Quantification of Lipid Content and Identification of the Main Lipid Classes Present in Microalgae Extracts Scenedesmus sp. for Obtaining Fatty Compounds for Biofuel Production

Microalgae biomass has been reported in the literature as one of the most 
promising sources for obtaining different products of industrial interest such 
as lipids, fatty acids, carotenoids, proteins and fibers. The lipid fraction of 
microalgae comprises neutral lipids, free fatty acids and polar lipids. It is 
of great importance to estimate the composition of the lipid fraction to define 
the potential for use, either as a raw material for the production of biofuels 
or for use for nutraceuticals and/or food purposes. The microalgae Scenedesmus sp. cultivated in a 
photobioreactor, the sky open raceway type, was 
evaluated for lipid content, identification and quantification of lipid 
components obtained from different extracts. In the quantification of the lipid 
content, extraction methods were proposed without chemical treatment (use of 
solvents only) such as chloroform:methanol (2:1 v/v)—Bligh & Dyer, Ethanol, 
Ethyl acetate:Hexane (1:1 v/v) and others with chemical treatment such as 
J-Schmid-Bondzynski-Ratzlaff (acid) and saponification (basic). For the 
identification of the main lipid components present in the extracts, the Thin 
layer chromatography (TLC) technique was used. This made it possible, using a 
simple and inexpensive method, to identify the compounds extracted by different 
extraction methods, that is, it was possible to verify the selectivity of the 
different extraction methods. In addition, it has been shown that using these 
methods, widely described in the literature as methods of extracting lipids in 
practice, extracts a wide diversity of compounds. The levels of lipids obtained via solvent 
extraction were up to 50% higher than those obtained with chemical treatment. 
In lipid extracts, obtained via solvent extraction, the presence of polar 
compounds, glycerides, carotenoids, pigments and sterols was identified, with 
up to 53% being composed of an unsaponifiable fraction, thus, 
presenting low selectivity for extracting fatty components. The acidic and 
basic treatments applied to the biomass of Scenedesmus sp. showed greater selectivity for obtaining fat components of 71.47% and 94.99%, 
respectively. The results showed that depending on the solvent/method used to 
quantify the lipids, the selectivity for obtaining the grease fraction, 
fundamental for conversion into biofuels, varies and the total lipid content 
may be overestimated.


Introduction
To develop large-scale microalgae cultivation, it is necessary to isolate and characterize the species, improving the genetic tools in search of specific characteristics. For example, in the cultivation of microalgae, some factors can influence the production of lipids such as: pH, concentration of nutrients, light intensity and temperature [1] [2] [3] [4]. These environmental conditions can be controlled and the species selected according to the desired fatty acid(s) [5].
The term "biomass" means any organic matter that is available on a recurring or renewable basis including, plants, agricultural waste, aquatic plants, wood and wood waste, animal waste, municipal waste and other waste used for industrial energy production, fuels, chemicals and materials [6] [7] [8].
An emerging alternative is the use of aquatic biomass; it is estimated that the global primary production of biomass is 50% aquatic and 50% terrestrial. To this day, government policies have focused almost exclusively on the use of terrestrial biomass, paying little attention to aquatic crops, taking as examples macro and microalgae [9]. Marine microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that can grow quickly due to their ability to convert CO 2 and transform into proteins, carbohydrates and lipids [1] [10] [11].
Currently, countries with emerging economies like China and India are working on the development of technologies for the production and commercialization of microalgae biodiesel, aware that the current sources have not supplied the energy required for planned economic growth [12] [13]. The cultivation of microalgae for the production of biomass is widely accepted as a probable ecocompatible option for the generation of biofuels.
The term "lipid" is broad, as it includes a highly diverse group of molecules with similar solubility in organic solvents. As will be discussed in this article, only some of these lipids are suitable for the production of biofuels, while others are potentially valuable in terms of nutrition [14] [15]. In the microalgae differ-  [18]. Saponifiable molecules are those that include at least one grease chain in their structure, which can be converted into soap from the saponification process and later into fatty acid [19].  [20]. The term "total lipids" is used primarily for analytical purposes, in addition to acylglycerols, the crude lipids obtained from microalgae often contain free fatty acids, hydrocarbons, ketones, sterols, carotenoids and chlorophylls [16]. For this reason, the crude lipid extract obtained from microalgae is often subjected to more than one fractionation step before being transesterified [21] [

