The study of microalgae Nannochloropsis salina fatty ...

Journal of Molecular Liquids 239 (2017) 96–100

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The study of microalgae Nannochloropsis salina fatty acid composition of the extracts using different techniques. SCF vs conventional extraction Aslan M. Aliev a,b, Ilmutdin M. Abdulagatov c,⁎ a b c

Institute of Physics DSC RAS, Yaragskogo Street, 94, Makhachkala 367003, Russia Mountain Botanical Garden DSC RAS, Yaragskogo Street, 75, Makhachkala 367030, Russia Dagestan State University, Physical Chemistry Department, Gadzhieva Str., 43a, Makhachkala 367000, Russia

a r t i c l e

i n f o

Article history: Received 8 April 2016 Received in revised form 29 July 2016 Accepted 5 August 2016 Available online 9 August 2016 Keywords: Acetone Carbon dioxide Chloroform Fatty acids Microalgae Nannochloropsis salina Supercritical extraction Supercritical fluid

a b s t r a c t The extraction of lipids and other valuable components from microalgae (Nannochloropsis salina) has been performed using various conditions: pure SC-CO2; SC-CO2 + acetone (at 32 MPa and 40 °C); Soxhlet apparatus with n-hexane; and Soxhlet apparatus with chloroform. Small amounts of co-solvents (3.2 wt.% of acetone) were added to modify the polarity and solvent strength of the supercritical CO2 to increase the solute solubility and to minimize operating costs of the extraction process. The extractions of lipids from algae were performed using a laboratory scale SCF extraction experimental apparatus. High pressure supercritical reactor with volume of 993 cm3, which made from stainless steel, was used. The flow rate of SCF was maintained constant (1.5 ± 0.05 kg/h). The extraction time was within 1.5 h. Four extracted lipid fractions were collected separately for each extraction conditions (for pure CO2 and CO2 + co-solvents) and analyzed using GC–MS. The experimental results showed that extraction technique is negligible small affecting on total extract yields and fatty acids content. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The population of many developing countries suffers from unbalanced nutrition and run short of food. A fatty acids deficiency in daily food raises the risk of cardio-vascular and autoimmune diseases, cancer, asthma, arthritis, depression, schizophrenia, and various pathologies in a child development [1–17]. For example, palmitoleic, frachidonic, timnodonic acids are used for treatment and prevention of diseases such as cardio-vascular, diabetes, and eye disorders, arthritis, contribute to the Alzheimer disease remission, facilitate to control the cholesterol and a blood pressure, and arthronosos, etc. Also, polyunsaturated fatty acids maintain the brain function, the formation of red blood cells, the immune system [18–22]. Microalgae have a short growth cycle and are unpretentious: necessary conditions for their cultivation are water, sunlight, and simple nutrient supply; they do not occupy fertile soils and have not seasonal constraints [23–25]. Microalgae are effective in a solution to environmental issues such as sewage treatment. They occlude carbon dioxide in large quantities (about 1.9 kg per 1 kg of dry biomass) [26]. The manufacturing of polyunsaturated fatty acids requires the elaboration of cost-efficient and renewable sources of their preparation as well as the design of technologies of extraction. Development of the methods for polyunsaturated fatty acids extraction is a ⁎ Corresponding author at: Geothermal Research Institute of the Russian Academy of Sciences, Thermophysical Properties, 39 A Shamil Ave, 367030 Makhachkala, Russia.

http://dx.doi.org/10.1016/j.molliq.2016.08.021 0167-7322/© 2016 Elsevier B.V. All rights reserved.

highly important issue for their degradation ability under high temperatures during the extraction process [27]. Although the extraction of oil from biomasses is usually performed using chemical processes (solvent extraction) with organic solvents such as n-hexane, acetone, methanol, and chloroform, supercritical CO2 (SC-CO2) is regarded with interest as an industrial process and has some advantages such as no environmental impact, high quality final product without any trace of toxic solvent, etc. [28]. Supercritical technology is very suitable for extraction of high-value bioactive compounds from microalgae (safer than organic solvent, negligible environmental impact, a short extraction time, and a high quality of final product than conventional n-hexane extraction) [14–17,29]. However, chemical method is difficult to scale and most recent studies have an increased focus on reducing the use of toxic and polluting solvents and the development of environmentally appropriate technologies for the extraction. New lipid–extraction technologies involve innovations in acoustics, sonication, mesoporous nanomaterials, and amphiphilic solvents. As a rule, supercritical fluid extraction processes are realized as a closed (continuous) extraction cycle with minimal emission of extractants to the environment (environmental protection), and no generate CO2. Therefore, a supercritical carbon dioxide technology considerably reduces the emission of CO2 into atmosphere and avoids the development of the GHG effect and global climate changing, increases the rate of the extraction process by 2 to 3 times and 2 to 2.5 times reduce energy consume at the regeneration process. Compared with

