High value pigment production from Arthrospira (Spirulina) platensis cultured

in seawater
J.T. Mary Leema
a,
*
, R. Kirubagaran
b
, N.V. Vinithkumar
a
, P.S. Dheenan
a
, S. Karthikayulu
b
a
Andaman Nicobar Centre for Ocean Science and Technology, NIOT R&D Complex, Dollygunj, Port Blair 744103, A&N Islands, India
b
National Institute of Ocean Technology, Pallikaranai, Chennai 600100, Tamil Nadu, India
a r t i c l e i n f o
Article history:
Received 22 January 2010
Received in revised form 23 June 2010
Accepted 26 June 2010
Available online 23 July 2010
Keywords:
Arthrospira platensis
Seawater
Phycocyanin
Lutein
Betacarotene
a b s t r a c t
The prospects of utilizing pretreated seawater for the culture of Arthrospira (Spirulina) platensis was eval-
uated under laboratory conditions with three seawater media and a control: (1) Zarrouk media (freshwa-
tercontrol) (2) seawater media SW 1 (3) seawater media SW2 and (4) seawater media SW 3. The relative
performance of these media were investigated with respect to their biomass production, pigment pro-
duction (phycocyanin, lutein and betacarotene), and biochemical composition. A. platensis grown in
media SW 2 had a biomass production (2.99 0.145 g L
1
) comparable to that of control media
(3.114 0.085 g L
1
); highest specic growth rate (0.255 d
1
) and lowest doubling time (2.720 days).
Phycocyanin content of the cells grown in seawater media SW 3(81.85%) was closer to that of control.
Similarly the purity ratio of phycocyanin produced from cells grown in seawater media SW 3 and control
were closer to 4, while the phycocyanin obtained from cells grown in other two media exhibited lower
purity ratios due to accumulation of lower molecular weight carbohydrates. The phycocyanin/Chl-a ratio
and the betacarotene/Chl-a ratio of the cells grown in seawater media were higher than control. The
lutein content of A. platensis cells grown in seawater media SW 2 was higher than that of control. The cells
grown in seawater media had a slightly modied biochemical composition than the control with a higher
carbohydrate and lower protein content. All the three seawater based media with fewer chemicals than
the control (Zarrouk media) supported the growth of A. platensis as good as the control.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
The cyanobacterium Arthrospira (Spirulina) platensis has gained
considerable attention worldwide as a source of several nutraceu-
ticals (Belay et al., 1993). Among the nutraceuticals, pigments have
attracted greater commercial interest (approximate price of food
grade phycocyanin is Australian $ 500/kg, Borowitzka, 1992)
due to their high end applications and relatively easy extraction
procedures. Pigments of microalgal origin which are currently
enjoying high market demand are the phycobiliproteins, b carotene
and lutein. b carotene produced from microlagae costs US$ 1000/
kg against US$ 500/kg for their synthetic equivalents but natural
b carotene is preferred in health market because it is a mixture
of cis and trans isomers with anticancer property whereas cis iso-
mer rarely gets expressed in synthetic ones (Downham and Collins,
2000). Phycobiliproteins are unique not only due to their exclusive
presence in cyanobacteria but also due to their wide range of com-
mercial applications (Moreno et al., 1995). Due to their distinctive
spectroscopic properties and non toxic nature they have found
additional applications in biomedical research, food, drug and cos-
metic industry replacing the toxic synthetic pigments (Cohen,
1986; Glazer, 1994). Phycocyanin contributes nearly 30% of the
biomass (Garnier and Thomas, 1993). However, culture conditions
determine the actual content of these pigments in cell (Mrquez-
Rocha et al., 1995).
