Int. J. Aquat. Biol. (2023) 11(2): 159-169 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2023 Iranian Society of Ichthyology Original Article Effects of different physical shocks and sampling time on lipid content and fatty acids composition of Nannochloropsis oculata Farzaneh Noori, Saeid Vahdat Artemia and Aquaculture Research Institute, Urmia University, Urmia, Iran. s Article history: Received 15 December 2022 Accepted 11 March 2023 Available online 2 5 April 2023 Keywords: Sampling time Fatty acid Physical shocks Biochemical composition Abstract: The microalga Nannochloropsis oculata is marine widely used in aquaculture systems as an essential source of protein, lipid, and polyunsaturated fatty acids. Day/night pH fluctuations driven by photosynthesis and respiration create an environment that exhibits changing pH ranges. The aim of this study was to find whether physical shocks could change the complete profile of nutrients (lipid, fatty acid, carbohydrate, chlorophylls, and proteins) in N. oculata. The algae were cultivated in 32 flasks of ten-liter for biomass production for 12 days using Guillard medium (f/2). The cells were reared at 0.5 molar salinity (29 ppt), under 3500 lux light intensity with a 12L:12D photoperiod and 21ºC temperature. After 12 days, when the cell density reached its stationary phase, they were centrifuged. The pellets were then re-suspended in fresh seawater thoroughly and transferred into thirty-two containers with 10-liter volumes, including eight treatments with four replicates. The algae in treatments 3, 4, and 8 were subjected to salinity (88 ppt), starvation, and pH (11) shocks, respectively and treatments 2, 5, 6, and 7 were subjected to salinity + pH + starvation, pH + starvation, salinity + pH and salinity + starvation shocks, respectively. The biochemical composition of N. oculata demonstrated that T3 at the end of the dark period, and T1 at the end of the light period, possessed significantly higher (51.51%), and lower (24.9%) lipid content, respectively. According to the results, EPA under pH shock, linoleic acid under pH + salinity + starvation shock, and DHA and omega-3 under pH + salinity shock at the end of the dark period revealed significant differences with the control group. The saturated fatty acids showed significantly higher value in the control group during the dark period. The monounsaturated fatty acids increased significantly under pH shock at the end of the light period on day 18. Based on the results, the best treatment to obtain more lipid production in N. oculata was using six-day salinity shock and harvesting algae at the end of the dark period, and for more EPA synthesis in N. oculata, pH shock for six days and harvesting the algae at the end of dark period is recommended. Introduction Some species of microalgae synthesize very long- chain fatty acids (carbon chains 20+ in length), including eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) (Bigogno et al., 2002; Guiheneuf et al., 2013). These omega-3 (ω-3) fatty acids are essential components of high-quality fish diets (Muhlroth et al., 2013). Fatty acids are the building blocks of lipids, but they are not distributed equally amongst different lipid classes. Algal polar lipids are located in structural and functional cell membranes, while neutral lipid triacylglycerols (TAGs) function as storage Correspondence: Farzaneh Noori DOI: http//doi.org/10.22034/ijab.v11i2.1809 E-mail: f.noori@urmia.ac.ir DOR: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.10.2 molecules (Hu et al., 2008). The