List of Publications

Page 163

List of publications

List of Publications

Page 164

List of publications

1. Dayananda, C., Venkatachalam, L., Bhagyalakshmi, N. and Ravishankar, G. A.

(2010). Assessment of genetic polymorphism among green microalgae

Botryococcus of distinct origin by RAPD. Genes, Genomes and Genomics. (In

press)

2. Dayananda, C., Kumudha, A., Sarada, R. and Ravishankar, G. A. (2010).

Isolation, characterization and outdoor cultivation of green microalgae

Botryococcus sp. Scientific Research and Essays. 5(17): 2497-2505

3. Dayananda, C., Sarada, R., Sila Bhattacharya and Ravishankar, G.A. (2005)

Effect of media and culture conditions on growth and Hydrocarbon production by

Botryococcus braunii. Process Biochemistry, 40: 3125–3131.

4. Dayananda, C., Sarada, R., Srinivas, P., Shamala, T.R. and Ravishankar, G.A.

(2006) Presence of methyl branched fatty acids and saturated hydrocarbons in

botryococcene producing strain of Botryococcus braunii. Acta Physiologiae

Plantarum, 28: 251-256.

5. Dayananda, C., Sarada, R., Usha Rani, M., Shamala, T.R and Ravishankar, G.A.

(2007) Autotrophic cultivation of Botryococcus braunii for the production of

hydrocarbons and exopolysaccharides in various media cultured in various media.

Biomass and Bioenergy, 31: 87-93.

6. Dayananda, C., Sarada, R., Shamala, T.R. and Ravishankar, G.A. (2006)

Influence of nitrogen sources on growth, hydrocarbon and fatty acid production

by Botryococcus braunii. Asian Journal of Plant Sciences, 5: 99-804.

7. Ranga Rao, A., Dayananda, C., Sarada, R., Shamala, T.R., Ravishankar, G.A.

(2006) Effect of salinity on growth of green alga Botryococcus braunii and its

constituents - hydrocarbons, fats, carbohydrates and carotenoids. Bioresource

Technology, 98: 560-564.

List of Publications

Page 165

Papers presented in symposia / conferences

1. Dayananda, C., Sarada R., Shamala, T. R. and Ravishankar, G.A.. Hydrocarbon

production by Botryococcus braunii under different nitrogen sources. Paper

presented at 44th

Annual Conference of Association of Microbiologists of India

held during 12-14th November 2003 at UAS, Dharwad, Karnataka.

2. Dayananda, C., Sarada R., Sila Bhattacharya, Usha Rani, M., Shamala, T.R. and

Ravishankar G. A. Optimization of culture conditions for growth and hydrocarbon

production from Botryococcus braunii. Paper presented at national symposium on

microalgal biotechnology held during 11- 13th

March, 2004 at Bharathidasan

University, Tiruchirapalli, Tamil Nadu.

3. Dayananda, C., Sarada, R., Shamala, T.R. and Ravishankar G. A. Botryococcus

the green alga as a source of hydrocarbons and polysaccharides. Paper presented

at 16th Indian convention of food scientists and technologists held during 9- 10

th

of December 2004 held at DFRL, Mysore.

4. Usha Rani, M., Dayananda, C., Sarada, R, Shamala, T.R, Ravishankar. G.A.

Optimized growth and recovery of hydrocarbon from Botryococcus braunii.

Paper presented at 5th International Food Convention held during 5 –8th

December 2003 at CFTRI, Mysore.

5. Dayananda , C., Sarada, R., Vinod Kumar, Shamala, T.R. and Ravishankar G. A.

Isolation and characterization of hydrocarbon rich green microalga Botryococcus

braunii occurring in fresh water bodies of south India. Oral presentation in 46th

annual conference of Association of microbiologists of India held during 8- 10th

December 2005 at Osmania University, Hyderabad, Andhra Pradesh. (Oral

presentations)

6. Dayananda, C., Ravishankar, G. A. and Sarada R. Fueling the Future:

Botryococcus braunii as a source of renewable energy. Oral presentation in

Second international conference on Environmental Sciences, held during 9-10th

July 2007 at Suez Canal University, Ismailia, Egypt. (Oral presentations)

Received: 24 March, 2010. Accepted: 14 May, 2010. Original Research Paper

Genes, Genomes and Genomics ©2010 Global Science Books

Assessment of Genetic Polymorphism among

Green Microalgae Botryococcus of Distinct Origin by RAPD

Dayananda Chandrappa • Venkatachalam Lakshmanan • Bhagyalakshmi Neelwarne • Ravishankar Gokare Aswathanarayana*

Department of Plant Cell Biotechnology, Central Food Technological Research Institute (CSIR), Mysore- 570 020, India

Corresponding author: * pcbt@cftri.res.in

ABSTRACT Genetic variation and relationships among 7 strains of Botryococcus sp. belonging to different geographical locations and different chemical races were evaluated using 35 decamer RAPD primers. Several RAPD primers were selected after preliminary screening for their ability to produce clear and multiple bands. The analyses resulted in the amplification of a total of 407 bands of 100-3000 bp, 380 of which were polymorphic, corresponding to 93.3% genetic diversity. The ability to distinguish genotypes and the Resolving power (Rp) of the primer showed a linear relationship. From these data, a genetic similarity matrix and dendrogram were obtained by using the unweighted pair group method with arithmetic means (UPGMA). The RAPD analysis produced genetic similarity coefficients ranging from 0.3312 to 0.7388. The study resulted in the identification of genetic relationship among various strains of Botryococcus sp. belonging to different climatic zones and origins. The study also revealed clear genetic distances between A race strains of B. braunii and B race strains of the same species. _____________________________________________________________________________________________________________ Keywords: Botryococcus sp., genetic diversity, polymorphism Abbreviations: PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA; Rp, Resolving power; SEM, scanning electron microscopy; UPGMA, unweighted pair group method with arithmetic mean INTRODUCTION Botryococcus is a colonial unicellular green microalgae re-cognized as a potential source of lipids and liquid hydrocar-bons. Botryococcus sp. were found to be present in geogra-phical regions belonging to different climatic zones like continental, temperate, tropical and alpine, that indicates their cosmopolitan nature (Wolf et al. 1985; Metzger et al. 1985; Okada et al. 2000). In addition, fossil studies re-vealed that Botryococcus sp. are the major hydrocarbon sources in a variety of oil-rich deposits across the world and are dated from Ordovician period to the present (Cane 1977). Botryococcus sp. were also reported to produce bio-active carotenoids like β-carotene, lutein, violaxanthin, echinenone, botryoxanthin-A, botryoxanthin-B, and α-botryoxanthin-A and many other such bioactive molecules of importance in pharmaceutical and nutraceutical applica-tions. The algae B. braunii is also known to produce large amounts (5-42% on dry weight) of lipids (Metzger and Lar-geau 2005), hydrocarbons (2-76%, w/w) and considerable amounts of exopolysaccharides (0.25-5.5 kg.m-3) (Casa-devall et al. 1985; Metzger et al. 1985; Fernandes et al. 1989; Allard and Casadevall 1990; Sawayama et al. 1994; Metzger and Largeau 2005; Dayananda et al. 2007; Ranga Rao et al. 2007; Eroglu and Melis 2010).

