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Applied Energy xxx (2011) xxx­xxx

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Applied Energy

journal homepage: www.elsevier.com/locate/apenergy

Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor

Sara Rasoul-Amini a,b,c, Nima Montazeri-Najafabady a,b, Mohammad Ali Mobasher a,b, Samira Hoseini-Alhashemi c, Younes Ghasemi a,b,

a b c

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran

a r t i c l e

i n f o

a b s t r a c t

The biodiesel production from a naturally isolated strain of Chlorella in 2 L bubble-column photobioreactor was studied. The microalgal strain was isolated from the rice paddy-field soil samples during a screening program. After 17 days, at the end of exponential phase of growth, the total content of the lipids was extracted. The extracted fatty acids were first esterified and then identified using GC/MS analysis. Several types of fatty acid methyl esters (FAMEs) were identified in the isolated microalga and the presence of saturated fatty acids in Chlorella sp. MCCS 040 was approved. The composition of fatty acids in the studied species of microalga was mainly palmitic acid methyl ester, myristic acid methyl ester, stearic acid methyl ester and undecanoic acid methyl ester. This strain because of its highly saturated fatty acids content can be an ideal candidate for biodiesel production. Ó 2010 Elsevier Ltd. All rights reserved.

Article history: Received 9 September 2010 Received in revised form 27 November 2010 Accepted 9 December 2010 Available online xxxx Keywords: Biodiesel Chlorella sp. Fatty acid methyl ester Bubble-column photobioreactor Production

1. Introduction Recently, fuel production from algae has been receiving considerable attention because of growing energy prices, emissions of green house gases [1] and gradual depletion of fossil fuels [2]. Microalgae due to their rapid biomass production, high photosynthetic efficiency [1] and ability to storage a large amount of lipid are ideal source of biodiesel [3]. Averagely, microalgae can produce biodiesel 10­20 times higher than that obtained from oleaginous seeds and/or vegetable oils [4]. To assess the feasibility of producing biodiesel, a large amount of microalgae biomass is needed [5]. Microalgae can be normally cultivated in the open-culture systems (lakes or ponds) and also in the closed-culture systems called photobioreactors (PBRs) [6]. Biomass production of up to 150 tons haÀ1 yearÀ1 has already been reached in photobioreactor [7]. Experimental photobioreactors are often designed as columns [8]. Vertical bubble-columns have good light­dark cycling, low surface/volume and a much more chaotic gas­liquid flow [6].

Chlorella is widespread in different locations such as fresh water, air and soil [9]. Chlorella sp. has good potential to accumulate more than 20% lipids, such as C 18:1, C 16:0 and C 18:3 [10]. In this study a naturally isolated strain of Chlorella collected from soil samples, was cultivated in 2 L bubble-column photobioreactor and investigated for biodiesel production.

2. Materials and methods 2.1. Bioreactor design The constructed photobioreactor has a total volume of 2 L (Fig. 1). The photobioreactor consisted of different parts. Glassmade cylinder, four plastic tubes with a diameter of 6 mm which were used for gas inlet, gas outlet, feeding and sampling, porous bulk stone which was used for bubbling the air, air pump, top head plastic cover with four ports for plastic tubes. 2.2. Growth and fatty acid composition of Chlorella The inoculation culture of Chlorella was cultivated in the appropriate BG-11 liquid medium in 250 mL Erlenmeyer flasks containing 100 mL culture. Then Chlorella sp. was grown under low illumination (37 lmol mÀ2 sÀ1) and unlimited aerated condition for one week. Temperature was adjusted at 25 ± 2 °C.

Corresponding author at: Department of Pharmaceutical Biotechnology, Faculty

of Pharmacy, Shiraz University of Medical Sciences, P.O. Box 71345-1583, Shiraz, Iran. Tel./fax: +98 711 2426729. E-mail address: [email protected] (Y. Ghasemi). 0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apenergy.2010.12.040

Please cite this article in press as: Rasoul-Amini S et al. Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor. Appl Energy (2011), doi:10.1016/j.apenergy.2010.12.040

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S. Rasoul-Amini et al. / Applied Energy xxx (2011) xxx­xxx Table 1 A summary of the identified fatty acid methyl esters (FAMEs) in the naturally isolated Chlorella strain. FAMEs Methyl Methyl Methyl Methyl Methyl Methyl Methyl Methyl tetradecanoate decanoate dodecanoate undecanoate hexadecanoate heptadecanoate octadecanoate pentadecanoate Common name Methyl myristate Methyl caprate Methyl lurate Methyl palmitate Methyl margarate Methyl stearate Formula C15H30O2 C11H22O2 C13H26O2 C12H24O2 C17H34O2 C18H36O2 C19H38O C16H32O2 FAME content (% total) 25.9 1.1 5.9 17.7 23.6 2.1 12.8 6.3

2.2.3. 18S rRNA PCR amplification 18S rRNA gene sequence of Chlorella sp. MCCS 040 was amplified using two sets of primers [11]. The applied PCR condition has been described before [11]. The PCR products were electrophoresed in a 1% (w/v) agarose gel. The sequence was determined by the CinnaGen Company with the primers.

