Due to increase in fuel cost and continuous depletion of crude oil associated with environmental issues there is an urge to search an alternative which is clean, renewable and sustainable. Recent studies established positive results in microalgal fuel with environment concern and fossil fuels dependence. Microalgae are promising source for high lipid content which has fast growing rate and can cultivate in waste surface. Moreover, a microalga does not compete with food feedstocks and makes it a stimulating substitute for popular food and non-food crops [1, 2].
Energy recovery from microalgae is a necessary footstep to enhance and improve the sustainability and economic competitiveness. Releasing the lipid from the complex structure of microalgal cell wall is a major tricky in the extraction process [3, 4]. Usually, microalgal biomass was subjected to taking out the lipids by mechanical pressing and organic solvents. In mechanical pressing, the yield of oil depends on the extraction technique employed such as screw / piston press, extruder and expander, pulverization using mortar, etc [5, 6]. The method of organic solvent extraction employs ‘like dissolving like’ concept of basic chemistry to extract lipid from microalgae using solvents [7].
In the cell membrane of microalgae, neutral lipids are found as a complex with polar lipids and are strongly linked with the proteins through hydrogen bonds. Ideal solvent must be highly specific and volatile in order to ensure low energy distillation for extracting lipid from the solvent [8]. The non-polar solvents used for lipid extraction from microalgae biomass include hexane, benzene, toluene, diethylether, chloroform, chloroethane and dichloromethane. Similarly, the polar solvents used are methanol, ethanol, acetone, ethyl acetate and 2-propanol. The non-polar solvents disrupt the hydrophobic interactions between non-polar and neutral lipids, whereas the polar solvent disrupts the polar lipids. Addition of both polar and non-polar organic solvent ensures, complete extraction of all neutral lipid (free standing globules and membrane associated complexes). The combinations of polar and non-polar solvent generally used are chloroform/methanol, n-hexane/ethanol, n-hexane/2-propanol, chloroethane/methanol, dichloroethane/ethanol and acetone/dichloromethane [9, 10]. However, chloroform/ methanol (1:2 v/v) is the mostly used organic solvent because of the reduced extraction time and high yield.
The remaining water in the algal cells acts as a ternary component, permitting complete extraction of both neutral and polar lipids. In this method complete drying of biomass is not needed, since separation happens by biphasic partitioning. The lower phase (chloroform and some methanol) contains lipid (neutral and polar), while the upper aqueous phase (water, methanol) contains non lipid components (proteins and carbohydrates) [10].
One hypothesis speculates that the presence of residual moisture in microalgal biomass unfavourably affects the lipid extraction efficiency. In such school of thought, the water forms a barrier and inhibits effective lipid mass transfer from cells to the organic solvent. So drying of microalgal concentrate is recommended prior to lipid extraction [9]. Another hypothesis postulates that the presence of residual moisture content in microalgal biomass will enhance lipid extraction efficiency. The principle is that the water swells the cells and allows better solvent access to the lipids and states that drying of microalgae before lipid extraction is unnecessary and may hinder the lipid extraction [11].
Soxhlet or hexane solvent extraction method can be used either alone or in combination with oil press method. Oil extraction in batch mode needs large amount of organic solvent, whereas continuous mode replenishes the biomass with organic solvent and reduces the solvent consumption [12, 9]. This method is quite expensive and has to be handled carefully as it involves chemicals. Benzene is a carcinogen and can lead to explosion hazard also. Hexane is less efficient than chloroform which is less toxic and has low affinity towards non lipid contaminants [13]. Bligh and Dyer’s method is used for both dry and wet mode of oil extraction [14] where the ratio of chloroform/methanol is maintained around 2:1. After mixing the solvent and the biomass, it is homogenized with another same quantity of solvent. The method of centrifugation separates the biphase layer (lipid in chloroform and methanol in water) in the process and the lipid is separated finally by fractional distillation [15, 16].
There is an emerging green technology for oil extraction where super critical carbondioxide (SCCO2) is used as the primary solvent at a critical pressure of 7.4 MPa and at lower critical temperature of 31.3ºC [17, 18]. The factors affecting this process include the pressure, the temperature and the fluid flow rate. SCCO2 has high solvatic power and is less toxic, but the high infrastructure requirements and the operating cost are the main challenges in this process [19, 20,]. A contrasting result has been reported in a comparative study between SCCO2 and Bligh and Dyer method for extracting heterotrophically cultured microalgae C. cohnii [21, 22]. The lipid yield obtained from Bligh and Dyer procedure is nearly double compared to this method. It is noted that the microalgae strains and culture conditions play a crucial role in the determination of appropriate lipid extraction method. The microalgal residues are rich in carbohydrates, proteins and pigment [23, 24, 25, 26, 27]. They can be further processed into biomethane, bioethanol, biobutanol, etc. [28, 29, 30, 31, 32, 33]. It is also used to produce valuable products like docosahexaenoic acid (DHA), carotenoids, drugs, food and feed additives [34, 35, 36].
In this work, microalgae Aphanothece halophytica was isolated from saltpan area and be cultivated in Jaworski’s medium. Then, the culture was mass propagated using organic and inorganic combo nutrients in open raceway ponds. The lipid was extracted by two ways 1) from wet biomass and 2) dry biomass. According to the polarity, solvents were selected for both methods. The extraction process parameters such as extraction time, temperature, biomass-to-solvent ratio and mixing intensity were optimized by response surface methodology (RSM). The lipid was quantified and proposed a better practice for commercialization. Finally, lipid was characterized by gas chromatography to obtain the fatty acid signature for further processes.