microalga Nannochloropsis sp. is a Eustigmatophyte widely used in marine aquaculture systems as an important source of vitamins, pigments, and PUFAs (Sukenik, 1991; Sukenik et al., 1993; Pal et al., 2011). This microalga has been widely studied due to its high PUFA content. As found in many other species of algae, nutrient depletion significantly increased FA accumulation in Nannochloropsis. Other studies have reported that Nannochloropsis salina reaches a total FA accumulation of 37.5% lipid/DW when arriving at the stationary phase and could grow well at 34 ppt 160 Noori and Vahdat/ Effects of different physical shocks and sampling time on lipid content and fatty acids salinity (Bartley et al., 2013). Similarly, Nannochloropsis gadiata grows at low irradiance demonstrating accumulation of up to 50% lipid/DW after a nutrient deprivation phase (Bartley et al., 2013). Doan and Obbard (2011, 2012) reported a total lipid production of 50 and 55% of DW in Nannochloropsis sp. using cell sorting and ethyl methanesulfonate, respectively. Nannochloropsis is a genus of robust, oleaginous microalgae that synthesizes EPA during balanced growth and is a promising candidate for commercial applications (Kilian et al., 2011; Radakovits et al., 2012; Li et al., 2014; Sharma et al., 2015). Research demonstrated that subjecting the culture to a temporary change of environmental factors such as pH can decrease unwanted organisms and improve final culture densities. Day/night pH fluctuations driven by photosynthesis and respiration create an environment that exhibits changing pH ranges (Richmond and Becker, 1986). Further, pH may affect the lipid composition (Griffiths et al., 2009, Rodolfi et al., 2009, Moheimani, 2013). Nitrogen and phosphorus starvation shifts the lipid metabolism from membrane lipid synthesis to neutral lipid storage. This, in turn, increases the total lipid content of green algae (Hu, 2004). Higher salinity increases the algae's lipid content (Fabregas et al., 1984; Zhila et al., 2011). Besides the pigment contents, other algal cell components such as carbohydrates and proteins can also be influenced by the light regime (Renaud et al., 1991). The aim of this study was to find out how different physical shocks, alone or in combination, can influence the synthesis of different nutrients such as lipids, fatty acids, carbohydrates, chlorophylls, and proteins by Nannochloropsis oculata. Materials and Methods Nannochloropsis oculata was obtained from Artemia and Aquaculture Research Institute (AARI), Urmia University, Iran. The algae were cultivated in ten-liter conical flasks for biomass production for 12 days using Guillard medium (f/2)1 (Guillard, 1973). The cells were reared at 0.5 molar salinity (29 ppt), 3500 lux light intensity with a 12L: 12D photoperiod and 21ºC temperature. After 12 days when the cell density reached its stationary phase, they were centrifuged (3500 rpm, 5 min). The pellets were collected and then re-suspended thoroughly, equally divided (0.2713 g.L-1 DW), and transferred into thirty-two 10-liter containers, including eight treatments and four replicates. Then the Nannochloropsis algae were exposed to different shocks for six days. Treatments 3, 4, and 8 were subjected to salinity of 88 ppt, nutrient starvation, and pH (11) shocks, respectively and treatments 2, 5, 6, and 7 were subjected to salinity + pH + starvation, pH + starvation, salinity + pH, and salinity + starvation shocks, respectively. However, the control group (T1) was cultured under normal conditions. Each container was aerated