Brazilian Journal of Chemical Engineering (2020) 37:41–48 https://doi.org/10.1007/s43153-020-00011-3

ORIGINAL PAPER

Evaluation of oleoabundans as sustainable source of oil‑rich biomass

Ivan A. Avila‑León1 · Marcelo C. Matsudo2 · Lívia S. Ferreira‑Camargo3 · Juliana N. Rodrigues‑Ract1 · João C. M. Carvalho1

Received: 15 July 2019 / Revised: 24 September 2019 / Accepted: 11 November 2019 / Published online: 3 February 2020 © Associação Brasileira de Engenharia Química 2020

Abstract In microalgae biotechnology, several strategies have been used aiming to increase biomass productivity and oil content, for example, by exploring novel bioreactor design and cultivation techniques. In the present study, the infuence of stressing agents (sodium thiosulphate, sodium chloride, and glycerol), under nitrogen starvation, were evaluated for lipid accumulation in . Additionally, diferent light regimes and the use of LED were also evaluated. The results showed that sodium thiosulfate, glycerol, and sodium chloride addition resulted in a reduction in biomass concentration, but, at the same time, an increase in lipid content. 2.5 mM of sodium thiosulfate provided the highest percentage of lipid content up to 44.7%. In a tubular photobioreactor, despite the twofold increase in biomass concentration, there was a decrease in lipid content, compared with cultures in Erlenmeyer fasks. Also, the use of white LED in combination with traditional fuorescent lamp (12 h:12 h) could efciently replace 24 h light with fuorescent lamp for producing oil-rich N. oleoabundans biomass.

Keywords Microalgae · Lipid · Bio-oil · Biomass · Biofuel

Introduction high amounts of lipid, that could be converted to biodiesel (Pandey 2017). Taking into account the unprecedented global energy crisis, Inside the cell, fatty acids and other lipids are essential mainly related to fossil fuel exhaustion, several research- constituents that have important functions, such as structural ers around the world are looking for alternative sources of components of biological membranes, nutrients storage, and renewable energy, mainly in those countries without con- energy production (Gurr et al. 2002; Guschina and Harwood ventional fuel resources. In this scenario, microalgal bio- 2013a, b). This cell lipid content can be increased by using technology seems to have high potential for biodiesel pro- biotechnological approaches that will result in values much duction (Huang et al. 2010; Chen et al. 2018). Microalgae higher than that observed in plants used for the production may be considered as an important alternative source of of biodiesel, like soybean and oil palm (Chisti 2008). Other biofuel, since some of their species are able to accumulate advantages of microalgae are: the fast growth that results in high biomass productivities in comparison with plants; possibility of cultivation in various environment conditions, * João C. M. Carvalho including non-arable land; and non-potable water may be [email protected] employed, even municipal or industrial wastewater (Gouveia and Oliveira 2009; Benedetti et al. 2018; Chen et al. 2018). 1 Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Despite the thousands of known microalgae species, only Paulo, Av. Prof. Lineu Prestes 580, Bl. 16, São Paulo, a few of them have been employed for commercial produc- SP 05508‑900, Brazil tion, such as Arthrospira (Spirulina) platensis, Chlorella 2 Institute of Natural Resources, Federal University vulgaris, Dunaliella salina, and Haematococcus pluvialis of Itajubá, Av. Benedito Pereira dos Santos, 1303, Itajubá, (Mobin and Alam 2017). Oil producing algae may be found MG 37500‑903, Brazil in diverse taxonomic groups, with varied total lipid content 3 Center of Natural and Human Sciences, Federal University in diferent species or strains. Most of the species studied of ABC, Av. dos Estados, 5001 Bl. B, Santo André, up to date belong to the green algae group, mainly because SP 09210‑580, Brazil

