hr

argunia

Received in revised form 23 June 2010Accepted 26 June 2010Available online 23 July 2010

Keywords:Arthrospira platensisSeawater

tercontrol) (2) seawater media SW 1 (3) seawater media SW2 and (4) seawater media SW 3. The relative

kg against US$ 500/kg for their synthetic equivalents but naturalb carotene is preferred in health market because it is a mixtureof 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 exclusivepresence in cyanobacteria but also due to their wide range of com-mercial applications (Moreno et al., 1995). Due to their distinctivespectroscopic properties and non toxic nature they have found

culture and make it more competitive for the production of valueadded products. Development of seawater culture of Spirulina isinevitable for propagating outdoor cultivation of these cyanobacte-ria in many tropical arid areas, where climatic conditions arefavourable 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-

* Corresponding author. Tel.: +91 9434266398; fax: +91 3192225089.

Bioresource Technology 101 (2010) 92219227

Contents lists availab

T

elsE-mail address: leemaroy2002@yahoo.com (J.T. Mary Leema).The cyanobacterium Arthrospira (Spirulina) platensis has gainedconsiderable attention worldwide as a source of several nutraceu-ticals (Belay et al., 1993). Among the nutraceuticals, pigments haveattracted greater commercial interest (approximate price of foodgrade phycocyanin is Australian $ 500/kg, Borowitzka, 1992)due to their high end applications and relatively easy extractionprocedures. Pigments of microalgal origin which are currentlyenjoying highmarket demand are the phycobiliproteins, b caroteneand lutein. b carotene produced from microlagae costs US$ 1000/

metic industry replacing the toxic synthetic pigments (Cohen,1986; Glazer, 1994). Phycocyanin contributes nearly 30% of thebiomass (Garnier and Thomas, 1993). However, culture conditionsdetermine the actual content of these pigments in cell (Mrquez-Rocha et al., 1995).

At present Spirulina is cultured mainly to quench the healthfood market utilizing a chemically dened medium (Belay andOta, 1994). Signicant share of the production cost is contributedby these chemicals. Hence, the use of low cost media like seawatermedia will bring down the production cost of commercial SpirulinaPhycocyaninLuteinBetacarotene

1. Introduction0960-8524/$ - see front matter 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.06.120performance of these media were investigated with respect to their biomass production, pigment pro-duction (phycocyanin, lutein and betacarotene), and biochemical composition. A. platensis grown inmedia SW 2 had a biomass production (2.99 0.145 g L1) comparable to that of control media(3.114 0.085 g L1); highest specic growth rate (0.255 d1) 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 controlwere closer to 4, while the phycocyanin obtained from cells grown in other two media exhibited lowerpurity ratios due to accumulation of lower molecular weight carbohydrates. The phycocyanin/Chl-a ratioand the betacarotene/Chl-a ratio of the cells grown in seawater media were higher than control. Thelutein content of A. platensis cells grown in seawater media SW 2was higher than that of control. The cellsgrown in seawater media had a slightly modied biochemical composition than the control with a highercarbohydrate and lower protein content. All the three seawater based media with fewer chemicals thanthe control (Zarrouk media) supported the growth of A. platensis as good as the control.

2010 Elsevier Ltd. All rights reserved.

additional applications in biomedical research, food, drug and cos-Article history:Received 22 January 2010

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-High value pigment production from Artin seawater

J.T. Mary Leema a,*, R. Kirubagaran b, N.V. VinithkumaAndaman Nicobar Centre for Ocean Science and Technology, NIOT R&D Complex, DollybNational Institute of Ocean Technology, Pallikaranai, Chennai 600100, Tamil Nadu, Ind

a r t i c l e i n f o a b s t r a c t

Bioresource

journal homepage: www.ll rights reserved.ospira (Spirulina) platensis cultured

a, P.S. Dheenan a, S. Karthikayulu b

j, Port Blair 744103, A&N Islands, India

le at ScienceDirect

echnology

evier .com/locate /bior tech

ment production and growth in seawater remains unknown(Materassi et al., 1984). The use of a suitable seawater media isan essential prerequisite for developing mass production systemsutilizing seawater. Mass production of A. platensis in seawater re-quires sorting out several physiological problems confronted inculturing these algae in seawater. Hence the present study aimsto assess the suitability of three different compositions of seawatermedia 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, Universityof Madras, Chennai, India. They were maintained in controlled con-ditions in Zarrouk medium (Zarrouk, 1966) with light intensity of140 lmol photon m2 s1 and temperature of 26 1 C. The strain

