Evaluation of green microalgae isolated from central and north coast of Sao Paulo as source of oil

Microalgae strains, newly isolated from freshwater in mangrove areas of Central and North Coasts of Sao Paulo State (Brazil), were evaluated regarding total protein and lipid content, and fatty acids profile. The biochemical composition was compared with that observed in strains obtained by UTEX Culture Collection (USA). Among seven identified green algae, Monoraphidium contortum (CCMA-UFSCar-701) presented the highest lipid content (43.60%), close to that observed in Botryococcus braunii (UTEX-2441; 48.85%). Protein content in isolated strains varied in the range of 13.90~23.60%. Finally, the most abundant fatty acids were palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), and y-linolenic acid (C18:3).Chlorella vulgaris (CCMA- UFSCar-704) may be highlighted for its high linoleic acid content (49%). On the other hand, Elakatothrix sp (CCMA- UFSCar-702) and Scenedesmus obliquus (UTEX-B2630) presented the highest content of oleic acid (41% and 43%, respectively), which is preferable for oils to be used as feedstock for biodiesel.


Evaluation of green microalgae isolated from central and north coast of Sao Paulo as source of oil.
Microalgae strains, newly isolated from freshwater in mangrove areas of Central and North Coasts of Sao Paulo State (Brazil), were evaluated regarding total protein and lipid content, and fatty acids profile. The biochemical composition was compared with that observed in strains obtained by UTEX Culture Collection (USA). Among seven identified green algae, Monoraphidium contortum (CCMA-UFSCar-701) presented the highest lipid content (43.60%), close to that observed in Botryococcus braunii (UTEX-2441;48.85%). Protein content in isolated strains varied in the range of 13.90~23.60%. Finally, the most abundant fatty acids were palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), and -linolenic acid (C18:3).Chlorella sp. (CCMA-UFSCar-697) may be highlighted for its high linoleic acid content (29%). On the other hand, Elakatothrix sp (CCMA-UFSCar-702) and Scenedesmus obliquus (UTEX-B2630) presented the highest content of oleic acid (41% and 43%, respectively), which is preferable for oils to be used as feedstock for biodiesel.

INTRODUCTION
Microalgae may be found in marine environments, freshwater or even in soil (Norton et al., 1996) and they are believed to be responsible for at least 60% of primary productivity on Earth (Chisti, 2004). Tens of thousands of microalgal species have been classified, and this high diversity may represent a promising source of new bioproducts and applications (Norton et al., 1996;Pulz and Gross, 2004).
Several studies focus on a diversity of applications of microalgal biotechnology in food, cosmetics and pharmaceutical industries, including: food and feed supplements, antioxidative compounds, carotenoids, polyunsaturated fatty acids, vitamins, and immunologically active polysaccharide (Derner et al., 2006;Gouveia et al., 2011;Olaizola, 2003;Pulz and Gross, 2004). More recently, microalgae have been highlighted as valuable alternative and sustainable source of third generation biofuels (Chen et al., 2014;Chisti, 2007).
It is important to note that unicellular photosynthetic organisms are capable of using light energy more efficiently, in comparison with higher plants (Janssen et al., 2003), being considered very efficient for carbon dioxide bio-fixation (Brown and Zeiler, 1993). Moreover, they can be cultivated in controlled conditions, presenting desired and stable composition (Dunstan et al., 1993;Sassano et al., 2010) and favoring industrial/ commercial operations.
This study aimed to evaluate the potential of green microalgae as a source of oil-rich biomass. Seven green algae strains were isolated from freshwater in mangrove areas of Central and North Coast of Sao Paulo State. The lipid content, fatty acids profile, and protein content were compared with that of green microalgae obtained from Culture Collection.

Sample collection and isolation
Using sterile glass bottles, freshwater samples (1 ~ 1.5 L) were collected in different mangrove areas within "Serra do Mar" State Park located in the cities of Cubatão and Ubatuba (Central and North Coast of São Paulo State -Brazil). Geographical coordinates are shown in table 1. Concomitantly with the collection of samples, temperature and pH (pH-indicator strips; Merck) were measured.
At the laboratory, microalgae containing water samples were used to inoculate enriched standard culture media: BOLD (UTEX, n.d.), CHU (UTEX, n.d.), Schlosser (Schlösser, 1982), and F/2 (Guillard and Ryther, 1962). When the culture became visually greenish, the isolation took place by combining micropipette method and streaking cell across agar plates (Andersen and Kawachi, 2005), in order to isolate unique species of microalga from the water samples.