At present Spirulina is cultured mainly to quench the health
food market utilizing a chemically dened medium (Belay and
Ota, 1994). Signicant share of the production cost is contributed
by these chemicals. Hence, the use of low cost media like seawater
media will bring down the production cost of commercial Spirulina
culture and make it more competitive for the production of value
added products. Development of seawater culture of Spirulina is
inevitable for propagating outdoor cultivation of these cyanobacte-
ria in many tropical arid areas, where climatic conditions are
favourable but freshwater is scarce (Materassi et al., 1984). Few re-
search works on the use of seawater as an alternative medium,
after pretreatment (Faucher et al., 1979) or after low level supple-
mentation with specic nutrients under laboratory conditions
(Materassi et al., 1984) or in outdoor raceways (Tredici et al.,
1986; Wu et al., 1993), have been reported. However, several phys-
iological aspects related to acclimatization, stress tolerance, pig-
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.06.120
* Corresponding author. Tel.: +91 9434266398; fax: +91 3192225089.
E-mail address: leemaroy2002@yahoo.com (J.T. Mary Leema).
Bioresource Technology 101 (2010) 92219227
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
ment production and growth in seawater remains unknown
(Materassi et al., 1984). The use of a suitable seawater media is
an essential prerequisite for developing mass production systems
utilizing seawater. Mass production of A. platensis in seawater re-
quires sorting out several physiological problems confronted in
culturing these algae in seawater. Hence the present study aims
to assess the suitability of three different compositions of seawater
media for the growth and pigment production from A. platensis.
2. Methods
2.1. Microorganism and growth conditions
A. platensis strain used in this study was obtained from the cul-
ture collection of Centre for Advanced studies in Botany, University
of Madras, Chennai, India. They were maintained in controlled con-
ditions in Zarrouk medium (Zarrouk, 1966) with light intensity of
140 lmol photon m
2
s
1
and temperature of 26 1 C. The strain
was acclimatized to grow in seawater for three generations prior to
its use in these experiments. Experiments were carried out in
500 mL Erlenmeyer asks with 250 mL of medium at 26 1 C un-
der 14:10 light/dark regime and continuous shaking (120 rpm) in a
temperature controlled orbital shaker (Orbitek LT, Scigenics, Chen-
nai, India) with light (light intensity 140 lmol photon m
2
s
1
).
Control was cultured with 250 ml of sterilized (121 C for
20 min) Zarrouk medium (with the following modication the
concentration of NaNO
3
was 3.0 g L
1
) in 500 mL Erlenmeyer
asks. Sea water was obtained from Aberdeen Bay, Andaman, India
(salinity 34.23 , pH 8.01). The seawater was pretreated with
NaHCO
3
in order to precipitate the excess divalent cations Ca
2+
and Mg
2+
according to the procedure described by Faucher et al.
(1979). The pretreated seawater was ltered through 0.22 lm cel-
lulose acetate lter (Millipore). Three different media prepared
with different ratios of seawater were tested. Details of the compo-
sition are furnished in Table 1. In the case of media SW1 (undiluted
seawater) the nutrients were separately autoclaved (121 C for
20 min) and added aseptically to pretreated, preltered, undiluted
sea water. The freshwater of media SW 2 (2 part seawater:1 part
freshwater, v/v) and SW3 (2 part freshwater:1 part seawater, v/v)
were autoclaved (121 C for 20 min) along with the nutrients, al-
lowed to cool and then aseptically added to pretreated, preltered,
sea water. The cultures were inoculated with 10% (v/v; average cell
concentration of 0.25 g L
1
dry weight) of exponentially growing
inoculums under aseptic condition.
2.2. Kinetic parameters
Samples were collected on alternate days and growth was mon-
itored turbidometrically (Leduy and Therien, 1977) by measuring
the optical density (O.D) at 560 nm with Unicam UV300 spectro-
photometer (Unicam, USA). The samples withdrawn were compen-
sated by the addition of equivalent quantity of fresh media. The
cell dry weight concentration was determined by drying the cells
at 80 C in an oven until constant weight. From the O.D. values dai-
ly biomass concentration was derived by using previously pre-
pared standard calibration curves for optical density against
biomass for each treatment. At the end of 25 days the maximum
biomass concentration designated as X
max
(g L
1
) was recorded
(Schmidell et al., 2001). Biomass (X) values and exponential regres-
sion were used to calculate the maximum specic growth rate
(l
max
, d
1
) during the logarithmic phase (Bailey and Ollis, 1986).