NaCl 1.0

9222 J.T. Mary Leema et al. / Bioresource TeMgSO47H2O 0.2 CaCl22H20 0.02 FeSO47H2O 0.01 0.01 0.01 0.01Na2EDTA 0.08Fe2EDTA 0.005 0.005 0.005A5 1 mL1

B6 1 mL1

pH 9.26 9.12 9.20 9.19

Solution A5 contains (g L1): H3BO3 2.85, MnCl4H2O 1.81, ZnSO47H2O 0.22,CuSO45H2O 0.079 and MoO3 0.015. Solution B6 contains (mg L1): NH4VO3 was acclimatized to grow in seawater for three generations prior toits use in these experiments. Experiments were carried out in500 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 atemperature controlled orbital shaker (Orbitek LT, Scigenics, Chen-nai, India) with light (light intensity 140 lmol photon m2 s1).Control was cultured with 250 ml of sterilized (121 C for20 min) Zarrouk medium (with the following modication theconcentration of NaNO3 was 3.0 g L1) in 500 mL Erlenmeyerasks. Sea water was obtained from Aberdeen Bay, Andaman, India(salinity 34.23, pH 8.01). The seawater was pretreated withNaHCO3 in order to precipitate the excess divalent cations Ca2+

and Mg2+ 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 preparedwith different ratios of seawater were tested. Details of the compo-sition are furnished in Table 1. In the case of media SW1 (undilutedseawater) the nutrients were separately autoclaved (121 C for20 min) and added aseptically to pretreated, preltered, undilutedsea water. The freshwater of media SW 2 (2 part seawater:1 partfreshwater, 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 cellconcentration of 0.25 g L1 dry weight) of exponentially growinginoculums under aseptic condition.

Table 1Element composition of the culture media used for the seawater culture of Arthrospiraplatensis.

Component Control* (g L1) SW1 (g L1) SW 2 (g L1) SW 3 (g L1)

NaHCO3 16.8 19.5 19.5 18.8K2HPO4 0.5 0.5 0.5 0.5NaNO3 3.0 3.0 3.0 3.0K2SO4 1.0 23, K2Cr2(SO4)42H2O) 96, NiSO47H2O 48, NaWO42H2O 18, Ti2(SO4)3 40,Co(NO3)26H2O 44.* Control = Zarrouk medium.2.2. Kinetic parameters

Samples were collected on alternate days and growth was mon-itored turbidometrically (Leduy and Therien, 1977) by measuringthe 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. Thecell dry weight concentration was determined by drying the cellsat 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 againstbiomass for each treatment. At the end of 25 days the maximumbiomass concentration designated as Xmax (g L1) was recorded(Schmidell et al., 2001). Biomass (X) values and exponential regres-sion were used to calculate the maximum specic growth rate(lmax, d1) during the logarithmic phase (Bailey and Ollis, 1986).The doubling time (td, days) was calculated as td = ln 2 (lmax)1.

2.3. Analytical methods

The protein content of the lyophilized biomass was determinedaccording to Lowry et al. (1951) with BSA as standard. Carbohy-drates were determined following the colorimetric phenol methodof Dubois et al.(1956) using glucose as standard. Lipids wereextracted from the lyophilized samples of biomass and determinedaccording to the method of Bligh and Dyer, 1959.

