Biomass 9 (1986) 37-48

Optimal Growth Conditions for lsochrysis galbana

Dr o r a Ka p l a n , Zvi Co h e n a n d Ah a r o n Ab e l i o v i c h
Laboratory for Environmental Applied Microbiology,
The Jacob Blaustein Desert Research Institute, Ben Gurion University,
Sede Boqer Campus 84990, Israel
(Received: 12 July, 1985)
A BSTRACT
Environmental and nutritional growth conditions of the unicellular
microalga Isochrysis galbana were studied under laboratory con-
ditions. The information obtained was used f or cultivating the alga in
outdoor miniponds. Outdoor cultures stayed monoalgal and free of
predators as long as the temperature did not fall below 19C and the
rate of dilution did not exceed 40 % of the culture's volume. Isochrysis
galbana grown in outdoor cultures provided lipid concentrations of
24-28 % of ash free dry matter.
Key words: Isochrysis galbana, optimal growth conditions, outdoor
cultivation, lipid accumulation.
I NTRODUCTI ON
Isochrysi s gal bana is a free living mar i ne uni cel l ul ar phyt ofl agel l at e of
t he or der Chrysomonadal es. Li ke ot her member s of this or der it is rich
in pol yunsat ur at ed fatty acids ~ whi ch are of nut ri t i onal val ue for mar i ne
fish l arvae 2 and j uveni l e stages of mol l us cs ) Li mi t ed exper i ence of grow-
ing this mi croal ga for feedi ng oyst er l arvae pr oved very successful. 4.5
The pur pose of t he wor k descr i bed her e was to defi ne opt i mal gr owt h
condi t i ons for out door cul t ure. The effects of vari ous envi r onment al and
nut ri t i onal par amet er s on t he growt h rat e of t he alga and its resi st ance to
pr edat or s wer e st udi ed.
37
Biomass 0144-4565/86/S03.50 - Elsevier Applied Science Publishers Ltd, England,
1986. Printed in Great Britain
38 D. Kaplan, Z. Cohen, A. Abeliovich
MATERI ALS AND METHODS
Isochrysis galbana (originally from the Hawaii Institute of Marine
Biology) was kindly provided by Dr Hillel Gordin (Limnological
Oceanographic Research Laboratory, Eilath) and used throughout the
study. Axenic cultures were used throughout all laboratory studies, and
monoalgal cultures in field studies.
Growth conditions
The following artificial sea water medium (ASW) was used (in g litre- ~):
NaCI 32.0; MgC12 7H20 6.7; MgSO46H20 5.5; CaCI 2 1.1; KNO 3 1.0;
KHzPO 4 0.07; NaHCO3 0.04; trace elements and vitamins (in/xg litre- 1):
ZnC12 4; (NH4)6MoyO244H20 36"8; CuC122H20 3.4; MnCIz4HzO 40;
H3BO3 60; FeCI36H20 240; EDTA (di-Na salt) 100; thiamine HCI 400:
Biotin 50; and B12 10. Unless otherwise stated, all growth experiments
were performed in batch cultures at 27C, with constant illumination of
150/ rE m- : s-~ at the surface of the flasks, provided by fluorescent
lamps (cool white) on a rotary shaker (180 rev mi n- 1), 100 ml in 250 ml
Erl enmeyer flasks. Larger volumes of algae were grown in 10 litre flasks
sparged with CO2 (l%)-enriched air maintaining a pH of 7.0-7"5 under
continuous light of 200/ ~E m- 2 s - 1 at the surface of the flasks. Effect of
temperature on growth was studied in an aluminium temperature block
maintaining a temperature gradient between 13-42C with 1.5C incre-
ments between adjacent test tubes containing the algal cultures. Light
was provided by fluorescent lamps (cool white) placed under the block,
providing light intensity of 11/~E m -2 s - l , at the bottom surface of the
tubes.
Outdoor growth experiments
Miniponds (1 m 2, 150 litres) were used for outdoor studies of algal
growth kinetics using the same growth medium. The cultures were
stirred by a paddle wheel so as to maintain a homogeneous suspension.
The pH was maintained at 7.2-7.9 by sparging pure CO z gas through the
culture.
