Bioresource Technology 98 (2007) 560���564 0960-8524/$ - see front matter �� 2006 Published by Elsevier Ltd. doi:10.1016/j.biortech.2006.02.007 EVect of salinity on growth of green alga Botryococcus braunii and its constituents A. Ranga Rao a, C. Dayananda a, R. Sarada a,��, T.R. Shamala b, G.A. Ravishankar a a Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 020, India b Food Microbiology Department, Central Food Technological Research Institute, Mysore 570 020, India Received 26 September 2005 received in revised form 23 January 2006 accepted 5 February 2006 Available online 19 June 2006 Abstract Growth of Botryococcus braunii (race ���A���) and production of its constituents viz, hydrocarbon, carbohydrate, fatty acid, and carote- noids were inXuenced by diVerent levels of salinity. Under salinity at 34 mM and 85 mM, 1.7���2.25-fold increase in the relative propor- tion of palmitic acid and two fold increase in oleic acid were observed. A twofold increase in carotenoid content was noticed at 85 mM salinity with lutein (75% of total carotenoid) as the major carotenoid followed by -carotene. The increase in biomass yields and changes in other constituents indicated the inXuence of salinity and the organism���s adaptability to the tested levels of salinity (17 mM to 85 mM). �� 2006 Published by Elsevier Ltd. Keywords: Botryococcus braunii Microalgae Hydrocarbon Biomass Salinity Carotenoids Carbohydrates Fatty acids 1. Introduction Botryococcus braunii is a green colonial fresh water microalga and is recognized as one of the renewable resource for the production of liquid hydrocarbons. B. braunii is classiWed into A, B and L races depending on the type of hydrocarbons synthesized. Race A produces C23 to C33 odd numbered n-alkadienes, mono-, tri-, tetra-, and pentaenes, which are derived from fatty acids (Metzger et al., 1990). Race B produces C30 to C37 unsaturated hydrocarbons known as botryococcenes and small amounts of methyl branched squalenes (Metzger and Lar- geau, 2005), whereas race L, produces a single tetraterpe- noid hydrocarbon known as lycopadiene (Metzger et al., 1990). Hydrocarbons extracted from the alga can be con- verted into fuel such as gasoline and diesel by catalytic cracking (Hillen et al., 1982). B. braunii (Races A and B) strains are also known to produce exopolysaccharides up to 250 g m��3, whereas L race produce up to 1 kg m��3 (Banerjee et al., 2002). However, the amount of exopoly- saccharides production varies with the strains and the culture conditions. Algae diVer in their adaptability to salinity and based on their tolerance extent they are grouped as halophilic (salt requiring for optimum growth) and halotolerant (having response mechanism that permits their existence in saline medium). In either case, the algae produce some metabolites to protect from salt injury and also to balance as per the surroundings osmotica (Richmond, 1986). Dunaliella, the unicellular green alga is an example for its ability to survive extreme salt stress and serve as a useful model to comprehend the strategies of cell response to high salt concentration. The present study focused on the adaptation of B. braunii (race A) to varied range of saline conditions and their eVect on the growth, hydrocarbon, carotenoid and carbohydrate production. * Corresponding author. Tel.: +91 821 2516 501 fax: +91 821 2517 233. E-mail address: pcbt@cftri.res.in (R. Sarada).