1 3 Vol.:(0123456789) 42 Brazilian Journal of Chemical Engineering (2020) 37:41–48 they are ubiquitous in varied habitats, can be easily isolated, Bench scale tubular photobioreactor and generally show better growth under artifcial conditions (Hu et al. 2008). Considering the runs in Erlenmeyer fasks, a scaling up In the present study, Neochloris oleoabundans was cul- to a tubular photobioreactor was performed to reproduce tivated in a bench scale tubular photobioreactor, with the the best results. aim of increasing cellular lipid content by evaluating the The tubular photobioreactor was built at the Laboratory addition of diferent components in the cultivation medium. of microalgal biotechnology at the University of Sao Paulo. Additionally, light emitting diode (LED) was evaluated as It is made of transparent glass tubes (internal diameter of energy source for oilirich microalgal biomass. 1.0 cm), with an inclination of 2%, connected by silicone tubes, and with a total volume of 3.5 L. In the lower part of the reactor, there is a Y shaped connector allowing the addition of pressurized air, which propels cells upwards Materials and methods to a fask that also act as degasser (Carlozzi and Pinzani 2005; Ferreira et al. 2012). A 0.2 µm flter membrane was Microorganism employed for sterilizing pressurized air. Fluorescent lamps (20 W) were used for providing a continuous light intensity The strain N. oleoabundans UTEX 1185 was acquired from of 60 µmol photons m−2 s−1. Room temperature was set at the UTEX Culture Collection of Algae. It was maintained in 25 ± 2 °C. pH values were maintained at 7.5 ± 0.3 with the BOLD 3N medium with 2% agar (UTEX n.d.). use of a solenoid valve coupled to a METTLER TOLEDO For inoculum preparation, N. oleoabundans was culti- M300 device, which monitored the culture pH with an elec- vated in 500 mL Erlenmeyer fasks containing 200–300 mL trode. When the medium pH increased, the solenoid valve of BOLD Standard medium (UTEX n.d.), previously auto- allowed ­CO2 to enter the photobioreactor. claved at 121 °C for 30 min. The culture was maintained Considering previous results, nitrogen was added dur- in an orbital shaker at 12 RPM, light intensity of 33 µmol ing the cultivation by a fed-batch process in accordance photons ­m−2 s−1 (with the use of 40 W fuorescent lamps), with daily productivity (Ávila-Leon 2014). and 25 °C (Tornabene et al. 1983). Additional cultivations were carried out with the aim of reducing energy consumption, by evaluating 12:12 h light/dark cycles or replacing 12 h dark regime by 12 h Cultivations for increasing oil content in N. illumination with red and white LED. Light inten- oleoabundans sity was 20 ± 2 µmol photons m−2 s−1 for red LED and 15 ± 2 µmol photons m−2 s−1 for white LED. Diferent compounds were evaluated for promoting an envi- ronmental stress condition for N. oleoabundans: sodium thi- osulfate ­(Na2S2O3), for increasing reducing power; sodium Analytical techniques chloride for increasing osmolarity (Table 1). Additionally, glycerol was used for providing a complementary organic Biomass concentration was measured by turbidimetry in a carbon source. spectrophotometer (FEMTO 700 Plus) at 495 nm, by using These cultivations were performed in 500 mL Erlen- a calibration curve correlating absorbance and biomass con- meyer fasks containing 350 mL of sterilized BOLD medium centration (dry weight; mg L−1) (Leduy and Therien 1977). (3N:3P) (UTEX) (Ávila-Leon 2014). Cultures were main- At the end of the cultivation, N. oleoabundans biomass tained at 25 ± 2 °C, 60 ± 5 µmol photons ­m−2 s−1, with the was harvested by centrifugation, washed with distilled addition of sterilized air. water, and dried at 55 °C for 12 h (Pelizer et al. 1999). Dried biomass was macerated and kept at 4 °C before the Table 1 Addition of diferent compounds for increasing lipid content biochemical composition analysis. Total lipid content was determined by extrac- Compound Final concentration Additional run (mM) tion with organic solvents. In a Soxhlet extractor chloroform:methanol (2:1 v/v) was refuxed until the liquid Sodium thiosulfate 1.2 2.5 3.8 Middle of exponential became clear (Piorreck et al. 1984; Olguín et al. 2001). growth Total protein content was determined by the Kjeldahl End of exponential method, considering 6.25 as the conversion factor from growth total nitrogen content (Association of Ofcial Analytical Sodium chloride 1.0 2.2 4.5 – Chemists 1984). Glycerol P.A 340 680 1020 1360 –

