Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 851-860

ISSN: 2319-7706 Volume 3 Number 4 (2014) pp. 851-860 http://www.ijcmas.com

Original Research Article Production of biofuel by using micro algae ( braunii)

Y.P.Nagaraja1*, Chandrashekhar Biradar1, K.S.Manasa1 and H.S.Venkatesh2

1Department of Biotechnology, Nagarjuna College of Engineering and Technology, Venkatagirikote, Mudugurki, Devanahalli Taluk Bangalore Karnataka, India 2Department of Chemical Engineering, Manipal Institute of Technology, Manipal, Karnataka, India

*Corresponding author

A B S T R A C T

Microalgae are sunlight driven cell factories that convert carbondioxide to K e y w o r d s potential biofuels. The micro algae culture Botryococcus braunii was obtained from Algae Depot, USA and was maintained in Chu 13 medium. Further, the Botryococcus culture was mass produced in a specially designed photo bioreactor. The physical braunii, and environmental factors were established for the proper growth of the culture. , Algal oil was extracted using Soxhlet apparatus through repeated washing or bubble column percolation with organic solvent such as hexane and petroleum ether for the photo 5weeks old culture. The final concentrated compound was separated using aqueous bioreactor, two phase extraction by mixing two incompatible polymers. Then it was purified biofuels, and subjected to gas chromatography for the analysis. The growth of the culture in a fabricated photo bioreactor was found to be best at a temperature of 23oC, a Saccharomyces 2 cerevisiae, light intensity of 60 W/M , with a light period of 12 hours per day, and a salinity of Soxhlet 0.15 molar NaCl and a pH of 7.5. In the laboratory, B.braunii is commonly grown apparatus. in cultures of Chu-13 media. The cell growth reached a maximum of 1.2g L-1 for B.braunii after 3 weeks in flasks. The growth was however higher in bubble column photobioreactor reaching a maximum of 4.1g L-1.

Introduction

Microalgae are photosynthetic, heterotrophic Under difficult agro-climatic conditions organisms that have an extraordinary and are able to produce a wide range of potential for cultivation as energy crops. commercially interesting byproducts such Microalgae are potential source of biomass, as fats, oils, sugars and functional which may have great biodiversity and bioactive compounds (Erika C Francisco consequent variability in their biochemical et al., 2010). composition (Satyanarayana et al., 2011).

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The Combustion of fossil fuels generates on cultural and physiological conditions sulfuric, carbonic, and nitric acids, which (Dayananda et al.,2006),these fuels were free fall to Earth as acid rain, impacting both from oxides of sulphur and nitrogen (SOX and natural areas and the built environment. NOX) after their combustion. Being a Monuments and sculptures made from photosynthetic organism, it can reduce CO2 emissions by 1.5 x 105 tons/yr and 8.4 x103 marble and limestone are particularly ha of microalgal cultivation area would be vulnerable, as the acids dissolve calcium necessary (Sawayama et al.,1999). carbonate. The algae oil is the best substitute for energy production and Zhang et al.,(2010) have used reactors energy drink (K.Vairavan et al., 2010). called raceway ponds systems and Botryococcus braunii The green colonial proposed closed type photobioreactors, for rich unicellular microalgae higher photosynthetic efficiency and Botryococcus braunii (Banerjee et al., .2002; temperature control of the culture medium. Metzger and Largeau, 2005) is widespread in Yusuf Christi et al.,(2008) found that, the freshwater, brackish lakes, reservoirs and roles algal cultures can play economically ponds. It is also widely distributed in with biofuel production and the long term reservoirs at temperate, tropical and arctic benefits. According to Katsumi et al., latitudes (Tyson, 1995). It is recognized as one (1987) Algae efficiently use CO2, and are of the potent renewable resource for the responsible for more than 40% of the production of liquid .B braunii is global carbon fixation. Thus this classified into A, B and L races based on the type of hydrocarbons in the range of C21 to investigation was undertaken to Grow the C33 odd numbered n-alkadiens,mono-, tri-, cultures of Botryococcus braunii in tetra-,and pentaenes and they are derived from photobioreactor and for the Biofuels fatty acids (Banerjee et al., 2002; Metzger et Production. al.,2005).The L race yields a single C40 isopropenoid hydrocarbon ,lycopa -14(E), Materials and Methods 18(E)-diene (Metzger et al; 1990).The B race produces two types of triterpenes called Sample collection Botryococcus of C30-c37 of general formula CnH2n-10 as mojor hydrocarbons and small amounts methyl branched squalene. Certain The culture of Botryococcus braunii was strains of B race also biosynthesize obtained from Algae Depot, USA .It was cyclobotryococcenes (David et al.,1988 cultivated in Chu-13 (1942) medium in ;Achitouv et al.,.2004). Also a feature small volumes in conical flasks for the common to all the three races is the presence stock culture and then it was inoculated to of a highly aliphatic ,non-hydrolysable and a shake flask after which it was inoculated insoluble bio macromolecule (algaenan) found to photobioreactor and was maintained at in their outer cell walls (Achitouv et al.,2004). a constant pH 7.5 and a set temperature The highly resistant nature of the B.braunii 23oC. algenn to degradation allows it to selectively preserves during fossilization, leading to fossil Shake Flask Culture B.braunii remains, a major contributor to a number of high oil potential sediments The microalgae obtained cultured in (Simpson et al., 2003). The algae B.braunii 250ml shake flask culture method. It was produces oil in the range 0f 29-75% (on dry maintained at room temperature (28+2) weight basis). This variation in the content of and, the light/dark hour cycle followed hydrocarbon is due to the difference in the was 12:12 in rotary shaker at 100rpm for strains , the race it belongs and also depends the adequate supply of oxygen. The