Fig. 1. Schematic diagram of a bubble-column photobioreactor: (a) side view and (b) top view of the photobioreactor. F: feeding port, S: sampling port, In: gas inlet, Out: gas outlet.

2.2.4. Multiple alignment analysis of the amplified sequence We applied the NCBI databases with BLAST search to search sequence similarity (http://blast.ncbi.nlm.nih.gov/Blast.cgi). 2.2.5. Fatty acid extraction, esterification and GC/MS analysis Fatty acids extraction, esterification and GC/MS analysis protocol was described in our previous study [12]. 3. Results The biomass concentration of Chlorella sp. at the end of exponential phase was approximately 1.9 g/L. It has doubling time of approximately 15 h during the exponential phase. The BLAST results showed 99% similarity to the 18S small subunit rRNA of some Chlorella species. The profile of FAMEs was identified through the comparison of their mass spectra with those in Wiley libraries. The results are displayed in Table 1. Several types of FAMEs were detected through GC/MS analysis. These are as follows: methyl tetradecanoate, methyl decanoate, methyl dodecanoate, methyl undecanoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecanoate, methyl pentadecanoate (Table 1). 4. Discussion The main goals of microalgae oil production are high lipid yield and high biomass productivity which can affect production costs [13]. The quality of biodiesel is dependent on composition of the fatty acid methyl esters [14]. Fatty acid methyl esters properties are determined by length of carbon chain, degree of unsaturation and the alcohol content of composed fatty acids [2]. Cetane number, cold-flow properties, oxidative stability and iodine value are the most important properties of biofuel which are derived from its fatty acid methyl esters structure [2]. Palmitic acid and stearic acid are known as the most common fatty acids contained in biodiesel [15]. Different types of saturated fatty acids were detected in the Chlorella sp. MCCS 040. Palmitic acid, stearic acid, myristic acid and undecanoic acid are the most fatty acid composition of the investigated strain. Also pentadecanoic acid, margaric acid, lauric acid and capric acid were determined in Chlorella sp. MCCS 040. Several types of fatty acids with different degree of unsaturation

2.2.1. Cultivation in photobioreactor After one week, 50 mL of an inoculation culture was transferred to 2 L bubble-column photobioreactor containing 1 L of BG-11 liquid medium. Filtered air was continuously pumped through multi nozzles bulk stone at the bottom of photobioreactor. The aeration was performed at flow rate of 9 L minÀ1 for 24 h. The photobioreactor was illuminated with 40 W white fluorescent lamps which were placed at three side of the column and the average light intensity was approximately 37 lmol mÀ2 sÀ1. This run lasted for 17 days until Chlorella sp. cells reached the stationary phase. After finishing cultivation, culture medium was centrifuged at 5000 rpm, 4 °C for 5 min, and the cell pellets were freeze-dried. Then alga pellets were prepared for fatty acid extraction. Moreover, the dry weight of the algal cells was measured. 2.2.2. Growth parameters The cell number and optical density was measured during the cultivation period in the photobioreactor. For cell count, each time 1 mL of algal suspension was removed through sampling tube and then direct count was performed using Neubauer hemocytometer and an Olympus Light Microscope. Also, optical density was measured at 670 nm using a UV/Visible spectrophotometer (PG instrument Ltd.). Growth curve is shown in Fig. 2.

Fig. 2. Growth profile in cell number and optical density of Chlorella sp.

Please cite this article in press as: Rasoul-Amini S et al. Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor. Appl Energy (2011), doi:10.1016/j.apenergy.2010.12.040

S. Rasoul-Amini et al. / Applied Energy xxx (2011) xxx­xxx

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in various microalgae have been reported [2,4,13­16]. Most of algal oils have high polyunsaturated fatty acids content with four or more double bonds [2]. As reported before 50­65% of microalgal (Chlorella vulgaris, Spirulina maxima, Nannochloropsis oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) fatty acids were mainly composed of unsaturated fatty acids [17]. In compare with our previous study, palmitic acid content (23.6%) is more than four times higher than that of Chlamydomonas MCCS 029(5.6%). Dodecanoic acid, tetradecanoic acid and pentadecanoic acid content of Chlorella sp. MCCS 040 was 5.9%, 25.9% and 6.3% respectively, which was higher than that of Chlamydomonas MCCS 029 (4.2%, 6.6% and 5.4%) [12]. Highly saturated fatty acids give an excellent cetane number and oxidative stability to biodiesel [17]. Biodiesel quality is directly related to cetane number which shows ignition quality in engine [18]. As the results show, only saturated fatty acids were detected in Chlorella sp. MCCS 040. Palmitic acid, stearic acid and myristic acid content in the microalgal strain is higher than Chlorella protothecoides 0710 [19,20], Chlorella vulgaris (UTCC 90), Dunaliella tertiolecta (UTCC 420), Phormidium sp., and Scenedesmus obliquus (UTCC 5) [21]. Capric acid which its presence can improve biodiesel properties [14] has the content of 1.1% in the studied strain. Its content is higher than Chlorella vulgaris (UTCC 90), Dunaliella tertiolecta (UTCC 420) and Phormidium sp. [21]. All of the oils extracted from algae are not suitable or compatible to use as biodiesel [2]. The most common vegetable oils that are used for biodiesel are C18 and C16 [2]. According to the results, Chlorella sp. MCCS 040 has some features that make this strain ideal for biodiesel production. These features are as follows: (1) All of its fatty acids are saturated which give good cetane number and oxidative stability to biodiesel. (2) Palmitic acid and stearic acid content which are known as the most common fatty acids contained in biodiesel, are present in this strain. (3) The hydrocarbon chain length of fatty acids is between C10 and C18. (4) This strain can be easily cultivated in photobioreactor with good biomass productivity.