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Fatty acids content was determined by frst extracting the lipid by the Soxhlet method. The lipid was recovered with petroleum ether, and the fatty acids were converted to the corresponding methyl esters (Hartman and Lago 1973). The analysis of fatty acid methyl ester was performed in a gas chromatograph (Agilent Model 7890 CX) in accordance with Rodrigues-Ract and Gioielli (2008) and Pérez-Mora et al. (2016), in which the components identifcation was performed by comparing their retention time with the stand- ard 37 FAME mix (Supelco).

Fig. 1 Infuence of diferent concentrations of sodium thiosulfate Results and discussion ­(Na2S2O3) on maximum biomass concentration (Xm), lipid content, and protein content in Neochloris oleoabundans cultivated in Erlen- meyer fasks Cultivations in Erlenmeyer fasks: addition of sodium thiosulfate 2.5 mM (3.8 mM), sodium thiosulfate seems to inhibit cell In a frst cultivation, 2.5 mM (Mandal and Mallick 2009) of growth (546 ± 127.5 mg L−1) with a decrease of 10.5% sodium thiosulfate was added on the 7th (middle of expo- in lipid content, in comparison with the cultivation with nential growth) or the 10th day of cultivation (fnal of expo- 2.5 mM. nential growth), and the results are presented in Table 2. Single addition of 2.5 mM sodium thiosulfate, in the mid- Cultivations in Erlenmeyer fasks: addition dle of exponential growth (7th day) helped to improve lipid of glycerol accumulation, but it slightly reduced biomass concentration, in comparison with the control run (without sodium thiosul- In Fig. 2, it is possible to see that the addition of glycerol fate addition). On the other hand, when sodium thiosulfate had a positive infuence on total lipid content (34–42%) in is added at the end of exponential growth, it can increase comparison with control culture (28%). Nevertheless, high the lipid content even more, while only a discrete decrease amounts of glycerol propitiated bacterial growth that could in cell growth is observed, in comparison with control run. have infuenced biomass concentration measurements. Par- Table 2 also shows at the same time a reduction in protein ticularly, with the addition of 1.36 M, a decrease in pH cor- content and an increase in lipid content. roborate the information that bacterial growth took place, Considering these results, in the following step, the total possibly resulting in competition for nutrients, nitrogen for amount of sodium thiosulfate (1.2, 2.5, and 3.8 Mm) was instance. fractionated in 5 additions from the end of exponential As the results for 1.02 M glycerol concentration were growth (10th day). The results in Fig. 1 show that the inhibi- very similar to the ones for glycerol concentration of 0.68 M, tion of cell growth by sodium thiosulphate could be avoided this last concentration was chosen for cultivation in a bench up to 2.5 mM. scale tubular photobioreactor. With the addition of 2.5 mM of sodium thiosulfate, it was possible to obtain 44.7% of total lipid, which was the best condition for biomass production (897.01 ± 21.6 mg L−1), similar to the control run (950.77 ± 64.1 mg L−1). Above

Table 2 Infuence of single sodium thiosulfate addition (2.5 mM, added on 7th or 10th day) on fnal biomass concentration (Xf), total lipid content, and total protein content in Neochloris oleoabundans cultivated in Erlenmeyer fasks

Run Addition Xf (mg L−1) Lipid (%) Protein (%)

A 7th ­daya 768.1 ± 28.3 41.2 14.3 B 10th ­dayb 854.9 ± 21.6 52.0 7.7 Control – 950.8 ± 77.8 28.9 15.4 Fig. 2 a Infuence of diferent glycerol concentrations on maximum Middle of exponential growth biomass concentration (Xm), lipid content, and protein content in b End of exponential growth Neochloris oleoabundans cultivated in Erlenmeyer fasks

