chakravarthy kavitha(1802 1807)

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy. Effect of sodium alginate on growth and lipid production of Botryococcus braunii kutzing for biodiesel production. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 October; 35(35): 1802-1807.

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Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

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CODEN JPBSCT NLM Title: J Pharm Biomed Sci.

Research article Effect of sodium alginate on growth and lipid production of Botryococcus

braunii kutzing for biodiesel production

Chakravarthy Kavitha*1, Senthil Chinnasamy2, Sailendra Bhaskar2, Ramasamy Rengasamy1.

Affiliation:- 1Centre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai-600025, Tamilnadu, India. 2Aban Infrastructure Private Limited (Biotechnology Division), Chennai 60008, Tamilnadu, India. The name of the Department and Institution to which the work should be attributed:- Department of Pharmacology, Centre for Advanced Studies in Botany, University of Madras, Guindy campus, Chennai-600025, Tamilnadu, India. Authors contributions: The major work was done by the Corresponding author, rest guided and supported for this work. *Corresponding author: C. Kavitha. CAS in Botany, University of Madras, Guindy campus, Chennai-600025.Tamilnadu, India. Telephone: 8754886646. Tel/Fax: +914422353309/22352494.

Abstract: Botryococcus braunii is a green colonial microalgae commonly found in fresh water, lakes and ponds. Due to its high lipid and hydrocarbon content, it is widely recommended for biodiesel production. The present study deals with the growth, lipid content, and fatty acid methyl ester (FAME) of Botryococcus braunii AP102 grown at different concentration of sodium alginate viz. 4mg, 8mg, 12mg, 16mg, and 20mg amended with Chu-13 medium and compared with control in order to find out the efficacy of sodium alginate in the growth and lipid production of B. braunii. The maximum growth and lipid production was obtained at 16mg concentration and it is most effective when compared to control. Thus sodium alginate helps greatly in promoting the growth of B.braunii for biodiesel production. Key Words: Botryococcus braunii; Fatty acid; Lipid; Mass cultivation; Sodium alginate.

Article citation:- Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy. Effect of sodium alginate on growth and lipid production of Botryococcus braunii kutzing for biodiesel production. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 October; 35(35):1802-1807. Available at http://www.jpbms.info

INTRODUCTION icroalgae are a diverse group of prokaryotic and eukaryotic photosynthetic microorganisms that

grow rapidly due to their simple structure. They can be employed potentially for biofuel production in an economically effective and environmentally sustainable manner and has the number of advantages, including higher photosynthetic efficiency, higher biomass production, and higher growth rates, as compared to other energy crops1. Microalgae with high oil productivities are desired for producing biodiesel. Depending on the species,

microalgae produce different kinds of lipids, hydrocarbons, and other complex oils2,3. The genus, Botryococcus, Kutzing has drawn the attention towards scientists in recent years due to its considerable amount of hydrocarbon4. It is one of the renewable sources for the production of bio fuel. This alga is characterized by a conspicuous ability to synthesize and accumulate wide variety of lipids. These lipid substances include numerous hydrocarbons, i.e. highly reduced compounds comprising only carbon and hydrogen as elements5,6 and a number of specific ether lipids7,8.