2.4. Pigment analysis

2.4.1. Extraction, quantication and purication of phycocyaninFor observing the time course of phycocyanin accumulation

phycocyanin was extracted from 5 mL algal samples once in 5 daysand quantied according to the method reported by Boussiba andRichmond, 1979. For determination of nal phycocyanin content(mg/g) and further purication the cells were harvested on day25 from different groups by centrifuging at 10,000g (Sigma coolingcentrifuge) for 30 min at 4 C. The harvested cells were washedtwice with distilled water and freeze dried (Lyodel). Phycocyaninwas extracted from 250 mg freeze dried cells suspended sus-pended in 25 mL of sodium phosphate buffer (0.1 M, pH 7) andquantied according to the method reported by Boussiba and Rich-mond (1979). For further purication crude phycocyanin obtainedfrom all the four groups were fractionated by precipitation with so-lid ammonium sulphate rst at 30% and then at 50% saturation. Theprecipitate 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 bufferovernight at 4 C. The crude phycocyanin fractions obtained werechromatographed 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 mLfractions were collected. The purity of the collected fractions wereevaluated according to the absorbance ratio (A620/A280 for c-phycocyanin and A655/A280 for allophycocyanin (Boussiba andRichmond, 1979). Visible and UV spectra of phycocyanin weremeasured in an UVVIS Unicam UV300 spectrophotometer.

2.5. Chlorophyll-a estimation

chnology 101 (2010) 92219227Chlorophyll-a (Chl-a) was determined by the method of Bennetand Bogorad (1973). Final Chl-a content (mg g1) was determined

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 mediacultivation conditions. Highest maximum specic growth rate(lmax) was obtained in the group cultured in seawater mediumSW 2. Even the group cultured with undiluted seawater had a lmaxclose to control group. However, SW 3 group had a signicantlylower maximum specic growth rate (lmax) than all other groups.The lmax obtained for A. platensis cultured in the seawater mediaare similar to the values reported by Costa et al. (2000) for thesame species in Zarrouks medium (lmax = 0.24 d1) with initialnitrogen of 0.003 M. This is very signicant because Zarrouks med-ium contains large number of chemicals. Hence using it for large

Table 2Specic growth rate (lmax), doubling time (td), correlation coefcient (r) andcondence level (P-level) for the culture of A. platensis in different seawater mediaand control (Zarrouks) media.

Media lmax(d1) td(day) r P-level

Control* 0.23 3.02 0.92 4.64 109SW 1 0.23 3.01 0.96 3.32 1010SW 2 0.26 2.72 0.90 7.99 108

e Technology 101 (2010) 92219227 9223in harvested and lyophilized cells according to the above method.The optical density was correlated with Chl-a content according toa standard calibration curve obtained using pure authentic Chl-astandard (Sigma Chemical Co., St. Louis, MO, USA).

2.6. Lutein extraction and quantication

Lutein was extracted from the algal cells using alkali digestionmethod and its concentration determined according to Shi et al.(1997) and Shi and Chen (1997). The whole process was carriedout in darkness. Lutein was analyzed by reverse phase High Perfor-mance Liquid Chromatography (Waters, Milford, USA) equippedwith a WATERS 515 HPLC pump, WATERS 2707 autosampler andprogrammable photodiode array detector (WATERS 2998). A re-versed-phase C-18 column WATERS Xterra (4.8 250 mm, 5 lmparticle 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 min1 at450 nm. Data were acquired three-dimensionally (absorbance-time-wavelength) using EMPOWER software. The column was keptin room temperature (2225 C). The samples and standard wereltered through a 0.22 lm syringe lter (acrodisc) prior to injec-tion. The lutein concentration in the microalga was calculated bycomparing 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 wereextracted with 3 mL of ethanol: hexane (v/v). To this 2 mL of waterand 4 mL of hexane were added and the mixture was shaken vig-orously and centrifuged at 1000g for 5 min. The hexane layerwas separated and evaporated under N2 at 30 C, redissolved indichloromethane 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 min1 using a WATERS Xterra re-versed-phase C-18 column (4.8 250 mm, 5 lm particle size) at450 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 mediabefore 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 (NikonDXM1200F) 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 seawatermedia on thedifferent parameterswereanalyzed using Oneway ANOVA followed by post-hoc analysis withNewmanKeuls multiple range test using the statistical programSPSS 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