The doubling time (t
d
, days) was calculated as t
d
= ln 2 (l
max
)
1
.
2.3. Analytical methods
The protein content of the lyophilized biomass was determined
according to Lowry et al. (1951) with BSA as standard. Carbohy-
drates were determined following the colorimetric phenol method
of Dubois et al.(1956) using glucose as standard. Lipids were
extracted from the lyophilized samples of biomass and determined
according to the method of Bligh and Dyer, 1959.
2.4. Pigment analysis
2.4.1. Extraction, quantication and purication of phycocyanin
For observing the time course of phycocyanin accumulation
phycocyanin was extracted from 5 mL algal samples once in 5 days
and quantied according to the method reported by Boussiba and
Richmond, 1979. For determination of nal phycocyanin content
(mg/g) and further purication the cells were harvested on day
25 from different groups by centrifuging at 10,000g (Sigma cooling
centrifuge) for 30 min at 4 C. The harvested cells were washed
twice with distilled water and freeze dried (Lyodel). Phycocyanin
was extracted from 250 mg freeze dried cells suspended sus-
pended in 25 mL of sodium phosphate buffer (0.1 M, pH 7) and
quantied according to the method reported by Boussiba and Rich-
mond (1979). For further purication crude phycocyanin obtained
fromall the four groups were fractionated by precipitation with so-
lid ammoniumsulphate rst at 30% and then at 50% saturation. The
precipitate from 30% ammonium sulphate saturation was dis-
carded and the supernatant was brought to 50% ammonium sul-
phate saturation and allowed to stand for four hours at 4 C.
Then it was recovered by centrifugation at 10,000g for 10 min.
The colourless, clear supernatant was discarded and the blue pre-
cipitate was reconstituted in a small volume of 0.0025 M Na-phos-
phate buffer (pH 7.0) and dialyzed against the same buffer
overnight at 4 C. The crude phycocyanin fractions obtained were
chromatographed on a DEAE Sepharose CL 6B column
(1.5 15 cm) in AKTA PURIFIER column chromatograph. The col-
umn was developed with linear increasing ionic concentration gra-
dient of NaCl solution (00.5 M) at a ow rate of 1 mL/min. 2 mL
fractions were collected. The purity of the collected fractions were
evaluated according to the absorbance ratio (A
620
/A
280
for c-
phycocyanin and A
655
/A
280
for allophycocyanin (Boussiba and
Richmond, 1979). Visible and UV spectra of phycocyanin were
measured in an UVVIS Unicam UV300 spectrophotometer.
2.5. Chlorophyll-a estimation
Chlorophyll-a (Chl-a) was determined by the method of Bennet
and Bogorad (1973). Final Chl-a content (mg g
1
) was determined
Table 1
Element composition of the culture media used for the seawater culture of Arthrospira
platensis.
Component Control
*
(g L
1
) SW1 (g L
1
) SW 2 (g L
1
) SW 3 (g L
1
)
NaHCO
3
16.8 19.5 19.5 18.8
K
2
HPO
4
0.5 0.5 0.5 0.5
NaNO
3
3.0 3.0 3.0 3.0
K
2
SO
4
1.0
NaCl 1.0
MgSO
4
7H
2
O 0.2
CaCl
2
2H
2
0 0.02
FeSO
4
7H
2
O 0.01 0.01 0.01 0.01
Na
2
EDTA 0.08
Fe
2
EDTA 0.005 0.005 0.005
A
5
1 mL
1
B
6
1 mL
1
pH 9.26 9.12 9.20 9.19
Solution A
5
contains (g L
1
): H
3
BO
3
2.85, MnCl
4
H
2
O 1.81, ZnSO
4
7H
2
O 0.22,
CuSO
4
5H
2
O 0.079 and MoO
3
0.015. Solution B6 contains (mg L
1
): NH
4
VO
3

23, K
2
Cr
2
(SO
4
)
4
2H
2
O) 96, NiSO
4
7H
2
O 48, NaWO
4
2H
2
O 18, Ti
2
(SO
4
)
3
40,
Co(NO
3
)
2
6H
2
O 44.