continuously at a rate of 758 mL.min-1. The samples were collected every three days and at the end of light and dark periods. Chlorophyll a, b and total carotene analysis: Fifty mL algae were frozen under -20°C and then defrost (for breaking down of cell wall), filtered with S&X filter (1 µm), and weighed. The pigments were extracted with 96% methanol (50 ml.g-1 algae). The samples were homogenized for one minute at 1000 rpm. The homogeneous mixture was filtered and centrifuged for 10 min at 2500 rpm. Supernatants were separated and the absorbance at 666, 653 and 470 nm was recorded using the microplate reader synergy HT. The following formula was used to calculate chlorophyll and total carotene: Ca= 15.65A666 – 7.340A653 Cb= 27.05A653 – 11.21A666 Cc= 1000A470 – 2.860Ca – 129.2Cb /245 Where Ca = chlorophyll a, Cb= chlorophyll b, and Cc= total carotene in terms of micrograms per gram of wet weight (Dereet al., 1998). Lipid and ash contents analysis: Laboratory methods were used to measure lipid content by ether extraction (AOAC, 2005). For this purpose, a certain amount of samples was weighted and held for 7 hours on soxhlet within diethyl ether (98%). To calculate the ash content samples were weighed and placed in an electric furnace for 6 hours at 550°C 161 Int. J. Aquat. Biol. (2023) 11(2): 159-169 (AOAC, 2005). Fatty acid profiles: The fatty acid composition of Nannochloropsis algae was determined based on Miquel and Browse (1992). In brief, 200 mg of algae was heated to 80°C in 1 ml of a mixed solution of H2SO4 2.5% and methanol 98% (1:40, v/v) for one hour in a Teflon lined screw cap glass tube. The mixture was cooled at room temperature and then 500 µl of hexane and 1.5 mL of NaCl 0.9% (w/v) were mixed and added to the samples. The samples were centrifuged for 10 min at 4000 rpm and supernatant (1 µl) was injected to Gas chromatograph (GC) to determine the fatty acid profiles (Miquel and Browse,1992). Protein content: The protein content was measured according to Slocombe et al. (2013). Five mg of dried algae was mixed with 0.2 ml trichloroacetic acid (TCA) 24% (w/v). The mixture was incubated for 15 min at 95°C and cooled at room temperature. The homogenate was centrifuged at 15000 g for 20 min at 4°C and the supernatant was discarded. The pellets were re-suspended in 0.5 mL Lowry reagent and were incubated for 3 hours at 55°C. The samples were cooled at room temperature and centrifuged at 15000 g for 20 min. The supernatant was placed for 30 min in Lowry reagent and absorption was read at 600 nm (Slocombe et al., 2013). The standard curve was prepared using bovine serum albumin (BSA) as standard. Total Carbohydrate content: The carbohydrate content of the samples was measured by adding 5 ml of 2.5 N HCl to 100 mg of algae. The samples were hydrolyzed to simple sugars by keeping them in a boiling water bath for three hours and then cooled at room temperature. The sample was neutralized by solid Na2CO3 until the effervescence ceases and then the volume was made to 100 mL with distilled water and centrifuged at 5000 rpm for 5 minutes. To 0.5 ml of supernatant, 0.5 mL of distilled water and 4 mL of 0.2% anthrone reagent were added and the mixture was heated for 8 min in boiling water bath. The sample cooled rapidly and the absorbance was read at 630 nm. The glucose standard solution was prepared at a concentration of 100 µ.mL-1 (Hedge and Hofreiter, 1962). Statistical analysis: The normality of all data was checked by the Kolmogorov-Smirnov test. Mean (±SD) and factorial multivariate were used. One- Way ANOVA, ANCOVA (Bonferroni test) and Post-hoc Tukey analysis were used at 5% (P<0.05). Calculated data and drawing charts were done by SPSS (version 22) and Excel (2013 version), respectively. Results Biochemical compositions and some fatty acid profiles of Nannochloropsis cultivated in f/2 medium on day 12 under laboratory conditions are shown in Table 1. The results (Fig. 1) showed that the protein content was affected by salinity and pH shock (T6) at the end of the dark period showing the lowest value, while the highest protein content was found in T4 at the end of the dark period on day 18 (P<0.05). Lipid content was significantly influenced by the dark period and significantly higher values were observed in T7 and T3 (P<0.05) and the lowest value was achieved in T1 (4.68%) at the end of the dark period on day 18 (P<0.05). The result of ash reveals a significantly higher value at the end of the light period in starvation + salinity shock (T7) on day 15 (P<0.05). Carbohydrate content was influenced significantly by starvation + salinity (T7) and salinity (T3) shocks at the end of the dark period and exhibited the highest and lowest values, respectively (P<0.05). The protein content was significantly decreased at the end of the light and dark period in T6 on day 18 and increased in T4 at the end of the dark period on day 18 (P<0.05). Moreover, at the end of the dark period on day 18, T3 showed a significantly higher lipid content (51.51%) (P<0.05). Carbohydrate content in T7 at the end of the dark period on day 18 showed the highest values (P<0.05). The chlorophyll a and b on day 18 and at the end of the dark photoperiod under pH + starvation + salinity shock revealed highly significant (P<0.05). The pH + starvation + salinity shock at the end of the dark period on day 18 increased total carotene 162 Noori and Vahdat/ Effects of different physical shocks and sampling time on lipid content and fatty acids level significantly (P<0.05) (Fig. 2). The results on day 18 showed pH + starvation + salinity shock at the end of dark period made chlorophyll a and b and total carotene to increased significantly (P<0.05) (Fig. 2). Based on the results, EPA in pH, DHA, and omega-3 in pH + salinity, and MUFA under starvation + salinity treatments at the end of the dark period on day 15 demonstrated significant Day 12 Protein (%) Lipid (%) Carbohydrate (%) Ash (%) Chlorophyll a (µg.g-1 fw) Chlorophyll b (µg.g-1 fw) Total carotenoid (µg.g-1 fw) End of Light 33.24±0.25 18.62±0.15 35.65±0.24 12.01±0.20 35.62±0.14 8.15±0.31 126.24±1.01 End of Dark 30.12±0.08 19.31±1.11 37.99±1.01 12.08±0.35 34.21±0.14 7.16±0.44 124.21±0.14 Day 12 ARA DHA EPA Linoleic acid Linolenic acid SFA MUFA PUFA Sum of ω3 Sum of ω6 End of Light ND ND 10.35±0.62 8.55±0.65 3.68±0.54 41.35±0.35 37.15±0.31 21.35±1.21 14.03±1.24 8.55±0.65 End of Dark ND ND 10.95±0.35 8.84±0.24 3.71±0.84 41.95±1.21 37.45±1.32 21.42±1.67 14.66±0.71 8.84±0.24 Table 1. Bio-chemical and fatty acid compositions of Nannochloropsis oculata cultivated in f/2 medium on day 12 under standard conditions. a a ab d bc a a a b a a a C c b a CD AB AB F B A CD CDD AB E C B A D C 0 5 10 15 20 25 30 35 P ro te in ( % ) L15 D15 D18 b f g c c c g ee bc bc a b d a cC D B E E E B D A F H C F E G C 0 10 20 30 40 50 60 Li p id ( % ) D15 L15 L18 D18 d c bc b a a b c d b a f e d c f CD D CD D C E E EF C C A F C B G C 0 5 10 15 20 25 30 35 40 45 50 T1 T2 T3 T4 T5 T6 T7 T8 Before Shock C a rb o h y d ra te ( % ) L15 D15 L18 D18 c d a bc b b ab a c bc d a bc c e a B C BC A C D AB C A A A D BC B A C 0 2 4 6 8 10 12 14 16 18 20 A sh ( % ) D15 L15 L18 D18 Figure 1. Mean (±SE) lipid, carbohydrate, ash and protein contents of Nannochloropsis oculata cultivated in f/2 medium on day 15 and 18 under different shocks (P<0.05). a, b, c and … indicate the significance differences on day 15 (dark and light) and A, B, C and … indicate the significance differences on day 18 (dark and light). 