Taking these facts into consideration, the organism Botryococcus is gaining importance as a potential source from food to biofuel. There are more than 13 species of the genus Botryococcus reported to date, and among them B. braunii is being characterized and worked out in detail. This is may be because of their ability to produce large amounts of lipids and liquid hydrocarbons. B. braunii is classified into A, B and L races based on the type of hydro-carbons produced (Metzger and Largeau 2005). Race – A strains produce C21 to C33 odd numbered n-alkadienes, mono-, tri-, tetra-, and pentaenes and they are derived from fatty acids (Metzger and Largeau 2005). The L race strains

produce a single tetraterpene hydrocarbon known as lycopa-diene (C40-C78). The B race strains produce two types of tri-terpenes called botryococcenes of C30-C37 of general for-mula CnH2n-10 as major hydrocarbons and small amounts of methyl branched squalene (Achitouv et al. 2004). Therefore there is an increasing quest to isolate newer species and strains of the genus Botryococcus which are capable to pro-duce lipids and other chemicals of industrial importance. Morphological heterogeneity and the chemical nature of hydrocarbons, and the degree of lipid and polysaccharide production were found to exist within the species and among the other species of the genus Botryococcus.

Therefore the present study focused on finding genetic polymorphism in different species of the genus Botryococ-cus as well as within the species belonging to different che-mical races of B. brauni using RAPD (random amplified polymorphic DNA) technique. RAPD technique is being used widely as an efficient technique in detecting genetic variations (Williams et al. 1990) even in closely related organisms such as two near isogenic lines (NIL). At present, RAPD markers have been successfully applied to detect the genetic similarities or dissimilarities in various plants, algae, fungi and bacteria, etc. (Carvalho et al. 2004; Comeau et al. 2004; Martins et al. 2004; Ramage et al. 2004; Modgil et al. 2005; Martinez et al. 2006; Touzet et al. 2007; Zhao et al. 2007, 2008; Olmos et al. 2009; Small et al. 2009; Tilman et al. 2009). RAPD only requires a small amount of DNA and is simpler, cheaper and faster. Therefore, the study was em-ployed here to differentiate the genetic variations among the different chemical races of B. braunii and between the indi-genous strains of Botryococcus.

®

Genes, Genomes and Genomics 4 (Special Issue 1), x-y ©2010 Global Science Books

MATERIALS AND METHODS Algal strains Botryococcus braunii (LB 572) was procured from the University of Texas, U.S.A. Botryococcus braunii (SAG 30.81) from the Sammlung von AlgenKulturen, pflanzenphysiologisches Institut, Universitat Gottingen, Germany. Botryococcus braunii (Strain 1) and Botryococcus braunii (Strain 2) from university of Berkeley, UK. Botryococcus sp. (DB-8) was obtained from the University of Pune, Pune, India and Botryococcus sp. (MCRC) from Murugappa Chattiar Research Centre, Chennai, India. Botryococcus sp. (CFTRI) was also used in this study. Stock cultures were main-tained routinely on both liquid and agar slants of modified Chu 13 medium (Largeau et al. 1980) by regular sub culturing at two-week intervals. Cultures were maintained at 25 ± 1°C with 1.2 ± 0.2 klux and a 16: 8 light: dark cycle. Scanning electron microscopy The algal cells were observed under scanning electron microscopy (SEM) according to the method of Fowke et al. for cellular details (Fowke et al. 1994). The samples were fixed in 2% glutaraldehyde in 0.2 M phosphate buffer (pH 6.8) for 12 hrs, dried in alcohol series up to 100%, sputter coated with gold and examined in a LEO Scanning Electron Microscope 435 VP (Leo Electron Mic-roscopy Ltd. Cambridge UK). Preparation of template DNA The genomic DNA from the two weeks grown cultures was extrac-ted by using the GenEluteTM Plant Genomic DNA Mini prep kit supplied by Sigma (USA). RNA contamination in all the samples

were removed using RNase-A by following the manufacturers protocol (100 μg mL-1; Bangalore Genei, India) for 30 min at 37°C. The quality and quantity of DNA were analyzed by standard spec-trophotometry and the samples were diluted to 25 ng μL-1. DNA amplification RAPD amplifications were performed using PCR mixture (25 μl) having 1 μl of genomic DNA as template, 1X PCR buffer, 200 μM dNTPs, 1 unit (U) of Taq DNA polymerase and 1 μM of each primer (Table 2) with an initial denaturation at 93ºC for 4 min followed by 40 cycles of 1 min denaturation at 94°C, 1 min annealing at 36°C and 2 min extension at 72°C with a terminal extension of 72°C for 10 min using a thermal cycler (Eppendorf Thermal cycler). The amplified fragments were eletrophoretically separated in 2% agarose gels in TAE and stained in ethidium bromide (0.001%) and documented in a gel documentation system (Hero-Lab GMBH, Germany). The size of the amplification prod-ucts was estimated from a 10-kb DNA ladder (Fermentas GMBH, Germany).

Table 1 Botryococcus strains used for the study. Algal strains Botryococcus brauni [Strain 1] Botryococcus brauni [Strain 2] Botryococcus brauni [LB-572] Botryococcus brauni [SAG 30.81] Botryococcus sp. [DB-8] Botryococcus sp. [MCRC] Botryococcus sp.[CFTRI]

Table 2 List of RAPD primers selected from Operon Technologies. Primer Primer sequence (5’_ 3’) TNB NPB %PB Rp AFS OPA 03 AGTCAGCCAC 15 15 100 7.71 250-3000 OPA 04 AATCGGGCTG 14 11 78.57 5.14 200-3000 OPA 09 GGGTAACGCC 16 14 87.50 7.14 250-2200 OPA 11 CAATCGCCGT 13 12 92.31 7.14 200-2500 OPC 06 GAACGGACTC 12 11 91.66 6.85 200-1500 OPC 08 TGGACCGGTG 11 11 100 6.28 200-1500 OPC 13 AAGCCTCGTC 11 11 100 5.43 350-1500 OPD 07 TTGGCACGGG 15 15 100 8.86 250-2000 OPD 08 GTGTGCCCCA 12 11 91.66 7.71 250-1800 OPD 11 AGCGCCATTG 13 9 69.23 5.14 250-3000 OPD 16 AGGGCGTAAG 11 10 90.91 6.29 400-2500 OPB 07 GGTGACGCGA 12 12 100 7.71 100-1500 OPC 03 GGGGGTCTTT 14 9 64.28 6.28 300-3000 OPF 12 ACGGTACCAG 14 11 78.57 6.00 300-3000 OPM 20 AGGTCTTGGG 11 11 100 6.57 400-2000 OPA 20 GTTGCGATCC 11 11 100 7.14 400-2500 OPJ 10 AAGCCCGAGG 13 13 100 6.86 200-2500 OPN 09 TGCCGGCTTG 12 11 91.67 7.14 200- 2000 OPN 10 ACAACTGGGG 10 9 90 5.71 350-1500 OPN 14 TCGTGCGGGT 5 5 100 3.43 700-3500 OPC 01 TTCGAGCCAG 11 11 100 7.43 200-2500 OPC 05 GATGACCGCC 10 10 100 5.71 350-1500 OPJ 18 TGGTCGCAGA 9 9 100 5.14 400-2000 OPJ 20 AAGCGGCCTC 10 10 100 6.00 250-1500 OPJ 09 TGAGCCTCAC 9 9 100 6.00 400-2000 OPJ 01 CCCGGCATAA 11 9 81.81 6.57 300-1300 OPJ 11 GTCCCGTGGT 13 13 100 8.29 250-2000 OPJ 19 GGACACCACT 11 10 90.91 5.71 200-1500 OPJ 13 CCACACTACC 10 10 100 6.00 200-2500 OPC 02 GTGAGGCGTC 11 11 100 6.57 200-2500 OPN 06 GAGACGCACA 12 12 100 4.29 400-2500 OPC 9 CTCACCGTCC 9 8 88.88 3.71 200-1200 OPJ 11 ACTCCTGCGA 14 14 100 8.29 400- 2500 OPN 4 GAGACGCACA 10 10 100 4.85 400-1500 OPC 7 GTCCCGACGA 12 12 100 8.85 300-3000 Total 407 380 93.3 223.94