J.T. Mary Leema et al. / BioresourcGrowth 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 mediaused in this study supported the growth of A. platensis. Biomassconcentration (as dry weight) of A. platensis cultured in seawatermedium 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 L1) andSW3 (2.26 0.06 g L1) were signicantly lower than the controlgroup and SW 2 group (P < 0.05). Similar to the present results Fau-cher et al. (1979) have also reported growth rates comparable orhigher than synthetic SOT (mineral standard medium) mediumfor A. maxima grown in seawater supplemented with phosphateand nitrate. Warr et al. (1985) have demonstrated that externalsea salt concentrations up to 150% seawater had little effect onthe growth yield of the strain of A. platensis. According to theseauthors the ability of A. platensis to withstand elevated salinitiesappears to be an important factor enabling it to survive and growin alkaline lakes and other similar waters.

The results presented in Table 2 were obtained by exponentialregression of each growth curve. The correlations were statistically

Fig. 1. Time course of biomass production by Arthrospira (Spirulina) platensiscultivated in different seawater media. Values are mean SE.SW 3 0.18 3.84 0.94 3.32 1010

* Control = Zarrouk medium.

scale production of Spirulina will be very expensive (Reinehr andCosta, 2006).

The A. platensis group cultured in seawater medium SW 2showed the lowest doubling time (td = 2.720 days) lower than thecontrol group (td = 3.016 days). Lower doubling times are preferredfor mass cultivation. As the doubling time increases the speed ofcell duplication declines and makes the commercial cultivationuneconomical (Reinehr and Costa, 2006). The doubling timereported for A. platensis cultured in seawater media (td = 2.73.0 days) was very close to the doubling time (td = 2.96 days) re-ported for A. platensis cultured in Zarrouks medium by Costaet al. (2000). The cost of nutrient accounts for about 1520% ofthe 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. Thenal 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 wasalmost linear throughout the entire culture period. Furthermorethe increase in Chl-a content in A. platensis grown in seawatermedia started to slow down after day 10. There was an inverse

Fig. 3 displays the time course of phycocyanin content in A. plat-ensis grown in different seawater media and the control group. Thephycocyanin content of the biomass also varied according to mediacomposition (Table 3). In the present study the Chl-a content of A.platensis cells grown in seawater media were 46.8957.65% lessthan the cells grown in control media while total phycocyanin con-tent of cells grown in seawater media ranged from 67.90% to81.83% of the control group. Hence it can be inferred that phycocy-anin content was affected to a lesser extent than Chl-a in seawatermedia.

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 saltconcentration of the media. Similar increase in total phycocya-nin/Chl-a ratio in salt adapted A. platensiswith 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 saltadapted cultures enhances the salt absorption by phycobilisomesrelative to that of Chl-a. Studies carried out on the effect of saltconcentration on physiological behaviour of A. platensis show con-tradictory results. Vonshak et al., 1996; Lu et al., 1999 and Lu andVonshak, 2002 have shown that an increase in salt concentrationleads to decrease in phycobilin/Chl-a ratio. Conversely, similar toour 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 contradictionsin 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 inbioenergetic processes in the cytoplasmic membrane, the thyla-

iffer

eigh

Lute

1.751.5

9224 J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227Fig. 2. Time course of phycocyanin content (mean SE) of A. platensis grown indifferent seawater media and control (Zarrouk) media.

Table 3Biomass (dry weight) and pigment content (mg/g dry weight) of A. platensis grown in dalphabets indicate signicance (P < 0.05) N = 6.

