SEARCH RE Indian Journal of Science, Volume 1, Number 2 ...Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India, Email:[email protected]

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Balakrishnaraja Rengaraju et al. Characterization of Algal Oil from Chlorella sp., and its growth optimization using Co-solvent systems, Indian Journal of Science, 2012, 1(2), 127-129, www.discovery.org.in http://www.discovery.org.in/ijs.htm © 2012 discovery publication. All rights reserved

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Balakrishnaraja Rengaraju1, Sathishkumar Palanisamy2, Ragupathy jagannathan3, Ramkumar

Boothalingam4, Sreehari Seenivasan5, Vasanthi NS6

1. Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India, Email:[email protected] 2. Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India. Email: [email protected] 3. Department of Biotechnology, Kumaraguru College of Technology, Coimbatore – 641 049, India. Email: [email protected] 4. Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India. Email: [email protected] 5. Department of Biotechnology, Indian Institute of Technology Hyderabad, Yeddumailaram - 502205, India. Email: [email protected] 6. Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India, Email:[email protected]

Received 15 October; accepted 22 November; published online 01 December; printed 16 December 2012

ABSTRACT Over the last two decades, increasing threat of global warming and the paucity of natural resources attributes the whole nation to focus on finding a novel technique to generate sustainable energy. Microalgae have been investigated as a potential source for biodiesel and they are considered as the third generation biofuels which may resolve world’s energy crisis. In the present study, Chlorella sp., was studied for the production of algal oil and characterization of the oil. In the intracellular membrane of the microalgae, lipids were tightly bound. These lipids are crushed using cell disruptors and the lipids are extracted using the organic solvents. Based on the maximal yield of protein content, cell disruption efficiency in French press for pressure (30 psi) was optimized. The solvent extraction was optimized for maximal yield of the lipid using chloroform quantitatively. The separated lipids were analysed using High Performance Liquid Chromatography with retention time at 10.23 minutes.

Key Words: Algal lipid extraction, Chlorella sp., Microalgae

1. INTRODUCTION Abatement of crude oil results into the hunt for alternate methods of fuel production. The basic feedstocks for the production of first generation biofuels are often grains, terrestrial crops traditionally used for food including sugar cane, maize, palm, rapeseed, and soybeans[5][8][14][16]. Second-generation biofuels are typically derived from dedicated energy crops, agricultural and forestry wastes and municipal solid waste includes waste biomass, the stalks of wheat, corn, or wood [19]. Due to the issues associated with the food crops and wastes generated from those food crops paves way to ponder renewable fuel generation from microalgae, which is known as third generation fuels [19]. For 2025 it is expected to have 6 billion gallons of algal biofuel production [21] Biodiesel is also energy efficient in that it can yield up to 320% more energy than is required in its production [20].

Microalgae containing 30% oil by weight of dry biomass could yield almost 587000 l/ha or 5000–15000 gallons/year [4]. Algal oils can be converted to diesel, gasoline and jet fuel using existing technology [2]. There are about 4000 species have been identified, which can be divided into several groups including cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow_green algae (Xanthophyceae), golden algae

(Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae) and ‘pico_plankton’ (Prasinophyceae and Eustigmatophyceae) [9]. The three most prevalent groups of algae targeted for biodiesel production include the diatoms, green algae and blue green algae having the oil levels between 20 and 75% on the basis of dry biomass weight. (Table 1) [1][23]. Pulz describes in a review paper, it was reported that the photosynthesis of most microalgae is saturated at about 30% of the total terrestrial solar radiation [15]. However, up to now, the commercial production of biofuels from microalgae has not been realised on an industrial scale in a cost effective manner. Microalgal biofuels are 4–10 times as expensive to produce as petroleum derived fuels or other biodiesels [2].

Homogenisation is the process of forcing biomass through an orifice which results in a prompt pressure change and high shearing action [13]. Hydrodynamic cavitation, which uses cavitation to disrupt cells, has also been proposed to break cell walls and enhance oil extraction [10]. Ultrasound has been applied to microalgae which utilize cavitation to disrupt the cell wall and extract oil from the cell [6]. Cavitation involves nucleation, growth and transient impulsive collapse of tiny bubbles in the liquid driven by bulk pressure variation due to ultrasound waves [17]. The organic solvents are used to extract the algal oil with the soxhlets apparatus / separating funnel. Effective solvent extraction requires that the solvent can firstly penetrate the solid matrix enclosing the