Total number of bands (TNB), number of polymorphic bands (NPB), percentage of polymorphic bands (%PB), resolving power (Rp) and amplified fragment size (AFS) obtained per RAPD primer

Genetic polymorphism among Botryococcus sp. Dayananda et al.

Resolving power (Rp) Resolving power (Rp) for each primer was calculated following the method of Prevost and Wilkinson’s (1999) for selecting pri-mers that can distinguish a maximal number of accessions. Resol-ving power (Rp) of a primer is = Ib where Ib (band informative-ness) takes the value of: Ib = 1 - [2 x |0.5 - p|], p being the propor-tion of the 7 genotypes (algal strains analyzed) containing the bands. Data analysis The well-resolved RAPD fragments ranging from 100 to 3000 bp were scored as present (1) or absent (0) for each primer analysis. Bands with the same migration distance were considered homo-logous. The data was computed and analyzed with NTSYS pc (Rohlf 1998) version 2.02 using the simple matching coefficient (Sokal and Michener 1958). Cluster analyses were obtained based on similarity matrices, using the unweighted pair group method with arithmetic mean (UPGMA), and relationships between acces-sions were visualized as dendrograms. RESULTS AND DISCUSSION Selection of primers and RAPD analysis Three indigenous strains of Botryococcus and four strains, two each of race-A and race-B were subjected for genetic analysis using RAPD markers (Table 1). Several decamer primers were screened for their ability to amplify DNA fragments. Based on the results of their ability to produce good number of distinct bands, 35 RAPD primers were selected. DNA samples from all the 7 strains of Botryococ-cus were amplified using the decamers listed in Table 2, where all the primers produced polymorphic bands. The majority of band positions varied between the strains. The polymorphisms were scored visually based on the presence or absence of amplified fragments. All the primers used in the study produced large numbers of polymorphic bands (Table 2). The total number of bands (TNB), number of polymorphic bands (NPB), percentage of polymorphic bands (%PB), Resolving power (Rp), amplified fragment size (AFS) obtained for each primer are detailed in Table 2. The total amplified products of 35 RAPD primers was 407 (average of 11.62 bands per primer) ranging from 100 to 3000 bp, of which 380 were polymorphic (93.3%). The number of bands for each RAPD primer varied from 5 (OPN 14) to 16 (OPA-09). The resolving power of the 35 RAPD primers ranged from 3.43 for primer OPN-14 to 8.86 for primer OPD-07 and the samples of RAPD analysis are visualized in Fig. 1. Molecular analysis and fingerprinting of Botryococcus strains The genetic similarity coefficients for 7 strains of Botryo-coccus species were obtained with RAPD markers ranged from 0.3312 to 0.7388 between the strains evaluated. The unweighted pair group method with arithmetic mean (UPGMA) analysis made it possible to discriminate all the genotypes of Botryococcus sp. used in this study. The den-drogram obtained based on RAPD data showed a clear distinction into two major clusters (Fig. 2). The dendrogram obtained using percent disagreement coefficient (Table 3) showed the presence of 2 main clusters (Cluster 1 and 2, having 2 and 5 strains in each, respectively). The cluster 1 had two strains (Strains 1 and 2) which belong to B race, while the A race strains LB -572 and SAG 30.81 formed a sub-cluster A1 in cluster 2 and the other 3 indigenous strains (CFTRI, MCRC and DB-8)formed an another sub cluster A2 of cluster 2.

The similarity coefficient matrix (Table 3) and the dendrogram data have shown clear distinction amomg all the strains of Botryococcus species studied and have also revealed a genetic relationship between the race A and race B strains. All the indigenous strains have shown their gene-

Fig. 1 RAPD profile of DNA from 7 strains of Botryococcus. Primers OPC-07 (A), OPC-09 (B) and OPN-04 (C). Lane marker represents 10 kbp GeneRuler DNA ladder.

Fig. 2 Dendrogram displaying the genetic distances among Botryo-coccus strains obtained from cluster analysis of RAPD data.

Genes, Genomes and Genomics 4 (Special Issue 1), x-y ©2010 Global Science Books

tic relationship with A race strains and among the indige-nous strains, Botryococcus sp. (DB-8) has shown its gene-tic diversity with that of the other two indigenous strains (MCRC and CFTRI). Morphological details of 7 strains of Botryococcus species (Fig. 3) shows that the features of A race strains, B race strains and indigenous strains were found to substantiate the observations of RAPD data as well. Gomez and Gonzalez (2004) have also used RAPD tech-nique to see the genetic variations among the several strains of microalgae Dunaliella salina. The detection of genetic variations of strains with industrial prospective has a great relevance in applied phycology because it allows the dif-ferentiation of phenotypic variation into environmental and genetic components. In addition genetic diversity studies will also give the details of exotic genotypes. RAPD has been successfully used to identify the genetic variation in both micro- and macroalgae (Neilan 1995; Nishihara et al.

1997; Bolch et al. 1999a, 1999b; Gomez and Gonzalez 2004; Martínez et al. 2006; Touzet et al. 2007; Zhao et al. 2007, 2008; Olmos et al. 2009; Small et al. 2009; Tilman et al. 2009).

The genetic diversity among the different strains of Botryococcus is a clear demonstration of the genome parti-cipation in determining the attributes related to production of different nature of hydrocarbons and morphological fea-tures. The results of the study are very important since the existence of genetically characterized strains will reduce incorrect assumptions about the biotechnological important traits of different strains, which could be due to phenotypic flexibility.