163 Int. J. Aquat. Biol. (2023) 11(2): 159-169 differences compared to other treatments (P<0.05). Furthermore, Linoleic acid (or omega-6) in T2 (pH + salinity + starvation) at the end of the dark period showed a significantly higher value compared to other treatments (P<0.05). At the end of the dark period, in T1, SFAs were increased significantly (P<0.05). Moreover, the PUFAs values under T8 and at the end of the dark period on day 15 increased significantly (P<0.05). Linolenic acid in pH + salinity at the end of the light period on day 15 showed a significant difference between all groups (P<0.05) (Table 2). According to the result obtained omega-6 and Linoleic acid in T4 (starvation), EPA in T4, and SFAs in T1 at the end of the dark period revealed significantly higher values than other treatments (P<0.05). The DHA in T2, PUFAs in T7, and linolenic acid in T3 at the end of the light period on day 18 showed significantly higher amounts (P<0.05). The MUFAs in T8 at the end of the light period exhibited significantly higher than other treatments (P<0.05). Moreover, the total omega-3 at the end of the dark period on day 18, in T8 was significantly higher than other treatments (P<0.05) (Table 3). Discussion Microalgae can accumulate considerable quantities of carbohydrates, proteins, and/or lipids (Ho et al., 2012, 2013). Changes in microalgae biochemical composition are likely to occur as a result of variations in pH (Khalil et al., 2010), temperature (Roleda et al., 2013), light, salinity (Ruangsomboon et al., 2013), and metal contents (Sun et al., 2014). ab ab a bc bc c ab d ab b a b c b ab c A CD AB A E CD AB D B C AB A F D A E 0 1 2 3 4 5 6 7 8 C h lo ro p h y ll b L15 D15 L18 D18 a ab a c e e ab g ab d a d f d ab f B C B A F D A E A C A A E C A D 0 5 10 15 20 25 C h lo ro p h y ll a L15 D15 D18 L18 b bc a e f g c i d f a f h f b h A B A A D B A B A B A A E B A C 0 500 1000 1500 2000 T1 T2 T3 T4 T5 T6 T7 T8 Before Shock T o ta l C a ro te n e L15 D15 L18 D18 Figure 2. Mean (±SE) Chlorophyll a, b and total carotenoids contents of Nannochloropsis oculata cultivated in f/2 medium on day 15 and 18 under different shocks (P<0.05). a, b, c and … indicate the significance differences on day 15 (dark and light) and A,B,C and … indicate the significance differences on day 18 (dark and light). 1 6 4 N o o ri a n d V ah d at / E ff ec ts o f d if fe re n t p h y si ca l sh o ck s an d s am p li n g t im e o n l ip id c o n te n t an d f at ty a ci d s S a m p li n g T im e C o n d it io n s (S h o c k ) A R A D H A E P A L in o le ic a c id L in o le n ic a c id S F A M U F A P U F A S u m o f ω 3 S u m o f ω 6 E n d o f d a rk (D a y 1 5 ) T 1 ( C o n tr o l) N D 0 .2 2 ± 0 .0 3 a 7 .0 8 ± 0 .3 5 a N D 2 5 .8 5 ± 1 .7 0 g h 3 4 .6 9 ± 0 .6 5 f 1 3 .4 9 ± 3 .6 9 a 3 3 .1 5 ± 1 .7 5 a 3 3 .1 5 ± 1 .7 5 a N D T 2 ( p H + S a li n it y + S ta rv a ti o n ) N D N D 3 3 .4 7 ± 1 .4 8 h 6 .5 5 ± 1 .4 1 4 .1 3 ± 0 .2 6 a 2 2 .6 6 ± 0 .1 3 c d e 1 6 .7 9 ± 1 .8 8 a b 4 4 .1 4 ± 3 .1 6 b c 3 7 .6 0 ± 1 .7 4 a b 6 .5 5 ± 1 .4 1 T 3 ( S a li n it y ) N D N D 3 1 .4 4 ± 0 .6 8 g h N D 7 .1 3 ± 0 .8 2 b 1 9 .8 2 ± 0 .3 2 b c d 3 4 .9 2 ± 3 .4 3 fg 3 8 .5 7 ± 1 .5 0 a b 3 8 .5 7 ± 1 .5 0 a b N