A.M. Aliev, I.M. Abdulagatov / Journal of Molecular Liquids 239 (2017) 96–100

ordinary organic solvents, supercritical fluid has a high selectivity extraction which related with their unusual properties. Solvent characteristics and selectivity of the supercritical solvent can be easy manipulated by a slight changing the thermodynamic conditions (temperature, pressure, and co-solvent concentration) of the extraction. Reactions in supercritical media and their applications, especially in “Green Chemistry”, are very attractive in scientific (research) and technological (ecologically clean and energy-efficiency) viewpoints in future. 2. Review previous studies of microalgae extractions with SCF Previously, some extractions of lipids from microalgae, Spirulina (Arthrospira) maxima; Spirulina (Arthrospira) platensis; Botryococcus braunii; Chlorella vulgaris; Ochronomas danica; Skeletonema costatum; Isochrysis galbana; and Nannochloropsis sp., with supercritical fluids were reported by other authors [14–17,29–41]. However, it is still very poorly explored the effect of operating conditions on the fatty acid composition of lipid extracts. The fatty acid composition of lipid extracts is very sensitive to the extraction conditions. Detailed and comprehensive experimental data are needed to develop predictive mathematical model of SCF extraction process, deeply understand, on the molecular level, the mechanism of the extraction process and the effect of chemical nature of SCF and co-solvent on the extracting components. Microalgae Nannochloropsis salina is one of the most promising EPA (eicosapentaenoic acid) producer [12,29,42–45]. Hu and Gao [45] optimized the growth and fatty acid composition of Nannochloropsis sp. under CO2-enriched photoautotropic or/and acetate-added mixotrophic conditions. These conditions gave the highest biomass yield (634 mg wt./L), the highest total lipid content (9% of dry wt.), total fatty acids (64 mg/g dry wt.), polyunsaturated fatty acid (35% total fatty acids), and eicosapentaenoic acid (EPA, 20:5n − 3, 16 mg/g dry wt. or 25% of total fatty acids). An addition of sodium acetate (2 mM) decreases the amounts of the total fatty acids and EPA. Elevation of CO2 in photoautotropic culture enhances growth and increasing the production of EPA in Nannochloropsis sp. [42,46,47]. Biktashev et al. [47] also used the same sample for supercritical CO2 and CO2 + ethanol extractions of lipids and other valuable components from microalgae (Nannochloropsis salina) at pressure 35 MPa and at temperature of 40 °C. The extraction of lipids from microalgae was performed using a laboratory scale batch-type SCFE experimental apparatus. High pressure supercritical reactor with volume of 8.47 cm3, which made from stainless steel, was used. The flow rate of SCF was maintained constant 1.0 g/min. The extraction time was within 5 to 8 h. In present work, we report the dependence of fatty acid profiles of Nannochloropsis salina extracts obtained using the various methods: pure SC-CO2, CO2 + acetone (co-solvent) extractions, and in the conventional Soxhlet apparatus using n-hexane and chloroform.