Analytical procedures
Algal growth was followed by measuring optical density at 540 nm, cell
counts in a haemocytometer, chlorophyll a concentration 6 and AFDM
(ash free dry matter). Lipids were extracted using a modified Bligh-Dyer
procedure: v freeze dried samples were extracted with 3 volumes of
chl oroform: met hanol :water ( 10: 5: 4 v/v) solution at room temperature
and centrifuged. Water and chloroform were added to the combined
Optimal growth conditions for Isochrysis galbana 3 9
super nat ant s so as t o f or m a c hl or of or m: me t ha nol : wa t e r mi xt ure of
10: 10: 9. Af t er ext ract i on t he chl or of or m phase was separ at ed and
evapor at ed to dr yness under N 2. Pr ot ei n was det er mi ned accor di ng to
Lowr y et aU
Quantitative determination of algal contaminants
Isochrysis, as do ot her chrysophyt es, cont ai ns fucoxant hi n as a maj or
xant hophyl l . Aci di fi cat i on of a fucoxant hi n ext ract f ol l owed by heating,
causes i somer i zat i on of t he 5, 6- epoxi de to t he 5, 8-epoxi de whi ch has a
st rong abs or bance in t he red, far beyond 700 nm. 9 The absor pt i on
spect r um of a met hanol i c ext ract of Isochrysis galbana pi gment s bef or e
and aft er aci di fi cat i on and heat i ng is shown in Fig. 1. Aci di fi cat i on and
heat i ng pr oduces a shift of t he Soret band and a shoul der at
7 0 0 - 7 5 0 nm, whi ch makes t he met hanol i c ext ract appear blue. In addi -
tion, a shift in chl or ophyl l a absor pt i on peak occurs f r om 670 to 660 nm.
Similar~ t r eat ment s of met hanol i c extracts of possi bl e cont ami nant s
such as Monolanthus sp. Chlorella sp., Scenedesus obliquus, and
Chroococcus sp. di d not pr oduce this effect. Thi s phe nome non enabl ed
us to use a si mpl e ' puri t y i ndex' for est i mat i ng cont ami nant s' concent r a-
tions in out door ponds, based on t he di fferences in xant hophyl l com-
posi t i on among algae. For pur e cul t ures of Isochrysis galbana
mg pr ot ei n
A71 o 100
= 1 " 4 0 " 3
Fig. l .
3.50
I i I [ I I 1 [ I
3 1 5
2 8 O
2 4 5
~ 175
~ 140
~ 105
07O
O35
O0
3 ~0 3 90 4 3 0 4 ~0 5 ~0 5 5 0 5 90 63 0 67o zto ~5 0
WBVELENGTH (nm)
Absorption spectrum of a methanolic extract from lsochrysis galbana. (A)
control, (B) after acidification and heating.
and t her ef or e
x - 1.4
1.4
T
o~
. J
I , I
. )
- - = % cont ami nat i on of cul t ure,
wher e x is t he abs or bance at 710 nm of t he t r eat ed met hanol i c ext ract of
an out door cul t ure. Obvi ousl y, this met hod cannot be appl i ed when t he
cont ami nant s, such as di at oms, cont ai n fucoxant hi n.
RESULTS AND DI SCUSSI ON
Laboratory studies
Temperature effects
The opt i mal t emper at ur e for achi evi ng highest algal yield was 27C (Fig.
2), whi l e t emper at ur es hi gher t han 32C or l ower t han 19C r educed
algal yield markedl y. Effect of t emper at ur e on growt h rat e was det er-
I O 6
1 0 5
40 D. Kaplan, Z. Cohen, A. Abeliovich
1 0 4 L ] k I L I ~ I I I I I
1 6 2 0 2 4 2 8 3 2 3 6
TEMPERATURE (C)
Fi g. 2 . Ef f ect of temper atur e on yi el d of lsochrysis galbana. Tubes contai ni ng 1 0 ml of
medi um were i nocul ated wi th algae ( 5 x 1 0 4 cells m] -~) and pl aced i n temper atur e
gradi ent bl ock . Cel l s were counted af ter al l owi ng gr owth f or ] 0 days.
Optimal growth conditions for Isochrysi s gal bana 41
mined for several selected temperatures (Fig. 3), and the temperature for
maximal growth rate was also 27C. However, it should be noted that the
low light intensity available for the alga under the experimental condi-
tions present in the temperature block accounts for the slow growth rates
measured (38 h doubling time at 27C).