Table 1 Microalgal Species & Lipid Content

Microalgal species Lipid Contenta Microalgae species

Lipid Conte

nta Ankistrodesmus species 28-40 Euglena gracillis 14-20 Anabaena cylindrica 4-7 Ellipsoldion sp., 27 Botryococcus braunii 25-86 Haemotococcus pluvialis 25 Chaetoceros muelleri 33 Hantzschia species 66 Chlamydomonas species 23 Isochrysis galbana 21.2 Chlorella emersonii 25-63 Monallantus salina 20-22 Chlorella minutissima 57 Nanochloropsis species 20-56 Chlorella protothecoides 14-57 Neochloris oleoabundans 35-65 Chlorella sorokiniana 22 Nitschia closterium 27.8 Chlorella vulgaris 14-56 Nitschia frustulum 25.9 Crypthecodinium cohnii 20-51 Pavlova lutheri 35 Cyclotella sp., 42 Phaeodactylum tricornutum 20-30 Dunaliella primolecta 23 Prostanthera incisa 62 Dunaliella salina 28.1 Prymnesium parvum 22-39 Dunaliella tertiolecta 36-42 Pyrrosia laevis 69.1 Skeletonema costatum 13-51 Spirulina plantensis 16.6 Scenedesmus dimorphus 16-40 Stichococcus species 33 Scenedesmus quadricauda 19.9 Tetraselmis suecia 15-23 Schizochytrium sp. 50-77 Thalassiosira pseudonana 20 Selenastrum species 21.7 Zitzschia sp. 45-47 a. % dry weight biomass

RESEARCH Indian Journal of Science, Volume 1, Number 2, December 2012

Characterization of Algal Oil from Chlorella sp., and its growth optimization using Co-solvent systems

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lipid, secondly physically contact the lipid and thirdly solvate the lipid [3]. Determining lipids is a challenge in any matrix. Algal Oil is characterized predominantly as well as precisely by either HPLC or GC using suitable column. Low UV absorbance and low volatility of larger lipids limit the sensitivity of existing HPLC and GC methods. Universal HPLC detectors are showing promise for the determination of these analytes, as compounds need not possess a chromophore, and they do not require derivatization. [17].

The lipids of some microalgal species are hydrocarbons, similar to those found in petroleum, while those of other species resemble seed oils, can be converted to diesel fuel by transesterification. Once the oil has been liberated from the algae, it can be transesterified along traditional biodiesel routes or hydro treated to produce hydrocarbons [7]. Wahlen et al. [24] conducted a comprehensive investigation into the lipid

characteristics of biomass from a number of different algal species and they determined the triglyceride content, the extractable lipid content and the Fatty Acid methyl Ester (FAME).

2. MATERIALS AND METHODS 2.1. Chemicals & Equipments Chloroform, Hexane, Methanol, Acetone, Basal salt Medium, Chlorella sp., French press, High performance Liquid chromatography, Rotary evaporator, Compact fluorescent lamp (12 V).

2.2. Microalgae Cultivation Chlorella sp., were propagated in BG 11 medium [18] for which the composition is given (table 2). Culture vessels were illuminated for 14 hours a day with compact fluorescent lamp (Fig.1).

2.3. Cell wall Disruption and Extraction Algae biomass was periodically observed and harvested after 14 days. French press was used to disrupt the algal cell wall. The lipids present in the cells were extracted using various organic solvents [3].

2.4. Optimization studies Mechanical method of disruption was optimized for biomass yield by changing pressure in the units of psi (10, 20, 30, 40 and 50) in French press. The cellular contents were measured in terms of protein released by standard protein assay [11]. Solvent system was also optimized for the maximum yield of

Table 2 Growth Medium BG – 11Composition Micronutrients Protein Concentration (g/l)

NaNO3 1.5 K2HPO4 0.04 MgSO4.7H2O 0.075 CaCl2.2H2O 0.036 Na2CO3 0.02 Citric acid 0.006 Ferric ammonium citrate 0.006 EDTA (Na2 Salt) 0.001 H3BO3 2.86 MnCl2.4H2O 1.81 ZnSO4.7H2O 0.22 Na2MoO4.2H2O 0.39 CuSO4 .5H2O 0.079 Co(NO3)2.6 H2O 0.049

Table 3 Estimation of protein yield Pressure (psi) Protein Concentration (mg/l)#

10 11.36 20 18.52 30 45.47 40 23.62 50 31 #Concentration absorbance taken in triplicates

Figure 1 Growth of Chlorella sp. After 9 days

Figure 2 Chromatogram of Algal extract

Figure 3 Standard Graph for Lowry’s method

Figure 4 Cell Disruption studies using French Press

Figure 5 Extraction using various Co-solvent Systems

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lipid content. The solvents used were acetone, methanol, chloroform, ethanol and diethyl ether.