852 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 851-860 culture was harvested after 3 weeks of Final weight (mg)-initial weight (mg) incubation and then transferred into Biomass yield content (mg ml-1) = photobioreactor. Sample taken (ml)

Bubble column Photobioreactor culture Lipid Estimation Test

The 3 weeks old culture obtained from the Extraction and fractionation of lipids shake flask was inoculated into bubble column photobioreactor specially designed The lipid extraction was done by using for the present investigation. The bubble Bligh and Dyer method 1.0g of the freeze- column photobioreactor consisted of two dried preparation was homogenized in circular concentric glass columns of 30mm 30ml of chloroform- methanol-water inside diameter and 65mm outside (1:2:0.8, v/v) with a Waring blender, and diameter. Height of 726 and 711mm/ the lipids were extracted after addition of 30 nominal working of 4.0L. The dispersion ml each of chloroform and water. The system for the reactor consisted of a 1.5cm homogenate was filtered through diameter air diffuser located at the center Whatmann filter paper no. 2 on a Buchner of the column. The culture was exposed to funnel. It was repeated for three times and the natural day and night photoperiod and the chloroform layer was evaporated by red blue light of different wavelengths for keeping on a water bath to dryness and the growth rate studies. The culture total lipids were measured gravimetrically. environment was maintained to a The silica gel column supplied by Amil temperature of 23oC, a light intensity of 60 was (Ixl5cm) packed with silica gel using W/M2 ,with a light period of 12 hours per 250ml of hexane, 150ml of chloroform, day, and a salinity of 0.15 molar NaCl at and 150ml of methanol to isolate pH of 7.5. The supplement of CO2 to the hydrocarbons, nonpolar lipids except culture would enhance the growth of the hydrocarbons and polar lipids, algae so, the photobioreactor was designed respectively to fractionation of total lipids. to supplement at a high rate of 1.5 The isolated lipids in each eluate were litres/min air supply through an air measured gravimetrically after evaporation sprayer/air stone to create a forced stress of the solvent. on the algae for exchange of gas. Analysis of lipids Hydrodynamic Stress The composition of each fraction was The hydrodynamic stress is brought about analyzed by thin-layer chromatography on by the use of an airlift PBR, which uses a silica gel 60F254 (Merck, 0.25 mm thick) sparger/air stone to propagate the algae plates using the following solvents: hexane from the dark zone to the dark zone to the for hydrocarbons; ; petroleum ether diethyl light zone with high degree of turbulence ether diethyl ether-acetic acid (90 : 10 : 1, offered by supplying air. v/v) for nonpolar lipids; chloroform- methanol-acetic acid-water (100 : 20 : 2 : Biomass Estimation 5, v/v) for polar lipids. The spots were visualized by spraying with sulfuric acid- The biomass yield of microalgae was ethanol (1 : 9, v/v) followed by charring. calculated by using the following Polar lipids were detected also by specific equation.

