**2.2 Oil productivity**

Oleaginous microalgae (**Table 1**) are a promising source for the production of renewable biofuels because of their efficient photosynthetic capabilities. Moreover, microalgal growth requires less area in comparison to the terrestrial plants, and they are capable to channel the majority of the acquired energy into cell division, which increases the biomass yield [23]. Microalgae can be subdivided into four different groups depending on the carbon source (inorganic and organic), namely, autotrophic, mixotrophic, heterotrophic, and photoheterotrophic [24].

The synthesis of triacylglycerol in microalgae takes place mostly in the chloroplast and endoplasmic reticulum through multiple enzymatic reactions [25]. Fatty acid synthesis in the chloroplast, assembly of glycerolipids in endoplasmic reticulum, and accumulation of TAGs into the oil bodies are the three major steps involved in the accumulation of lipids in the microalgae [26]. It has been proven that facilitate the synthesis of high amounts of lipids is influenced by different stress conditions


#### **Table 1.**

*Some prominent oleaginous microalgae.*

such as physical, chemical, or environmental, individually or in combination [27]. Under the aforementioned stress conditions, microalgae can switch their metabolism towards the synthesis of neutral lipids in the form of TAGs, which serves as a form of carbon and energy storage [28–30]. Microalgae employ the de novo pathway to synthesize lipids. It starts in the chloroplast by CO2 fixation into sugars, which are further metabolized to acetyl-CoA, which acts as a precursor of fatty acid synthesis [31].

Marine microalgae have a higher content of PUFA in comparison to the freshwater species because they need to produce more unsaturated fatty acids to survive in the salty marine environment [32]. Thus, cultivation of marine microalgae can render higher economic interest to the cultivars. According to reported literature, the marine oleaginous diatom Fistulifera solaris when cultivated in photoautotrophic conditions can produce 135.7 mg/(L·day) EPA. On the otherhand, the heterotrophic growth of the marine diatom Nitzschia laevis, when supplemented with glucose, resulted in EPA production of 174.6 g/(L·day) [33].

## **2.3 Extraction of oil**

During lipid extraction, the microalgal biomass is exposed to an organic eluting solvent which extracts the lipids out of the cell cytoplasm. A lipid extraction technology for microalgal oil production needs to be highly specific towards the lipids in order to avoid the co-extraction of non-lipid contaminants, viz. protein and carbohydrates. The lipid extraction technology should be more selective towards acylglycerols than other lipid fractions as they are not readily convertible to biodiesel, such as polar lipids and non-acylglycerol neutral lipids (free fatty acids, hydrocarbons, sterols, ketones, carotenes, and chlorophylls) [34]. Moreover, the technology should be efficient (both time and energy saving), non-reactive with the lipids, relatively cheap (capital cost and operating cost), and safe (environmentally and mechanically) [35]. Dewatering of the microalgal biomass beyond a paste consistency (200 g dried microalgal biomass/L culture) is energy consuming, so, it will be economically friendly if the selected lipid extraction technology is effective for the wet feedstock, i.e. concentrate or disrupted concentrate with concentrations between 10 and 200 g dried microalgal biomass/L culture [36].

### *2.3.1 Solvent extraction*

The principles underlying solvent extraction of microalgal lipids are based on the concept of chemistry 'like dissolving like'. The long hydrophobic fatty acid chains interact with neutral lipids through weak van der Waals forces, thus forms globules in the cytoplasm [37]. The mechanism for organic solvent extraction is depicted in **Figure 1**. When a microalgal cell is exposed to a non-polar organic solvent, such as hexane or chloroform, the organic solvent penetrates through the cell membrane into the cytoplasm and interacts with the neutral lipids trough van der Waals forces to form an organic solvent-lipids complex. This organic solvent–lipids complex, driven by a concentration gradient, diffuses across the cell membrane. The neutral lipids are thus extracted out of the cells and remain dissolved in the non-polar organic solvent. However, some neutral lipids remain as a complex with polar lipids in the cytoplasm. The complex is strongly linked via hydrogen bonds to the proteins in the cell membrane. The van der Waals interactions formed between non-polar organic solvent and

#### **Figure 1.**

*Schematic representation depicting the solvent extraction of oil from oleaginous microalgae. 1. Penetration of the solvent inside the cell cytoplasm. 2.Solvent interaction with the lipids. 3. Formation of lipid-solvent complex. 4. Diffusion of the complex through the cell membrane. 5. Diffusion of the complex in the surrounding bulk organic solvent.*

neutral lipids are inadequate to disrupt these membrane-based lipid–protein associations. On the other hand, polar organic solvent (viz. methanol or isopropanol) is able to disrupt the lipid–protein associations by forming hydrogen bonds with the polar lipids in the complex [38].

**Figure 2** depicts the extraction steps generally, undertaken for laboratory-scale production of microalgal oil and finally trans-esterified to biodiesel using an organic solvent mixture.
