Extraction of oils from microalgae is a much more difficult problem than extraction of oils from oilseeds. Microalgae are single-cell organisms with extremely tough cell walls that can be difficult to disrupt [1
]. The most common techniques for lipid extraction from microalgae in current use involve chloroform/methanol mixtures or hexane in solvent or Soxhlet extraction. These flammable and toxic organic solvents cause adverse health and environmental effects.
-hexane, derived from petroleum, is the solvent of choice for extraction of oils. Its chemical properties provide ideal functionality as an extraction solvent for oils. n
-Hexane is a light paraffinic petroleum fraction, has a fairly narrow boiling point range of 69 °C and is an excellent solvent in terms of oil solubility and ease of recovery. However, this solvent can be emitted during extraction and recovery and has been identified as an air pollutant since it can react with other pollutants to produce ozone and photochemical oxidants [2
]. Safety, environmental and health concerns have increased the interest in alternatives to n
-hexane in order to reduce emissions of volatile organic compounds into the atmosphere. Due to the new emphasis on environmental protection and the development of green chemistry, such solvent use is to be avoided as much as possible. The green chemistry is based on twelve principles [4
] such as “design less hazardous chemical syntheses” (use substances with little or no toxicity to humans and the environment) or “use safer solvents and reaction conditions” (avoid using solvents). The twelve principles provide a good basis for researchers who want to develop new, more environmentally acceptable experiments. The most feasible alternative to n
-hexane as solvent for extraction seems to be the replacement of this solvent by bio-solvents such as terpenes which as recognized as environmentally safer. In this study d
-limonene, α-pinene and para
-cymene were used in order to replace n
Terpenes are natural solvents existing both in citrus fruits and in many other plants, with extraordinary technical and chemical properties. They include hydrocarbons with C5
isoprene units and are derivable chiefly from essential oils, resins, and other vegetable aromatic products. Many terpenes are acyclic, bicyclic, or monocyclic, and differ somewhat in physical properties. They represent an optimal alternative to petroleum solvents in many industrial applications. d
-Limonene is a low cost, low toxicity biodegradable terpene present in agricultural wastes derived from citrus peels. As such this reagent can be considered as an economical renewable feedstock. It’s a very versatile chemical which can be used in a wide variety of applications. The growing interest of limonene has emerged since its cleaner and degreaser qualities were recognized and taken into consideration [5
]. Recently, as an alternative to organic solvent extraction, the extraction of oil from oil-containing materials with d
-limonene has been investigated [6
]. The yield and quality of crude oil obtained from the d
-limonene extraction were almost similar to those obtained using n
-hexane. α-Pinene is a natural terpene hydrocarbon obtained from gum turpentine, a kind of essential oil distilled from pine gum. Gum turpentine, a renewable resource, has become a very important material as a solvent which is used to thin oil based paints and for producing varnishes. Gum turpentine obtained from pine forests, it used to produce high quality α-pinene, β-pinene, pine oil, terpineol and other terpenes. p
-Cymene is an aromatic hydrocarbon that occurs widely in tree leaf oils [9
]. It’s an important product and valuable intermediate in the chemical industry. Among others, it is used as a solvent for dyes and varnishes, as a heat transfer medium, as an additive in fragrances and musk perfumes, and as a masking odor for industrial products. To our knowledge, this work represents the first time that terpenes were used as solvent in extraction of oil-containing materials such as microalgae.
The relevant properties of those three terpenes as compared to n
-hexane as solvent are listed in Table 1
. The terpenes have similar molecular weights and structures to substitute n
-hexane. Solubility parameters of solvents have been studied by means of Hansen Solubility Parameters (HSP) [10
]. HSP were developed by Charles M. Hansen and provide a way to describe a solvent in terms of its non-polar, polar, and hydrogen bonding characteristics. The HSPs work on the idea of “like dissolves like” where one molecule is defined as being ‘like’ another if it bonds to itself in a similar way. The overall behavior of a solvent is characterized by three HSP parameters: δd
, the energy from dispersion bonds between molecules, δp
, the energy from dipolar intermolecular force between molecules and δh
, the energy from hydrogen bonds between molecules. n
-Hexane and terpenes have similar values of the three descriptive terms; they likely behave similarly in practice. From this point of view, the terpenes are as effective as hexane to dissolve oils. However, considering their dielectric constants terpenes are slightly more polar and have more dissociating power than n
-hexane. From the security point of view, terpenes have higher flash points than n
-hexane, so they are less flammables and hazardous. The major drawback of using terpenes is their high viscosity and density, and also the higher energy consumption related to solvent recovery by evaporation due to their higher boiling point (155 °C and 176 °C) and higher enthalpies of vaporization (37–39 kJ/mol) compared to n
-hexane (Bp = 69 °C, ΔHvap = 29.74 kJ/mol).
