Extractions were performed in a 50 mL flask, starting from 5 g of dry biomass with 15 mL of the mixture chloroform: methanol (2:1 v/v), following the methodology described in the literature [25] [26] [27] [28] [29]. 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. The lipid extracts were analyzed by Thin Layer Chromatography (TLC) and compared with the available lipid standards.

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 [30]. The experiments were conducted in triplicate. The lipid extracts were analyzed by Thin layer chromatography (TLC) and compared with the available lipid standards.

For identification of the lipid classes of interest present in the extracts of the microalgae Scenedesmus sp., was used Thin layer chromatography (TLC). In this analysis, 0.001 g of the extracted lipid fraction was dissolved in 600 µL of chlorofor plate m. The equivalent of 0.03 µL of the solution was applied to a 60 TLC (Merck) silica gel with the aid of an automatic pipette. In addition to the sample to be analyzed, equal amounts of triolein, diolein, monoolein, fatty acid, ergosterol and cholesteryl oleate were applied to the plates for comparison and quantification. To ensure efficient separation and obtain acute bands of nonpolar lipids, the separation was performed as described below. First, the solvent mixture composed of petroleum ether/diethyl ether/acetic acid (70:30:2 v/v) was used to separate the lipids, until reaching 2/3 the height of the silica plate (7 cm). After drying the plate, the separation continues in the same direction using the solvent mixture composed of petroleum ether/diethyl ether (100:2 v/v) until it reaches the top of the plate (9 cm) (Figure 1) [21].

The chromatographic plate after elution was revealed with iodine vapor and the stain retention (Rf) factor of the standards and sample components was determined.

To achieve an irreversible staining of nonpolar lipids and sterols in the TLC plate, it was kept in a solution of 0.63 g of MnCl₂∙4H₂O, 60 mL of water, 60 mL of methanol and 4 mL of sulfuric acid for 10 seconds, followed by heating to 105˚C. The intensity of the color depends on the heating time, which must be a minimum of 30 min. With the aid of the Image Master Total Lab version 1.11 program, was used to identify and quantify the lipid classes present in the extracts. The methodology previously described was used to identify the lipid classes present in the extracts obtained by the different extraction methods evaluated in this work. The main objective of this analysis was to assist in the selection of the procedure that allows a more selective extraction of lipids convertible into biodiesel.

The microalgae Desmodesmus sp. and Scenedesmus sp. were evaluated for lipid accumulation capacity under the same cultivation conditions. The average values obtained in the extraction of lipids, following the different methods, are shown in Figure 2 and were expressed as a percentage in relation to the ash-free dry weight biomass.

It is observed that the lipid yields showed statistically significant differences (p < 0.05). According to the literature, as expected, the extraction method directly influences the content of recovered lipids [3] [31] [32] [33]. In the microalgae Desmodesmus sp. the recovery of lipids using the Bligh-Dyer method was 9.18% ± 0.33% slightly higher than that obtained for the microalgae Scenedesmus sp.

(8.16 ± 0.42). The recovery of lipids for the microalgae Scenedesmus sp. using the method J: Schmid-Bondzynski-Ratzlaff was 12.46% ± 0.38%, showing that the presence of lipids in this polarity range was quite similar for both microalgae. The presence of polar solvents such as chloroform and methanol helped the extraction of lipids such as phospholipids and glycolipids, as they break these associations, forming hydrogen bonds with the polar lipids of the complex, this behavior can be partially explained due to the presence 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 [34]. Therefore, the addition of a polar solvent helps the extraction of neutral lipids associated with the membrane.

To identify the lipid classes present in the extracts, a thin layer chromatography analysis of the Bligh & Dyer and J: Schmid-Bondzynski-Ratzlaff extracts was performed (Figure 3). The plates show the presence of fatty acids, triglycerides, diglycerides and sterols according to the Rf (retention factor) of the added standards and the order of elution of each compound.

The chromatograms generated from Figure 3 referring to the lipid extracts of the microalgae Desmodesmus sp. and Scenedesmus sp. can be seen in Figures 4-7.

According to the percentages of fatty compounds identified in thin layer chromatography (Figure 8), it is possible to observe a variation in relation to the percentage of triglycerides present in Bligh & Dyer extracts when compared to extracts J: Schmid-Bondzynski-Ratzlaff.

The Bligh & Dyer extract showed the highest concentration of triglycerides in the lipid fraction with 18.26% in the microalgae Scenedesmus sp. (Figure 8) and the use of this method allowed to quantify the accumulation of triglycerides in the biomass in its integral form. The microalgae evaluated in this work showed a reduced amount of triglycerides when compared to conventional oilseeds such as palm and sunflower that present amounts above 90% [35] [36].

