*2.4. Bioethanol*

Bioethanol is considered a substitute for conventional petroleum, as they both have the same chemical and physical properties [96–98]. Microalgae biomass, in particular, has lately received a lot of interest as a viable renewable source for the production of biofuels. Thirdgeneration bioethanol made from microalgae biomass could also be an environmentally beneficial fuel. As discussed earlier, microalgae are rich in lipids, enabling it to produce biodiesel. Similarly, some species of microalgae can store large amounts of carbohydrates, such as triacylglycerol and starch, within their cells. These carbohydrates can be used as a carbon source or substrate during fermentation to generate bioethanol [99,100]. Proteins can also be accumulated within the cells, along with carbohydrates and lipids under restriction of nitrogen or starvation [101]. Microalgae breakdown the complex nitrogen molecules into protein. Variation in salinity, light intensity, and temperature can also accumulate carbohydrates. Microalgae also lack lignin, and have low hemicellulose levels, making hydrolysis and fermentation yields more efficient [102].

There are three different routes to produce bioethanol from microalgae:(i) The first route is the conventional method, in which the biomass is pretreated, hydrolyzed enzymatically, and fermented using yeas<sup>t</sup> [103]. (ii) The second route operates in the dark condition, and uses metabolic pathways to redirect photosynthesis to create hydrogen, acids, and ethanol [104]. (iii)The third method is to use photofermentation, which is impossible in nature [105]. (iv) The last route necessitates the use of genetic engineering to reroute microalgaes pre-existing metabolic pathways for more subjective and efficient bioethanol synthesis. Bioethanol production from microalgae and cyanobacteria is a viable technical advancement, as they have shown to be more productive than crops such assugarcane and corn. Light is used as an energy source by genetically engineered strains to produce bioethanol from carbon dioxide and water in a single process [106].

#### 2.4.1. Bioethanol by Hydrolysis and Fermentation

This route is entirely based on the production of microalgae biomass in photobioreactors, achieved by way of pretreatment steps which involve the hydrolysis of the biomass and breakdown of the cell walls. The biomass is preferably pretreated using enzymes. These treated biomass are further fermented with *Sacchromyces cerevisae* or bacteria to yield bioethanol.

#### 2.4.2. Bioethanol by Dark Fermentation

In this route of dark fermentation, the organic biomass is converted into biohydrogen. Fermentative and hydrolytic microorganisms hydrolyze complex natural polymers into monomers which are, in the end, converted into a combination of low molecular weight organic acids and alcohols such as acetic acid and ethanol.

In the absence of light, certain microalgae and cyanobacteria are capable of expelling ethanol through the cell wall via an intracellular process. Some of the species include *Chlamydomonas moewusii*, *C.vulgaris*, *C.reinhardtii*, *Oscillatoria limnetica*, *O. limosa*, *Chlorococcum littorale*, etc. [102,107]