Isolates identification
Isolated strains were observed under Olympus system microscope model BX51 and images were captured with camera Olympus SC30. Microscopic identifications were based on the morphology of the individual cells and colonial characteristics.

Cultivation of isolate strains
Isolated microalgae strains were cultivated in the BOLD medium. Microalgae were grown in 6 L Erlenmeyer flasks containing 3 L of culture medium. Filtered air was continuously injected with the use of air stone diffuser. Room temperature was adjusted to 25 o C, and 30W fluorescent lamps were positioned above the flasks for providing light intensity of 60 µmol photons.m -2 .s -1 . Initial pH was adjusted to 7.0.
Cell growth was indirectly measured by optical density at 550 nm (Becker, 1994) using a spectrophotometer (Femto 700 Plus), and the cultivation was finished when there was no further daily increase in optical density. Furthermore, four microalgae strains (Chlorophyceae) obtained from The Culture Collection of Algae at the University of Texas at Austin (UTEX) were cultivated in the same conditions: Botryococcus braunii (Utex 2441), Chlorella vulgaris (Utex 2714), Neochloris oleoabundans (Utex 1185), and Scenedesmus obliquus (Utex B2630).

Microalgal biomass evaluation
At the ended of each cultivation, biomass was recovered by centrifugation, washed twice with distilled water, and dried at 55 o C for 12h. Dry biomass was submitted to the determination of total lipids, employing organic solvents (chloroform: methanol 2:1, v/v) in Soxhlet extractor (Olguín et al., 2001;Piorreck et al., 1984). Lipid fraction, recovered with petroleum ether, was submitted to analysis of fatty acids content, after conversion to corresponding methyl esters (Hartman and Lago, 1973). The analysis of fatty acid methyl esters (FAME) was performed in a gas chromatograph (Agilent Model 7890 CX) in accordance with Rodrigues-Ract and Gioelli (Rodrigues-Ract and Gioielli, 2008) and Perez-Mora et al. (Pérez-Mora et al., 2016). FAME components were identified by comparing their retention time with the standard 37 FAME mix (Supelco).
Furthermore, total protein content in dry biomass was analyzed by the Kjeldahl method, and factor 6.25 was adopted to convert from total nitrogen content (AOAC, 1984).