When we compare the levels of triglycerides obtained in this work by the microalgae Desmodesmus sp. and Scenedesmus sp. they were lower than the levels of triglycerides present in the microalgae Parietochloris incisa which presented 42.9% and Pavlova lutheri with 40.3% [35]. For Scenedesmus sp. according to Yao [19] the content of triglycerides did not exceed 4.1% in the lipid extract. The presence of fatty acid in the lipid extracts of the microalgae Desmodesmus sp. and Scenedesmus sp. obtained by the Bligh & Dyer method represented 24.38% and 24.51% respectively, in relation to the other lipid classes separated by thin layer chromatography (Figure 8). The amount of water present in the biomass after collection (85% humidity) and the time of 24 hours, associated with a temperature of 100˚C in the drying of the biomass, probably contributed to the oxidation of triglycerides, increasing the amount of free fatty acids. It is known that the drying temperature may have contributed to microbial degradation and consequently lipid oxidation. In addition, chlorophyll can undergo photooxidation generating O²⁻ radicals in the cell that can promote the degradation of lipids [37]. According to the literature, the microalgae Phaeodactylum tricornutum 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 biomass of microalgae in natura, without any type of enzymatic inactivation with a high amount of water, lipolysis occurs naturally.

At the points of origin of application of the sample under the chromatography plate, in the lipid extracts of Bligh & Dyer of both strains evaluated in this work,

they indicated a higher concentration of polar compounds (Figure 9). These compounds retained at the base of the plate demonstrated that they did not show sufficient affinity to interact with the mobile phase composed of petroleum ether and ethyl ether, having more affinity with the stationary phase (Silica). In this case, the greater the affinity of the compounds with the stationary phase, the greater the polarity of the compounds. It was possible to evaluate that the amount of polar compounds present in the Bligh & Dyer extracts from the microalgae Desmodesmus sp. and Scenedesmus sp. represented 15.59% and 15.55% of the total lipids identified. In the presence of chloroform and methanol, used in Bligh & Dyer, a greater number of polar compounds may be extracted due to the affinity to the extracting solvents. According to Chen [39], microalgae grown in autotrophic form presented 70.6% of polar lipids, while microalgae cultivated in a heterotrophic manner, only 19% of total lipids were polar lipids. No additional separation of the polar lipids has been performed, however, according to Yao [15], the vast majority of polar lipids present in microalgae include phospholipids and glycolipids.

In the acid treatment of biomass, structures such as proteins, polysaccharides and lipids due to the presence of H₃O⁺ ions in water undergo hydrolysis, causing decomposition into amino acids, simple sugars and fatty acids [40] [41]. It can be seen in Figure 9 that in the extracts of J: Schmid-Bondzynski-Ratzlaff there is an increase in the amount of free fatty acids and a decrease in triglycerides and polar compounds. According to Figure 9, it was possible to observe that in the extracts of Desmodesmus sp. and Scenedesmus sp. with acid treatment, the

amount of polar lipids decreased to 5.76% and 5.82%, respectively. The amount of triglycerides in extracts with acid treatment for the biomass of Desmodesmus sp. and Scenedesmus sp., this amount decreased to 6.52% and 1.73%, respectively. The increase in the concentration of fatty acids in the extracts of J: Schmid-Bondzynski-Ratzlaff in the microalgae Desmodesmus sp. and Scenedesmus sp. of 48.03% and 35.22%, respectively, was higher than that reported by Petrok [35] for Chlorella sp. of 31% - 32% after the biomass hydrolysis. This fact can be partially explained by the presence of HCl used to break these bonds that keep the more complex triglycerides and lipids that are hydrolyzed to simpler lipids like free fatty acids [42]. An carbonyl, for example, from a triglyceride, undergoes an electrophilic attack from H⁺, forming a carbocation. This carbocation then undergoes a nucleophilic attack by a water molecule, forming a tetrahedral intermediary. Then, the elimination of a diglyceride and a fatty acid occurs, together with the regeneration of the H⁺ species. By similar processes monoglycerides and glycerol will be formed. The presence of diglycerides in lipid extracts can also be seen as one of the intermediates of the reaction (Figure 9). Another grease species that was detected in thin layer chromatography was fatty esters. The formation of these components in greater quantity in the extracts of J: Schmid-Bondzynski-Ratzlaff can be explained due to the presence of H⁺ in the reaction medium by the protonation of carbonyl (C=O) present in the structure of the lipids, followed by an attack by the nucleophilic of ethanol (CH₃CH₂OH) present in the mixture of solvents used in the extraction [43] [44]. The product of this reaction is the fatty esters formed from the lipid transesterification/esterification reaction. The acidic treatment of biomass using 8% sulfuric acid at 160˚C for 45 min, for lipid extraction was reported by Halim [7] using the microalgae Chlorococcum sp. which resulted in an increase in the fatty acid content. Other studies also reported the use of an acid in the treatment of biomass resulted in an increase in the extraction process [45].