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Alginic acid and its salts (alginates) occur mainly in marine brown algae (Pheophyta) comprising most of their polysaccharides and averaging 40% of the dry weight9. Alginate occurs in the cell walls of seaweeds as a mixed salt with the major cations being Na, Ca, Mg, and K together with a number of minor metal counterions10. Ishii et al.11 observed that alginate oligosaccharides, produced by enzymatic degradation of alginic acid mainly extracted from brown algae, significantly stimulated hyphal growth and elongation of arbuscular mycorrhizal (AM) fungi and triggered their infectivity on trifoliate orange seedlings. Alginates, as well as agars and carrageenans, are characterized by unique functional properties. Thickening and gelling are the most important of their functions. Over the past decade, alginates have become widely adopted as dietetic food hydrocolloids. Sodium alginate was introduced into the US Pharmacopoeia as early as 1938. Sodium salt of alginic acid is well water-soluble; due to its high molecular weight (from 100 to 500 kDa, depending on the raw material), it forms viscous solutions. In the food industry, sodium alginate is used as thickeners and stabilizers in the production of fruit candy, sweets, soft drinks, and in juice settling12. Several investigators have concluded that the alginate oligomers have the growth promoting activity in higher plants and microbes. In the present study, different concentrations of sodium alginate were amended with chu-13 medium in order to find out its efficacy in growth and lipid production of B.braunii and mass cultivation of B.braunii using the selected concentration of alginate in an open raceway pond for biodiesel production. MATERIALS AND METHODS Microalgae The Botryococcus braunii culture AP102 (Accession no: JQ585724) was obtained from Algal culture collection, CAS in botany, University of Madras, Chennai. It was grown in modified CHU-13 medium (Composition : KNO3- 371mg, K2HPO4- 80mg, MgSO4.7H2O-200mg, CaCl2.6H2O-107mg, Ferric Citrate - 20mg, Micronutrients; H3BO3 - 2.86g/L, MnCl2.4H2O -1.81g/L, ZnSO4.7H2O - 0.22g/L, Na2MoO.2H2O - 0.39g/L, CuSo4.5H2O- 0.08g/L, Co (NO3)2.6H2O- 0.05g/ L) amended with different concentrations of sodium alginate viz., 4mg, 8mg, 12mg, 16mg, and 20mg under laboratory conditions kept under 30E m-2 s-1 light intensity, 12/12 light dark cycle and at

241C. The study was carried out for a period of 30days and all the growth parameters were analyzed and recorded at every 5 days interval. Determination of pigment13 Five ml of culture sample was taken and centrifuged at 5000 rpm for 10 min and the supernatant was discarded. The algal pellet was then added with 5mL of 80% acetone and homogenized in a sonicator. Then it was covered with black paper and kept overnight at 4C. The sample was then centrifuged at 5000rpm for 10 minutes. The supernatant was collected and the optical density was measured at 644.8 , 661.6 and 470 in Milton Roy UV - Visible spectrophotometer. Extraction and estimation of total protein14 Five ml of algal culture was taken and centrifuged at 5000 rpm for 10 minutes. The pellet was homogenized in 5ml of 0.1M sodium phosphate buffer at pH 7.0 in a sonicator and then centrifuged at 5000 rpm for 10 minutes. The supernatant was taken for the estimation of total protein. To 0.2 ml of sample protein 5ml of CBB reagent (100 mg of CBBG-250 dissolved in 50ml of 93% ethanol. To this 100 ml of 85% Phosphoric acid was added and diluted to 1000 ml with glass distilled water) was added and mixed thoroughly. The absorbance was read at 595 nm against a reagent blank . The amount of protein was calculate by using a standard graph with Bovine Serum albumin ranging from 10 to 100 g ml-1. Extraction and estimation of total carbohydrate15 Five ml of algal culture was taken and centrifuged at 5000 rpm for 10 minutes. The pellet was homogenized with 5ml of 0.1M sodium phosphate buffer at pH 6.8 in a sonicator and then centrifuged at 5000 rpm for 10 minutes . The supernatant was collected for the estimation of carbohydrate. To the 1ml of sample 1ml of 5% phenol and 5ml of H2SO4 was added and mixed thoroughly. The solution was allowed to stand at room temperature for 30 minutes. The Optical Density was read at 490nm. Standard graph was prepared with different concentrations of D-glucose ranging from 10 to 100gml-1. Extraction and estimation of total lipid 16 Five ml of culture was taken and centrifuged at 5000 rpm for 5minutes. The pellet was


