiv. of Microbiol. 142: 66-71.
Strickland, J. D., Parsons, T. R.: 1972, A practical handbook of
seawater analysis. Bull. Fish. Res, Bd. CAn. 167.
Zarrouk,
C.
1966. Contribution a 1 etude d'une cyanophycee. In-
fluence de divers facteurs physiques sur la crossance et la
photosynthesis de Spirulina
m a x i m a .
(France).
Thesis, University of Paris
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A P P E N D I X
4 4
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ORfG1FUAL PAGE
tS
OF
POUR QUALtTY
Three billion Year old exoert
grants
immunity
"I
am the
immortal
descendant of the original
life
form.
Over 3 billion years
ago. blue-green algae produced our
Dxygen atmosphere
so
life could
:valve. Today,
I
offer your life
imely health benefits.
Your own technology and life-
style threaten you. Pollution. toxic
:hemicals. radiation. disease , stress.
irugs and processed foods anack
your immune system. Damage to
your body and its cells can cause
premature aging and cancer.
Natural Life Preserver
Vitamin supplements help
out.
but with two limitations. First.
synthetic mega-doses are wasted if
your b o d y can't absorb them and may
even be toxic. But my whole food
spirulina vitamins and minerals arc
casily absorbed.
Second, because your bod is not
simply a mechanism, it needs
more
than isolated nuuicnts. Living cells
need specific information to remem-
ber their function.
DNA
molecular
codes
i n
natural foods contain genet-
ic memories of successful life forms
for millions of years. I have reju-
venated myself since the beginning.
My
3
billion years
of
cell memories
can help your body remember iu
powers and renew itself. When
y ou
need a natural life preserver. put mc
IOwork inside your body.
Unusually Concentrated
Within me is a powerful concen.
tmtion of nutrients, unlike any
other single plant. p i n . food or
herb. 1 flourished in the nutrients of
the original primordial soup. Many
of these nutrients arc strongly
recommended by scientists to build
the immune system. New medical
research has focused on
my
effects
0
choles.ternl reduction. cancer and
immune system reaponse.
Protective Nutrients
I
have more
beta
carotene than
chlorella algae and ten times more
than any other food. Beta carotene is
known
to
lower the incidence of
cancer and protect against UV radia-
tion.
l
am the only food that con-
tains significant amounts of gamma
linolenic acid (GLA). the beneficial
numcnt in evening primrose oil. 1
am the best source of vitamin B-12.
Extra iron and trace minerals added
to my ponds make Eanhrise Spiru-
lina the best iron food.
My living cells are dehydrated in
seconds by low temperature spray
drying, best preserving valuable
nutrients. Then I'm freshly scaled in
anti-oxidant containers.
Eanhrise
is
the only company that can guarantee
quality from my living ponds to my
consumers.
sometimes deficient in vegetarian
diets. I am rich in iron, essential for
healthy red blood cells and a strong
immune system.
I
have the highest protein content
(60-70'70). ll the essential amino
acids. and RNA and DNA nucleic
acids I'm
1
cleansing chlorophyll ~ ~ b , , h ~
and 15 sfrenethenine Dhvcocvanin.
a unique blue &ynen;fouAd only in
blue-green algae.
Clean
Green nergy
Enjoy my energy every day- p a t
between meals and before swenuous
aturattv Diaestible
1
am so old ;hat IFvolved betore
hard cellulose cell walls. My cell
walls are naturally soft proteins.
My younger cousin. chlorella.
requirch additional factory procehh-
ing
to
break down its hard cell
walls. But I'm already perfect
-
95%
digestible. The most digestible food.
Many people say the feel my
energy within minutes.
activity for quick nutrition without
feeling too full.
I'm
also helpful
for dieting and cleansing your body.
Once known as
il
/ nod o/ [he
/rtrurc.
I
ani already growing in
villages in the developing world.
I'm
called 'green medicine' food by
the children.
So. panake of my immortal body
each dav. Eat 3 billion
wars
of cell
memory' and a conccntrihon of pro-
tective nutrients. Reneu your own
health. reneu your connection with
your SISICN and brothers in the third
world. and with the ongins of life."
1
S p i ~ l i ~
t healtn tood stores
1
I
1 m m 1 m 1 1 1 1
You
can
iind
best
selling Earthw e
I or order 11direct
from
Earthrise. I
Earthrise Quality Standard I Please send
me
f r ee literature on: I
introduced as a health food supple- I Name
I
ment in
1979
by Earthrise. Now I'm
enjoyed by millions world wide-
athletes. vegetarians, dieters, health
praciitioners. and people of all ages.
Earthrise i s one o my most ad-
vanced farms. Here. I'm grown free
of
pcsticidcs. hcrhicides. additives
and prcacrvalivca. and 100% purc.
L L ~ J
an Ralaei CA 94915
Earthrise Spirulina - nature's most protective
food