Media Xmax (g/L) Pigment content (mg/g dry w

Chl-a (mg/g)

Control* 3.11 0.09ab 16.73 0.59ab

SW 1 2.44 0.18abc 7.85 0.16abcrelationship 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 weresignicantly 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. platensisgrown in 0.5 M to 1.0 M NaCl.SW 2 2.99 0.15bc 8.58 0.66c 2.2SW 3 2.26 0.06ac 9.64 0.09ac 1.8koid membrane and the cytoplasm, which are mainly, achieved

ent seawater media and control (Zarrouk) media after 25 days of cultivation. Different

t)

in (mg/g) Detacarotene (mg/g) Phycocyanin (mg/g)

1 0.02ab 3.76 0.08ac 48.44 1.94abc

7 0.01abc 2.35 0.04ab 32.90 0.97abc

Fig. 3. Time course of chlorophyll-a content (mean SE) of A. platensis grown indifferent seawater media and control (Zarrouk) media.8 0.04c 2.75 0.04ac 37.68 1.91abc

1 0.02ac 3.54 0.13bc 39.64 2.00ab

Fig. 4. Total phycocyanin/Chl-a ratio of A. platensis grown in different seawatermedia and control (Zarrouk) media. Data are mean SE.

J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227 9225by using available components from the tool-box with differentexpression levels.

Phycocyanin extracted from A. platensis cultured in differentseawater media and control (Zarrouk) media were puried byammonium sulphate precipitation followed by elution throughDEAE Sepharose column. After elution through the DEAE Sepharosecolumn the purity ratio of c-phycocyanin extracted from the con-trol group was >4.0 and that of diluted seawater media SW3 was3.84 (Table 5). But the purity of c-phycocyanin extracted from A.platensis grown seawater media SW 1 and SW 2 were less than4.0. 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 extractedfrom A. platensis cultured in seawater media requires more numberof purication steps to attain the purity ratio of 4.

As shown in Table 3 the lutein content of A. platensis culturesgrown in seawater media (SW2 and SW 3) were higher than theTable 5Purication yields of phycocyanin from seawater cultured Arthrospira platensis.

Purication Yield purity ratios

Control SW 1

620/280 655/280 620/280 655/2

Step 1 0.82 0.68 0.78 0.36Step 2 0.90 0.75 0.96 0.41Step 3 2.21 1.83 1.36 1.16Step 4 4.10 3.93 2.61 2.30

Step 1: crude extract.Step 2: fractional precipitation with (NH4)2SO4 30%.Step 3: fractional precipitation with (NH4)2SO4 50%.Step 4: DEAE sepharose.

Table 4Protein, lipid and carbohydrate content of A. platensis grown in different seawatermedia and control (Zarrouk) media. Different alphabets indicate signicance(P < 0.05) N = 6.

Media Protein (% DW) Carbohydrates(%DW) Lipids (%DW)

Control* 71.17 1.00abc 15.01 0.61abc 13.79 0.41ab

SW 1 59.87 2.10abc 26.97 0.50abc 8.04 0.62abc

SW 2 65.21 1.57abc 24.08 0.16abc 10.61 1.68abc

SW 3 66.96 0.42abc 18.81 1.04abc 12.14 0.45bcontrol (P < 0.05). However, the cultures grown in medium con-taining undiluted seawater (SW 1) had lower lutein content thanthe control cultures (P < 0.05). The lutein content observed in thecultures grown in seawater medium SW 2 (2.278 0.044 mg g1)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 g1 vs. 3.759 0.076mg g1). 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 seawatercultures of A. platensis. This is also an indication that the strain ofA. 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

Fig. 5. Total b-carotene/Chl-a ratio of A. platensis grown in different seawater mediaand control (Zarrouk) media. Data are mean SE.is possibly a response to protect the cells against the higher osmot-icum in the seawater media. Similar increase in betacarotene/Chl-aratio was observed by Lamela and Mrquez-Rocha (2000) in theseawater culture of Arthrospira maxima.

3.3. Effect of different seawater media on the nal chemicalcomposition of biomass

A decrease in protein content and a concurrent accumulation ofcarbohydrates was observed (Table 4) with increase in salinity of