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Neochloris oleoabundans does not assimilate glycerol as allows the increase of each cell size, as stressed by Takagi carbon source under strict heterotrophic growth (in mineral and Yoshida (2006) in cultivations of Dunaliella. media under dark conditions) (Morales-Sánchez et al. 2013). Diferent mechanisms have been observed in microalgae However, this organic compound can be assimilated under cells as response to increasing osmotic strength, such as cell mixotrophic conditions (Das et al. 2011; Perez-Garcia and size decrease, glycerol excretion and lipid accumulation. Bashan 2015). Inside the cell, by means of several enzy- Baldisserotto et al. (2012), cultivating N. oleoabundans in matic activities, glycerol may be converted to intermediates high salinity conditions, observed modifcations in the cell of glycolysis, yielding energy by this glycolytic pathway and wall, and accumulation of starch and polyphosphate gran- entering the TCA cycle (Perez-Garcia et al. 2011). Moreo- ules. Furthermore, according to Xu and Beardall (1997), an ver, glycerol, as a substrate, may favor triacylglycerol (TAG) increase in medium salinity allows the production of satu- synthesis (Sharma et al. 2016). rated fatty acids that in turn provokes the reduction in fuid- ity and permeability of cell membranes. Cultivations in Erlenmeyer fasks: addition Takagi and Yoshida (2006) observed that the addition of of sodium chloride NaCl (0.5 and 1 M), in the middle of exponential growth, had a signifcant efect, increasing the total lipid content Still in Erlenmeyer fasks, N. oleoabundans was cultivated in marine microalgae Dunalliela cells. However, over this with the addition of diferent concentrations of sodium chlo- threshold (1 M) high salinity inhibited cell growth. ride, aiming to accumulate lipid by inducing osmotic stress. In Fig. 3, it is possible to observe that diferent concen- Cultivations in a bench scale tubular trations of NaCl have aninfuence on cell growth. Although photobioreactor 2.2 mM of NaCl had a negative infuence on cell growth, the lipid content obtained in this condition was higher than that The best conditions observed in the previous cultivations obtained in control cultures or other concentrations of the (2.5 mM sodium thiosulfate, 0.68 M glycerol, and 2.2 mM same salt. That is why this condition was selected for culti- sodium chloride) were separately applied in bench scale vation in the bench scale tubular photobioreactor. The high tubular photobiorreactor cultivations. Additionally, since cell growth observed with the addition of 4.5 mM of NaCl 1.2 mM of sodium thiosulfate concentration resulted in high may be justifed by the fact that higher osmotic pressure cell growth and relatively high lipid content for experiments carried out in Erlenmeyer fasks, this condition was also applied in the bench tubular photobioreactor. Sodium thiosulfate was added at the end of exponential growth (10th day), and it is possible to observe that this component negatively infuenced the fnal biomass con- centration (Table 3). In fact, 2.5 mM of sodium tiosulphate resulted in Xf of 757 mg L−1, a value much lower than that obtained in the control culture (1902 mg L−1). Sodium thio- sulfate toxicity is lower when 1.2 mM is added, allowing the fnal biomass concentration of 1338 mg L−1. Previous studies have already shown the negative efect of sodium thiosulfate on biomass concentration and bio- mass productivity when a concentration above 2.5 mM Fig. 3 Infuence of diferent concentrations of sodium chloride on maximum biomass concentration (Xm), lipid content, and protein was applied for Chlorella (Feng et al. 2005) and when content in Neochloris oleoabundans cultivated in Erlenmeyer fasks

Table 3 Results of cultivations Compound Xm (mg L−1) Xf (mg L−1) Lipid (%) Protein (%) in a bench scale tubular photobioreactor with the Control 2192.5 ± 65.5 1902.8 ± 18.1 11 37 addition of diferent lipid Na S O 1.2 mM 1894.6 ± 167.0 1338.6 ± 123.9 21 40 accumulation inducers 2 2 3 Na2S2O3 2.5 mM 1709.9 ± 93.6 757.0 ± 33.9 15 18 Glycerol 0.68 M 1976.8 ± 99.8 1511.2 ± 260.8 17 35 NaCl 2.2 mM 2226.0 ± 164.7 1203.8 ± 29.8 25 17