Materials and Methods
The biomass of Scenedesmus sp., provided by UFRN (Federal University of Rio Grande do Norte), was grown for 5 days in an open Raceway photoborractor with a volume of 5.000 L (Figure 1), using 30% PP medium. Growth monitoring was performed by optical density, as described by Lee [27], making absorbance

Solvent 2-Ethanol
In order to exhaust all the grease fraction present in the microalgae biomass, the Sohxlet extraction technique was used, performed through successive washes of the biomasses from the continuous cycle of evaporation and condensation of the organic solvent. This cycle was repeated until the total removal of crude lipids  [2]. To do this, 10 grams of the dry biomass of microalgae, stored in a cellulose cartridge, inserted inside the Soxhlet apparatus, and 200 mL of ethanol were added in the balloon located at the bottom of the equipment, which was subsequently heated to approximately 80˚C for 2 hours. The flask containing the solvent and the solubilized content was taken to a rotary evaporator in order to recover all the solvent, and then to an oven at 60˚C until constant weight. The experiments were carried out in triplicates and the extraction yields were determined as a percentage, in relation to the dry biomass mass free of ash.

Solvent 3-Ethyl Acetate:Hexane (1:1 v/v)
The extractions were carried out in a 50 mL flask, starting from 5 g of dry biomass with 15 mL of the mixture Ethyl acetate: hexane (1:1 v/v). A solvent: charge ratio of 3:1 (mL of solvent: g of biomass), extraction time of 2 hours at a temperature of 60˚C and 200 rpm stirring on a magnetic stirrer were used. For the separation of residual biomass from the liquid phase (lipid fraction + solvent), a filter paper filtering process was carried out. Then the biomass was washed with 30 mL of the solvent mixture selected for the extraction step and filtered again. The solvents were removed from the liquid phase by vacuum evaporation and the lipid fraction (not volatile under operating conditions) was dried to constant weight in an oven at 60˚C. The experiments were carried out in triplicates and the extraction yield was determined in percentage, in relation to the dry biomass mass.

Acid Treatment-J:Schmid-Bondzynski-Ratzlaff [32]
The extraction was performed starting from 5 g dry biomass-in a 50 mL -10 mL HCl 8 M falcon tube; Hydrolysis: 10 minutes water bath at 60˚C; 1 st Extraction: 10 mL absolute ethanol; 25 mL ethyl ether; 25 mL petroleum ether; Separation of the phases in a funnel. 2 nd and 3 rd Extraction: 10 mL absolute ethanol; 25 mL ethyl ether; 25 mL petroleum ether; Washing of the "solvent" phase with distilled water until pH of the water = 7 (to remove HCl residues); Evaporation of the solvent; Kiln drying at 60˚C.

Basic Treatment-Saponification "In Situ"
Saponification "in situ" was carried out by mixing 5 g of dry biomass with 50 mL

Identification of Lipid Components-Thin Layer Chromatography (TLC)
In

Lipid Content of the Biomass of Scenedesmus sp.
The average values obtained in the extraction of lipids, following the different solvents/methods, are presented in Figure 2 and were expressed as a percentage in relation to the dry biomass mass free of ash.
The lipids showed statistically significant differences (p < 0.05  of neutral lipids inside the cell, which bind strongly to proteins located in the cell membrane, via hydrogen bonds, forming a complex with polar lipids. Van der Waals interactions between the nonpolar solvent and lipids are not able to break this membrane, based on lipid-protein associations. Polar solvents, such as methanol or ethanol, break these associations, forming hydrogen bonds with the polar lipids in the complex. In addition, the use of polar solvents such as acetone, ethanol, methanol increases the affinity for pigments, sugars, and polar lipids, leading to an increase in these compounds in the extract [33]. Therefore, the addition of a polar solvent facilitates the extraction of neutral lipids associated with the membrane. However, the use of more polar solvents leads to overestimated total lipid yields, when compared to methods using less polar solvents, as in the case of the ethyl acetate:hexane mixture (1:1 v/v). The content of lipids obtained by the acid treatment of biomass (3.21% ± 0.32%) was statistically equal to that obtained in the basic treatment (3.44% ± 0.28%). When biomass was applied with an acid or basic treatment, there was a reduction of 49.84% and 53.41%, respectively, in the amount of lipids extracted when compared to that obtained via solvent, chloroform:methanol, 2:1 v/v-Bligh & Dyer [28].