A.R. Rao et al. / Bioresource Technology 98 (2007) 560���564 561 2. Methods 2.1. Algal culture The strain Botryococcus braunii (LB 572) was obtained from University of Texas, USA. This organism was identiWed as race-A based on its hydrocarbon proWle. Stock cultures were maintained regularly on both liquid and agar slants of modiWed Chu 13 medium (Largeau et al., 1980). 2.2. Media and culture conditions A set of 500 ml Erlenmeyer conical Xasks were taken and 200 ml of modiWed Chu 13 medium was distributed and sodium chloride was added in the range of 17 mM to 85 mM to the Xasks and inoculated. Two weeks old culture of B. braunii LB 572 grown in modiWed Chu 13 was used as inoculum at 20% (v/v). The culture Xasks were incubated at 26 �� 1 ��C temperature under 1.2 �� 0.2 klux light intensity and 16:8 h light dark cycle. All the experiments were carried out in triplicates. 2.3. Analytical methods 2.3.1. Biomass, chlorophyll and carotenoid estimation The cultures were harvested by centrifugation at 5000 rpm and the cells were washed twice with distilled water. Then the pellet was freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight (g L��1). To estimate chlorophyll, a known volume of B. braunii culture was cen- trifuged and the residue was extracted with methanol repeatedly. The chlorophyll content in the pooled extract was estimated spectrophotometrically by recording absor- bance at 652 and 665 nm and quantiWed using the method of Lichtenthaler (1987). To estimate carotenoids, known amount of freeze dried algal biomass was extracted with acetone and absorbance was measured at 450 nm and the concentration of carotenoid was determined using Davies (1976) method. 2.3.1.1. HPLC analysis of carotenoids. The carotenoid pro- Wles of stress induced cultures were analysed by HPLC using a reversed phase C18 column with an isocratic solvent system consisting of acetonitrile/methanol/dichlo- romethane (70:10:20) at a Xow rate of 1.0ml/min and detected at 450 nm. Lutein, -carotene, violoxanthin and zeaxanthin were identiWed using authentic standards (Sigma). 2.3.2. Carbohydrate and protein estimation Known amount of cell free (spent) medium was analysed for total carbohydrate by phenol���sulphuric acid method (Dubois et al., 1956). Protein content in the cell free (spent) medium was analysed by Bradford protein assay (Zor and Selinger, 1996). 2.3.3. Hydrocarbon extraction and analysis The dry biomass was homogenised in mortar and pestle with n-hexane for 15 minutes and centrifuged. The extrac- tion process was repeated twice and supernatant was transferred to pre-weighed glass vial and evaporated under the stream of nitrogen to complete dryness. The quantity of residue was measured gravimetrically and expressed as dry weight percentage (Dayananda et al., 2005). The crude extracts were puriWed by column chromatography on silica gel with n-hexane as an eluent. The eluent was concentrated and analysed by GC using BP-5 capillary column as described by Dayananda et al. (2005). 2.3.4. Fatty acid analysis The lipids were extracted with chloroform���methanol (2:1) and quantiWed gravimetrically. The lipid sample was dissolved in benzene and 5% methanolic hydrogen chloride (95mL chilled methanol + 5 mL of acetyl chloride) was added and shaken well. The mixture was reXuxed for 2 h then 5% sodium chloride solution was added and the fatty acid methyl esters (FAME) were extracted with hexane. The hexane layer was washed with 2% potassium bicarbon- ate solution and dried over anhydrous sodium sulphate (Christie, 1982). FAME were analysed by GC���MS (Perkin- Elmer, Turbomass Gold, Mass Spectrometer) equipped with FID using SPB-1 (poly(dimethysiloxane)) capillary column (30m �� 0.32 mm ID �� 0.25 m Wlm thickness) with a temperature programming 130��C to 280 ��C at a rate of 3 ��C/min. The FAME were identiWed by comparing their fragmentation pattern with authentic standards (Sigma) and also with NIST library. 3. Results 3.1. EVect of sodium chloride on growth B. braunii was able to grow in all the tested concentra- tions of sodium chloride (17 mM to 85 mM). The biomass yields increased with increasing concentration of sodium chloride and maximum biomass was achieved in 17 mM and 34mM salinity (Fig. 1). The variation in pH from 8.5 to 9.5 observed during the experimental period was indepen- dent of sodium chloride concentration. It was evident from the data (Fig. 1) that the decrease in phosphate was due to its utilization by the alga, which was not inXuenced by salinity concentration. 3.2. EVect of salinity on metabolite production Hydrocarbon content in B. braunii was similar to growth pattern. The content varied in the range of 12��� 28% in diVerent salinities and maximum hydrocarbon content was observed in 51 mM and 68 mM of salinity (Fig. 2). The hydrocarbon proWle as analysed by GC