D T 4 ( S ta rv a ti o n ) N D N D 1 2 .2 6 ± 1 .7 8 a b c N D 2 4 .8 9 ± 1 .4 4 fg h 2 3 .7 8 ± 1 .3 6 c d e f 2 5 .6 8 ± 2 .7 5 c d e 3 7 .1 5 ± 0 .3 3 a b 3 7 .1 5 ± 0 .3 3 a b N D T 5 ( p H + S ta rv a ti o n ) N D N D 2 4 .2 9 ± 1 .0 8 e fg N D 1 3 .2 5 ± 0 .5 3 c 2 4 .0 1 ± 0 .3 9 c d e f 3 1 .8 0 ± 0 .3 2 e fg 3 7 .5 4 ± 1 .6 2 a b 3 7 .5 4 ± 1 .6 2 a b N D T 6 ( S ta rv a ti o n + S a li n it y ) N D N D 2 0 .8 3 ± 1 .0 2 d e f N D 1 6 .4 9 ± 1 .3 2 c d 1 7 .7 2 ± 0 .5 5 a b c 3 8 .8 2 ± 1 .0 2 g 3 7 .3 2 ± 0 .3 0 a b 3 7 .3 2 ± 0 .3 0 a b N D T 7 ( P H + S a li n it y ) N D 4 .7 5 ± 0 .2 3 b 9 .7 4 ± 3 .5 6 a b N D 1 9 .9 2 ± 1 .7 6 d e f 2 5 .5 0 ± 1 .1 2 d e f 3 0 .8 1 ± 0 .0 9 d e f 3 4 .4 2 ± 1 .5 6 a b 3 4 .4 2 ± 1 .5 6 a b N D T 8 (p H ) N D N D 4 7 .2 0 ± 0 .5 0 i N D 1 5 .8 9 ± 0 .8 4 c d 1 4 .8 9 ± 1 .6 3 a b 2 0 .9 4 ± 0 .9 4 a b c 6 3 .0 9 ± 0 .3 3 e 6 3 .0 9 ± 0 .3 3 e N D E n d o f li g h t (D a y 1 5 ) T 1 ( C o n tr o l) N D N D 1 6 .2 9 ± 0 .5 7 b c d N D 1 8 .4 9 ± 0 .5 0 c d e 2 7 .5 2 ± 3 .0 8 g 2 2 .6 4 ± 0 .2 2 b c d 3 4 .7 8 ± 0 .0 7 a b 3 4 .7 8 ± 0 .0 7 a b N D T 2 ( p H + S a li n it y + S ta rv a ti o n ) N D N D 4 3 .4 7 ± 4 .2 1 i N D 5 .3 7 ± 1 .4 8 a b 2 1 .1 9 ± 0 .3 0 c d e 2 2 .7 9 ± 2 .7 3 b c d 4 8 .8 3 ± 2 .7 3 c d 4 8 .8 3 ± 2 .7 3 c d N D T 3 ( S a li n it y ) N D N D 5 5 .3 4 ± 3 .0 9 k N D N D 2 0 .5 6 ± 3 .6 8 b c d 1 7 .1 5 ± 2 .3 9 a b 5 5 .3 4 ± 3 .0 9 d e 5 5 .3 4 ± 3 .0 9 d e N D T 4 ( S ta rv a ti o n ) N D N D 1 5 .4 8 ± 1 .5 1 b c d N D 2 0 .8 2 ± 2 .0 3 d e fg 3 0 .4 4 ± 2 .9 7 e f 3 2 .1 8 ± 3 .1 4 e fg 3 6 .2 9 ± 3 .5 4 a b 3 6 .2 9 ± 3 .5 4 a b N D T 5 ( p H + S ta rv a ti o n ) N D N D 2 7 .4 4 ± 2 .1 2 fg h N D 1 6 .0 9 ± 3 .1 8 c d 2 3 .0 1 ± 0 .7 7 c d e 2 6 .1 1 ± 1 .2 2 c d e 4 3 .5 2 ± 5 .3 0 b c 4 3 .5 2 ± 5 .3 0 b c N D T 6 ( S ta rv a ti o n + S a li n it y ) N D N D 1 5 .0 0 ± 0 .2 4 b c d N D 2 3 .3 6 ± 0 .1 4 e fg 2 2 .8 5 ± 0 .8 5 c d e 2 5 .5 7 ± 0 .4 4 c d e 3 8 .3 7 ± 0 .3 9 a b 3 8 .3 7 ± 0 .3 9 a b N D T 7 ( p H + S a li n it y ) N D N D 2 1 .6 9 ± 1 .7 8 d e f N D 2 8 .2 8 ± 2 .3 2 h 1 9 .2 7 ± 1 .5 8 b c d 2 9 .0 0 ± 2 .3 8 c d e f 4 9 .9 7 ± 4 .1 1 c d 4 9 .9 7 ± 4 .1 1 c d N D T 8 (p H ) N D N D 1 7 .4 8 ± 0 .1 0 c d e N D 1 9 .1 7 ± 0 .3 9 d e f 1 3 .0 1 ± 2 .2 4 a 3 0 .0 2 ± 0 .5 4 d e f 3 6 .6 5 ± 0 .2 8 a b 3 6 .6 5 ± 0 .2 8 a b N D T ab le 2 . M ea n ( ± S E ) so m e fa tt y a ci d c o m p o si ti o n s o f N a n n o ch lo ro p si s o cu la ta c u lt iv at ed i n f /2 m ed iu m o n d ay 1 5 u n d er d if fe re n t sh o ck s. T h e d at a sh o w n r ep re se n t th e m ea n v al u es o f th re e re p et it io n s. S a m p li n g T im e C o n d it io n s (S h o c k ) A R A D H A E P A L in o le ic a c id L in o le n ic a c id S F A M U F A P U F A S u m o f ω 3 S u m o f ω 6 E n d o f d a rk (D a y 1 8 ) T 1 ( C o n tr o l) N D 5 .7 4 ± 0 .7 2 b 1 7 .1 5 ± 2 .3 2 a b N D 6 .1 9 ± 1 .1 2 c d 3 7 .4 4 ± 4 .0 2 d e 3 0 .9 7 ± 0 .4 7 b 2 2 .5 8 ± 1 .6 6 b c d 3 0 .9 7 ± 0 .4 7 b N D T 2 ( p H + S a li n it y + S ta rv a ti o n ) N D N D 1 7 .8 8 ± 5 .0 5 a b N D 1 5 .0 9 ± 3 .5 4 g h 1 0 .8 2 ± 2 .4 6 a 3 2 .9 7 ± 1 .5 0 b 2 7 .7 6 ± 3 .1 9 c d e 3 2 .9 7 ± 1 .5 0 b c N D T 3 ( S a li n it y ) N D N D 3 0 .8 1 ± 0 .3 8 d e N D 4 .1 2 ± 0 .6 5 b c 4 3 .4 9 ± 2 .3 3 e 3 4 .9 4 ± 0 .2 7 b c 1 5 .1 4 ± 0 .8 5 a 3 4 .9 4 ± 0 .2 7 b c d N D T 4 ( S ta rv a ti o n ) N D N D 4 4 .3 6 ± 1 .1 8 f 4 .7 3 ± 0 .9 2 c N D 1 2 .4 4 ± 0 .3 5 a 4 9 .0 8 ± 2 .1 0 d e 2 7 .8 6 ± 0 .7 2 c d e 4 4 .3 6 ± 1 .1 8 e f 4 .7 3 ± 0 .9 3 c T 5 ( p H + S ta rv a ti