4.1. Lipids Extraction Methods

Lipids are essential compounds in the microalgae biomass as they comprise triacylglycerols (TAG), phospholipids and glycolipids [2,5,68], which are essential for metabolic activities and biofuel production. Usually, microalgae were associated with high costs for biomass downstream processing for oil extraction. Thus, it is important to extract lipids from microalgae cells using suitable technology that is cost-effective and environmentally friendly. Several methods were applied to quantify lipids from microalgae such as near infrared spectroscopy at specific wavelengths, electroporation, the supercritical fluid extraction method (SEF), pressurized solvent extraction, organic solvent and also osmotic shock [2,64,75] as in Table 5.

Techniques used to quantify lipids in microalgae [64].

Kumar et al. [64] stated that electroporation, pressurized solvent extraction and the supercritical fluid extraction method are the most efficient technologies to extract lipids from microalgae biomass. However, they required high energy with high operational costs. Generally, cost-effective methods are used for lipid extraction such as osmotic shock, organic solvents through Soxhlet extraction, Bligh–Dyer method and Folch method [18,24,75].

According to King, [76] the supercritical fluid extraction method (SEF) can be used to extract lipids without using any organic solvent for analysis. This SEF method requires high energy consumption that is expensive for commercialization. The SEF method can separate lipids according to properties, enrichment, hydrogenation and hydrolysis. Figure 4 illustrates microalgae lipid extraction using SEF methods. Santana et al. [77] reported the use of the SEF method to extract lipids from Botryococcus braunii for biodiesel production. They observed that the lipid yield decreased with an increase in the temperature of extraction. However, the pressure enhancement has a positive influence on lipids productivity. The optimum operating conditions for SEF was in the range of 220–250 bar for pressure and 50 °C for temperature, respectively. Similar results were carried out in Nannochloropsis salina, Scenedesmus obliquus and Scenedesmus obtusiusulus for extraction of lipids to generate biodiesel [52,78]. SEF optimum conditions to generate 92% of lipids was performed at 12 MPa, 20 °C, and a CO₂ to biomass ratio of 100 [52].

Microalgae supercritical fluid extraction method.

Other than SEF, the most common methods used for lipid extraction are modified Folch and Bligh and Dyer methods as shown in Figure 5. The harvested microalgae biomass was added with methanol and chloroform (2:1 v/v) for lipid extraction. Then, lipid extracts were washed with 0.9% saline followed by vortex and separation phase either using thin layer chromatography (TLC) or chromatography (GC-MS). Zienkiewicz et al. [8] followed this method for Nannochloropsis oceanica: total lipid quantification and after centrifugation separation, lipids were visualized by thin layer chromatography (TLC). Similarly, Vooren et al. [24] had measured lipid content in Nannochloropsis oculata using the Bligh and Dyer extraction method followed by high performance thin layer chromatography (HP-TLC). Vooren et al. [24] showed that C. zofingiensis cultivated under nitrogen starvation conditions produced triglycerides in the range of 31% to 43% due to a shift in cell metabolism. Zhu et al. [3] used GC-MS to analyze lipid content in C. zofingiensis. The Folch et al. [79] and Bligh and Dyer [80] methods were used for total lipid recovery and extraction was used; it was widely performed by researchers due to the simple procedures, time efficiency, energy savings and suitability for a large sample size.

Microalgae lipid extraction by Bligh and Dyer and Folch methods.

Another promising method for lipid extraction was an osmotic shock that degrades microalgae cell structures to increase lipid yield efficiency. Gonzalez-Gonzaález et al. [81] studied the effect of osmotic shock on Dunaliella salina and Chaetoceros muelleri for biogas (methane) generation. The study reported that C. muelleri lipid recovery efficiency for methane production was higher than D. salina with 72% and 21%, respectively. Microalgae biomass can be used for biomethane generation through anaerobic digestion or the biomass gasification and methanation process. Biomass produced in limited nitrogen culture media was more stable with low inhibitory substances such as ammonia which caused acidosis. Microalgae with high lipids content will yield greater volumes of biomethane than microalgae rich in carbohydrates and protein [82]. Klassen et al. [83] reported that high biomethane productivity was achieved under nitrogen-limited biomass conditions with 462 ± 9 mLN CH₄ g⁻¹VS day⁻¹ and the biomass-to-biomethane energy conversion efficiency was up to 84%.

Similarly, Yoo et al. [75] reported that osmotic shock enhanced the lipid recovery by two-fold in wet biomass of Chlamydomonas reinhardtii. In this technique, microalgae biomass was mixed with hexane, chloroform and methanol solvent in the Teflon-sealed tubes for 10 min for better lipid recovery since hexane and methanol required low heat for evaporation and rupture of microalgae cells [75]. It concluded that an osmotic shock is a promising technique for microalgae lipid extraction recovery from wet biomass.