Effect of pH
Within the range of 5.0-9.0 the pH in itself had no significant effect on
yield and growth rate of the alga, provided that there was sufficient con-
centration of Fe 3+ available in the growth medium (Fig. 4). There was a
marked pH effect on algal yield whenever supply of Fe 3 + was limited, so
that no growth occurred at >i pH 9.1. This effect of pH is enhanced by
the presence of carbonates in the medium (Fig. 5).
Effect of light
The effects of increasing light intensity from 50 to 150 ktE m - 2 s - 1 are
presented in Table 1. Growth rate was doubled by increasing light
intensity 3.
Effect of salinity
Isochrysis galbana exhibited resistance to a wide range of NaCI concen-
trations. Onl y a slight reduction in growth rates was observed upon dilut-
T
O9
__1
__.1
t.~ o
1 05 [ L i , I ~ 1 , I , I
0 2 4 6 8 L O
T I M E ( d a y s )
Fig. 3. Effect of t emperat ure on growt h rate of lsochrysis galbana. Experi ment al con-
ditions as descri bed in Fig. 2, except that cell density was moni t or ed daily. O, 15C and
35C; e, 17*C; o, 22C; x , 27C; , 33C.
42 D. Kaplan, Z. Cohen, A. Abeliovich
1
7 I0;'
q_)
l0 ~
, % ' ~ ' ~ , ~, ~ ' , ~' , ~' ~, , ~, ,~
T I M E (doys)
Fi g. 4. Effect of pH on growt h of lso-
chrys& galbana. The pH of the growt h
medi um was moni t ored and adjusted
daily. At the times indicated by arrows
1' 2 mg litre -~ of FeC13.6H20 was added to
the medi um, e, pH 5.7; &, pH 6.8; m, pH
8"2; V, pH 9"1.
7
O3
.J
W
~D
107
106
1 0 5 I I I I I I
4 5 6 7 8 9 I 0
pH
Fi g. 5. Effect of pH and addition of
car bonat es on algal yield in Fe 3 limited
culture. See Materials and Met hods for
experimental conditions. The pH was
moni t ored and adjusted daily. Yield is
present ed after 10 days of growth. (e),
wi t hout NaHCO3; (0), with 42 mg litre -~
NaHCO3, in the growt h medi um.
TABLE 1
Effect of Light Intensity on Gr owt h u of lsochrysis galbana
Light intensity Time (days)
( pE m -2 s -l)
0 4 5 7
50 3"0 105 2"0 106 3-7 106 6"1 x 106
150 3-0 105 3' 0 106 5-0 106 1' 2 x 107
aGrowt h is present ed as cells ml-L For growt h condi t i ons see Materials and Met hods.
Optimal growth conditions for I s o c hr ys i s gal bana 43
10 7
"7
10 6
/
tad
t.)
I0 i [ i I t I J I ~ I
0 2 4 6 8 I 0
T i me (dQys)
F i g . 6 . Ef f e c t o f c hang i ng N a Cl c o n c e n -
t rat i on i n g r o wt h me d i u m o n g r o wt h rat e
o f I sochrysis galbana. S e e M at e r i al s and
M e t h o d s f or g r o wt h c o ndi t i o ns . AS W
me d i u m c o nt a i ne d 5 ( t ) , 30 ( o) and 60
( x ) g l i t re -l NaC1.
iO 7
T
10 6
_A
LO
t . . )
1 I
' 0 5 0 i
TI ME (doys)
F i g . 7 . Ef f e c t o f agi t at i on
I I
5 6
on g r owt h
rate. Cul t ur e s we r e g r o wn s t at i onary, e ; or
o n rot ary s hak e r A, 150 re v rain -~ at
i de nt i c al l i ght r e g i me ( s e e M e t ho ds ) .
i ng t he growt h me di u m to 1 5 % sea water, whi l e i nc re as i ng sal i ni ty to
t wi c e that of sea wat e r had no ef f ect ( Fi g. 6 ) .