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homogenized in a sonicator with 6ml of chloroform: methanol (2:1). It was then transferred to a separating funnel and added with 2ml of 0.9% NaCl solution and mixed well. This mixture was left undisturbed for overnight. Then from the lower chloroform phase, 0.5ml was collected in a clean vial and the solvent was allowed to evaporate at room temperature and the pellet was collected. To the pellet 0.5ml of concentrated sulfuric acid was added and mixed well. The tubes were closed with glass marbles kept in a boiling water bath for 10min and allowed to cool at room temperature. To 0.2ml of sample 5ml of vanillin reagent (0.2g vanillin in 80ml 0f orthophosphoric acid and 20ml of distilled water) was added and mixed well. It was allowed to stand for 30minutes and the colour developed was read at 520nm. Standard graph was prepared using cholesterol ranging from 5.0 to 50g/ml and the values are expressed as gml-1. Mass cultivation of Botryococcus braunii in open raceway pond The Botryococcus braunii was mass cultivated in an open raceway pond in Modified CFTRI medium (Urea -0.4 g-L-1, DAP - 0.016 gL-1, Potash - 0.234 gL-1, NaHCO3 1 gL-1, MgSO4 - 0.5 gL-1) amended with optimized sodium alginate concentration at pH 7.5 for a period of 15 days. The experiment was conducted in an open raceway pond of 2000L capacity. An initial inoculum of 200L of B.braunii grown in mini raceway pond was transferred to 1800L of Modified CFTRI medium .The culture was mixed using paddle wheel fixed to the pond in order to prevent settling and also to increase the Carbon dioxide concentration. The experiment was conducted for a period of 15days and at every 5days interval the pigments, total protein, carbohydrates and lipids were analyzed. The biomass was harvested for further study. Harvesting The biomass was harvested on 15th day through auto flocculation, shade dried for 3 days and pulverized using mixer- grinder. A known quantity of dry biomass was taken and extracted for total lipid using Chloroform: Methanol 2:1 in Soxhlet apparatus. Total lipid extraction and fatty acid analysis Ten grams of dried algal sample was taken and extracted using chloroform: methanol (2:1) and the lipid content was quantified gravimetrically. The lipid sample was dissolved in benzene and 5%

methanolic hydrogen chloride (95ml of chilled methanol and 5ml of acetyl chloride) and vortexed thoroughly. The mixture was refluxed for 3hour at 65 C and 5% NaCl was added. The FAME (Fatty Acid Methyl Ester) was extracted using hexane and the hexane layer was washed with 2% potassium bicarbonate and dried over anhydrous sodium sulfate. The FAME was analyzed using GC-MS (Perkin-Elmer, Turbomass Gold, Mass Spectrometer) equipped with FID using SPB-1 (Poly-dimethysiloxane) capillary column (30m 0.32mm ID 1m film thickness) with a temperature programming 130-280 C at a rate of 2 C/min. The FAME was identified by comparing their fragmentation pattern with authentic standards (Sigma) and also with NIST library17. RESULTS Effect of sodium alginate on Chlorophyll a, b, and carotenoids production of B.braunii under laboratory conditions: The effect of sodium alginate on pigment content of B. braunii AP102 strain was studied. The alga when grown at different concentrations of sodium alginate showed maximum growth at the concentration of 16mg. The maximum amount of chlorophyll a 20.09mgL-1 chlorophyll b 11.03mgL-1 and Carotenoids 8.20mgL-1 were observed at 16mg concentration on 20th day whereas in control the maximum amount of chlorophyll a 9.62mgL-1, Chlorophyll b 6.53mgL-1 and carotenoids 5.173mgL-1 was observed on 10th, 25th and 25th day respectively (Figure 1, 2 &3). The concentration above 20mg retards the growth of microalgae and hence the experiment was restricted up to 20mg. Yokose18 reported that alginate has the ability to promote the growth rate of Nanochloropsis oculata during the exponential phase and showed increased cell density in stationary phase. Effect of sodium alginate on protein, carbohydrate and lipid production of B.braunii under laboratory conditions: The total protein, carbohydrate and lipid content of B.braunii were also found to be increased when the medium is amended with sodium alginate. The highest protein content of 14.17mgL-1 was recorded at 12mg concentration on 25th day which was higher than that of control 10mgL-1 on 15th day. The carbohydrate showed maximum of 26.27mgL-1 at the concentration of 12mg on 20th day where as in control only 14.43mgL-1 was obtained on 20th day. The maximum lipid