853 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 851-860 spray reagents: Dragendorff and each Immobilization of lipase on fabric spots were compared with the literature. membrane For measurement of relative percentage of individual components, the lipids were Lipase was immobilized by using an chromatographed on silica gel-coated established immobilization procedure. glass plates. Briefly, 0.1g of fabric (approximately 9cm2) is presoaked for 1hour in 10ml of co It was also calculated by using the immobilization solution consisting of 0.5g following formula, of gluten, 0.2g of lecithin, 0.2g of polyethylene glycol 6000 and 0.1g of Final weight (mg)-initial weight (mg) magnesium chloride. Lipid content (mg ml-1) = Sample taken (ml) Fabric membranes were dried at room temperature and used as supports for the Determination of Alcohols by Gas immobilization of lipase. Membranes were Chromatography added into 10ml of enzyme solution (5,000 to 10,000 Uw/ml), stirred for 2 to 3 hours, The algal sample was analyzed by gas- lt was taken out and dried at room liquid chromatography the test was temperature under a vacuum. The activity conducted at Azyme Biosciences of immobilized lipase was determined by Bangalore for the determination of alcohol using an olive oil emulsion method after by gas chromatography. grinding at 0°C, was 10,000 Uw/g membrane. Production of Biofuels Production of Bioethanol The extraction thimble was closed with a Extractive fermentation fat free cotton swab. The thimble was inserted into the soxhlet extractor. The The fermentor, input, output and sampling solvent such as hexane, petroleum ether, was sterilized by autoclaving. The lipid was filled into the solvent vessel. The oil extracted algae debris after soxhlet extract was extracted at a temperature 110- extraction and other nutrients were 130oC for 10-20extraction cycles. The sterilized separately and added aseptically solvent was drained into a container by to the reactor before inoculation of opening the spigot to the soxhlet extractor. Saccharomyces cerevisiae (Anuradha The solvent vessel was heated until all the Karunanidhi 2008). solvent evaporated and condensed in soxhlet extractor. The vessel containing After the sterilized medium cooled to the the fat residue was placed in a drying oven ambient temperature, it was inoculated at 103oC and heated to constant weight. with 6%v/v of inoculums growth with 1kg.m-3 biomass of algae. Working Lipase catalyzed esterification volume of the reactor was maintained 1liter. Fatty acids after the soxhlet exraction was esterified using immobilized lipase The experiments were conducted in the membrane in 50ml stoppered flasks batch mode. The temperature was without organic solvent. maintained at 30oC and the aeration at

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0.5VVM. The initial pH was adjusted braunii in shake flasks and bioreactor are between 6.5-6.8 with 1M NaOH. The depicted in Fig.4..the cell growth reached control pH was maintained at 5.0.The a maximum of 1.2g L-1 for B.braunii agitation was set at 160rpm.These after 3 weeks in flasks. The growth was conditions were maintained throughout the however higher in photobioreactor chemostat runs. ,reaching a maximum of 4.1g L-1. The results are concurrent with the earlier Results and Discussion reports made by Chisti, Y. 2008.

From the present investigation the Lipid Estimation following results were obtained. The microalgae sample Fig.1a had adapted to The total lipid was measured the Chu-13 culture medium with gravimetrically by using following microscopic view of the culture Fig. 1b formula.and the data was obtained as after 3 weeks of incaution in rotary shaker shown in the Table.4.1. Fig.2, which was monitored in terms of its increase in biomass after which, it was From the Table-1 It was found that, transferred to the photobioreactor. The increase in the lipid content as the days results were confirmed with the earlier increases. Maximum lipid content was studies made by David et al., 1988. The found to be 78% later on the percentage culture was found to be yield well and the of lipid content was showing constant as yield was measured by taking the total the increasing age of culture. The graph weight of the biomass and was found to be plotted, when compared with the 1.2g/l from the shake flask studies. investigation done by Katsumi et al., 1987 was found to be 71.6% whereas in our Bubble column Photobioreactor culture investigation it is observed to be 78% of lipid content from the B. braunii grown in Botryococcus braunii inoculated to the a bubble column photobioreactor photobioreactor showed much increase in supplemented with Chu 13 medium after the growth rate and the lipid content five weeks of culture. compared to that of shake flask culture. However, the apparatus designed and fabricated for the study was found to be Determination of Alcohols by Gas suitable because the light intensity, Chromatography temperature, humidity and co2 supply was found to be more appropriate for the yield The five weeks old sample when subjected of total biomass from the algal sample to Gas chromatography, chromatogram of when compared to the shake flask studies the sample was found similar to that of Fig. 3a & b. The result found to be 4.1g/l. methanol chromatogram (Fig.6 & Fig.7). The peak area of the sample was Growth rate calculated by comparing to the peak area of methanol and the percentage of The growth rate curves of Botryococcus

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Fig.1a. Algal sample obtained from US depot. b. Microscopic image of Botryococcus braunii

Fig.2 Weeks old Shake Flask Culture

Fig.3 Weeks old Bubble column Photobioreactor culture

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Fig.4 Biomass growth rate curve of Botryococcus braunii

Table.1. Showing the percentage of oil in terms of grams

Age of culture Dry weight Lipid extract weight % Lipid (days) (grams) (grams) content 0 5 0 0 7 5 0.45 9 14 5 0.91 18.2 21 5 1.8 36 28 5 2.89 57.8 35 5 3.9 78 42 5 3.8 78 Fig.5 Percentage v/s time in days, tested for lipid content

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Fig.6 Chromatogram of Methanol

