1. Introduction
In the field of renewable energy, the development of biofuel production, particularly biodiesel production by using microalgae, has received great attention. A microalga is one of the non-edible plants which are referred to as the third generation feedstock because they do not compete for land like other terrestrial plants [1]. It is easy to cultivate and easily grows on non-arable land, saline water and even in contaminated water. It contains oil (TAG) of around 1~75% of its dry weight depending on the species [2] and it’s also able to produce ten-times more oil than other plants [2,3]. This plant is considered to be one of the most promising alternatives sources of biodiesel [3].
The biodiesel production process, which puts microalgae to use, is made up of several steps: cultivation, harvesting, lipid extraction and transesterification reaction. Among these, lipid extraction is regarded as the greatest obstacle in biodiesel production because of its cost and low extraction rate [2]. The methods of extracting lipids from microalgae can be divided into mechanical and chemical. These two groups also contain several methods, such as press, Soxhlet extraction, ultrasonic-assisted extraction and ionic liquid extraction [4]. However, these methods are ineffective and very slow. For instance, solvent based extraction takes a relatively long period of time and depends merely on the solvent used. Moreover, the chemical solvents used in performing it are toxic, inflammable and have bad effects on the environment together with people’s health. Solvent extraction is a costly process which requires a large amount of solvent and extra energy for recovering the solvent from the lipids after extraction [5]. Therefore, the extraction of lipids from intracellular locations should be efficient in the usage of time and energy to ensure the quick production of microalgae oil.
In this study, the use of Soxhlet extraction and microwave for extracting lipids was evaluated, and microwave-assisted extraction (MAE) was developed as an alternative method to minimize the requirements for solvents and time. Due to its ability to penetrate materials and excite molecules homogeneously, MAE is superior in minimizing the requirements for solvents and time.
2. Materials and Methods
2-1. Materials
Dried Spirulina platensis powder was obtained from BBAP Takalar in Indonesia. Methanol andn-hexane were of analitycal grade and obtained from Chemical supplier Brataco Chem in East Java, Indonesia.
2-2. Soxhlet extraction
Microalgae lipids were extracted into a Soxhlet apparatus using heating mantel as a heating source. Ten grams of dry algae were kept over a Whatman filter paper and placed in a Soxhlet apparatus. The oil in the algae was extracted over 14 h using 100 mL hexane as the solvent. After the solvent was vaporized, the weight of the microalgae lipids was determined and used to calculate the maximum recovery of yield. The schematic representation of extraction using Soxhlet apparatus is shown in Fig. 1.
Fig. 1. Schematic representation for extraction of lipid from Spirulina platensis using Soxhlet.
2-3. Microwave-assisted lipid extraction
The extraction process was conducted using microwave Elextolux EMM2007X with minimum power of 800 W. The equipment used was designed as shown in Fig. 2. The microwave was used for lipid extraction because this heating method is more beneficial than the conventional one. The lipid extraction was in a 500 mL flat bottom flask with equipped reflux condenser and stirrer. Ten grams of dried Spirulina platensis powder was loaded into the flask with solvent and heated with different extraction times while stirring. An extraction study was conducted using varying solvents and extraction times. The solvents used were methanol and n-hexane, which were modified using ratio 1:2, 1:1 and 1:0 (v/v).
Fig. 2. Schematic representation for extraction of lipid from Spirulina platensis using microwave.
The effects of using varying extraction times and solvents on the yield of microalgae lipid were investigated. After extraction, the mixture of biomass with solvent was filtered to separate filtrate and residue. Lipid from Spirulina platensis was obtained after the remaining solvents were distilled and evaporated.
3. Results and Discussion
3-1. The comparison of lipid extraction using Soxhlet extraction and microwave-assisted extraction methods
Table 2 shows the yield of lipid extracted from Spirulina platensis biomass using Soxhlet extraction and microwave-assisted extraction methods. The yield of lipids is presented as the total amount of extracted lipid per dry mass of microalgae.
Table 2. Fatty acid contents and composition of lipids from Spirulina platensis
Microwave-assisted extraction (MAE) produced better results than Soxhlet extraction. The yield of lipid extraction was 12.530% per dry mass of algae produced by extraction using a mixture of methanol-hexane solvent (1:2) at 600 W of microwave power for a period of 40 min, whereas the yield by Soxhlet method was just 1.293%.
While using microwave irradiation, energy transfer into materials takes place rapidly through ionic conduction and interfacial polarisation mechanism. Ranjan et al. (2010) [6] reported that oil extraction using microwave involves 1) Diffusion by lipid through cell walls into the solvent due to selectivity and solubility; and 2) Cell wall destruction with the contents being released into the solvent. Energy is transferred by the microwave into the material through dipolar polarization, ionic conduction and interfacial polarization, which brings about heating localization in materials and accelerates the reaction [7].