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production of 200mgL-1 at 16mg concentration was observed on 20th day which was higher when compared to control 69.80mgL-1 on 25th day (Figure 4, 5 & 6). The lipid content was slightly decreased at 20mg concentration. This reveals

that the sodium alginate has the optimum concentration to enhance the lipid production of B.braunii.

Figure 1. Effect of sodium alginate on chlorophyll a production of B.braunii

Figure 2. Effect of sodium alginate on chlorophyll b production of B.braunii

Figure 3. Effect of sodium alginate on carotenoids production of B.braunii

Figure 4. Effect of sodium alginate on protein production of B.braunii

Figure 5. Effect of sodium alginate on carbohydrate production of B.braunii

Figure 6. Effect of sodium alginate on lipid production of B.braunii


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Effect of sodium alginate on the growth of B.braunii under open raceway pond: The B.braunii when grown with different concentration of Sodium alginate under laboratory conditions revealed best growth and lipid production at the concentration of 16mg. Hence the above concentration of sodium alginate was selected for mass cultivation. The B.braunii was mass cultivated in an open raceway pond with modified CFTRI medium amended with sodium alginate. The algal biomass was harvested, dried and analyzed for total lipid, total carbohydrate, total protein and total ash content. The lipid content in the B.braunii dry biomass shows up to 18% (w/w). The carbohydrate and protein content showed 40% and 17% (w/w) respectively. The results were shown in (Table1). Table 1. Biochemical Composition of B. braunii dry biomass

Fatty acid methyl ester (FAME) analysis The fatty acid composition of B.braunii biomass was analyzed through GC-MS. The fame analysis revealed the presence of oleic, palmitic, lauric, stearic, linoleic, linolenic, elaidic, lignoceric, margaric and myristic acid (Table 2). Among that Oleic acid (C18:1) and Palmitic

acid (C16:0) were found to be the major fatty acids from the B.braunii oil. The oleic acid contains 41.63% and the palmitic acid contains 19.12%. Similar observations were made in B.braunii by Rai et al. 19 in which the oleic acid constitutes 38.5% and palmitic acid 17.6%. Dayanandha et al.20 reported that the lipid content in organism would be 22% (w/w) of those the oleic acid and palmitic acid acquires the major percentage of 22.3% and 40.6% respectively. The biomass to biodiesel conversion showed 22% (w/w) in this study.

Table 2. Fatty acid profile of B. braunii AP102 DISCUSSION Alginates are natural polysaccharide which is abundant in nature. Alginates have wider spread application in the food and beverage, pharmaceutical and bioengineering industries21. Alginates are the major components of brown seaweed cell walls made up of D-glucuronic acid and D-mannuronic acid units22. The brown alga sargassum is the main source of sodium alginate and it is eco friendly in nature23. In the present study different concentration of sodium alginate was tested to enhance the growth and lipid production of B.braunii. A number of studies have been reported regarding the growth promoting effect of alginates. Mollah24 reported the growth enhancing effect of gamma irradiated sodium alginate on red amaranth with a total dose of 37.5 k G y at dose rate 3.5kGy/h; applied in 150 ppm solutions. Yokose et al.18 achieved maximum growth promoting effect of Nanochloropsis oculata at 20mgml-1 concentration with alginate oligosaccharides. Yokose et al.25 also achieved maximum growth promoting effect of alginate oligosaccharides in Chaetoceros gracilis at a concentration of 125g ml-1. Naeem et al.26 achieved best plant growth promoting effect of Mentha arvensis L at the concentration of 100 mgL-1 of irradiated sodium alginate. Interestingly, oligo-alginates also stimulate the growth of marine and fresh water green microalgae by enhancing

nitrogen and carbon assimilation providing antimicrobial effect and, increase the content of fatty acids which is useful for biodiesel production22. CONCLUSION Alginate oligosaccharides enhances the growth of many terrestrial plants and microalgae helping in