SW 2 SW 3

80 620/280 655/280 620/280 655/280

0.78 0.32 0.80 0.640.92 0.46 0.87 0.671.42 1.34 1.87 1.722.87 2.44 3.84 3.77

25 C. The protein content observed for A. platensis maintained in

no signicant difference (P > 0.05) in the lipid content of A. platen-

Jeeji Bai, N., 1985. Competitive exclusion or morphological transformation? A case

e Tesis grown in different seawater media when compared with thecontrol group. The lipid content of A. platensis grown in SW 1 med-ium alone had a slightly lower (P < 0.05) lipid content than othergroups. Hence, the effect of salinity on the chemical compositionof cells to be used as a source of biomass plays a very signicantrole in determining the suitability of seawater or brackishwaterfor 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 thecultures grown with different seawater media, there were somedifference in the length of trichomes and degree of helicity. Inthe rst generation immediately after acclimatization there wasmore number of short but closely coiled trichomes. But after threegeneration of acclimatization in seawater unusually long tric-homes dominated the cultures grown in seawater media SW-2and SW-1 however shorter straight laments and coiled trichomesdominated the culture grown in SW-3 media. Jeeji Bai (1985) andLewin (1980) have shown that increase in salinity above the basallevel inhibits the growth of helicoidal morphome, while thestraight morphomes growth behaviour remains unaffected. Dhiabet al. (2007) have also shown that the change in the morphology ofthe 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 growthmedia.

4. Conclusions

Though Arthrospira (Spirulina) platensis is a freshwater organismthe strain used in the present study adapted well to seawater cul-ture conditions. All the three seawater media supported thegrowth of A. platensis. There was no signicant difference in thediluted seawater (SW 2, 65.20% and SW 3, 66.96%) falls withinthe 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. platensisgrown 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 therewas 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 othergroups. A. platensis grown in undiluted seawater (SW-group) hadthe highest carbohydrate content (Table 4). This might be due tolow-molecular-weight carbohydrates accumulated by A. platensisas osmoprotectors during its acclimatization process to high saltenvironment. Reed et al. (1984) have reported low-molecular-weight carbohydrates, glucosyl-glycerol and trehalose as the twomajor organic osmoticum accumulated by Spirulina platensis inproportion to the external salinity particularly when it is grownin brackish and saline waters. Vonshak et al., 1988 have also showncarbohydrate as a major solute for osmotic adaptation. There wasthe media. The protein content of A. platensis grown in (Zarrouksmedium) control group was signicantly higher than the othergroups (P < 0.05). The protein content observed for A. platensisgrown in (Zarrouks medium) (71.16%) is very close to the valuesreported (68.01%) by Oliveira et al. (1999) for the same speciesgrown in mineral medium described by Paoletti et al. (1985) at

9226 J.T. Mary Leema et al. / Bioresourcgrowth, doubling time and biomass production of A. platensisgrown in seawater medium SW2 and control (Zarrouk) medium.A. platensis cells grown in seawater medium (SW 2) had signi-study with Spirulina fusiformis. Arch. Hydrobiol. 191 (Suppl. 71, Algal Studies),3839.

Lamela, T., Mrquez-Rocha, F.J., 2000. Phycocyanin production in seawater cultureof Arthrospira maxima. Cienc. Mar. 26, 607619.

Leduy, A., Therien, N., 1977. An improved method for optical density measurementof the semi-microscopic blue algae Spirulina maxima. Biotechnol. Bioeng. 19,12191224.

Lewin, R.A., 1980. Uncoiled variants of Spirulina platensis (Cyanophyceae:Oscillatoriaceae). Arch. Hydrobiol. 26 (Suppl. 60, Algal Studies), 4852.

Lowry, O.H., Rosebroug, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement withFolin phenol reagent. J. Biol. Chem. 193, 265275.

Lu, C.M., Vonshak, A., 2002. Effect of salinity on photo system II functions incyanobacterial Spirulina platensis cells. Physiol. Plant. 114, 405413.

Lu, C.M., Torzillo, G., Vonshak, A., 1999. Kinetic response of photosystem IIphotochemistry in cyanobacterium Spirulina platensis to high salinity iscantly higher lutein content than control medium. Efforts are alsounderway for the mass culture of this strain under outdoor cultureconditions.

Acknowledgements

The authors are grateful to the Director, National Institute ofOcean Technology, and the Ministry of Earth Sciences, Governmentof India for providing adequate research funding and facilities forcarrying out this work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2010.06.120.