Extraction through microwave is generally based on mass transfer, properties of solvent and solute, the selectivity of solvents and the level of convection in the medium [8]. It is also determined by the dielectric constant and the dielectric properties of the solvent used. The result obtained agrees with that of a related research from Balasubramanian et al. (2011) [8], which revealed that microwave assisted extraction yielded higher lipids in comparison to extraction using Soxhlet method. Lee et al. (2010) [9] also reported better lipid extraction from Scenedesmus sp with 1:1 (v/v) chloroform-methanol by microwave for 5 min.
The yield obtained while using Soxhlet method for the extraction and n-hexane as the solvent was just 1.293%. In this method, nonpolar solvents disrupt hydrophobic components by interacting with the neutral lipids in microalgae [4]. The yield obtained while using this method was lower when compared to the one gotten from the microwave assisted extraction because this method only relies on the ability of the solvents to penetrate the cell structure and attract oil stored in the cell matrix.
Lipid extraction of microalgae is highly dependent on the nature of solute and solvent, selectivity of solvent and the level convection of the medium. The major mechanism in Soxhlet extraction is diffusion, and it does not involve mechanical application to the biomass. Ultimately, this is a slow process which results in low yield and shows no improvement in yield, even when a polar solvent is used [10].
Table 1. Yield of lipid extracted from Spirulina platensis by two different methods
In the extraction processes using microwave, the use of solvent mixture modification (n-hexane/methanol) resulted in higher yield than the other extraction process. It is a non-polar solvent that is highly soluble to oils; thus, it is able to solve and attract oil content from the matrix cells of microalgae. It is also able to increase the absorption of energy, while methanol as the polar solvent allows microalgae to absorb more microwave energy; thus the rate of mass transfer and disruption of cells rises during microwave extraction. The extractable yield obtained from microalgae while using the mixing solvent was noticeably higher than the yield obtained with hexane
The result of using microwave in the extraction process (MAE) is strongly influenced by the microwave power used and the length of the extraction time [11-13]. Fig. 3 shows the yield of lipids extracted while using different solvents. In this study, the highest yield was obtained at 600 W over a period of 40 min using a modification of methanol and hexane solvent 1:2. The use of solvent modification nhexane/methanol for the extraction of microalgae lipids while using microwave produced a higher yield than other extraction processes. N-hexane as a non-polar solvent is highly soluble to oils; therefore, it is able to solve and attract oil content from the matrix cells of microalgae. It is also a medium which has the ability to increase the absorption of energy, while methanol is the polar solvent which allows microalgae to absorb more microwave energy; thus, the rate of mass transfer and disruption of cells rises during extraction with microwave. The lipid yield from microalgae using the mixing solvent was noticeably higher than the yield obtained with hexane.
Fig. 3. Yield of lipid from extraction of Spirulina platensis using microwave at any time (process condition: mass of dried Spirulina platensis of 10 g and microwave power of 600 W).
The extraction process was conducted using high microwave power to obtain optimum yield of microalgae lipid. Microwave power acts as a driving force in the extraction process and brings about a heating localization to ease the disruption of the matrix cell of microalgae, thus making it easier to diffuse solutes out and dissolve in the solvent. However, the increase of microwave power increases the temperature and causes the yield of lipids to be constant or to decrease.
3-2. Fatty acid component of lipid from Spirulina platensis
Microalgae lipids can be grouped into neutral and polar lipids. Neutral lipids consist of triglyceride, hydrocarbon, fatty acid and sterol, whereas polar lipids are made up of phospholipid, glycolipid and carotenoid. Triglyceride (TAGs) is the highest component in microalgae cells and it can be reached 80% from the total fat. It is often used as the main raw material in the production of biodiesel.
GC-MS analysis of fatty acid composition revealed that the fatty acid components of Spirulina platensis varied from C16-C18. The fatty acid composition of Spirulina platensis displayed the potential for biodiesel production.
From the composition of Spirulina platensis in Fig. 4 and Table 2, it can be seen that the fatty acid component consists of saturated fatty acid (SAFA) and polyunsaturated fatty acid (PUFAs). The fatty acid component of SAFAs contains palmitic acid with a percentage of around 76.25%, whereas the percentage of PUFAs contained in Spirulina platensis lipid was just 7.43%. The fatty acid component of PUFAs consists of a-linoleic acid, elaidic acid and linoleic acid.
Fig. 4. Chromatogram of lipid from Spirulina platensis.
4. Conclusions
Microwave assisted extraction was developed for the extraction of lipids from microalgae. This method has many advantages when compared to control, resulting in increased extraction yields. More than 12% lipid was extracted in 40 min in comparison to 1.293% extracted in 14 h using Soxhlet extraction. The profile of microalgae lipid shows more saturated fatty acids which have the largest quantities in the fatty acid component of microalgae that can potentially be developed for algal biodiesel production.
Acknowledgments
This research was partially supported by the Indonesian Ministry of Research, Technology and Education through Applied Research University Grant. The authors are thankful to BBAP Takalar for providing dried Spirulina platensis.
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