*
Control = Zarrouk medium.
9222 J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227
in harvested and lyophilized cells according to the above method.
The optical density was correlated with Chl-a content according to
a standard calibration curve obtained using pure authentic Chl-a
standard (Sigma Chemical Co., St. Louis, MO, USA).
2.6. Lutein extraction and quantication
Lutein was extracted from the algal cells using alkali digestion
method and its concentration determined according to Shi et al.
(1997) and Shi and Chen (1997). The whole process was carried
out in darkness. Lutein was analyzed by reverse phase High Perfor-
mance Liquid Chromatography (Waters, Milford, USA) equipped
with a WATERS 515 HPLC pump, WATERS 2707 autosampler and
programmable photodiode array detector (WATERS 2998). A re-
versed-phase C-18 column WATERS Xterra (4.8 250 mm, 5 lm
particle size) was used for pigment analysis. Elution was per-
formed with isocratic solvent methanol/dichloromethane/acetoni-
trile/water (67.5:22.5:9.5:0.5, v/v) at a ow rate of 1 mL min
1
at
450 nm. Data were acquired three-dimensionally (absorbance-
time-wavelength) using EMPOWER software. The column was kept
in room temperature (2225 C). The samples and standard were
ltered through a 0.22 lm syringe lter (acrodisc) prior to injec-
tion. The lutein concentration in the microalga was calculated by
comparing the peak area with that of authentic lutein standard
(Sigma Chemical Co., St. Louis, MO, USA).
2.7. Beta carotene extraction and quantication
For beta carotene analysis 10 mg of lyophilized algal cells were
extracted with 3 mL of ethanol: hexane (v/v). To this 2 mL of water
and 4 mL of hexane were added and the mixture was shaken vig-
orously and centrifuged at 1000g for 5 min. The hexane layer
was separated and evaporated under N
2
at 30 C, redissolved in
dichloromethane and analyzed by HPLC (Waters, Milford, USA)
according to the method described by Ben-Amotz et al. (1988). Elu-
tion was performed with isocratic solvent of methanol: acetonitrile
(9:1 v/v) at ow rate of 1 mL min
1
using a WATERS Xterra re-
versed-phase C-18 column (4.8 250 mm, 5 lm particle size) at
450 nm. The betacarotene concentration in the microalga was cal-
culated by comparing the peak area with that of standard betacar-
otene (Sigma Chemical Co., St. Louis, MO, USA).
2.8. Morphological studies
Trichome morphology of A. platensis cultured in seawater media
before and after acclimatization was determined by (Olgun et al.,
1997) observing 50 trichomes using a phase contrast microscope
(Nikon Eclipse E600, Japan) equipped with a digital camera (Nikon
DXM1200F) and software at 40 magnication.
2.9. Statistical analysis
Data presented are the mean of three independent experiments,
each consisting of two cultures running in parallel for each treat-
ment. The effect of seawater media onthe different parameters were
analyzed using One way ANOVA followed by post-hoc analysis with
NewmanKeuls multiple range test using the statistical program
SPSS ver.17. Signicant levels for all analyses were set to p < 0.05.