Effect of aeration
Agi t at i on of t he cul t ures had a st rong ef f ect bot h on growt h rate and on
al gal yi el d, as s e e n i n Fi g. 7 . S pargi ng of cul t ures wi t h CO2( 1 %) e nri c he d
air was e v e n mor e e f f e c t i ve i n i nc re as i ng growt h rate and yi el d, as l ong as
t he c ul t ure me d i u m was s u ppl e me nt e d wi t h e x c e s s Fe 3+ ( Fig. 8) , ot her-
wi se, Fe 3 st arvat i on de v e l ope d rapi dl y. Appare nt l y e x c e s s CO2 c aus e s
pre c i pi t at i on of Fe 3 as i ns ol ubl e carbonat es. Addi t i on of saturati ng c on-
c e nt rat i ons of Fe 3+ c he l at e s rest ores hi gh growt h rates. Thi s phe no-
me n o n mi ght al so e x pl ai n t he de c re as e growt h rates at hi gh pH s whe n
hi gh N a H CO 3 c onc e nt rat i ons are pre s e nt i n t he growt h me di um, s hown
i n Fi g. 5 .
44 D. Kaplan, Z. Cohen, A. Abeliovich
dO ~
iO 7
l0 6
1 2 3 4 5 678 91 0 [ 2
T I M E ( d a y s )
Fig. 8. Effects of aeration with and
without CO 2 and Fe 3+ on growth rate of
lsochrysis galbana. Algae were grown in
10 litre flasks with (closed symbols) and
without (open symbols) Fe 3+ supplement.
Cultures were sparged with air (A, ) or
with air with 1% CO 2 (e, o).
bO 7
T
l0 6
IO 15
TI ME ( days)
Fig. 9. The effect of initial concentra-
tions of KNO 3 o n growth rate and yield of
Isochrysis galbana. Cultures were grown
in 10 litre flasks on improved ASW
medium (x5 [Fe3+]) and were sparged
with air enriched with 1% CO2. e, 1000
mg litre-]; o, 200 mg litre -~ KNO 3.
Nitrate vs ammoni a as nitrogen source
Th e ef f ect s of i ncr easi ng initial c onc e nt r a t i ons of KNO 3 in t he gr owt h
me d i u m on gr owt h r at e and yi el d of Isochrysis galbana ar e pr e s e nt e d in
Fi g. 9. Ini t i al gr owt h r at es we r e onl y sl i ght l y af f ect ed by t he di f f er ence in
NO~ c onc e nt r a t i ons , but t ot al yi el d at t he e nd of l ogar i t hmi c pha s e of
gr owt h s hows di f f er ences of a b o u t an o r d e r of magni t ude. NH 3 can
r epl ace NO3 as ni t r ogen s our ce, but speci al at t ent i on mus t b e gi ven t o
t he r el at i on b e t we e n p H and c onc e nt r a t i on of NH 3 s uppl i ed, as t he c om-
bi nat i on of hi gh p H a nd hi gh NH 3 c onc e nt r a t i on was f o u n d t o be t oxi c t o
Isochrysis galbana (Fig. 10), as al r eady de s c r i be d in pr evi ous l y r e p o r t e d
cases, l Gr o wt h r at es on NH 3 we r e sl i ght l y l owe r t han t he c or r e s pondi ng
f i gur es f or NO~.
Effects of addi t i on of mi croel ement s on growth
Of all mi c r oe l e me nt s t es t ed ( Mn 2 - ; Z n 2 + ; Mo 6 + ; Cu 2 + ; B 3 + and F e 3 ) by
i ncr easi ng t hei r c onc e nt r a t i on up t o 5 t i mes t hei r c onc e nt r a t i on in t he
Optimal growth conditions for I sochrysis galbana 45
pH
7
!
/
I ~ ' ' ~ ' ' ' ' , ' o
N H4 CL (uM }
Fig. 1 0 . Growth of I sochrysis galbana
at various combinations of pH and NH4CI
concentrations. Algae were grown in
Ehrlenmeyer flasks as described in
Material and Methods. The numbers rep-
resent concentration of cells ml -t x 1 0 6 at
the 1 0 th day of growth. S haded area indi-
cates optimal growth conditions with
respect to pH and N H 3 concentrations.
"6
E
6.
5 0-
4 0-
3 0-
2C-
~0
2 4
i I I l ~ J I I I i ~ J I
6 8 I 0 1 2 1 4 t 6
TI ME ( d a y s )
~q
0,4 N ~
~ r
'0.2 ~
g
0 ~
Fig. I t . Effect of nitrogen starvation on lipid, protein and chlorophyll a content.