In conclusion, RAPD markers having high resolution power appear to offer many advantages in establishing genetic distances among the microalgae. They were found to be effective for assessing genetic variation in different species and strains of Botryococcus belong to various geo-graphic locations and climatic zones as well. This study has revealed a molecular classification of Botryococcus strains of different chemotypes and between two strains belonging to different climatic zones and different geographical loca-tions. Thus diversity analysis by RAPD technique effici-ently discriminate phenotypic and environmentally acquired characteristics. RAPD primers have rarely been applied for establishing diversity of algal forms. ACKNOWLEDGEMENTS The author DC thanks Council of Scientific and Industrial Re-search, New Delhi, for Senior Research fellowship. The authors thank Prof. Joseph Chappel, University of Kentucky and Dr. Amy Lau, University of Berkeley., Director of MCRC, Chennai, Dr. Aditi Pant, Emeritus professor, Department of Botany, University of Pune for providing Botryococcus cultures. We express our sincere thanks to Prof. Jiri Komarek, Institute of Botany, Trebon, Czech Republic for providing scientific literature. REFERENCES Achitouv E, Metzger P, Rager MN, Largeau C (2004) C31–C34 methylated

squalenes from a Bolivian strain of Botryococcus braunii. Phytochemistry 65, 3159-3165

Allard B, Casadevall E (1990) Carbohydrate composition and characterization of sugars from green microalga Botryococcus braunii. Phytochemistry 29, 1875-1878

Bolch CJS, Blackburn SJ, Hallegraeff GM, Vaillancourt RE (1999b) Gene-tic variation among strains of the toxic dinoflagellate Gymnodinium cate-natum (Dinophyceae). Journal of Phycology 35, 356-367

Bolch CJS, Orr P, Jones GJ, Blackburn SJ (1999a) Genetic, morphological and toxicological variation among globally distributed strains of Nodularia (Cyanobacteria). Journal of Phycology 35, 339-355

Cane RF (1977) Coorongite, balkashite and related substances- an annotated bibliography. Transactions of the Royal Society of South Australia 101, 153

Carvalho LC, Goulão L, Oliveira C, Gonçalves JC, Amâncio S (2004) RAPD assessment for identification of clonal identity and genetic stability of in vitro propagated chestnut hybrids. Plant Cell, Tissue and Organ Culture 77, 23-27

Casadevall E, Dif D, Largeau C, Gudin C, Chaumont D, Desanti O (1985) Studies on batch and continuous cultures of Botryococcus braunii: hydrocar-bon production in relation to physiological state, cell structure, and phosphate nutrition. Biotechnology and Bioengineering 27, 286-295

Comeau AM, Short S, Suttle CA (2004) The use of degenerate-primed ran-dom amplification of polymorphic DNA (DP-RAPD) for strain-typing and inferring the genetic similarity among closely related viruses. Journal of Virological Methods 118, 95-100

Table 3 Genetic similarity coefficient among the 7 strains of Botryococcus species based on RAPD analysis. Strains Strain 1 Strain 2 SAG-0.81 LB-572 CFTRI MCRC DB-8 Strain 1 1 Strain 2 0.7388 1 SAG-30.81 0.3846 0.3806 1 LB-572 0.4359 0.3918 0.5254 1 CFTRI 0.3896 0.3766 0.4333 0.4481 1 MCRC 0.3980 0.3940 0.4474 0.4430 0.4947 1 DB-8 0.3485 0.3312 0.3595 0.3628 0.4256 0.4765 1

Fig. 3 Scanning electron microscopic images of different strains of Botryococcus sp. used for RAPD analysis.

Genetic polymorphism among Botryococcus sp. Dayananda et al.

Dayananda C, Sarada R, Usha Rani M, Shamala TR, Ravishankar GA (2007) Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass and Bio-energy 31, 87-93

Eroglu E, Melis A (2010) Extracellular terpenoid hydrocarbon extraction and quantitation from the green microalgae Botryococcus braunii var. Showa. Bioresource Technology 101, 2359-2366

Fernandes HL, Tome MM, Lupi F, Filho AM, Sá-Correia I, Novais JM (1989) Biosynthesis of high concentrations of an exopolysaccharides during the cultivation of the microalga Botryococcus braunii. Biotechnology Letters 11, 433-436

Fowke LC, Stephen MA, Pat JR (1994) Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss). Plant Cell Reports 13, 612-618

Gomez PI, Gonzalez MA (2004) Genetic variation among seven strains of Dunaliella salina (Chlorophyta) with industrial potential, based on RAPD banding patterns and on nuclear ITS rDNA sequences. Aquaculture 233, 149-162

Largeau C, Caradevall E, Berkaloff C, Dhamliencourt P (1980) Sites of accumulation and composition of hydrocarbons in Botryococcus braunii. Phytochemistry 19, 1043-1051

Martínez R, Anibarro C, Fernández S (2006) Genetic variability among Alexandrium tamarense and Alexandrium minutum strains studied by RAPD banding pattern analysis. Harmful Algae 5, 599-607

Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropro-pagated almond plantlets as assessed by RAPD and ISSR markers. Plant Cell Reports 23, 492-496

Metzger P, Berkaloff C, Caradevall E, Coute A (1985) Alkadiene and botryo-coccene producing races of wild strains of Botryococcus braunii. Phytoche-mistry 24, 2305-2312

Metzger P, Largeau C (2005) Botryococcus braunii: a rich source for hydro-carbons and related ether lipids. Applied Microbiology and Biotechnology 66, 486-496

Modgil M, Mahajan K, Chakrabarti SK, Sharma DR, Sobti RC (2005) Molecular analysis of genetic stability in micropropagated apple rootstock MM106. Scientia Horticulturae 104, 151-160

Neilan BA (1995) Identification and phylogenetic analysis of toxigenic cyano-bacteria by multiplex randomly amplified polymorphic DNA PCR. Applied and Environmental Microbiology 61, 2286-2291

Nishihara H, Mina H, Watanabe M, Nagashima M, Yagi O, Takamura Y (1997) Random amplified polymorphic DNA (RAPD) analysis for discrimi-

nating genotypes of Microcystis cyanobacteria. Bioscience, Biotechnology and Biochemistry 61, 1067-1072

Okada S, Devarenne TP, Chapell J (2000) Molecular characterization of squa-lene synthase from the green microalga Botryococcus braunii, race B. Ar-chives of Biochemistry and Biophysics 373, 307-317

Olmos J, Ochoa L, Paniagua-Michel J, Contreras R (2009) DNA fingerprin-ting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world. Saline Systems 5, 5

Ramage CM, Borda AM, Hamill SD, Smith MK (2004) A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish banana (Musa spp. AAA). Scientia Horticulturae 103, 145-151

Ranga Rao A, Dayananda C, Sarada R, Shamala TR, Ravishankar GA (2007) Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresource Technology 98, 560-564

Rohlf FJ (1998) NTSYS-pc. Numerical taxonomy and multi-variate analysis system, Version 2.02, Exeter software. State University of New York at Stony Brook, New York

Sawayama S, Inoue S, Yokoyama S (1994) Continuous culture of hydrocar-bon-rich microalga Botryococcus braunii in secondarily treated sewage. Ap-plied Microbiology and Biotechnology 41, 729-731

Small HJ, Shields JD, Haas LW, Vogelbein WK, Moss J, Reece KS (2009) Genetic variation among strains of Pseudopfiesteria shumwayae and Pfies-teria piscicida (Dinophyceae). Journal of Phycology 45, 1315-1322

Sokal RR, Michener CD (1958) A statistical method for evaluating systematic relationships. The University of Kansas Science Bulletin 38, 1409-1438