microalgae samples were crushed in electro-coffee grinder to powder with particles size of 0.5 to 1.0 mm. The following chemicals were used: liquid carbon dioxide (99.5 wt.%) and acetone (99.5 wt.%). 3.2. Supercritical fluid extraction The process of the supercritical extraction has a number of advantages by contrast to the extraction in the Soxhlet apparatus: the high speed of the process, the low-temperature extraction, an oxygen-contact-free extracting that permits obtaining of thermolabile compounds, and environmental friendliness. The measurements were made using an experimental supercritical fluid apparatus (see updated version in Fig. 2, some details also reported in our previous works [49,50]). The apparatus allows for complex researches of extracting processes at pressures up to 40 MPa and temperature from 25 to 100 °C with the maximum flow of supercritical fluid of 1.7 (±0.05) kg/h. Raw material grinded (80 g) is loaded into a cylindrical extractor-1 (70 mm in inside diameter and 0.993 L of internal volume), where a SC fluid is fed through a power cylinder-3, and infused for 10 min. Then, a SC fluid with dissolved extract is delivered into a separator-2 simultaneously feeding pure SC fluid into the extractor from the power cylinder-3. In the separator-2, a temperature and a pressure are supported within 20 °C and 0.5 MPa being optimum for separation of an extract from gaseous CO2. The pressure transducer P1 (see Fig. 2) provides the measurements and the control isobaric process of the extraction, while the pressure transducer P2 is controlling the separator's pressure. The pressure transducers P3 and P4 were also used for the pressure control in the various parts of the extraction apparatus. The temperature transducers T1 and T3 are measuring and controlling the temperature in the extractor and power cylinder, respectively, while the T2 is controlling the separator's temperature. The heaters H1 to H4 were used for heating and to maintain isothermal conditions of the extraction process. A pressure in the system (extractor) is created by a pump-8 with an automatic electronic system of desired pressure maintain, i.e., isobaric process in the SC reactor was maintained automatically [49,50]. Pump-8 feeds distilled water from a reservoir-7 into the power cylinder-3 where CO2 or CO2 + cosolvent is compressed to achieve a desired pressure. In the power cylinder-3, a plunger-4 separate water from CO2. The extraction time (1.5 h), a rate of SC fluid flow (1.5 ± 0.05 kg/h), temperature (40 °C), and pressure (32 MPa) were kept constant during all processes. Temperature and pressure sensors were installed inside the extractor. Co-solvent was completely evaporated from the obtained

3. Materials and methods 3.1. Raw material The Nannochloropsis salina microalgae samples used in the experiments were obtained from a strain of the Solix BioSystems (USA, Colorado). The microalgae sample was grown in outdoor photobioreactor and salt water; the obtained specimen was at once frozen. More detailed description of microalgae growing is reported in the work [48]. During the microalgae storage period, the container was keeping in darkness (refrigerator). The frozen microalgae were cream-like mass with dark-green color (see Fig. 1). Just before start the extraction experiment the sample was defrosted at room temperature, then applied thin (fine) layer with thickness of 1 to 2 mm on a tray and dried. The drying period at atmospheric pressure and temperature 25 °C was 16 h. The initial moisture of the microalgae sample was 68.63% and the final moisture (after drying is complete) was 4.12%. Prior to extraction, the dried

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Fig. 1. Freeze microalgae (Nannochloropsis salina).

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Fig. 2. Schematic diagram of the laboratory-scale experimental setup for extraction with supercritical SC CO2: 1-extractor; 2-separator; 3-power cylinder; 4-separating plunger; 5-cylinder with CO2; 6-filter for CO2 clearing; 7-reservoir with distilled water; 8-dosing high pressure pump; 9-valves. P1, P2, P3, and P4 are the pressure transducers; T1, T2, and T3 are the temperature transducers; H1, H2, H3, and H4 are heaters.

was 4 h. The same procedure, recommended by Kates [51], was used to estimate the fatty acid contents in the Soxhlet extracts.

extracts using rotary evaporator at 40 °C and the extract was weight. Set parameters provide a total extraction for every process. The percentage content of fatty acids in the SC-CO2 (and modified SC-CO2) extracts were determined as the mass of the free fatty acids obtained by saponification of the extracts with 4% solution of KOH in 80%, ethanol according the method recommended by Kates [51].

3.4. Compositional analysis of extracts The compositional analysis of extracts was carried out using Agilent 6890 N/JMS GCmate II chromatograph-mass spectrometer in DB5-MS column (30 m × 0.32 mm × 0.25 μm) in “split” mode. High-purity helium (99.9999%) with a flow rate of 1 mL/min was used as a carrier gas. The column temperature was raised from 40 °C (the hold time was 1 min) to 280 °C at a rate of 6 °C/min, hold time 15 min. The temperature of an injector was 280 °C, an interface and a detector was 250 °C. The ionization was performed by an electron impact with electron energy of 70 eV.

3.3. Extraction in Soxhlet apparatus The microalgae samples were also extracted in the Soxhlet apparatus during 4 h. n-Hexane of 99.95 wt.% purity and chloroform of 99 wt.% purity were used as solvents. The extract was separated from the solvent by a rotary evaporator. The extraction time for this method

12

340

11

CO2 + acetone PC / MPa

TC / K

332 324 316 308 300 0.00

10 9 8 7

0.03

0.06

6 0.00

0.09

0.03

x / mole f raction

0.06

0.09

x / mole f raction

15

PC / MPa

12 9 6 3 0 180

240

300

360

420

480

TC / K Fig. 3. Critical curve data for binary CO2 + acetone mixture reported by various authors together with vapor-pressure curves for pure components CO2 and acetone in various projections [52]. ●-[53,54]; ○-[55]; ×-[56]; □-[57,58]; ▲-[59]; ■-[60]; Δ-[61].