3. Results and discussion
3.1. Effect of different seawater media on growth parameters
Growth kinetics (Fig. 1) of A. platensis grown with three differ-
ent seawater media and Zarrouks medium (control) were evalu-
ated during 25 days of cultivation. All the three seawater media
used in this study supported the growth of A. platensis. Biomass
concentration (as dry weight) of A. platensis cultured in seawater
medium SW 2 was not signicantly different (P > 0.05) from Zar-
rouk medium (control). But the biomass concentration of A.
platensis grown in seawater media SW 1 (2.44 0.18 g L
1
) and
SW3 (2.26 0.06 g L
1
) were signicantly lower than the control
group and SW 2 group (P < 0.05). Similar to the present results Fau-
cher et al. (1979) have also reported growth rates comparable or
higher than synthetic SOT (mineral standard medium) medium
for A. maxima grown in seawater supplemented with phosphate
and nitrate. Warr et al. (1985) have demonstrated that external
sea salt concentrations up to 150% seawater had little effect on
the growth yield of the strain of A. platensis. According to these
authors the ability of A. platensis to withstand elevated salinities
appears to be an important factor enabling it to survive and grow
in alkaline lakes and other similar waters.
The results presented in Table 2 were obtained by exponential
regression of each growth curve. The correlations were statistically
signicant (P < 0.01) for all curves. High values of correlation ob-
tained for the regression, indicate that A. platensis exhibited expo-
nential growth, showing rapid adaptation to the seawater media
cultivation conditions. Highest maximum specic growth rate
(l
max)
was obtained in the group cultured in seawater medium
SW 2. Even the group cultured with undiluted seawater had a l
max
close to control group. However, SW 3 group had a signicantly
lower maximum specic growth rate (l
max)
than all other groups.
The l
max
obtained for A. platensis cultured in the seawater media
are similar to the values reported by Costa et al. (2000) for the
same species in Zarrouks medium (l
max
= 0.24 d
1
) with initial
nitrogen of 0.003 M. This is very signicant because Zarrouks med-
ium contains large number of chemicals. Hence using it for large
Fig. 1. Time course of biomass production by Arthrospira (Spirulina) platensis
cultivated in different seawater media. Values are mean SE.
Table 2
Specic growth rate (l
max
), doubling time (t
d
), correlation coefcient (r) and
condence level (P-level) for the culture of A. platensis in different seawater media
and control (Zarrouks) media.
Media l
max
(d
1
) t
d(day)
r P-level
Control
*
0.23 3.02 0.92 4.64 10
9
SW 1 0.23 3.01 0.96 3.32 10
10
SW 2 0.26 2.72 0.90 7.99 10
8
SW 3 0.18 3.84 0.94 3.32 10
10
*
Control = Zarrouk medium.
J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227 9223
scale production of Spirulina will be very expensive (Reinehr and
Costa, 2006).
The A. platensis group cultured in seawater medium SW 2
showed the lowest doubling time (t
d
= 2.720 days) lower than the
control group (t
d
= 3.016 days). Lower doubling times are preferred
for mass cultivation. As the doubling time increases the speed of
cell duplication declines and makes the commercial cultivation
uneconomical (Reinehr and Costa, 2006). The doubling time
reported for A. platensis cultured in seawater media (t
d
= 2.7
3.0 days) was very close to the doubling time (t
d
= 2.96 days) re-
ported for A. platensis cultured in Zarrouks medium by Costa
et al. (2000). The cost of nutrient accounts for about 1520% of
the total costs for cultivation on a large scale (Vonshak, 1997).
Hence utilization of seawater media for the cultivation of A. platen-
sis will reduce the production cost considerably.
3.2. Effect of different seawater media on the pigments
Fig. 2 shows the time course of Chl-a accumulation in A. platen-
sis grown in different seawater media and the control group. The
nal Chl-a content of A. platensis grown in control media with low-
est salinity were signicantly higher than the cells grown in sea-
water media (P < 0.05). Chl-a content in the control group was
almost linear throughout the entire culture period. Furthermore
the increase in Chl-a content in A. platensis grown in seawater
media started to slow down after day 10. There was an inverse
relationship between the salinity content of the media and the -
nal concentration of chlorophyll-a in the biomass (Table 3). The -
nal Chl-a content of A. platensis grown in seawater media were
signicantly lower than the control group (P < 0.05). Similar de-
crease in Chl-a content in A. maxima grown in seawater was re-
ported by Lamela and Mrquez-Rocha (2000). Vonshak et al.