2.5 Algal Oil Characterization The separated lipids were analysed using High performance liquid Chromatography with hexane as a diluents and hexane diethyl ether as a mobile phase in the ratio of (9:1) in Thermo scientific Normal Phase column.

3. RESULTS AND DISCUSSION 3.1 Algal Cultivation Microalgae are grown and periodically the flasks were checked for the growth by just visualization. The growth characteristics of the algal strains were already studied and it was optimized as 14 days. The culture vessels were hand shaken atleast twice a day to keep organisms in homogeneous state. The absorbance value was taken after 14 days to ensure its exponential phase and it was taken for the further studies and oil extraction. At the exponential phase the oil content is maximum. The algal culture was mantained at 14 hours per day in compact fluorescent lamp (12 V) which emits 9.8 W m-2 light intensity at the surface of the culture vessel. In general, lower oil strains grow faster than high oil strains [22]. Microalgae containing 30% oil grow 30 times faster than those containing 80% oil [1]. Another challenge is that microalgae usually accumulate oil under stress conditions with slow growth rate.

3.2. Optimization studies The cell wall forms a barrier to the penetration of the solvent into the cell. This was achieved by cell disruption techniques. In this study, the french press was used to break the algal cell wall. The French press was operated in different pressures at 10, 20, 30, 40 and 50 psi. Samples from french press were collected based on protocols given as per the standard operating procedure. The proteins released from cell content were measured using Lowry’s method at 750 nm and found the maximum protein content was released at 30 psi. Thus the cell disruption efficiency were calculated by measuring the protein at 750 nm. (Fig. 4)

The extractions of the disrupted samples were studied using co-solvent systems. The different co-solvent systems used for the study were Acetone: Methanol, Diethyl ether: Methanol, Methanol: Water, Chloroform: Methanol with the ratio 2:1 using a separating flask method. In CHCl3: CH3OH system, the oil yield was comparitively better than other solvent systems on the volume basis (v/v). The organic layer (Chloroform+lipid) was subjected to distillation using rotary evaporator system. Samples were distilled at 600C and the final extract was collected. Methanol and chloroform form a co-mixture that dissolves the lipids (Fig. 5). The chloroform dissolves the neutral lipids (triglycerides) while the methanol dissolves the polar membrane lipids [3]. Water is then added to separate the polar methanol phase from the non-polar chloroform phase.

3.3. Algal Oil Characterization The samples were analysed by High Performance Liquid Chromatography and the results showed that the algal supernatant having the retention time of 10.33 which matches with the standard fatty acid profile which was eluted near 10 minutes retention time for the standard reference material (Algal oil from Thermoscientific co.,) (Fig.2).

4. CONCLUSION The increasing fuel demand and the global warming urge the technologists to find an alternate source to mitigate energy crisis. Microalgae based biofuel production is promising and potential alternate to overcome fuel demand. The algal oil production was confirmed. The highly remarkable portion of the study was optimization of disruption technique and co-solvent systems which decides the fuel oil generation efficiency. In future studies, algae oil shall be transesterified using the suitable solvent system and transforming the technology to be adopted for industries. It can be blend with the fossil fuel and used to mitigate energy crisis.

ACKNOWLEDGMENT The authors would like to thank Dr V Sivasubramanian, Director, Vivekanandha Institute of Algal Technology, Chennai for providing Microalgal culture. The authors would like to extend their heartfelt gratitude to the Principal and Management of Bannari Amman Institute of Technology Sathyamangalam, Erode District, India for their financial support.