Xm: maximum biomass concentration Xf: fnal biomass concentration

1 3 Brazilian Journal of Chemical Engineering (2020) 37:41–48 45 concentrations above 3.8 mM were applied for Scenedesmus in dark condition or with LED (red or white), for saving (Mandal and Mallick 2009). energy in biomass production. Glycerol and sodium chloride also negatively infuenced In Table 4, it is possible to see the infuence of diferent the fnal biomass concentration, but in a lower intensity in light supply conditions on cell growth. As expected, 24 h comparison with 2.5 mM of sodium tiossulfate. In the pre- illumination with fuorescent lamps (control) provided bet- sent study, the addition of sodium chloride resulted in fnal ter growth. The use of 12 h illumination with LED (red or biomass concentration (Xf) lower than that obtained with white) allowed a cell growth better than in the condition glycerol. On the other hand, lipid accumulation was more of 12 h in the dark, but slightly lower than the control efcient when sodium chloride was added to the cultiva- culture. This result is in accordance with Kula and Rys tion. In fact, sand dune isolated N. oleoabundans showed (2014) who observed that classic fuorescent lamps were tolerances to growth in medium with higher salinity values a better source of light for the growth of the green algae (Baldisserotto et al. 2012). C. vulgaris. The addition of 1.2 mM of sodium thiosulfate allowed an It is also possible to observe in Table 4 that the evident increase of 100% in lipid content (21%) in comparison with negative efect of 12 h dark condition on maximum bio- the control run (11%), while with the addition of 2.5 mM, mass concentration (Xm) and fnal biomass concentration the increase was only about 50% (Table 6). Sodium thiosul- (Xf) is probably because of the shift from carbon dioxide fate acts as a reducing agent able to promote the enhance- fxation (photosynthesis) to organic carbon consumption ment of the NADH pool, which in turn inhibits citrate (respiration), in accordance with Carvalho et al. (2011). synthase enzyme. Therefore, instead of being incorporated The use of Red or White LED provided similar val- into the Citric Acid Cycle, Acetil-CoA may be deviated to ues of maximum biomass concentration (Xm). However, Malonil-CoA, which increases the fux to lipid biosynthesis after the 8th day of cultivation, biomass concentration (Feng et al. 2005; Mandal and Mallick 2009; Ngangkham decreased with the use of the red LED, whereas fnal bio- et al. 2012). mass concentration (Xf) was maintained with the use of Still in Table 3, it is possible to observe that the addi- the white LED, probably because the white LED includes tion of sodium chloride and glycerol allowed an increase light around 436 nm and 460, which is the favorable range of almost 130% (25% of lipid in dry biomass) and 50% of for absorption of light by chlorophylls a and b, respec- lipid (17% of lipid in dry biomass), respectively, in compari- tively (Matthijs et al. 1996). son with the control run (11% of lipid in dry biomass). The Regarding lipid content, the use of LED clearly allowed positive efect of inducer addition on microalgal biomass the increase in total lipid content, and the use of white LED oil accumulation is supported by several published studies provided the highest value (27%). White LED seems to bet- (Baldisserotto et al. 2012; Band et al. 1992; Santos et al. ter stimulate the microalgal photosystem by including the 2012). Moreover, as already stressed by Wang et al. (2011), blue spectrum (Kula and Rys 2014; Okumura et al. 2015). when the microorganism accumulates lipids, a reduction in In the condition of nitrogen deprivation (after the 8th day protein content takes place. of cultivation), it was probably possible to synthesize lipids using the energy from photophosphorylation and the carbon Cultivations in bench scale tubular photobioreactor assimilated from ­CO2. with the use of LED Cultivation of Arthrospira showed better growth condi- tions using red LED (Wang et al. 2007; Chen et al. 2010). In this last step, different forms of light supply were However, the present study showed that the white LED pro- evaluated. A 12 h:12 h regime was established: 12 h with vided the best results concerning not only cell growth, but fuorescent lamps (simulating natural sunlight) and 12 h also lipid content in N. oleoabundans.