Parameters Open raceway pond

Total protein (%) 17 Total carbohydrate (%) 40

Total lipid (%) 18 Total ash (%) 25

Fatty Acid Wt %

Oleic acid (18:1) 41.63 Palmitic acid (16:0) 19.12 E-11-Hexadecenoic acid (18:1) 10.08 Palmitelaidic acid (16:1) 7.08 Stearic acid (18:0) 5.81 Linoleic acid (18:2) 2.71 Cyclohexane (C12) 2.63 Heptadecane (C17) 1.44 Myristic acid (14:0) 1.36 Homo-- linolenic acid (20:3) 0.26 Eicosatrienoic acid(20:4) 1.04 Lignoceric acid (24:0) 1.87 Pentadecanoic acid (15:0) 0.84 Elaidic acid (9-18:1) 0.57 Margaric acid (17:0) 0.57 Lauric acid (12:0) 0.36 2-Pentadecanone,6,10,14-trimethyl 0.83 6-Octen-1-ol, 3,7-dimethyl acetate 0.28 1,3-Cyclohexanediamine 0.21 (1R,4S)-1,7,7-Trimethylbicycloheptan-2-yl 3-Chlorobenzoate

0.84

2-Methylthio-5-nitro anisole 0.26 Ethyl 4,8,12-trimethyl-tridecanoat 0.24


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nitrogen assimilation, enhancing photosynthesis, basal metabolism and cell division and also increases the fatty acid content providing protection against pathogen due to its antimicrobial activity. In this study, the growth and lipid content of Botryococcus braunii was evaluated using different concentration of sodium alginate under laboratory conditions. The best growth and lipid production was achieved at the concentration of 16mgL-1. Thus the sodium alginate plays a vital role in the growth and lipid production of green alga B.braunii for biodiesel production. ACKNOWLEDGEMENTS The authors are thankful to the management of Aban Infrastructure Pvt., Ltd., Chennai for the financial support provided for this study. REFERENCES 1.Huang GuanHua,Chen Feng,Wei Dong,Zhang XueWu, GuChen. Biodiesel production by microalgal biotechnology.Appl Energy 2010; 87:38-46. 2.Lin Lin, Cunshan Zhou,Vittayapadung Saritporn, Xiangqian Shen, Mingdong Dong. Opportunities and challenges for biodiesel fuel. Appl Energy 2011; 88:1020-31. 3.Metzger P,Largeau C.Botryococcus braunii:a rich source for hydrocarbons and related ether lipids.Appl Microbiol Biotechnol 2005; 66:486-96. 4.Borowitzka M A.Fats, oils and hydrocarbons.In: Microalgal Biotechnology:Eds.Borowitzka & Borowitzka. Cambridge University Press, Cambridge 1988;Pp.257-287. 5.Brown AC and Knights BA. Hydrocarbon content and its relationship to physiological state in the green alga Botryococcus braunii. Phytochemistry 1969; 8: 543-547. [6] Knights BA, Brown AC, Conway E and Middleditch BS. Hydrocarbons from the green form of the freshwater alga Botryococcus braunii. Phytochemistry 1970; 9: 1317-1324. 7.Metzger P and Casadevall E.Botryococcoid ethers, ether lipids from Botryococcus braunii. Phytochemistry 1991; 30: 1439-1444. 8.Metzger P and Largeau C.Chemicals of Botryococcus braunii.In:Cohen Z (ed)Chemicals from micro algae. Taylor and Francis London.1999;Pp:205-260. 9.Rossel KG and Srivastava LM.Seasonal Variation in the Chemical Constituents of the Brown Algae Macrocystis integrifolia and Noveocystic luetkeana.Can J Bot 1984; 62: 2229-2236. 10.Rioux LE,Turgeon SL,Beaulieu M.Characterization of polysaccharides extracted from brown seaweeds.Carbohydrate Polym 2007;69:530-537. 11.Ishii T, Aikawa J,Kirino S,Kitabayashi H,Matsumoto I, Kadoya K. Effects of alginate oligosaccharide and polyamines on hyphal growth of vesicular-arbuscular mycorrhizal fungi and their infectivity of citrus roots. In: Proceedings of the 9th