References

Bailey, J.E., Ollis, D.F., 1986. Biochemical Engineering Fundamentals, second ed.McGraw-Hill, Singapore.

Belay, A., Ota, Y., 1994. Production of high quality Spirulina at earthrise farms. In:Phang, S.M., Borowitzka, M.A., Whitton, B. (Eds.), Algal Biotechnology in theAsia-Pacic region. University of Malaya, Kuala Lumpur.

Belay, A., Ota, Y., Miyakawa, K., Shimamatsu, H., 1993. Current knowledge onpotential health benets of Spirulina. J. Appl. Phycol. 5, 235241.

Ben-Amotz, A., Lers, A., Avron, M., 1988. Stereoisomers of b-carotene and Phytoenein the alga Dunaliella bardwil. Plant. Physiol. 86, 12861291.

Bennet, J., Bogorad, I., 1973. Complementary chromatic adaptation in a lamentousblue-green alga. J. Cell Biol. 58, 419435.

Berry, S., Bolychevtseva, Y.V., Rgner, M., Karapetyan, N.V., 2003. Photosyntheticand respiratory electron transport in the alkaliphilic cyanobacteriumArthrospira (Spirulina) platensis. Photosynth. Res. 78, 6776.

Borowitzka, M.A., 1992. Algal biotechnology products and processes: matchingscience and economics. J. Appl. Phycol. 4, 267279.

Boussiba, S., Richmond, A.E., 1979. Isolation and characterization of phycocyaninsfrom the blue-green alga Spirulina platensis. Arch. Microbiol. 120, 155159.

Chen, T., Zheng, W.J., Yang, F., Bai, Y., Wong, Y.S., 2006. Mixotrophic culture of highselenium enriched Spirulina platensis on acetate and the enhanced productionof photosynthetic pigments. Enzyme Microb. Technol. 39, 103107.

Cohen, Z., 1986. Products from microalgae. In: Richmond, A. (Ed.), CRC Handbook ofMicroalgal Mass Culture. CRC Press, Boca Raton, Florida.

Costa, J.A.V., Linde, G.A., Atala, D.I.P., Mibielli, G.M., Kruger, R.T., 2000. Modelling ofgrowth conditions for cyanobacterium Spirulina platensis in microcosms. WorldJ. Microb. Biot. 16, 1518.

Dhiab, R.B., Ouada, H.B., Boussetta, H., Franck, F., Elabed, A., Brouers, M., 2007.Growth, uorescence, photosynthetic O2 production and pigment content ofsalt adapted cultures of Arthrospira (Spirulina) platensis. J. Appl. Phycol. 19, 293301.

Downham, A., Collins, P., 2000. Coloring our foods in the last and next millennium.Int. J. Food Sci. Technol. 35, 522.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetricmethod for determination of sugars and related substances. Anal. Chem. 28,350355.

Faucher, O., Coupal, B., Leduy, A., 1979. Utilization of seawater-urea as a culturemedium for Spirulina maxima. Can. J. Microbiol. 25, 752759.

Garnier, F., Thomas, J.C., 1993. Light regulation of phycobiliproteins in Spirulinamaxima. In: Doumenge, F., Durand-Chastel, H., Toulemont, A. (eds.) SpirulinaAlgae of Life. Spirulina Algae de Vie. Monaco Musee Oceanographique NS 12, pp.4148.

Glazer, A.N., 1994. Phycobiliproteins a family of valuable widely useduorophores. J. Appl. Phycol. 6, 105112.

chnology 101 (2010) 92219227characterized by two distinct phases. Aus. J. Plant Physiol. 26, 283292.Mrquez-Rocha, F.J., Sasaki, k., Nishio, N., Nagai, S., 1995. Inhibitory effect of oxygen

accumulation on the growth of Spirulina platensis. Biotechnol. Lett. 17, 225238.

Materassi, R., Tredici, M., Balloni, W., 1984. Spirulina culture in seawater. Appl.Microbiol. Biotechnol. 19, 384486.