Relevant properties of n-hexane and terpenes.
In order to resolve this problem of evaporation (energy and temperature), it is important to know how these bio-solvents have been extracted from the plant matrix. Terpenes are the primary constituents of the essential oils of many types of plants and flowers which are commonly extracted from their matrix by using water hetero-azeotropic distillation. The Clevenger apparatus has been used for decades in hydrodistillation in order to extract and measure essential oils contained in plants [11
]. This process allows the extraction of compounds at low temperature, about 97–98 °C at atmospheric pressure and less if reduced pressure is applied, as compared to the high boiling point of terpenes contained in essential oils (150 to 300 °C). Based on this fact this way can be used for the recovery of terpenes such as d
-pinene and para
-cymene, resulting from the extraction step of oil from microalgae (Figure 1
Extraction procedure using terpene solvents.
Extraction procedure using terpene solvents.
The aim of the study was to evaluate the possible extraction of oils from microalgae using terpenes as alternative solvents to n-hexane. Extraction step of oils from microalgae was thus investigated using Soxhlet extraction and the step of elimination of the solvent from the medium was carried out using Clevenger distillation. Extracted oils were then compared with oils obtained with n-hexane in terms of crude extract (quantitative results) and fatty acid composition (qualitative comparison).
3.1. Chemical and Reagents
Solvents used during extraction experiments (d-limonene, α-pinene, para-cymene or n-hexane) were of analytical grade and were supplied by VWR International (Darmstadt, Germany). Methanol, sulfuric acid, BHT, toluene and NaCl used for the preparation of fatty acid derivatives were all of analytical grade and were also purchased from VWR International. Various glasswares, Soxhlet apparatus and extraction thimbles used in extractions and fatty acid methyl ester preparations were supplied by Legallais (Montferrier-sur-Lez, France).
3.2. Microalgae Cultivation and Harvesting
Commercial dry Chlorella vulgaris was obtained from Alphabiotech Company (Asserac, France). The microalgae were grown in raceway, with ambient air. After cultivation, the biomass was harvested by membrane filtration, and then centrifuged to obtain microalgae paste. All the microalgal paste was stored at −80 °C then freeze-drying.
3.3. Determination of the Total Lipid Content
The content of total lipids in the microalgae was determined by mixing with chloroform-methanol (1:2 v/v) using the Bligh and Dyer method [12
]. The mixture was agitated during 15 min in an orbital shaker at room temperature. The lipid fraction was then separated and the solvent evaporated under a nitrogen stream. The lipids obtained were weighted and calculated (% dry weight) as standard for the following calculation.
3.5. Preparation of Fatty Acid Methyl Ester Derivatives
The modified Morrison and Smith method was used to prepare fatty acid methyl ester (FAMEs) derivatives [18
]. An acid catalysis was employed during derivatization procedures by using a defined amount of 5% methanolic sulfuric acid solution (1 mL) added to a specific amount of extracted oil. Internal standard used was glyceryl triheptadecanoate (C54
). The mixture was then heated during 90 min at 85 °C. After, the flask was removed from heat and sodium chloride (1.5 mL, 0.9%) solution and n-hexane (1 mL) were added. The flask was stoppered and shaken vigorously during 30 s, then centrifuged at 4,000 rpm during 2 min. A small amount of the organic layer was removed and transferred in a vial before being injected directly in a gas chromatograph.
3.6. Chromatographic Analysis of Fatty Acids
FAMEs were separated, quantified and identified by gas chromatography coupled with mass spectrometry (GC/MS). Analyses were performed by using a Shimadzu QP2010 (Kyoto, Japan) instrument equipped with a UB-Wax capillary column 30 m × 0.25 mm × 0.5 μm (Varian) and the velocity of the carrier gas (He) was at 35 cm/s. Injection of 2 µL of the various samples were carried out with a splitless mode and the injector temperature was set at 250 °C. Oven temperature was initially 50 °C for 1 min and then progressed at a rate of 20 °C/min from 50 °C to 190 °C and then increased from 190 °C to 230 °C at a rate of 2 °C/min. The temperature was then held at 230 °C for 15 min. The mass spectra were recorded at 3 scan/s from 50 to 380 a.m.u and the ionization mode was e.i at 70 eV. Identification of common fatty acids was a performed using the NIST’98 [US National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA] mass spectral database.
3.7. Microscopic Observations
The impacts of the extracting methods on the structure of microalgae cells were examined using light microscopy. Microalgae residues were collected before (native microalgae) and after extractions and introduced in 70% ethanol at 6 °C. Microscopic observations were performed with a Leica DM 2000 Microscope (Wetzlar, Germany) equipped with DFC 30F digital camera (LAS software).