Table 4 Results of cultivations Light supply Xm (mg L−1) Xf (mg L−1) Lipid (%) Protein (%) in bench scale tubular photobioreactor with the Control: 24 ­Lighta 2981.1 ± 114.7 2360.5 ± 197.3 11 36 diferent light supply conditions 12 h ­Lighta: 12 h dark 1961.0 ± 28.5 1665.2 ± 17.5 12 39 12 h ­Lighta: 12 h Red LED 2350.3 ± 160.2 1836.2 ± 58.4 19 33 12 h ­Lighta: 12 h White LED 2415.5 ± 111.6 2335.0 ± 15.9 27 34

a Light supplied by fuorescent lamps Xm: maximum biomass concentration Xf: fnal biomass concentration

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Fatty acids profle be signifcantly changed depending on the growth phase (exponential or stationary), nutrient availability, tempera- Lipid fractions of biomass produced in the tubular pho- ture, salinity, pH, and light intensity (Dunstan et al. 1993). tobioreactor were recovered and submitted to fatty acids Therefore, cultivation conditions may be controlled for esterifcation and the corresponding fatty acid methyl ester obtaining biomass with desired characteristics. components were analyzed by gas chromatography. The fatty acids profles of the lipids obtained in the According to Becker (1994), lipids in microalgae are present study are presented in Table 5 (cultivations with usually constituted by glycerol and sugars or bases esteri- the addition of lipid accumulation inductor) and Table 6 fed to fatty acids composed of carbon numbers in the range (cultivations under diferent light supply conditions). It is C12–C22. Microalgal fatty acids may be either saturated possible to observe that palmitic acid (C16:0) and oleic or unsaturated, and the triglycerides are the most abundant acid (C18:1n9) are always present in high percentages form of stored lipid (up to 80%). The fatty acids profle may (above 19% and 10%, respectively, in Table 6), which is in

Table 5 Neochloris Fatty acid (%)a Control Na S O 1.2 mM Na S O 2.5 mM NaCl 2.2 mM Glycerol 0.68 M oleoabundans fatty acids 2 2 3 2 2 3 content in cultivations with lipid C11:0 – 1.46 ± 0.05 1.17 ± 0.00 – – accumulation inductor C12:0 – 5.50 ± 2.10 – 3.43 ± 0.04 – U.C.b – 1.93 ± 0.15 – – – C16:0 21.39 ± 0,52 19.02 ± 0.31 21.01 ± 0.17 23.74 ± 0.06 25.47 ± 1.57 U.C.b 6.05 ± 0,11 6.71 ± 0.39 4.03 ± 0.46 3.76 ± 0.02 8.63 ± 1.99 C16:1 – 4.01 ± 0.11 – – – U.C.b 5.48 ± 0,11 5.55 ± 0.39 2.73 ± 0.46 4.96 ± 0.01 – C17:1 7.57 ± 0,40 3.83 ± 0.59 6.21 ± 1.13 4.87 ± 0.01 9.86 ± 0.63 C18:1n9 10.01 ± 0,49 19.22 ± 0.76 22.79 ± 0.02 16.88 ± 0.03 10.77 ± 0.04 C18:2n6 35.09 ± 0,10 24.08 ± 1.63 15.56 ± 1.71 31.96 ± 0.08 28.10 ± 1.99 C18:3n6 14.42 ± 0,17 8.7 ± 1.05 24.52 ± 0.80 10.43 ± 0.00 21.02 ± 0.82

a Percentage of fatty acids relative to the total content (weight/weight) b Unidentifed compound. Absent in the standard 37 FAME mix C11:0 undecanoic acid; C: 12: decanoic acid; C16:0 palmitic acid; C16:1 palmitoleic acid; C17:1 cis- 10-heptadecenoic acid; C18:1n9 oleic acid; C18:2n6 linoleic acid; C18:3n6 γ-linolenic acid –: Not detected