Identification of Lipid Components-Thin Layer Chromatography
For the identification of the lipid classes present in the extracts, obtained by the different methods evaluated in this work, the thin layer chromatography technique was used. The main objective of this identification was to select more selective extraction procedures for the fatty compounds of interest for the production of biofuels [34]. As it was possible to verify in Figure 3, the extracts presented fatty acids, triglycerides, diglycerides, sterols and carotenoids identified according to with Rf (Retention Factor)-order of elution of each compound.  Performing a detailed analysis of the main fatty components present in lipid extracts it is possible to verify that, even when methods without chemical conversion are used, the triglyceride content was low and did not exceed 2.1% as in the case of Bligh & Dyer. The Scenedesmus sp. evaluated in our work showed a reduced amount of triglycerides when compared to conventional oilseeds used for biodiesel production. Conventional oilseeds such as palm, soy and sunflower have amounts greater than 90% of triglycerides in the oil [34].
The triglyceride content present in Scenedesmus sp. evaluated in our work was also inferior to those reported for the microalgae Parietochloris incisa, which presented 42.9% and for Pavlova lutheri with 40.3% [35]. For Scenedes- nutum also showed high levels of free fatty acids, which probably resulted from lipid degradation during the storage and processing time of the biomass [38].
According to Ryckebosch [38], when storing microalgae biomass in nature, with high amount of water and without any type of enzymatic inactivation, lipolysis occurs naturally.
As can be seen by thin layer chromatography plates, the methods and solvents used in extracting the lipids from Scenedesmus sp. they did not include only fatty components. Pigments and sterols were observed (Figure 3). In the lipid ex- Chemical treatment contributed to the hydrolysis and esterification of complex lipids such as phospholipids and glycolipids in fatty acids and fatty esters, respectively [40] [41].
In the extract produced via saponification, 38.48% of ethyl esters (biodiesel) were observed (Figure 3). The presence of these compounds was also identified in the extract obtained via J-Schmid-Bondzynski-Ratzlaff, but in a smaller amount (22.53%). It is known that the presence of water in the reaction medium, can inhibit the esterification reaction. The ethyl esters formed come from the transesterification/esterification reaction of triglycerides and fatty acids through the presence of ethanol and catalyst (acid or basic). The direct saponification of biomass can be an interesting process for the production of biodiesel since it eliminates the lipid extraction step; however a greater amount of chemical inputs may be necessary.
The lipid extracts obtained via solvent (without chemical treatment), showed a large amount of unsaponifiable compounds such as: sterols, carotenoids and pigments, demonstrating that only the use of organic solvents was not selective to obtain fatty components. In this sense, a chemical refining of these extracts  The lipid extracts obtained from the extraction via solvents presented between 38.58% and 46.86% of fatty material (saponifiable) while those obtained through chemical treatment presented between 71.47% and 94.99%. In Figure 5, it was possible to evaluate the different methods used to obtain the lipids in relation to the amount of saponifiable material (fatty) generated from 100 grams of dry biomass of Scenedesmus sp.
The amount of fatty material did not exceed 3.43%, this result being lower when we consider conventional oilseeds, such as soybeans, the main raw material for the production of biodiesel in Brazil, which has an average of 18% oil. The   To obtain a fraction rich in fatty components, the extracts will require an additional chemical refining step to separate saponifiable and unsaponifiable.

Conclusions
The chemical treatment methodologies applied to biomass, J:Schmid-Bondzynski-Ratzlaff and saponification, showed the same amount of extracted lipids in the conversion of the most complex lipids into free fatty acids. Among the methods evaluated in this study, the basic hydrolysis of biomass via saponification, was proved to be the most selective to obtain a 94.99% grease fraction.
Therefore, starting from a lipid fraction, with a more homogeneous composition in fatty components, the definition of the technological route to be applied in its conversion into biofuels will be facilitated.