Tilman JA, Urban T, Bank B, Allan DC, Uwe J (2009) Phenotypic variation and genotypic diversity in a planktonic population of the toxigenic marine dinoflagellate Alexandrium tamarense (Dinophyceae). Journal of Phycology 46, 18-32

Touzet N, France JM, Raine R (2007) Characterization of nontoxic and toxin-producing strains of Alexandrium minutum (Dinophyceae) in Irish coastal waters. Applied and Environmental Microbiology 73, 3333-3342

Wolf FR, Nanomura AM, Bassham JA (1985) Growth and branched hydrocarbon production in a strain of Botryococcus braunii. Journal of Phy-cology 21, 388-396

Zhao F, Wang X, Liu J, Duan D (2007) Population genetic structure of Sar-gassum thunbergii (Fucales, Phaeophyta) detected by RAPD and ISSR mar-kers. Journal of Applied Phycology 19, 409-416

Zhao F, Liu F, Liu J, Ang Jr. PO, Duan D (2008) Genetic structure analysis of natural Sargassum muticum (Fucales, Phaeophyta) populations using RAPD and ISSR markers. Journal of Applied Phycology 20, 191-198

Scientific Research and Essays Vol. 5(17), pp. 2497-2505, 4 September, 2010 Available online at http://www.academicjournals.org/SRE ISSN 1992-2248 ©2010 Academic Journals

Full Length Research Paper

Isolation, characterization and outdoor cultivation of green microalgae Botryococcus sp.

C. Dayananda, A. Kumudha, R. Sarada and G. A. Ravishankar*

Department of Plant Cell Biotechnology, Central Food Technological Research Institute [Constituent laboratory of CSIR

(Council of Scientific and Industrial Research)], Mysore- 570 020, India.

Accepted 2 July, 2010

Samples of the green, colonial, unicellular microalgae Botryococcus sp. were collected from freshwater ponds in Mahabalipuram, Tamil Nadu, India. Specimens were isolated and examined for morphological features using microscopic and scanning electron microscopic images and was found to be Botryococcus sp. The hydrocarbon analysis of its hexane extracts showed hexadecane (10.15%), heptadecane (17.82%) and pentacosane (18.74%) as its major hydrocarbons. Further, the 18S rRNA sequence (GU182893.1) analysis has confirmed its taxonomical relationship to the order Trebouxiophyceae and has shown similarities with the reported species of the genus Botryococcus and in particular to Botryococcus braunii. Based on morphological features and 18S rRNA sequence analysis, the Indian isolate was designated as Botryococcus mahabali. Biomass analysis of B. mahabali showed 19% proteins, 18% carbohydrates and 14% lipid. It was found that its oil comprised mainly of hexadecadienoic acid (16:2), oleic (18:1), linoleic (18:2), and linolenic acids (18:3) as its major fatty acids. Oleic acid is recognized to be the major fatty acid in most of the reported species of the genus Botryococcus and thus it can serve as another significant chemical signature for the Indian isolate. Its pigment profile exhibited lutein (41.57%) and �-carotene (37.96%) as major carotenoids. In view of its chemical profile, the algae was scaled up in open air raceway ponds in batch mode and the biomass yields were found to be 2 g/L (w/w) up on two weeks growth in outdoor raceway ponds. In conclusion, the results of the study reveal that an Indian isolate B. mahabali can be of relevance for its prospective applications from food to biodiesel feed stock. Key words: Carotenoids, fatty acids, hydrocarbons, lipids, lutein, mass cultivation, raceway pond.

INTRODUCTION There is an increasing quest all over the world for the exploration and exploitation of potential microalgae for various industrial applications from nutraceutical to bio-diesel feed stock. The green colonial lipid rich microalgae Botryococcus is predominant in freshwater, brackish lakes, reservoirs and ponds (Metzger and Largeau, 2005). Botryococcus is characterized by its ability to synthesize and accumulate very high levels of lipids. These lipid substances include numerous hydro-carbons, that is, highly reduced compounds comprising only *Corresponding author. E-mail: pcbt@cftri.res.in. Abbreviations: GC-MS, Gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; rRNA, ribosomal RNA; SEM, scanning electron microscope.

carbon and hydrogen as elements (Brown et al., 1969). Being a photosynthetic organism, it has been reported to reduce CO2 emissions by 1.5 × 105 tons/yr/8.4 × 103 ha (Sawayama et al., 1999) and thus it offers an eco-friendly process for production of lipid and other bioactive compounds along with its carbon dioxide mitigation credits.

The existence of Botryococcus sp. in USA, Ivory Coast, Portugal, Bolivia, Morocco, India, Philippines, Thailand, France and West Indies has confirmed its wide distribution (Chandra, 1964; Metzger et al., 1985; Wolf et al., 1985; Okada et al., 2000; Dayananda et al., 2010). Further, these geographical regions belong to different climatic zones like continental, temperate, tropical and alpine, indicating its ability to grow in varied climatic conditions (Tyson, 1995). Because of its potential to produce large amounts of lipids and hydrocarbons,

2498 Sci. Res. Essays exploration for newer strains and species of the genus Botryococcus is increasing day by day and to date more than 60 Botryococcus strains were cultivated in laboratory (For a non-exhaustive list, see Metzger and Largeau, 1999) and the exploration is still on.

Most of the Botryococcus strains cultivated in labora-tory and wild samples collected from lakes are analyzed for their lipid production (Metzger and Largeau, 1999) and are reported to produce 5 - 42% of lipids and 0.1 - 61.0% hydrocarbons on their dry weight (Metzger and Largeau, 1999, 2005; Dayananda et al., 2007a; Ranga Rao et al., 2007; Hai-Linh et al., 2009; Ela and Anastasios, 2010; Chiara et al., 2010). Therefore, the exploration for a suitable strain for biotechnological exploitation for lipid production is on in various algal research laboratories and industries of various countries across the world. However, there were no reports available regarding the mass cultivation of Botryococcus sp. either in raceway or in any other type of mass cultivation systems. In view of its importance, the present study aims to isolate the indigenous species of Botryococcus for production of lipids and other bioactive compounds of commercial importance and an attempt was also made to scale up in open air raceway ponds. MATERIALS AND METHODS Isolation and purification The algal samples were collected from the blooms in freshwater ponds of Mahabalipuram (Latitude 12°37' in the North to Longitude 80°14' in the East.) Tamil Nadu, India. The samples were cultured in modified Chu 13 medium and subjected to purification by serial dilution. The individual colonies were microscopically observed for their colonial and morphological features. Pure culture was obtained from single colony and the culture was established in both liquid and agar slants of modified Chu 13 medium, incubated at 25 ± 1°C under 1.2 ± 0.2 klux irradiance with 16:8 h light dark cycle. The purity of the culture was ensured by repeated plating and by regular observation under microscope. The Indian isolate was analyzed for its 18S rRNA sequence along with hydrocarbons, carotenoids and fatty acids profiles. Light microscopy and scanning electron microscopy The algal cells were observed under light microscope for their morphological features and for other cellular details, the cells were further studied using scanning electron microscope (SEM) according to the method of Fowke et al. (1994). The samples were fixed in 2% glutaraldehyde in 0.2 M phosphate buffer (pH 6.8) for 12 h, dried in alcohol series up to 100%, sputter coated with gold and examined in a LEO Scanning Electron Microscope 435 VP (Leo Electron Microscopy Ltd. Cambridge UK). Hydrocarbon extraction and analysis by GC-MS Hydrocarbons were extracted by homogenizing the dry biomass with n-hexane for 30 min intermittently. Supernatant recovered was evaporated to complete dryness under the stream of nitrogen. Hydrocarbon content was measured gravimetrically and expressed