(1996) have also shown a decrease in Chl-a content in A. platensis
grown in 0.5 M to 1.0 M NaCl.
Fig. 3 displays the time course of phycocyanin content in A. plat-
ensis grown in different seawater media and the control group. The
phycocyanin content of the biomass also varied according to media
composition (Table 3). In the present study the Chl-a content of A.
platensis cells grown in seawater media were 46.8957.65% less
than the cells grown in control media while total phycocyanin con-
tent of cells grown in seawater media ranged from 67.90% to
81.83% of the control group. Hence it can be inferred that phycocy-
anin content was affected to a lesser extent than Chl-a in seawater
media.
The effect of seawater media on the total phycocyanin/Chl-a ra-
tio is summarized in Fig. 4. There was a signicant (P < 0.05) in-
crease in the total phycocyanin/Chl-a with increase in the salt
concentration of the media. Similar increase in total phycocya-
nin/Chl-a ratio in salt adapted A. platensis with increase in salt con-
centration (17500 mM) was demonstrated by Dhiab et al. (2007).
They have also suggested that higher Phycobilin/Chl-a ratio in salt
adapted cultures enhances the salt absorption by phycobilisomes
relative to that of Chl-a. Studies carried out on the effect of salt
concentration on physiological behaviour of A. platensis show con-
tradictory results. Vonshak et al., 1996; Lu et al., 1999 and Lu and
Vonshak, 2002 have shown that an increase in salt concentration
leads to decrease in phycobilin/Chl-a ratio. Conversely, similar to
our results Dhiab et al. (2007) have demonstrated an increase in to-
tal phycocyanin/Chl-a ratio in salt adapted A. platensis with in-
crease in salt concentration (17500 mM). These contradictions
in results might be attributed to genetic difference in the A. platen-
sis strains used in different studies and environmental factors
(Dhiab et al., 2007). According to Berry et al. (2003), special adap-
tation strategies in strains like A. platensis leads to difference in
bioenergetic processes in the cytoplasmic membrane, the thyla-
koid membrane and the cytoplasm, which are mainly, achieved
Fig. 2. Time course of phycocyanin content (mean SE) of A. platensis grown in
different seawater media and control (Zarrouk) media.
Table 3
Biomass (dry weight) and pigment content (mg/g dry weight) of A. platensis grown in different seawater media and control (Zarrouk) media after 25 days of cultivation. Different
alphabets indicate signicance (P < 0.05) N = 6.
Media X
max
(g/L) Pigment content (mg/g dry weight)
Chl-a (mg/g) Lutein (mg/g) Detacarotene (mg/g) Phycocyanin (mg/g)
Control
*
3.11 0.09
ab
16.73 0.59
ab
1.751 0.02
ab
3.76 0.08
ac
48.44 1.94
abc
SW 1 2.44 0.18
abc
7.85 0.16
abc
1.57 0.01
abc
2.35 0.04
ab
32.90 0.97
abc
SW 2 2.99 0.15
bc
8.58 0.66
c
2.28 0.04
c
2.75 0.04
ac
37.68 1.91
abc
SW 3 2.26 0.06
ac
9.64 0.09
ac
1.81 0.02
ac
3.54 0.13
bc
39.64 2.00
ab
Fig. 3. Time course of chlorophyll-a content (mean SE) of A. platensis grown in
different seawater media and control (Zarrouk) media.
9224 J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227
by using available components from the tool-box with different
expression levels.