RESULTS AND DISCUSSION
In table 1, it is possible to see data from collection sites. pH values were within 6.5 and 7.0, and temperature values were in the range 25.5 and 30.0. In fact, higher temperature (30.0) observed at Cotia -Pará Park may be attributed to the shallow water (approximately 5.0 cm). In the same day at the laboratory, collected samples were used to inoculate different culture medium, generally used for photosynthetic microorganisms: BOLD (UTEX, n.d.), CHU (UTEX, n.d.), Schlosser (Schlösser, 1982), and F/2 (Guillard and Ryther, 1962). After 2 to 3 weeks, BOLD medium containing flasks showed the most intense green color and these cultures were employed for isolation of 7 microalgae strains.
The identification of isolated strains was performed following the classification system by microscopic identifications based on morphological characteristics. As it is possible to see in table 2, all isolates belong to the Chlorophyceae class.
Table 2 -List of strains isolated from Nucleo Itutinga Pilões (Cb) in the city of Cubatão (S.P.) and Nucleo Picinguaba (P) in the city of Ubatuba (S.P.).
Solitary or colonial cells. Random colony shape determined by cell arrangement. Hyaline and thin cell membrane. Fusiform cells with one parietal chloroplast.   All these isolates and the four microalgae strain obtained from Culture Collection (UTEX) were cultivated in 6L Erlenmeyer flasks containing 3L of BOLD Basal Medium for obtaining sufficient biomass for biochemical analysis: total protein content, total lipid content, and fatty acids profile. Table 3 shows results from the analysis of total lipid and total protein content. Total lipid content varied from 18.07 to 43.60% in isolated strains and from 21.40 to 48.85% in microalgae strains obtained from Culture Collection (UTEX) The fatty acid profile of each strain was obtained by analysis of fatty acid methyl esters (FAME) by gas chromatography. The results in table A1 (Annex 1) presented palmitic acid (16:0), oleic acid (18:1n9), linoleic acid (C18:2n6), and -linolenic acid (C18:3n6) as the most abundant fatty acids for these strains. The isolated Monoraphidium contortum (CCMA-UFSCar 701) and the purchased Botryococcus braunii (UTEX 2441) presented the highest values of lipid content (43.60 % and 48.85 %, respectively). The lack of nutrients at the end of the cultivation may have contributed to the increase in lipid content, as a result of the well-known phenomenon of triacylglycerol synthesis in response to stress condition (Liu and Benning, 2013). However, further studies have to be performed for a specific strain, since lipid metabolism seems not to be uniform in the algal realm (Liu and Benning, 2013). Mahmoud et al. (Mahmoud et al., 2015) evaluated microalgae strains isolated in different locations in Egypt. Among fifteen isolates, they highlighted Chlorella vulgaris, Scenedesmus quadricauda and Trachelomonas oblonga as the most suitable candidates for oil production, with 37%, 34% and 29% of lipid (w/w), respectively.
Several studies have been done, not only submitting microalgae to stress conditions but also with genetic approaches. For example, increases in lipid accumulation were observed when the starch synthesis pathway was blocked in mutants of Chlamydomonas (Li et al., 2010) and Chlorella (Ramazanov and Ramazanov, 2006).
After the extraction of oil, defatted biomass may contain hydrophilic compounds with commercial interests, such as sugars and proteins (Bellou et al., 2014). Microalgal proteins may be used in pharmaceutical products, cosmetics or mainly for food and feed supplements (Derner et al., 2006;Pulz and Gross, 2004). In the present study, total protein content varied in the range 13.90~23.60% in biomass of isolated microalgae strains and 14.05~29.28% in biomass of microalgae obtained from Culture Collection (UTEX).
The values of protein content obtained in this study were much lower than the values observed among the data presented by Barka & Blecker (Barka and Blecker, 2016), where some species of Chlorophyceae strains present protein content higher than 40% or even higher than 50%. However, to produce Single Cell Protein, for example, it would worth it to evaluate the physico-chemical condition for increasing the biosynthesis of these biomolecules, mainly light intensity and nitrogen content. In this context, there are several studies showing the possibility of increasing protein content in Arthrospira platensis in different types of photobioreactor by fed-batch process (Cruz-Martínez et al., 2015); repeated fed-batch (Matsudo et al., 2009) or continuous process (Avila-Leon et al., 2012;Matsudo et al., 2012) Concerning fatty acids profile, linoleic and -linolenic acid, commonly known as -6 fatty acids, are precursors of arachidonic acid, which is important in the synthesis of eicosanoids in human body (Verlengia and Lima, 2002). These eicosanoids may play a role as mediators in processes associated to inflammation, immune system modulation, and platelet aggregation (Kus et al., 2011).
In the case of -linolenic acid, Chlorella sp. (CCMA-UFSCar 697), Chlorella vulgaris (CCMA-UFSCar 698), and Neochloris oleoabundans (UTEX 1185) presented the highest values (23, 22, and 19%, respectively) (Table A1 -Annex 1). It is important to note that, the consumption of this fatty acid may be useful in the case of deficiency in delta-6-dessaturase, responsible for its ordinary synthesis in the human body (Horrobin, 1992). In this sense, it is worth it using one of these strains for further studies aiming to increase unsaturated fatty acids. For example, Ronda & Lele (Ronda and Lele, 2008) observed that increasing light intensity and decreasing temperature have a positive effect on the content of -linolenic acid.
Palmitic acid was above 20% in all the evaluated strains, but the highest contents were observed in that from UTEX culture collection, Chlorella vulgaris (46%) and Scenedesmus obliquus (34%) (Table A1 -Annex 1). The abundance of this fatty acid was also found by Molino et al. (Molino et al., 2018) in the green algae Chlorella vulgaris and Dunaliella salina.
The most abundant FAME, presented in the strains evaluated in the present study, are within the most common fatty esters in biodiesel, in accordance with Knothe (Knothe, 2008). Moreover, Elakatothrix sp (CCMA-UFSCar 702) and Scenedesmus obliquus (UTEX B2630) presented the highest content of oleic acid (41% and 43%, respectively), which is preferable for oils to be used as feedstock for biodiesel (Knothe, 2008;Mahmoud et al., 2015).

Annex 1
Table A1 -Fatty acids profiles found in the isolated species and microalgae from Culture Collection (UTEX)