Table 6 Neochloris Fatty acid (%)a Control 24 h light (FL) 12 h:12 h LIGHT 12 h:12 h Light 12 h:12 h Light oleoabundans fatty acids (FL): dark (FL): red LED (FL): blue LED content in cultivations with diferent light supply conditions C11:0 – – – – C12:0 – – – – U.C.b – – – – C16:0 21.39 ± 0.52 18.05 ± 0.14 17.77 ± 0.81 14.46 ± 0.0 U.C.b 6.05 ± 0.11 5.56 ± 0.81 10.64 ± 0.18 9.23 ± 0.0 C16:1 – – – – U.C.b 5.48 ± 0.11 5.98 ± 0.13 7.41 ± 0.08 8.42 ± 0.0 C17:1 7.57 ± 0.40 9.65 ± 0.23 9.72 ± 0.21 8.39 ± 0.0 C18:1n9 10.01 ± 0.49 8.89 ± 0.79 3.17 ± 0.03 12.84 ± 0.0 C18:2n6 35.09 ± 0.10 34.73 ± 1.46 33.44 ± 1.22 24.99 ± 0.1 C18:3n6 14.42 ± 0.17 17.23 ± 0.39 16.31 ± 0.07 16.02 ± 0.1

a Percentage of fatty acids relative to the total content (weight/weight) b Unidentifed compound. Absent in the standard 37 FAME mix C11:0 undecanoic acid; C: 12: decanoic acid; C16:0 palmitic acid; C16:1 palmitoleic acid; C17:1 cis- 10-heptadecenoic acid; C18:1n9 oleic acid; C18:2n6 linoleic acid; C18:3n6 γ-linolenic acid; –: Not detected

1 3 Brazilian Journal of Chemical Engineering (2020) 37:41–48 47 accordance with Tornabene et al. (1983) and Santos et al. Compliance with ethical standards (2012), who analyzed the fatty acids profle in N. oleo- abundans cultivated in nitrogen deprivation and alkaline- Conflict of interest The authors declare that they have no confict of saline conditions, respectively. interest. When 1.2 mM of sodium thiosulfate was added, it was possible to detect short chain fatty acids, most likely because of the rapid mechanism of capturing the excess References of electrons from photophosphorylation, as a result of a reducing environment. Furthermore, the use of LED did Association of Ofcial Analytical Chemists (1984) Ofcial methods of not show a considerable infuence on the fatty acids profle analysis of the Association of Ofcial Analytical Chemists, 14th (Table 6). edn. AOAC, Arlington Considering the purpose of using this lipid fraction as Ávila-Leon IA (2014) Estudo da produção de biomassa e lipídios no biodiesel, one has to pay attention to the linolenic acid cultivo de Neochloris oleoabundans sob diferentes condições de estresse nutricional e físico. University of Sao Paulo, Sao Paulo concentration (C18:3), since European Standards (EN Baldisserotto C, Ferroni L, Giovanardi M, Boccaletti L, Pantaleoni L, 14214) limit the content of this component to 12%, for Pancaldi S (2012) Salinity promotes growth of freshwater Neo- achieving satisfactory oxidative stability (Knothe 2006). chloris oleoabundans UTEX 1185 (, ): In the present study, only the cultivations with 1.2 mM of morphophysiological aspects. Phycologia 51:700–710. https://doi.​ org/10.2216/11-099.1 sodium thiosulfate and 2.2 mM of NaCl meet the level of Band CJ, Arredondo-Vega BO, Vazquez-Duhalt R, Greppin H (1992) EN 14214. 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Bioresour Technol 102(4):3883–3887. oleoabundans could be employed for biodiesel production https​://doi.org/10.1016/j.biort​ech.2010.11.102 after hydrogenation reactions. Dunstan GA, Volkman JK, Barrett SM, Garland CD (1993) Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture. J Acknowledgements This work was supported by CAPES (Coorde- Appl Phycol 5(1):71–83. https​://doi.org/10.1007/BF021​82424​ nação de Aperfeiçoamento de Pessoal de Nível Superior), Finance Feng F, Yang W, Jiang G, Xu Y, Kuang T (2005) Enhancement of Code 001, and FAPESP (Fundação de Amparo à Pesquisa do Estado fatty acid production of Chlorella sp. () by addi- de São Paulo), Grant No. 10/51503-4. tion of glucose and sodium thiosulphate to culture medium.

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