Phycocyanin extracted from A. platensis cultured in different
seawater media and control (Zarrouk) media were puried by
ammonium sulphate precipitation followed by elution through
DEAE Sepharose column. After elution through the DEAE Sepharose
column the purity ratio of c-phycocyanin extracted from the con-
trol group was >4.0 and that of diluted seawater media SW3 was
3.84 (Table 5). But the purity of c-phycocyanin extracted from A.
platensis grown seawater media SW 1 and SW 2 were less than
4.0. This might be due to the accumulation of low-
molecular-weight carbohydrates for osmotic protection in the sea-
water media (Vonshak et al., 1996). Hence phycocyanin extracted
from A. platensis cultured in seawater media requires more number
of purication steps to attain the purity ratio of 4.
As shown in Table 3 the lutein content of A. platensis cultures
grown in seawater media (SW2 and SW 3) were higher than the
control (P < 0.05). However, the cultures grown in medium con-
taining undiluted seawater (SW 1) had lower lutein content than
the control cultures (P < 0.05). The lutein content observed in the
cultures grown in seawater medium SW 2 (2.278 0.044 mg g
1
)
is very close to the lutein content reported by Chen et al. (2006)
for A. platensis cultured in selenium enriched mixotrophic cultures.
The betacarotene content of the control culture was signi-
cantly higher (P < 0.05) than the cultures grown in seawater media
(Table 3). But there was no signicant difference in the betacaro-
tene content of the cultures grown in seawater medium (SW 3)
and the control cultures (3.535 0.129 mg g
1
vs. 3.759 0.076
mg g
1
). The lack of signicant increase in beta carotene concen-
tration in seawater cultures relative to the control (Zarrouks med-
ia) shows the absence of osmotic or salinity stress in seawater
cultures of A. platensis. This is also an indication that the strain of
A. platensis used in this study adapted well to seawater culture.
There was a signicant increase (P < 0.05) in the betacarotene/
Chl-a ratio in the cultures grown in seawater media (Fig. 5). This
is possibly a response to protect the cells against the higher osmot-
icum in the seawater media. Similar increase in betacarotene/Chl-a
ratio was observed by Lamela and Mrquez-Rocha (2000) in the
seawater culture of Arthrospira maxima.
3.3. Effect of different seawater media on the nal chemical
composition of biomass
A decrease in protein content and a concurrent accumulation of
carbohydrates was observed (Table 4) with increase in salinity of
Fig. 4. Total phycocyanin/Chl-a ratio of A. platensis grown in different seawater
media and control (Zarrouk) media. Data are mean SE.
Table 5
Purication yields of phycocyanin from seawater cultured Arthrospira platensis.
Purication Yield purity ratios
Control SW 1 SW 2 SW 3
620/280 655/280 620/280 655/280 620/280 655/280 620/280 655/280
Step 1 0.82 0.68 0.78 0.36 0.78 0.32 0.80 0.64
Step 2 0.90 0.75 0.96 0.41 0.92 0.46 0.87 0.67
Step 3 2.21 1.83 1.36 1.16 1.42 1.34 1.87 1.72
Step 4 4.10 3.93 2.61 2.30 2.87 2.44 3.84 3.77
Step 1: crude extract.
Step 2: fractional precipitation with (NH
4
)
2
SO
4
30%.
Step 3: fractional precipitation with (NH
4
)
2
SO
4
50%.
Step 4: DEAE sepharose.
Fig. 5. Total b-carotene/Chl-a ratio of A. platensis grown in different seawater media
and control (Zarrouk) media. Data are mean SE.
Table 4
Protein, lipid and carbohydrate content of A. platensis grown in different seawater
media and control (Zarrouk) media. Different alphabets indicate signicance
(P < 0.05) N = 6.