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Table 1 Content of fatty acids in Nannochloropsis salina extracts derived from various methods: 1 – pure SC-CO2 (32 MPa and 40 °C); 2 – SC-CO2 + acetone (32 MPa and 40 °C); 3 – extract yielded using the Soxhlet apparatus with n-hexane; 4 – extract yielded using the Soxhlet apparatus with chloroform.

Fig. 4. The yield of the total extract from Nannochloropsis salina samples and fatty acid contents in the extracts using various extraction techniques. 1 – pure SC-CO2 extract; 2 – SC CO2 + acetone extract; 3 – extract using the Soxhlet apparatus with n-hexane, 4 – extract using the Soxhlet apparatus with chloroform.

4. Results and discussion The extraction was performed by pure SC-CO2 (a yield was 6.25% (± 0.02)) and acetone modified SC-CO2 in the ratio of 96.9:3.1 wt.% (or 0.024 mol% fraction of acetone) (a yield was 6.38% (±0.018)) at a constant temperature of 40 °C and pressure of 32 MPa for all SCF extraction processes. The CO2 + acetone mixtures with concentrations of 0.0237 mol% at the extraction conditions of (40 °C and 32 MPa) were in the supercritical homogenous phase before pass through the SCF cell (see Fig. 3). The yields of the total extracts from Nannochloropsis salina using various extraction techniques are presented in Fig. 4 together with percentage contents of fatty acids. As one can see, the yields of the total extract from the microalgae sample using Soxhlet apparatus with n-hexane solvent is 5.53% (±0.019), while with chloroform solvent is 4.62% (± 0.017). The yield was defined as the ratio of studied dried raw material mass to the obtained extract (wt.%). A maximum yield (6.38%) was obtained at acetone modified SC-CO2, which is not much differ from the value (6.25%) of pure SC-CO2 extraction. As one can note from Fig. 4, amount of fatty acids in each type of extract approximately the same (varying within 3.65% to 4.12%). However, in the SC-

Acid esters

1

2

3

4

Caproic acid, methyl ester Caprylic acid, methyl ester Capric acid, methyl ester Lauric acid, methyl ester Tridecanoic acid, methyl ester Myristic acid, methyl ester cis-10-Pentadecenoic acid, methyl ester Pentadecanoic acid, methyl ester Palmitoleic acid, methyl ester Palmitic acid, methyl ester cis-10-Heptadecenoic acid, methyl ester Margaric acid, methyl ester g-Linolenic acid, methyl ester Oleic acid methyl, ester Stearic acid, methyl ester Arachidonic acid, methyl ester cis-5,8,11,14,17-Eicosapentaenoic acid, methyl ester cis-8,11,14-Eicosatrienoic acid, methyl ester cis-11,14-Eicosadienoic acid, methyl ester Nervonic acid, methyl ester Lignoceric acid, methyl ester Cerotic acid, methyl ester

Tract 0.03 0.01 0.42 0.03 2.71 0.05 0.50 25.12 17.87 0.06 0.56 5.32 19.47 1.22 5.88 13.09 1.78 0.06 0.65 3.12 2.05

0.01 0.03 0.02 0.4 0.02 2.7 0.05 0.05 27.34 17.5 0.04 0.5 5.3 19.4 1.1 5.7 12.5 1.64 0.05 0.7 3 1.95

Tract Tract Tract 0.39 0.01 3.2 0.03 0.2 26.61 18.62 0.02 0.5 5.92 19.58 1.23 5.98 12.83 1.45 0.06 0.3 1.67 1.4

Tract Tract Tract 0.27 0.02 3.06 0.01 0.15 27.11 18.98 0.05 0.17 6.34 19.2 1.11 5.88 12.48 1.65 0.03 0.27 1.45 1.77

CO2 extracts we found more byproducts (2.21%) than in the extracts obtained with Soxhlet apparatus using n-hexane and chloroform (0.7%). In the extracts obtained with Soxhlet apparatus using n-hexane and chloroform the fatty acids contents slightly lower than in SC-CO2 extracts. This is can be explained due to partly decomposition of the fatty acids since the boiling temperature of n-hexane (68.86 °C) and chloroform (61.2 °C) relative high. The organic compounds present in the each microalgae extracts were analyzed using GC–MS (see, for example Fig. 5). In Table 1, relative components contents as a ratio of the pick area to the total area all of the picks in percent (%) are presented. The table provides the fatty acid compositions of Nannochloropsis salina samples lipids as a function of the extraction conditions. The results showed that each extract was the multicomponent mixture of preferable containing polyunsaturated and saturated fatty acids. As one can see from Table 1, the compositional content of fatty acids in the extracts is slightly dependent on the applying extraction technique.