Cultures were grown in 1 0 litre flasks on AS W medium containing 2 0 0 mg litre -~ KNO3.
Growth ceased at day 5 because of N depletion, o, lipids; o, proteins; i , chlorophyll a.
original growth medium, the algae responded only to the addition of
Fe 3 + ( see above) . All the rest were ineffective in enhancing growth rate.
Effects of nitrogen starvation
According to Fogg, H nitrogen starvation affects not only algal yields, but
also shifts anabolic pathways from protein synthesis to carbohydrate or
lipid accumulation. When I sochrysis galbana was subjected to conditions
of nitrogen starvation, concentration of lipids increased from 24% to
47 % of total AF DM . Thi s increase in lipid concentration was coupled to
a decrease in both chlorophyll a and total protein content. Carbohydrate
concentration did not change significantly ( Fig. 1 1 ).
46 D. Kaplan, Z. Cohen, A. Abeliovich
Ou t do o r c ul t ur e o f I sochrysis galbana
Growth conditions
Out door growth experiments in miniponds were started by inoculating
them with 10% of their volume by indoor cultures. Out door light inten-
sities reached 2500/ ~E m -2 s -1 at noon throughout the summer. To
protect the low-light-adapted cultures the ponds were covered by
screens cutting 50% of the light intensity on the surface of the ponds.
The screens were gradually removed as the culture density increased.
After initial adaptation new ponds could be started without using any
protective means against photo-oxidative damage. The ponds were
operated as semicontinuous reactors: when cultures reached the
stationary phase of growth they were diluted by replacing up to 40% of
the pond' s volume with fresh medium. Doubling time of Isochrysis
galbana in outdoor cultures was 6 days, and maximal cell density was
5 10 7 cells ml-~, which was the highest density obtained under any
experimental conditions we tried, suggesting that light was the limiting
factor preventing higher growth rates and higher population densities in
the laboratory.
Cultures were maintained essentially monoalgal for over one month,
providing yields of 11-12 g m- 2da y -1 as determined spectrophoto-
metrically (see Methods), and by microscopic observation.
Effect of nitrogen starvation on outdoor cultures
Algal lipid content in outdoor cultures was 22- 25% of AFDM, similar
to what was found for cultures grown indoors. However, nitrogen deple-
tion had only a marginal effect on lipid accumulation by outdoor cultures
when maintained in nitrogen-free medium.
After a period of about 2 weeks without the addition of NO3 nitrogen
was exhausted from the medium, causing cessation of growth and reduc-
tion in chlorophyll a content, but no significant increase in lipid content.
Lipid content of outdoor cultures, after 30 days of nitrogen starvation
was 27- 28% of AFDM. The discrepancy between indoor and outdoor
cultures in respect to nitrogen starvation might possibly be a result of
conditions suitable for the development of a nitrogen fixing microbial
population.
Predators
Out door cultures of Isochrysis galbana were found to be sensitive to
various predators. Among these, ciliates such as Paramecium sp.,
Euplotes sp., as well as various rotifers were identified. Infections
developed primarily whenever growth conditions deteriorated; in
Optimal growth conditions for Isochrysis galbana 47
part i cul ar high light i nt ensi t y shocks or pr ol onged low ni ght t emper a-
tures ( < 18-19C) afftected t he cultures adversely. It shoul d be poi nt ed
out her e t hat Thomas et al. 12 wer e also careful t o mai nt ai n cultures of
Isochrysis at > 22C.
CONCLUSI ON
The series of exper i ment s descr i bed her e wer e ai med at defi ni ng opt i mal
condi t i ons for out door mass cul t ure of Isochrysis galbana. The organi sm
was f ound sensitive to Fe 3+ starvation and t emper at ur es 4 19C. Low
light adapt ed cultures wer e sensitive to phot odynami c damage. Ther e-
fore, if gr owt h condi t i ons are pr oper l y desi gned and t hese precaut i ons
are t aken into consi derat i on, t he alga can be grown in out door ponds at
good yields with high lipid concent r at i ons ( 24- 28% of AFDM) wi t hout
any part i cul ar difficulty.
ACKNOWLEDGEMENTS
Thi s wor k was suppor t ed by Grant No. XK-4-0410-1 f r om SERI.
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