Media Protein (% DW) Carbohydrates(%DW) Lipids (%DW)
Control
*
71.17 1.00
abc
15.01 0.61
abc
13.79 0.41
ab
SW 1 59.87 2.10
abc
26.97 0.50
abc
8.04 0.62
abc
SW 2 65.21 1.57
abc
24.08 0.16
abc
10.61 1.68
abc
SW 3 66.96 0.42
abc
18.81 1.04
abc
12.14 0.45
b
J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227 9225
the media. The protein content of A. platensis grown in (Zarrouks
medium) control group was signicantly higher than the other
groups (P < 0.05). The protein content observed for A. platensis
grown in (Zarrouks medium) (71.16%) is very close to the values
reported (68.01%) by Oliveira et al. (1999) for the same species
grown in mineral medium described by Paoletti et al. (1985) at
25 C. The protein content observed for A. platensis maintained in
diluted seawater (SW 2, 65.20% and SW 3, 66.96%) falls within
the range reported for Spirulina cultured in enriched seawater
(65.61%) (Olgun et al., 1997) and salt enriched media (5165%)
(Tredici et al., 1986). Similarly the protein content of A. platensis
grown in undiluted seawater (59.87%) was comparable to the val-
ues reported for S. maxima (55.459.4%) cultured in seawater
(Materassi et al., 1984). In contrast to the protein content there
was increase in carbohydrate content with increase in salinity.
The carbohydrate content of A. platensis grown in (Zarrouks med-
ium) control group was signicantly lower (P < 0.05) than the other
groups. A. platensis grown in undiluted seawater (SW-group) had
the highest carbohydrate content (Table 4). This might be due to
low-molecular-weight carbohydrates accumulated by A. platensis
as osmoprotectors during its acclimatization process to high salt
environment. Reed et al. (1984) have reported low-molecular-
weight carbohydrates, glucosyl-glycerol and trehalose as the two
major organic osmoticum accumulated by Spirulina platensis in
proportion to the external salinity particularly when it is grown
in brackish and saline waters. Vonshak et al., 1988 have also shown
carbohydrate as a major solute for osmotic adaptation. There was
no signicant difference (P > 0.05) in the lipid content of A. platen-
sis grown in different seawater media when compared with the
control group. The lipid content of A. platensis grown in SW 1 med-
ium alone had a slightly lower (P < 0.05) lipid content than other
groups. Hence, the effect of salinity on the chemical composition
of cells to be used as a source of biomass plays a very signicant
role in determining the suitability of seawater or brackishwater
for mass culturing S. platensis .
3.4. Effect of different seawater media on the morphology of trichomes
Though the helical shape of A. platensis was maintained in the
cultures grown with different seawater media, there were some
difference in the length of trichomes and degree of helicity. In
the rst generation immediately after acclimatization there was
more number of short but closely coiled trichomes. But after three
generation of acclimatization in seawater unusually long tric-
homes dominated the cultures grown in seawater media SW-2
and SW-1 however shorter straight laments and coiled trichomes
dominated the culture grown in SW-3 media. Jeeji Bai (1985) and
Lewin (1980) have shown that increase in salinity above the basal
level inhibits the growth of helicoidal morphome, while the
straight morphomes growth behaviour remains unaffected. Dhiab
et al. (2007) have also shown that the change in the morphology of
the trichome (from the helicoidal to the straight form) as a modi-
cations of physiological behaviour of Arthrospira (Spirulina) plat-
ensis in response to the increase in NaCl concentration in growth
media.
4. Conclusions
Though Arthrospira (Spirulina) platensis is a freshwater organism
the strain used in the present study adapted well to seawater cul-
ture conditions. All the three seawater media supported the
growth of A. platensis. There was no signicant difference in the
growth, doubling time and biomass production of A. platensis
grown in seawater medium SW2 and control (Zarrouk) medium.
A. platensis cells grown in seawater medium (SW 2) had signi-
cantly higher lutein content than control medium. Efforts are also
underway for the mass culture of this strain under outdoor culture
conditions.
Acknowledgements
The authors are grateful to the Director, National Institute of
Ocean Technology, and the Ministry of Earth Sciences, Government
of India for providing adequate research funding and facilities for
carrying out this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2010.06.120.
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