Fig.7 Chromatogram of Algae Sample

858 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 851-860 methanol in algae sample was found out References by the formula. Achitouv, Etienne, Metzer, Pierre, Rager, Production of Biofuels Marie-Noëlle and Largeau, Claude. 2004. C31-C34 methylated squalene Algae oil extraction by Soxhlet from a Bolivian strain , Botryococcus extraction method braunii. Phytochem. 65(23): 3159- 3165. After the extraction, the solvent was Anuradha Karunanidhi. 2008 : drained into a suitable container by Extractive Fermentation of Ethanol opening the spigot on the soxhlet inan Aqueous Two-Phase system by extractor. The solvent vessel was heated Saccharomyces cerevisiae NCIM until the solvent was evaporated and the 3288 : Advanced Biotech.11-14. algae oil was obtained. Banerjee, A., R. Sharma, Y. Chisty and Banerjee, U.C. 2002 : Botryococcus Lipase catalyzed esterification braunii: A renewable source of hydrocarbons and other chemicals , There was no separation of aqueous and Critical Rev. Biotechnol. 22( 3): 245- organic phases when it was left 279. undistributed after stirring slowly for Chisti Y., 2008. from 30minutes. The production of biodiesel by microalgae beats bioethanol , Trends esterification was not possible for the in Biotechnology : 26(3):126 131. Botryococcus braunii oil and it was later Chu. S.P., 1942. The Journal of Ecology, studied Aquatic Species Program (ASP) 30( 2): 284-325 report that the Botryococcus oils are not David, M., P. Metzer and Casadevall, E. fatty acid trigycerides but they are 1988. Two cyclobotryococcenes from triterpenes and they lack the free oxygen the B race of the green alga , atom needed for esterification process. Botryococcus braunii. Phytochem. 27 Later the Botryococcus oil was used as a (9) : 2863-2867. feedstock for hydrocracking. Dayananda, C., R.Sarada, P. Srinivas, T.R. Shamala and Ravishankar, G.A. 2006. In conclusion, the biomass yielded in a Presence of methyl branched fatty bubble column photobioreactor system. acids and saturated hydrocarbons in Subjected to extractive fermentation, for botryococcene producing strain of the lipid content and methanol production Botryococcus brauni , Acta Physiol. was found to be successful in production Plantarum.28 (3): 251-256. of biofuel from the microalgae Erica C. Francisco, Debora B. Neves, Botryococcus braunii. Eduardo Jacob-lopes and Telma T. Franco.2010. Microalgae as feedstock Acknowledgement for biodiesel production Carbon dioxide sequestration, lipid production We thank US Depot. for supplying the and biofuel quality , J.Chem. Technol. culture microalgae Botryococcus braunii. Biotechnol. 85:395-402. Further, we are also thankful to VGST, Katsumi Yamaguchi, Hiroshi Nakano, Bangalore, Karnataka, India for the Masahiro Murakami, Shoji Konosu, financial support Osamu Nakayama, Midori Kanda,

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Akhiro nakamura and Hiroaki Zhang, J., A. Meeson, A.E. Welchman, Iwamotto. 1987. Lipid composition of and Kourtzi, Z. 2010. Learning alters green algae Botryococcus brauni . the tuning of functional magnetic Agri. Biol. Chem. 51(2): 493-498. resonance imaging patterns for visual Metzger, P., and Largeau, C. 2005 forms . J. Neurosci.30 (42): 14127 Botryococcus braunii: a rich source 14133. for hydrocarbons and related ether lipids , Appl. Microbiol. Biotechnol. 66(5):486-496. Satyanarayana, K.G., A.B. Mariano and Vargas, J.V.C. A review on microalgae, a versatile source for sustainable energy and materials . Inter. J. Energy Res. 35:291-311. Sawayama, Shigeki, T.Minowa and Yokoyama, Shin-Ya. 1999. Possibility of renewable energy production and carbon dioxide (CO2) mitigation by thermochemical liquefaction of microalgae . Biomass Bioenergy. 17( 1): 33-39. Scragg, A.H., 2002. Growth of microalgae with increased calorific values in a tubular bioreactor , Biomass. Bioenergy.23(1):67 73. Sharma,O. P. 1986 : Textbook of Algae, Tata McGraw-Hill Education. Simpson, J. Andre, Zhang, Xu, Kramer, Robert and Hatcher. Patrick G. 2003. New insights on the structure of algaenan from Botryococcus braunii race A and its hexane insoluble botryals based on multidimensional NMR spectroscopy and electrospray- mass spectrometry techniques . Phytochem. 62( 5) : 783-796. Tyson, R.V., 1995. Sedimentary organic matter: Organic facies and palynofacies , Chapman and Hall, New York, ISBN 0-41-236350-X. 400. Vairavan, K., P. Thukkayannan, M. Paramathma, P.Venkatachalan and Sampathrajan, A. BIOFUEL CROPS :3-9. Vairavan. K., 2009: Global Energy Review , Enerdata Publication.

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