as percentage dry weight (Sawayama et al., 1999). The hydro-carbon samples were analyzed on SPB-5 column (30 m × 0.32 mm ID × 0.25 µm film thickness) using GCMS equipped with FID and were identified by comparing their fragmentation pattern with standards (Sigma chemicals USA) and with NIST library (Dayananda et al., 2005). Total Lipid estimation and fatty acid analysis Total lipids were extracted with chloroform-methanol (2:1) and quantified gravimetrically. The fatty acid methyl esters (FAME) were prepared by following the procedure of Christie (1982). FAME were analyzed by GC-MS (PerkinElmer, Turbomass Gold, Mass spectrometer) equipped with FID using SPB-1 (poly (dimethysilo-xane)) capillary column (30 m × 0.32 mm ID × 0.25 µm film thickness) with a temperature programming of 150°C(3’) to 280°C (5’) at a rate of 5°C/min. The FAMEs were identified by comparing their fragmentation pattern with authentic standards (Sigma) and with NIST library. HPLC analysis of carotenoids The acetone extract of the alga Botryococcus mahabali was analyzed by HPLC using a reversed phase C18 column (4.5 cm × 150 mm) with an isocratic solvent system consisting of acetonitrile/ methanol/dichloromethane (7:1:2) at a flow rate of 1.0 ml/min and the compounds were detected at 450 nm. Lutein, �-carotene were identified using standards (Dayananda et al., 2007b). Extraction of genomic DNA and 18S rRNA amplification Genomic DNA was extracted from the lyophilized algal biomass using the GenElute™ Plant Genomic DNA Mini prep kit (Sigma, St. Louis, USA). The RNA contamination was removed by digesting the extract with 10 �g of RNase-A (Bangalore Genei, Bangalore, India) for 30 min at 37°C. Quality and quantity of DNA preparations were checked by standard spectrophotometry and the samples were diluted to a concentration of 25 ng/µl and used for PCR reactions. The 18S rRNA gene specific primers for the Botryococcus sp. were designed from the reported sequences at NCBI data base, 5’-CTGTGAAACTGCGAATGGC-3’as FP and 5’CTCCAATCCCTAGTCGGCATCG-3’ as RP. PCR reaction was performed in thermo-cycler using a PCR programme with 4 min initial denaturation at 94ºC and 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 52ºC and 30 s at 72ºC for extension with final extension at 72ºC for 10 min. The PCR products were separated on agarose gels and stained with ethidium bromide and the gels were documented with a Hero Lab gel documentation system. The PCR was performed at least thrice to check the reproducibility and then the PCR product was purified by the purification kit supplied by Sigma and was again checked by gel electrophoresis. The PCR product was cloned in T/A cloning vector (kit supplied by Invitrogen) and was sequenced after confirmation by both PCR and restriction digestion. The sequence was submitted to NCBI data base and was accorded with an accession number GU182893.1. Acclimatization of Botryococcus to outdoor conditions B. mahabali established in agar plates were inoculated into 150-ml Erlenmeyer flasks and incubated for two weeks at 25 ± 1°C under 1.2 ± 0.2 klux light intensity with 16:8 h light dark photoperiod. The cultures were sub-cultured at an intervals of two weeks and such sub-culturing were done at least ten times prior to its scale up in

500, 1000 and 2000-ml Erlenmeyer flasks in modified Chu 13 medium.

The seed cultures were exposed to open air environments in Corbouy of 10 - 20 L capacity for 4 - 8 weeks. And then cultures were inoculated into glass tanks of size (26 cm height × 76 cm long × 26 cm wide) with a culture holding capacities of 10 - 15 L. The algae in glass tanks were subjected for adaptation to open air condition for more than 4 cycles (sub-culturing at an intervals of two weeks) at ambient temperature and were covered with transparent glass plates having four to five holes of size 1 - 2 cm to facilitate aeration and to avoid condensation. Cultures were observed under microscope periodically for any possible contaminants. Algal cultures in carbouys and glass tanks were used as starter cultures for open air circular (cultures in circular tanks were mixed twice a day) ponds which in turn served as a starter culture for 1000 L capacity raceway pond. Cultivation in raceway pond Modified Chu 13 media was prepared by using potable water supplied by CFTRI water facility, and the pH was tentatively adjusted to 7.0 - 7.5. The culture was inoculated at 30 - 35% (v/v) in to raceway pond of 1000 L capacity and the volume was made up to 800 ± 5 L. The paddle wheel was set to 15 rpm to provide the aeration from 10 am to 5 pm daily. Light irradiance, pH, chlorophyll, carotenoids and biomass yields were recorded on daily basis. Periodically cells were observed under microscope for any possible contamination. Strainer was used to remove any dust and particulate matters from raceway tanks. Cultures were monitored for their growth for two weeks in outdoor conditions and the biomass was harvested by online centrifugation and lyophilized biomass was analyzed for its chlorophyll, carotenoids and lipid yields. Biomass estimation The known volume of cultures was harvested by centrifugation at 5000 rpm for 5 min and the pellet was washed at least twice with distilled water and freeze dried. The dry weight of algal biomass was determined gravimetrically and growth was expressed in terms of dry weight gram per liter. Harvesting of algal biomass The algal culture in raceway tank was allowed to settle by density for 2 - 3 h and then the upper clear algal medium was removed. The biomass settled at the bottom was collected and was fed to bowl centrifuge used at a speed of 5000 rpm (M/s West Folia, Germany). Thirty liter of algal culture was manually fed at a time to the centrifuge. The rotor speed was 5000 rpm with flow of the culture adjusted to 5 L/h. Biomass collected in a cone shaped rotor was recovered, lyophilized and stored at -20°C for further use. Freeze-drying Algal biomass was spread uniformly in a stainless steel tray and was lyophilized using a freeze drier (Model-10XB, Lyophylization Systems Inc. USA). The freeze-dried samples were analyzed for chlorophyll, carotenoids and lipid yields. Chlorophyll and carotenoids estimation A known volume of culture was centrifuged (5000 rpm) for 5 min and the pellet was treated with known volume of methanol (1:1) and