Acknowledgement This work was supported by a grant from the Research Council of Shiraz University of Medical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran. References

[1] Xiong W, Gao C, Yan D, Wu C, Wu Q. Double CO2 fixation in photosynthesis­ fermentation model enhances algal lipid synthesis for biodiesel production. Bioresour Technol 2010;101:2287­93. [2] Damiani MC, Popovich CA, Constenla D, Leonardi PI. Lipid analysis in Haematococcus pluvialis to assess its potential use as a biodiesel feedstock. Bioresour Technol 2010;101:3801­7. [3] Johnson MB, Wen Z. Development of an attached microalgal growth system for biofuel production. Appl Microbiol Biotechnol 2008;85:525­34. [4] Gouveia L, Oliveira AC. Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 2009;36:269­74. [5] Chisti Y. Biodiesel from microalgae beats bioethanol. Trends Biotechnol 2008;26:126­31. [6] Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renew Sustain Energy Rev 2010;14:217­32. [7] Sialve B, Bernet N, Bernard O. Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol Adv 2009;27:409­16. [8] Eriksen NT. The technology of microalgal culturing. Biotechnol Lett 2010;30:1525­36. [9] Wu HL. Identification of Chlorella spp. isolates using ribosomal DNA sequences. Bot Bull Acad Sinica 2001;42:115­21. [10] Scarsella M, Parisi MP, D'Urso A, De Filippis P, Opoka J, Bravi M. Achievements and perspectives in hetero- and mixotrophic culturing of microalgae. Chem Eng Transac 2009;17:1065­70. [11] Rasoul-Amini S, Ghasemi Y, Morowvat MH, Mohagheghzadeh A. PCR amplification of 18S rRNA, single cell protein production and fatty acid evaluation of some naturally isolated microalgae. Food Chem 2009;116:129­36. [12] Morowvat MH, Rasoul-Amini S, Ghasemi Y. Chlamydomonas as a ``new'' organism for biodiesel production. Bioresour Technol 2010;101:2059­206. [13] Rodolfi L, Zittelli GC, Bassi N, Padovani G, Biondi N, Bonini G, et al. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol Bioeng 2009;102:100­12. [14] Yoo C, Jun SY, Lee JY, Ahn CY, Oh HM. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 2010;101:71­4. [15] Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM. Comparison of several methods for effective lipid extraction from microalgae. Bioresour Technol 2010;101:75­7. [16] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294­306. [17] Chinnasamy S, Bhatnagar A, Hunt RW, Das KC. Microalgae cultivation in a wastewater dominated by carpet mill effluents for biofuel applications. Bioresour Technol 2010;101:3097­105. [18] Van Gerpen J. Biofuel. Methods in molecular biology. In: Jonathan R. Mielenz editor. Biodiesel: small scale production and quality requirements. Humana Press, a part of Springer Science and Business Media, New Jersey; 2009. p. 281­90. [19] Xiong W, Li X, Xiang J, Wu Q. High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl Microbiol Biotechnol 2008;78:29­36. [20] Li X, Xu H, Wu Q. Large-scale biodiesel production from microalgae Chlorella protothecoides through heterotrophic cultivation in bioreactors. Biotechnol Bioeng 2007;98:764­71. [21] Francisco, EC, Neves DB, Jacob-Lopes E, Franco TT. Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality. J Chem Technol Biotechnol 2009;85:395­403.

5. Conclusion The results of this study show that Chlorella sp. MCCS 040, according to its fatty acid content is an ideal candidate for biodiesel production. It is for the first time that such microalgae with highly saturated fatty acids and none monounsaturated and none polyunsaturated fatty acids has been reported. So that further investigation should be done on this strain to make it as a commercial biodiesel producer.

Please cite this article in press as: Rasoul-Amini S et al. Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor. Appl Energy (2011), doi:10.1016/j.apenergy.2010.12.040

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