In addition, according to Figure 10, the presence of hydrochloric acid together with the polarity of the solvents: petroleum ether, ethyl ether, ethanol in the extraction process via J: Schmid-Bondzynski-Ratzlaff in both strains evaluated in this study increased extraction of unsaponifiable compounds such as sterol esters. The presence of the acid facilitated the breaking of the micro-algal cell wall and in the case of Desmodesmus sp. 10% of sterols were detected in the lipid extract via J: Schmid-Bondzynski-Ratzlaff when compared to the extract obtained via Bligh & Dyer where this compound was not identified. Phytosterols in microalgae are found in several configurations, including free sterols, fatty acid sterol esters and glycoside sterol.

According to the literature, sterols are important constituents of cell membranes, being intertwined with phospholipid bilayers in all eukaryotes. Through this existence, sterols play significant roles in maintaining cellular structural stability and acclimatization to the membrane temperature. Sterols help maintain fluidity and permeability by controlling the movement of fatty acid chains within the membrane [46] [47]. However, it has been reported in the literature that changes in growth conditions such as: renewal rate of semi-continuous cultures [48], concentration of salt in the growth medium [49] [50] [51], type of photobioreactor [52], amount of light and phosphorus [53], temperature and silicate content [54] and growth stage of microalgae [55], can affect the production of sterols in microalgae.

In the lipid extracts of the microalgae Desmodemus sp. and Scenedesmus sp. the presence of hydrocarbons with double conjugated bonds was detected in the same time of carotene retention in amounts of up to 2% in relation to the lipid extract. For example, during photosynthesis, microalgae absorbs sunlight, nutrients and CO₂ that are converted into biomass and, at the same time, produces molecular oxygen [56] [57]. As, oxygen is easily activated by ultraviolet radiation or by the heat of sunlight transforming itself into reactive oxygen species, highly oxidizing, plants and microalgae have developed a protection mechanism against these reactive oxygen species, which consists in the production of numerous and efficient antioxidant compounds such as carotenoids.

In the Bligh & Dyer extracts of the Desmodesmus sp. and Scenedesmus sp. biomasses, the significant presence of chlorophylls was detected in concentrations of 37.03% and 27.91%, respectively (Figure 11). The use of polar solvents such as acetone, ethanol, methanol increases the affinity of these molecules with the solvent, resulting in an increase of these compounds in the extract [34]. After the acid treatment (method J: Schmid-Bondzynski-Ratzlaff), the pigments suffered a reduction of 9.18% and 7.21% of the total pigments of the Desmodesmus sp. and Scenedesmus sp. microalgae, respectively. After the addition of acid (H⁺), a phenomenon occurs which is known as pheophytinization, where the magnesium in the center of the chlorophyll molecule is replaced by 2 hydrogens (H⁺) [58]. According to the literature, the presence of chlorophyll acts as a photosensitizer due to the production of O₂ (singlet) promoting self-oxidation in vegetable oils [59]. As there is a large amount of chlorophylls, carotenoids and sterols, it was higher than that found in vegetable oils, the relative amount of triglycerides must be lower [35].

After the individual analysis of the components, according to Figure 12 in this work, the amount of saponifiable and unsaponifiable material in each of the extracts was also evaluated. The components were classified as fatty the sum of the lipid components: content of fatty esters + fatty acids + triglycerides + diglycerides. As unsaponifiable, not greasy, carotenes + sterol ester + chlorophylls + polar compounds were considered. In this case, the possible conversions of the polar compounds in free fatty acids present in the extracts were neglected in order not to overestimate the results. According to Figure 12, it can be seen that acid hydrolysis contributed to the increase in fatty components of the microalgae Desmodesmus sp. and Scendesmus sp. the amount of fatty components represented 72.84% and 76.66% in relation to the total content of lipids. The amount obtained in our study was higher than that reported by Petkov [35] where the authors stated that it was possible to obtain 50% of fatty compounds after hydrolysis of the lipid extract. For the Bligh & Dyer extracts from Desmodesmus sp. and Scendesmus sp. strains this amount was 44.74% and 50.96% of fatty components in relation to the total amount of material extracted.

From 100 grams of dry biomass of Desmodemus sp. (Bligh & Dyer and J: Schmid-Bondzynski-Ratzlaff method) and Scenedesmus sp. (Bligh & Dyer method) the results were statistically equal and did not exceed 4% of fatty material (Figure 13). When we evaluate only the percentage of fatty material obtained from the method of J: Schmid-Bondzynski-Ratzlaff from 100 grams of dry biomass of Scenedesmus sp. this amount was 9.55% ± 0.38%. Indicating that from the microalgae Scenedesmus sp. under the same growing conditions as the microalgae Desmodesmus sp. it was possible to obtain a percentage of fatty components in relation to dry weight (Figure 13).

As described even after acid treatment of biomass, concentrations of other non-fatty compounds still remained in the lipid extract. Therefore, an additional step of separation and concentration of the fatty components will improve the quality of these extracts [35]. Using a purer fraction of fatty components, it will be easier to convert to biofuels, for example. The other components, such as sterols and carotenoids, can be used as high added value [60] [61] accumulation products.