Dayananda et al. 2499 kept in water bath for 30 min at 60°C. Absorbance of the pooled extracts was measured at 450, 652 and 665 nm and total chlo-rophyll and total carotenoids were estimated using Lichtenthaler equations (Lichtenthaler, 1987). Total protein analysis Total protein of the lyophilized algal biomass was estimated by Kjeldahl method (AOAC, 2000). Total carbohydrate estimation Total carbohydrate from the lyophilized algal biomass was estimated spectroscopically using phenol-sulphuric method and glucose as the standard, as indicated by Dubois et al. (1956). Estimation of moisture and ash Moisture and ash contents were determined by following official AOAC methods (AOAC, 1984). RESULTS AND DISCUSSION Isolation and identification Colony characteristics and morphological features of the Indian isolate have demonstrated its close similarity with the genus Botryococcus. The individual cells of the colonies were in the range of 3 - 11 µm and the colonies were found to be between 25 - 150 µm. However, even bigger aggregates of colonies were also observed in natural habitat as well as during their stationary growth phase (Figure 1). Cells are spherical in shape and the variation in colonial size of the Indian isolate is depending upon the daughter colonies which remain attached to one another. Cells are generally green to yellowish green, and under some stressed conditions, they also show orange yellow colouration as well. Similar observations were made by Chandra in Miocene lignites of Kerala, India (Chandra 1964). And further, the 18S rRNA sequence (GU182893.1) analysis was carried out to know the taxonomical identity of the Indian isolate and the studies revealed its taxonomical relationship to the order Trebouxiophyceae, and further the sequence analysis has shown more than 90% similarities with the reported 18S rRNA sequences of genus Botryococcus and in particular to the species B. braunii. The species of the genus Botryococcus were mainly distinguished based on colony size and details of cell shape. Therefore, based on its distinctive morphological and cellular features with that of the reported Botryococcus species, the Indian isolate was designated as B. mahabali.

The production of hydrocarbons is one of the characteristic features of Botryococcus species and hence hydrocarbons analysis was also carried out to understand the nature of hydrocarbons produced by the Indian isolate. Hydrocarbons of B. mahabali were

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Figure 1. Scanning electron microscopic and light microscopic images of B. mahabali.

Table 1. Hydrocarbons of B. mahabali.

Hydrocarbons Relative % C13 0.04 C14 3.64 C15 7.64 C16 10.15 C17 17.82 C18 6.2 C19 4.19 C20 8.21 C21 2.57 C22 5.8 C23 6.35 C24 4.81 C25

C26 18.74 4.33

identified by comparing their mass spectra with standard hydrocarbons (Sigma) and also with the NIST library. The types of hydrocarbons produced by the Indian isolate were identified as saturated hydrocarbons in the range of C13 to C26 (Table 1). The Indian isolate has produced hexadecane (10.15%), heptadecane (17.82%) and pentacosane (18.74%) as its major hydrocarbons (Table 1). Similar types of saturated hydrocarbons (C21 to C31) were also reported by Yang et al. (2004) from the Chinese strain of B. braunii with heptacosane as its major constituent. Volova et al. (2003) has also reported the presence of saturated straight-chain, branched-chain (C14–C28) and long-chain linear aliphatic (C20–C27) hydrocarbons. Dennies and Kolattukudy (1992) have

demonstrated the alkane biosynthesis by decarbonylation of aldehyde catalyzed reactions in the absence of oxygen in the microsomal preparations of B. braunii. Audino et al. (2000) identified the macrocyclic alkanes (ranging from C15 to C34) and their methylated analogues (ranging from C17 to C26) in B. braunii rich sediment (torbanite). These hydrocarbons served as another chemical marker for the Indian isolate B. mahabali to assign to the genus Botryococcus.

B. mahabali was evaluated for its lipid content and was found to be 14% on dry weight basis and its fatty acids analysis has revealed hexadecadienoic acid (16:2), oleic (18:1), linoleic (18:2), and linolenic acids (18:3) as its major fatty acids (Table 2). Similarly, Fang et al. (2004)

Dayananda et al. 2501

Table 2. Fatty acid composition* of B. mahabali.

Fatty acids Relative % 14:0 0.05 ± 0.01 15:0 0.1 ± 0.08 16:0 28.9 ± 5.43 16:1 1.92 ± 0.32 16:2 2.9 ± 1.64 18:0 2.35 ± 1.33 18:1 22.81 ± 3.7 18:2 14.94 ± 3.71 18:3 25.28 ± 2.56 20:0 0.32 ± 0.37 20:1 0.1 ± 0.11 22:0 0.31 ± 0.19

*Values represents Mean ± SD of three replicates.

Minites Figure 2. HPLC profile of carotenoids from B. mahabali.

have also reported palmitic acid and oleic acids as major components in the Botryococcus sp. (Fang et al., 2004). The algal biomass was also analyzed for total carotenoids contents (2.5 �g/g DW) and constituted predominantly lutein (41.57%) and carotene (37.96%) (Figure 2). These carotenoids are well known for their various pharmaceutical, nutraceutical and cosmetic applications. Scale up of B. mahabali in raceway ponds In view of its chemical profile, the algae was attempted

for scale up in open air raceway ponds. Therefore, as a prerequisite, B. mahabali was gradually scaled up in 500-ml, 1 and 2-L flasks and then in 10 and 20 L carbouys. Rectangular glass tanks with a culture holding capacity of 10 - 15 L (26 cm height × 76 cm long × 26 cm wide) covered with a glass plate with 4 - 5 holes of size 1 - 2 cm to facilitate aeration as well as to avoid condensation were used to acclimatize the algae for outdoor conditions. The culture was observed under microscope periodically for any possible contaminants. Glass tanks with 10 -15 L algal culture were subjected to acclimatization for more than 4 cycles (sub-culturing at an intervals of two weeks) with open air conditions at ambient temperature. Thus

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Figure 3. B. mahabali cultivated in raceway pond.

Figure 4. Online centrifuge showing the harvesting of B. mahabali.

developed starter cultures were inoculated into open air circular ponds (cultures were mixed twice a day) with a culture holding capacities of 100 - 200 L wherein the algae was gown for a week and that seed culture was utilized for raceway ponds cultivation studies (Figure 3).

Raceway ponds were maintained in natural environmental conditions with ambient temperature and light. 600 ± 15 L modified Chu 13 media was prepared with the potable water supplied by CFTRI water facility and inoculated with 200 ± 5 L of green culture developed in circular pond to get an at least initial OD of 0.5 - 580

nm. The cultures were daily mixed by paddle wheels with 15 rpm between 10 AM to 5 PM, in order to prevent settling of cells at the bottom of the tank. B. mahabali was grown for a period of two weeks, and the culture was allowed to settle by gravity, then the clear supernatant media was removed by HDPE tube by vacuum succession. The biomass was washed with potable water and was again allowed to settle; the process was repeated thrice and fed to online centrifuge (Figure 4). Wet biomass was dried by using a Freeze drier (Model-10XB, Lyophylization Systems Inc. USA). The freeze

Dayananda et al. 2503

Figure 5. Biomass yields of B. mahabali cultivated in raceway pond.

Figure 6. Total chlorophyll and total carotenoids yields of B. mahabali cultivated in raceway pond.

dried samples were analyzed for carotenoids and lipid yields. B. mahabali has shown exponential growth (Figure 5) up to 14 days and similar profiles were also observed by chlorophyll and carotenoids evaluation as well (Figure 6). The biomass yields were found to be 2.0 ± 0.09 g/L (w/w) up on two weeks growth in outdoor raceway ponds (Figure 5). During the first week of its growth, pH of the culture media was steadily increased from 7.5 to 9.3 and in the second week, it was increased up to 10.3. Contamination by any other algal species was checked periodically by microscopic observations, and no algal contaminations were witnessed. This may be due to

the alkaline pH of the algal medium or may be due to other chemical profiles of the algae. The Indian isolate have shown relatively high contents of protein, carbo-hydrate and fat (Table 3).