Fig. 5. GC–MS chromatogram of microalgae (Nannochloropsis salina) extract derived with SC-CO2 + acetone at pressure of 32 MPa and temperature 40 °C.

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5. Conclusion The reported in this work experimental data reveals that applying various extraction techniques for microalgae slightly influences on the total extract yield and fraction of fatty acids. Also, we found small difference in the compositional contents of fatty acids in the obtained extracts. However, extraction with SC-CO2 has some advantages (no environmental impact, high quality final product, low extraction time, and low temperature extraction, for example) in comparing with extraction using Soxhlet apparatus. Thus, SC-CO2 can be recommended for fatty acid extraction from microalgae samples. The GC–MS analyzing results confirm that palmitoleic acid, oleic acid-19.6, palmitic acid-18.0, cis-5,8,11,14,17-eicosapentaenoic acid, arachidonic acid, and g-linolenic acid are the major fatty acid compounds in the microalgae (Nannochloropsis salina) extracts. We found high fatty acids content in the present microalgae sample which allows to use them in medical and pharmaceutical industries. Acknowledgment This work was supported by a Russian Scientific Fund grant No. 1419-00749. We also would like thanks Solix BioSystems (USA, Colorado) for providing microalgae (Nannochloropsis salina) samples. References [1] N. Rubio-Rodríguez, S. Beltrán, I. Jaime, S.M. de Diego, M.T. Sanz, J.R. Carballido, Innovative Food Sci. Emerg. Technol. 11 (2010) 1–12. [2] W.E. Lands, FASEB J. 6 (1992) 2530–2536. [3] I. Gill, R. Valivety, Trends Biotechnol. 15 (1997) 401–409. [4] R.A. Gibson, M. Makrides, Adv. Exp. Med. Biol. 501 (2001) 375–383. [5] L.A. Horrocks, Y.K. Yeo, Pharm. Res. 40 (1999) 211–225. [6] F. Shashidi, U.N. Wanasundara, Trends Food Sci. 9 (1998) 230–240. [7] A.P. Simopolous, Am. J. Clin. Nutr. 70 (1999) 560–569. [8] J.E. Kinsella, Adv. Food Nutr. Res. 35 (1991) 1–184. [9] B.C. Zyriax, E. Windler, Eur. J. Lipid Sci. Technol. 102 (2000) 355–365. [10] W.O. Richter, Eur. J. Lipid Sci. Technol. 103 (2001) 42–45. [11] E.A. Trautwein, Eur. J. Lipid Sci. Technol. 103 (2001) 45–55. [12] Z. Wen, F. Chen, Nitzschia laevis, Biotechnol. Lett. 22 (2000) 727–733. [13] W. Yongmanitchai, O.P. Ward, Proc. Biochem. 24 (1989) 117–125. [14] A. Zinnai, C. Sanmartin, I. Taglieri, G. Andrich, F. Venturi, J. Supercrit. Fluids 116 (2016) 126–131. [15] S. Millaoa, E. Uquiche, J. Supercrit. Fluids 116 (2016) 223–231. [16] J. McKennedy, S. Önenç, M. Pala, J. Maguire, J. Supercrit. Fluids 116 (2016) 264–270. [17] A. Mouahid, C. Crampon, S.-A. Toudji, E. Badens, J. Supercrit. Fluids 116 (2016) 271–280. [18] D. Mozaffarian, H. Cao, I.B. King, R.N. Lemaitre, X. Song, D.S. Siscovick, G.S. Hotamisligil, Am. J. Clin. Nutr. 92 (2010) 1350–1358. [19] Z. Amtul, M. Uhrig, L. Wang, R.F. Rozmahel, K. Beyreuther, Structural Insight Neurobiology of Aging, 33, 2012 21–31. [20] E.L. Schaeffer, O.V. Forlenza, W.F. Gattaz, Psychopharmacology 202 (2009) 37–51. [21] M. Huan, K. Hamazaki, Y. Sun, M. Itomura, H. Liu, W. Kang, S. Watanabe, K. Terasawa, T. Hamazaki, China Biol. Psychiatry 56 (2004) 490–496. [22] J.G. Martins, J. Am. Coll. Nutr. 28 (2009) 525–542.

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