**7. Synthesis of biodiesel**

*Biotechnological Applications of Biomass*

in the filter; a short process time.

**6. Extraction of lipids**

be reached in the order of 95% [10].

known as "osmotic shock" [39].

liquid medium [37].

ciency [20].

oil [39].

The filtration method consists of passing the culture medium through a small pore membrane that retains the microalgal cells and allows only the passage of the

According to Lourenço [20], in the filtration of small volumes, a filtration apparatus such as a Kitasato flask or similar is applicable, however, on a large-scale cultivation, the separation of huge volumes of microalgae and biomass processing is only feasible if the species have large or filamentous cells, which are easily retained

Regarding the harvest time, gravimetric sedimentation is a slow and simple process which involves the separation of microalgae cells from the culture medium by the action of gravitational force. The downside of this process is the low effi-

addition of chemicals, preserving its original characteristics [20].

process; solvents such as hexane and methanol are widely used [24].

On the other hand, centrifugation, cell sedimentation equipment that acts by the action of centrifugal force, is a fast process of biomass recovery, however, it requires high energy consumption. By this technique, biomass is concentrated without the

Microalgae oils can be extracted similarly to other oilseed biomasses, which usually use physical extraction by adding a chemical solvent to improve the extraction

The extraction of oil with solvent is a process of transferring soluble constituents (oil) from an inert material (biomass) to a solvent, in a purely physical process, without any chemical reaction. Solvent extraction is currently the most economical method and the use of hexane and chloroform has made it the fastest and most efficient method for lipid extraction from the most diverse biomasses. However, the presence of these chemical solvents can affect the lipid composition of the extracted

Mechanical presses such as screw and piston, among others, can rupture the cells and release the oil, but these operation lead to low efficiency. On the other hand, when combined with the use of organic solvents, oil extraction efficiency can

High concentrations of salts can cause a sudden change in the osmotic pressure, which can lead to the rupture of cells and the release of oil from inside; a method

A method that has received notable attention is supercritical extraction, which uses fluids above the critical point of temperature and pressure, providing properties such as low viscosity and high diffusivity, allowing to achieve greater efficiency in the lipid extraction step. In this context, the use of CO2 as a supercritical fluid is highly attractive due to its high selectivity, non-toxicity and short processing period. The only drawback is the high cost associated with installing adequate equipment to operate under safe conditions at high temperature and

In the search for environmentally friendly processes, enzymatic extraction is highly recommended, since it uses enzymes such as pectinase and cellulase, to degrade the cell wall, providing highly efficient extractions without affecting the microalgal lipid composition, unlike the method that uses organic chemical solvents [39]. On the other hand, these methods are still used only on a laboratory scale, due

to the high costs involved with the acquisition of enzymes [24].

**392**

pressure [10].

Biodiesel is the natural and renewable substitute fuel for petroleum diesel. It is produced from vegetable oils such as soy, palm, sunflower and jatropha and/or animal fat, such as beef tallow and chicken fat. In other words, any sources that have lipids can, in theory, be useful in the production of biodiesel [6].

First generation raw materials such as soy, palm and sunflower are not considered socially sustainable because they compete with the demand for food. In contrast, second generation raw materials such as beef tallow, residual frying oils and chicken fat, stand out as alternatives to produce biodiesel. Physic nut, which grows in semiarid regions, where other sources of oilseeds are not able to grow and, therefore, do not compete with the food chain can also be used to produce biodiesel [6].

Microalgae, as well as cyanobacteria, belong to the group of third generation raw materials. These are a promising alternative, as they are able to accumulate high levels of lipids combined with high growth rates and biomass productivity compared to conventional oilseeds, and they do not compete with the food chain [40].

Biodiesel can be defined as a mixture of alkyl esters of fatty acids obtained by transesterification of triacylglycerides from vegetable oils or animal fat, or by the esterification reaction of free fatty acids resulting in alkyl esters of fatty acids and water. Both reactions occur in the presence of alcohols and catalyst [41].

Alcohols are considered to be transesterification agents, and may be methyl alcohol (methanol), ethyl alcohol (ethanol), propyl, butyl or amyl. Methanol is the most widely used alcohol due to its low cost and its physical–chemical properties (polarity and lower carbon chain), while ethanol has stood out for its potential for low toxicity and easy availability [37].

Transesterification is a multi-stage reaction, including three reversible stages in series, so that triglycerides are converted to diglycerides, then diglycerides are converted to monoglycerides and finally monoglycerides are converted to esters (biodiesel) and glycerol (co-product). Stoichiometrically the transesterification reaction requires 3 moles of alcohol for every mole of triacylglyceride, with an excess of alcohol being used to shift the balance towards the formation of the products, as it is a reversible reaction. The esterification reaction requires 1 mole of alcohol to 1 mole of fatty acid and requires excess alcohol to favor the direction of product formation [42].

The biodiesel synthesis reactions can be catalyzed homogeneously or heterogeneously. Homogeneous catalysts can be acidic (sulfuric, sulfonic and hydrochloric acid) or alkaline (sodium or potassium hydroxides), while heterogeneous catalysts can be enzymes or metallic compounds. In the catalyst determination, the acidity index is the main characteristic of the oil to be observed since high amounts of fatty acids do not allow alkaline catalysis (soap formation in the reaction medium). In this case, the use of acid catalysts is suggested [18].

On enzymatic catalysis, enzymes such as lipases, present in several organisms including animals, plants, fungi and bacteria have the biological function of accelerating the hydrolysis of fats and vegetable oils, releasing fatty acids, monoglycerides, diglycerides and glycerol [43].

Enzymes have some advantages when compared to the chemical catalytic process, such as high selectivity, reaction temperature in the range of 30–40°C and pH between 4 and 9. On the other hand, it requires long reaction periods to achieve high conversions and, therefore, costly [43].

Leung, Wu and Leung [44] explained that in general there will be advantages and disadvantages associated with the choice of the type of catalyst. The main


#### **Table 3.**

*Catalysts used in biodiesel reactions for Chlorella microalgae.*

advantages of alkaline catalysts are high catalytic activity, low cost and moderate operating conditions. However, it requires a low acidity index to prevent soap formation. Acid catalysts have the advantage of preventing soap formation. However, these catalysts corrode the equipment and require long reaction times. Heterogeneous catalysts (enzymes and metallic compounds) are highly selective and allow recycling, but at high costs.

The oil extracted from marine microalgae has a high acidity index, indicating acid catalysis as the most suitable for biodiesel production, as can be seen in **Table 3** which highlights the research done in the process of biodiesel synthesis via acid catalysis [45].

The synthesis of biodiesel from microalgal oils is carried out in a reactor. The mixture of alcohol and catalyst reacts with the triglyceride and/or fatty acids present in the microalgal oil. After the reaction, the mixture is transferred to a separation tank to guarantee the formation of the upper layer consisting of methyl ester, excess of alcohol and catalyst (acid or base) and the lower layer, predominantly glycerol [24].