Further, its fatty acids profile has shown relatively high levels of unsaturated fatty acids and hence it could be exploited for human or animal nutrition. However, further detailed studies are required to study the changes in its chemical profile using varied culture conditions, since various algae are reported to accumulate high levels of secondary metabolites under various stress conditions (Banerjee et al., 2002).

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Table 3. Proximate compositiona of B. mahabali.

Proximate composition Outdoor Indoor Protein 19 20.5 Carbohydrate 18.74 18.78 Fat 14.3 13.7 Hydrocarbon 6.3 9.1 Moisture 5.41 4.93 Ash 39.73 33.15

aMeans of triplicate determinations based on alga dry matter.

In conclusion the Indian isolate B. mahabali was culti-vated for the first time in open air raceway ponds and the same can be exploited for its lipid and protein rich biomass for various applications from food to biodiesel. Indigenous species are most likely desirable for any successful industrial exploitation and hence the present investigation has used the new Indian isolate for outdoor cultivation. However, further studies are required to optimize culture parameters which are necessary for high yields of biomass and other secondary metabolites production for its industrial prospects. ACKNOWLEDEGMENTS Authors are grateful to the Department of Biotechnology, Government of India for their financial support. CD is thankful to Council of Scientific and Industrial Research (CSIR), Government of India for Research Fellowship. Authors thank Mr. Shivaswamy, Scientist and Mr. K. Anabalagan, Scientist of CIFS, CFTRI, Mysore for their help in GCMS and SEM analysis respectively. REFERENCES American Association of Cereal Chemists (2000). Approve methods of

AACC, (10th Ed), St. Paul, Minnesota, USA. Association of Official Analytical Chemists (1984). Official method of

analysis (14th Ed), Washington, DC, USA. Audino M, Kliti G, Robert A, Robert IK (2000). Macrocyclic-alkanes: a

new class of biomarker. Organic Geochem., 32: 759-763. Banerjee A, Sharma R, Chisti Y, Banerjee UC (2002). Botryococcus

braunii: a renewable source of hydrocarbons and other chemicals. Crit. Rev. Biotechnol., 22: 245-79.

Brown AC, Knights BA, Ellisie C (1969). Hydrocarbon content and its relationship to physiological state in the green alga Botryococcus braunii. Phytochemistry, 8: 543 - 547.

Chandra A (1964). The note on the occurrence of Botryococcus in the Miocene lignites of Kerala. Cur. Sci., 214-215.

Chiara S, Cristian T, Giulia S, Daniele F, Paola G, Franca G, Rossella P, Emilio T (2010). Extraction of hydrocarbons from microalga Botryococcus braunii with switchable solvents. Bioresour. Technol., 101: 3274-3279.

Christie WW (1982). Lipid analysis, 2nd ed. Pergamon press, New York, p. 338.

Dayananda C, Sarada R, Bhattacharya S, Ravishankar GA (2005). Effect of media and culture conditions on growth and hydrocarbon production by Botryococcus braunii. Process Biochem., 40: 3125-

3131. Dayananda C, Sarada R, Usha Rani M, Shamala TR, Ravishankar GA

(2007a) Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass Bioenergy, 31: 87-93.

Dayananda C, Sarada R, Vinod Kumar, Ravishankar GA (2007b) Isolation and characterization of hydrocarbon producing green alga Botryococcus braunii from Indian freshwater bodies. Elect. J. Biotechnol., 10: 1-14.

Dayananda C, Venkatachalam L, Bhagyalakshmi N, Ravishankar GA (2010) Assessment of Genetic Polymorphism among Green Microalgae Botryococcus of Distinct Origin by RAPD. Genes, Genomes and Genomics (in press).

Dennies MW, Kolattukudy PE (1992). A cobalt-porphirin enzyme converts a fatty aldehyde to a hydrocarbon and CO. Proc. Natl . Acad. Sci. USA., 89: 5306-5310.

Dubois M, Gilles K, Hamilto J, Rebers P, Smith F (1956). Colorimetric method for the determination of sugars and related substances, Anal. Chem., 18: 350-356.

Ela E and Anastasios M (2010). Extracellular terpenoid hydrocarbon extraction and quantitation from the green microalgae Botryococcus braunii var. Showa. Bioresour. Technol., 101: 2359-2366.

Fang JY, Chiu HC, Wu JT, Chiang YR, Hsu SH (2004). Fatty acids in Botryococcus braunii accelerate tropical delivary of flurbiprofen into and across skin. Int. J. Pharmaceut., 276: 163 -173.

Fowke LC, Stephen MA, Pat JR (1994). Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss). Plant Cell Reports. 13: 612-618.

Hai-Linh T, Ji-Sue K, Choul-Gyun L (2009). Optimization for the growth and the lipid productivity of Botryococcus braunii LB572. J. Biosci. Bioeng., 108: 52-55.

Lichtenthaler HK (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzymology. Packer L, Douce R. Academic press. 148: 350-382.

Metzger P, Berkaloff C, Caradevall E, Coute A (1985). Alkadiene and botryococcene producing races of wild strains of Botryococcus braunii. Phytochem., 24: 2305-2312.

Metzger P, Largeau C (1999). Chemicals of Botryococcus braunii. In: Cohen Z (Ed) Chemicals from microalgae. Taylor & Francis, London, pp: 205-260.

Metzger P, Largeau C (2005). Botryococcus braunii: a rich source for hydrocarbons and related ether lipids. Appl. Microbiol. Biotechnol., 66: 486-496.

Okada S, Devarenne TP, Chapell J (2000). Molecular characterization of squalene synthase from the green microalga Botryococcus braunii, race B. Archives Biochem. Biophys., 373: 307-317.

Ranga Rao A, Dayananda C, Sarada R, Shamala TR, Ravishankar GA (2007). Effect of salinity on growth of green alga Botryococcus braunii and its constituents. Bioresour. Technol., 98: 560-564.

Sawayama S, MinowaT, Yokayama S (1999). Possibility of renewable energy production and carbon dioxide mitigation by thermochemical liquefaction of microalgae. Biomass Bioenergy. 17: 33-39.

Tyson RV (1995). Sedimentary Organic Matter: Organic Facies and Palynofacies Chapman and Hall, London.

Volova TG, Kalacheva GS, Zhila NO (2003). Specificity of lipid

composition in two Botryococcus strains, the producers of liquid hydrocarbons. Russian J. Plant Physiol., 50: 627-633.

Wolf FR, Nanomura AM, Bassham JA (1985). Growth and branched hydrocarbon production in a strain of Botryococcus braunii. J. Phycol., 21: 388-396.

Dayananda et al. 2505 Yang S, Wang J, Cong W, Cai Z, Ouyang F (2004). Effects of bisulfite

and sulfite on the microalga Botryococcus braunii. Enzyme Microbial Technol., 35: 46-50.