Moreno, J., Rodriguez, H., Vargas, M.A., Rivas, J., Guerrero, M.G., 1995. Nitrogen-xing cyanobacteria as source of phycobiliprotein pigments. Composition andgrowth performance of ten lamentous heterocystous strains. J. Appl. Phycol. 7,1723.

Olgun, E.J., Galicia, S., Camacho, R., Mercado, G., Prez, T.J., 1997. Production ofSpirulina sp. in sea water supplemented with anaerobic efuents in outdoorraceways under temperate climatic conditions. Appl. Microbiol. Biotechnol. 48,242247.

Oliveira, M.A.C.L., Monteiro, M.P.C., Robbs, P.G., Leite, S.G.F., 1999. Growth andchemical composition of Spirulina maxima and Spirulina platensis biomass atdifferent temperatures. Aquac. Int. 7, 261275.

Paoletti, C., Pushparaj, B., Tomaselli, L., 1985. Ricerche sulla nutrizione minerale diSpirulina platensis. In: Atti Cong. Naz. Soc. Ital. Microbiol., 17, Padova, SocietItaliana di Microbiologia, Rome, Italy, pp. 833839.

Reed, R.H., Richardson, D.L., Wan, S.R.C., Stewart, W.D.P., 1984. Carbohydrateaccumulation and osmotic stress in cyanobacteria. J. Gen. Microbiol. 130, 14.

Reinehr, C.O., Costa, J.A.V., 2006. Repeated batch cultivation of the microalgaSpirulina platensis. World J. Microb. Biot. 22, 937943.

Schmidell, W., Lima, A.U., Aquarone, E., Borzani, W., 2001. Biotecnologia Industrial,vol.2. Edgard Blcher LTDA, So Paulo.

Shi, X.M., Chen, F., 1997. Stability of lutein under various storage conditions.Nahrung. 41, 3841.

Shi, X.M., Chen, F., Yuan, J.P., Chen, H., 1997. Heterotrophic production of lutein byselected Chlorella strains. J. Appl. Phycol. 9, 445450.

Tredici, M., Papuzzo, T., Tomaselli, L., 1986. Outdoor mass culture of Spirulinamaxima in sea-water. Appl. Microbiol. Biotechnol. 24, 4750.

Vonshak, A., 1997. Spirulina platensis (Arthrospira): Physiology, Cell biology andBiotechnology. Taylor and Francis Ltd., London.

Vonshak, A., Guy, R., Guy, M., 1988. The response of the lamentouscyanobacterium Spirulina platensis to salt stress. Arch. Microbiol. 150, 417420.

Vonshak, A., Kancharaska, N., Bunnag, B., Tanticharoen, M., 1996. Role of light andphotosynthesis on the acclimation process of the cyanobacterium Spirulinaplatensis to salinity stress. J. Appl. Phycol. 8, 119124.

Warr, S.R.C., Reed, R.H., Chudek, J.A., Foster, R., Stewart, W.D.P., 1985. Osmoticadjustment in Spirulina platensis. Planta 163, 424429.

Wu, B., Tseng, C.K., Xiang, W., 1993. Large scale cultivation of Spirulina in seawaterbased culture medium. Bot. Mar. 36, 99102.

Zarrouk, C., 1966. Contribution ltude dune cyanophyce. Inuence de diversfacteurs physiques et chimiques sur la croissance et photosynthse de Spirulinamaxima Geitler. Ph.D. Thesis. University of Paris.

J.T. Mary Leema et al. / Bioresource Technology 101 (2010) 92219227 9227

High value pigment production from Arthrospira (Spirulina) platensis cultured in seawaterIntroductionMethodsMicroorganism and growth conditionsKinetic parametersAnalytical methodsPigment analysisExtraction, quantification and purification of phycocyanin

Chlorophyll-a estimationLutein extraction and quantificationBeta carotene extraction and quantificationMorphological studiesStatistical analysis

Results and discussionEffect of different seawater media on growth parametersEffect of different seawater media on the pigmentsEffect of different seawater media on the final chemical composition of biomassEffect of different seawater media on the morphology of trichomes

ConclusionsAcknowledgementsSupplementary dataReferences