REFERENCES 1. E.W.Becker, J.Baddiley, I.J.Higgins, and W.G.Potter, 1994, Microalgae: Biotechnology and Microbiology, Cambridge: Cambridge Univ. Press. 2. Chisti Y, 2007, Biodiesel from Microalgae, Biotechnol. Adv., vol. 25, no. 3, pp. 294–306. 3. Cooney M. Young G. and Nagle N, 2009, Extraction of bio-oils from microalgae, Sep Purif Rev., 38:291–325. 4. Deng X. Li Y. and Fei X, 2009, Afr. J. Microbiol. Res.,vol. 3, no. 13, pp. 1008–1014. 5. Doornbosch R. and Steenblik R, 2007, Biofuels: Is the Cure Worse than the Disease?, in Round Table on Sustainable Development. Organisation for Economic

Cooperation and Development (OECD), Paris. 6. Echevarria Parres, 2011, AJDJDSJB Process and apparatus for extracting biodiesel from algae, US Patent No. 2011/0189741 A1. 7. Egeberg R G. Michaelsen N H. and Skyum L, 2012, Novel hydrotreating technology for production of green diesel, Lyngby, Denmark Haldor Topsoe A/S,

Available from: http://www.topsoe.com/business_areas/refining/~/media/PDF%20files/Refining/novel_hydrotreating_technology_for_production_of_green_ diesel.ashx (Accessed 11 July 2012).

8. Eisentraut A, 2010, Sustainable production of second generation biofuels OECD/IEA 2010: International Energy Agency. 9. Hu Q. Sommerfeld M. Jarvis E. Ghirardi M. Posewitz M. Seibert M. and Darzins A, 2008, Microalgal triacylglycerols as feedstocks for biofuel production:

perspectives and advances, Plant J., vol. 54, pp. 621–639. 10. Larach MC, 2010, Continuous cultivation, harvesting and oil extraction of photosynthetic cultures, PCT Patent No. WO 2010/017243 A1. 11. Lowry O H. Rosebrough N J. Farr A L. and Randall R J, 1951, Protein measurement with the Folin phenol reagent, J Biol Chem., 193(1):265-75. 12. Marc Plante. Bruce Bailey. and Ian Acworth, 2009, The use of charged aerosol detection with HPLC for the measurement of lipids, Lipidomics, Vol. 579, 469-

482. 13. Mercer P. and Armenta R E, 2011, Developments in oil extraction from microalgae, Eur J Lipid Sci Technol, 113:539–547. 14. Mittelbach. and Remschmidt, 2006, Biodiesel - the comprehensive handbook, Graz: Selbstverlag des Verfassers, 230 S. - ISBN 3-200-00249-2. 15. Pulz, 2001, Photobioreactors: production systems for phototrophic microorganisms, Appl Microbiol Biotechnol, 57, pp. 287–293. 16. Oil World, December 12th No. 50 Vol. 51. Oil World Monthly, Available from: ISTA Mielke GmbH, 2008. 17. Ranjan A. Patil C. and Moholkar V S, Mechanistic assessment of microalgal lipid extraction, 2010 Ind Eng Chem Res, 49:2979–2985. 18. Rippka. Rosmarie, Josette Deruelles. John B Waterbury. Michael Herdman. and Roger Y Stanier, 1979, Generic Assignments, Strain Histories and Properties of

Pure Cultures of Cyanobacteria, Journal of General Microbiology, Vol. 111, p. 1-61. 19. Ryan C, 2009, Cultivation Clean Energy: The Promise of Algae Biofuels, Hartley A, Ed., Terrapin, Bright Green, pp. 9–62. 20. Sheehan J. Camobreco V. Duffield J. Graboski M. and Shapouri H, 1998a, Life cycle inventory of biodiesel and petroleum diesel for use in an urban, bus. Tech.

Rep. NREL/SR-580- 24089, U.S. Department of Energy and U.S. Department of Agriculture. 21. Thurmond W, 2012, Algae 2020: Biofuels Markets, Business Models, Strategies, Players and Commercialization Outlook, 2012–2020. 22. Vasudevan P.T, and Briggs M, 2008, Biodiesel production-current state of the art and challenges, J. Int. Microbiol. Biotechnol., vol. 35, pp. 421–430. 23. Verma N M. Mehrotra S. Shukla A, and Nath Mishra, B, 2010, Afr. J. Biotechnol ,vol. 9, no. 10, pp. 1402–1411. 24. Wahlen B D. Willis R M. and Seefeldt L C, 2011, Biodiesel production bysimultaneous extraction and conversion of total lipids from microalgae, cyanobacteria,

and wild mixed-cultures, Bioresour Technol, 102:2724–2730.

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SEARCH RE Indian Journal of Science, Volume 1, Number 2 ...Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam – 638 401, India, Email:[email protected]