International Society of Citriculture Congress,Orlando,FL,3-7 December 2000;1030-1032. 12.Khotimchenko Yu S,Kovalev VV,Savchenko OV,Ziganshina O A. Physical-Chemical Properties,Physiological Activity, and Usage of Alginates,the Polysaccharides of Brown Algae. Russian Journal of Marine Biology 2001;27:53-64. 13.Lichtenthaler HK. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. In: PACKER,L. and DOUCE, R. eds .Methods in Enzymology. Washington,Academic Press 1987;148:350-382. 14.Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976; 72:248-254. 15.Dubois M, Giles KA,Hamilton JK,Robeus RA, Smith E. Calorimetric methods for determination of sugars and related substance. Anal Biochem 1956;28:305-365. 16.Folch J,Lees M and Sloane-Stanley GH.A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1956; 226: 497-509. 17.Dayananda C, Sarada R, Srinivas P, Shamala TR and Ravishankar GA. Presence of methyl branched fatty acids and saturated hydrocarbons in botryococcene producing strain of Botryococcus braunii. Acta Physiologiae Plantarum 2006; 28: 251-256. 18.Yokose T,Nishikawa T,Yamamoto Y,Yamazaki Y,Yamaguchi K, Oda T. Growth-promoting effect of alginate oligosaccharides on a unicellular marine microalga, Nannochloropsis oculata. Biosci Biotechnol Biochem 2009; 73:450-453. 19.Rai UN, Dwivedi S,Baghel VS,Tripathi RD,Shukla OP and Shukla MK.Morphology and cultural behavior of Botryococcus protuberans with notes on the genus.J.of Environmental Biology 2007;28(2):181-184. 20.Dayananda C, Sarada R, Usha Rani M, Shamala TR,Ravishankar GA.Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass Bioenergy 2007; 31:87-93. 21.Gacesa P, Alginates.Carbohydr Polym 1988; 8: 161-182. 22.Alberto Gonzalez,Jorge Castro, Jeannette Vera, and Alejandra Moenne. Seaweed Oligosaccharides Stimulate Plant Growth by Enhancing. Carbon and Nitrogen Assimilation, Basal Metabolism, and Cell Division. J Plant Growth Regul 2013: 32:443-448. 23.Aziz A, Nurul Islam AKM, Pervin R. Marine algae of St. Martins Island,Bangladesh. I. New records of Sargassum sp. Bangladesh J.Bot 2001; 30 (2):135-140. 24. Mollah MZI, Mubarak A Khan, Ruhul A Khan. Effect of gamma irradiated sodium alginate on red amaranth (Amaranthus cruentusL.) as growth promoter. Radiation Physics and Chemistry 2009; 78: 61-64. 25.Yokose T,Yamasaki Y, Nishikawa T, Jiang Z, Wang Y, Yamaguchi K,Oda T.Effects of alginate oligosaccharides on the growth of various mammalian cell lines,unicellular phytoplankters and marine bacteria. Jpn J Food Chem Saf 2010; 17:2735. 26.Naeem M,Mohd Idrees,Tariq Aftab, Masroor M,Khan A, Moinuddin and Lalit Varshney.Irradiated sodium alginate improves plant growth, physiological activities and active constituents in Mentha arvensis L.Journal of Applied Pharmaceutical Science 2011; 02(05):28-35.

Copyright 2013 Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar, Ramasamy Rengasamy. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

chakravarthy kavitha(1802 1807)

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