o n ) N D N D 3 9 .3 7 ± 0 .8 9 e f N D 9 .2 2 ± 0 .2 5 d e 1 2 .4 2 ± 1 .0 8 a 4 8 .5 9 ± 0 .6 3 d 2 7 .4 3 ± 0 .4 9 c d e 4 8 .5 9 ± 0 .6 3 f N D T 6 ( S ta rv a ti o n + S a li n it y ) N D N D 2 8 .3 1 ± 1 .4 2 c d N D 7 .6 3 ± 0 .6 8 c d 2 3 .4 8 ± 1 .7 5 b c 3 9 .0 4 ± 2 .6 5 b c 2 1 .0 5 ± 3 .5 3 b c d 3 9 .0 4 ± 2 .6 5 c d e N D T 7 ( p H + S a li n it y ) N D N D 1 8 .9 6 ± 0 .4 3 a b c N D 1 2 .4 6 ± 0 .8 0 fg 2 0 .5 8 ± 1 .2 9 b 3 1 .4 2 ± 0 .3 6 b 4 4 .4 0 ± 2 .0 2 f 3 1 .4 2 ± 0 .3 6 b c N D T 8 (p H ) N D N D 6 3 .4 6 ± 1 .2 4 h N D 1 .6 4 ± 0 .7 3 a b 1 1 .5 7 ± 0 .0 9 a 6 5 .0 9 ± 0 .5 1 f 1 7 .9 5 ± 0 .0 8 a b 6 5 .0 9 ± 0 .5 1 h N D E n d o f li g h t (D a y 1 8 ) T 1 ( C o n tr o l) N D 3 .8 2 ± 0 .9 8 a 1 5 .7 0 ± 1 .6 1 a N D 2 .8 2 ± 0 .0 8 a b 5 2 .0 7 ± 0 .1 4 f 2 2 .3 4 ± 2 .5 1 a 1 4 .8 8 ± 1 .2 1 a 2 2 .3 4 ± 2 .5 1 a N D T 2 ( p H + S a li n it y + S ta rv a ti o n ) N D 7 .6 3 ± 0 .5 8 c 3 4 .0 9 ± 2 .7 7 d e N D 2 .5 2 ± 0 .4 9 a b 3 6 .1 9 ± 1 .0 9 d 4 2 .3 5 ± 2 .6 8 c d 1 5 .0 3 ± 0 .2 8 a 4 2 .3 5 ± 2 .6 8 d e f N D T 3 ( S a li n it y ) N D N D 1 7 .6 6 ± 1 .4 0 g h N D 1 7 .6 4 ± 1 .4 9 h 2 6 .6 0 ± 0 .2 1 b c 3 5 .3 1 ± 0 .0 9 b c 2 7 .8 0 ± 0 .0 7 c d e 3 5 .3 1 ± 0 .0 9 b c d N D T 4 ( S ta rv a ti o n ) N D N D 5 4 .3 2 ± 5 .4 8 g N D 2 .7 9 ± 0 .0 1 a b 2 0 .7 8 ± 1 .9 6 b 5 7 .1 0 ± 5 .4 8 e f 2 1 .2 5 ± 3 .5 8 b c d 5 7 .1 0 ± 5 .4 8 g N D T 5 ( p H + S ta rv a ti o n ) N D N D 1 9 .0 5 ± 2 .6 2 a b c N D 1 3 .5 2 ± 1 .3 2 fg 2 7 .1 0 ± 0 .5 0 b c 3 2 .5 6 ± 1 .2 9 b 2 8 .4 7 ± 3 .0 4 d e 3 2 .5 6 ± 1 .2 9 b c N D T 6 ( S ta rv a ti o n + S a li n it y ) N D N D 4 8 .2 3 ± 0 .0 4 fg 2 .6 6 ± 0 .1 6 b 8 .7 7 ± 0 .3 7 d e 8 .9 2 ± 1 .2 0 a 5 9 .7 2 ± 0 .4 8 f 2 9 .0 0 ± 0 .9 5 d e 5 7 .0 5 ± 0 .3 2 g 2 .6 6 ± 0 .1 6 b T 7 ( p H + S a li n it y ) N D N D 2 5 .2 2 ± 0 .6 0 b c d N D N D 2 8 .1 1 ± 0 .5 9 c 3 2 .1 7 ± 0 .4 6 b 3 5 .7 4 ± 6 .5 8 e f 3 2 .1 7 ± 0 .4 6 b c N D T 8 (p H ) N D N D 3 0 .7 4 ± 1 .7 0 d e 1 .0 9 ± 0 .0 4 a 1 .6 0 ± 0 .5 4 a 4 0 .1 3 ± 1 .5 2 d e 3 3 .4 4 ± 2 .2 9 b 1 8 .7 8 ± 1 .8 6 b c 3 2 .3 4 ± 2 .2 5 b c 1 .0 9 ± 0 .0 4 a T ab le 3 . M ea n ( ± S E ) so m e fa tt y a ci d c o m p o si ti o n s o f N a n n o ch lo ro p si s o cu la ta c u lt iv at ed i n f /2 m ed iu m o n d ay 1 8 u n d er d if fe re n t sh o ck s. T h e d at a sh o w n r ep re se n t th e m ea n v al u es o f th re e re p et it io n s. 165 Int. J. Aquat. Biol. (2023) 11(2): 159-169 Regarding the effects of starvation, studies demonstrated that N and/or P limitation in growth media causes metabolism alterations that induce lipid accumulation (Liang et al., 2013; Li et al., 2014). Despite several studies made in an attempt to understand the potential effects of N and/or P limitations on microalgae biochemical composition changes, the phenomena seem to be species- dependent (Shifrin and Chisholm, 1981). Nitrogen and phosphorus starvation shifts the lipid metabolism from membrane lipid synthesis to neutral lipid storage. This, in turn, increases the total lipid content of green algae (Hu, 2004). Major effects of nitrogen deficiency in algal culture include the enhanced biosynthesis and accumulation of lipids (Thompson, 1996, Shifrin and Chisholm, 1981; Converti et al., 2009; Demirbas, 2010) and triglycerides (Takagi et al., 1999; Stephenson et al., 2010) with a concomitant reduction in protein content (Fogg, 1956; Morris et al., 1974; Kilham et al., 1997; Heraud et al., 2005), resulting in a higher lipid/protein ratio (Converti et al., 2009). In this study, a high alkaline pH + starvation + high salinity increased carbohydrate and decreased lipid and protein contents of algae, however high salinity in combination with any other shock caused higher lipid production, especially on day 18. This could be explained that under salinity conditions the cell would release nitrogen from the photosynthetic pigments and utilize the same for the metabolic processes. Higher salinity increases the algae lipid content (Fabregas et al., 1984; Zhila et al., 2011). Dunaliella, a marine alga, exhibited an increase in saturated and monounsaturated fatty acids with an increase in NaCl concentration from 0.4 to 4 M (Xu and Beardall, 1997). In another study with Dunaliella tertiolecta, an increase in intracellular lipids (60 to 67%) and triglyceride concentration (40 to 56%) with an increase in NaCl concentration from 0.5 (freshwater concentration) to 1.0 M was observed (Takagi and Karseno, 2006). In this work, when the N. oculate biomass was cultured under normal conditions and transferred to high salinity, they showed different levels of lipid accumulation in the cell. Among all conditions and sampling time, the most favorable treatment to achieve high lipid content per cell was at the end of the dark period on day 18. Nannochloropsis oculata, showed a profound increase in lipid content under salinity shock at the end of the dark period on day 18 (51.51%). The lipid accumulation is enhanced by a combination of salinity shock. This inference is achieved on the bases of the results obtained from treatment 3 (end of light, day 15, and end of dark, day 18), where a significant increase in the cellular lipid content of N. oculate was observed under salinity shock individually. In a study conducted by Ben-Amotz, the lipid content of Botryococcus braunii cultured in 0.50 M NaCl was higher compared to media without the addition of NaCl, but protein, carbohydrates, and pigments levels were lower (Ben-Amotz et al., 1985). Another study with the same alga reported a decrease in protein content with unchanged carbohydrate and lipid content with an increase in salinity (Vazquez-Duhalt et al., 1991). Research on Tetraselmis suecica also reported a reduction in protein content per cell of up to 20% with an increase in salinity (Fabregas et al., 1984), but protein in all shocks decreased to less than 25%. Variations in salinity also influence several biochemical and physiological mechanisms such as lipid production and growth which are essential in marine organisms (Fava and Martnini, 1988). In the current study, the treatments of 2 (salinity + pH + starvation), 7 (salinity + starvation), and 8 (pH) on day 15 at the end of the light period showed the highest lipid level, while the other treatments revealed different action, that it would be due to different physiological mechanisms in a specific algal. Lipids in general (as a percentage of particulate organic matter or as absolute amounts) and particularly fatty acids have been targeted as important variables determining the food quality of algae (Ahlgren et al., 1990; Coutteau and Sorgeloos, 1997; Weers and Gulati, 1997a). The characteristic types of fatty acids produced by diatoms, green algae and/or cryptomonads have been studied (Piorreck et al., 1984; Cranwell et al., 166 Noori and Vahdat/ Effects of different physical shocks and sampling time on lipid content and fatty acids 1989; Ahlgren et al., 1990). The general pattern observed is that green algae rarely produce fatty acids in excess of eighteen carbons, while diatoms and cryptophytes make many long-chained (18C) polyunsaturated fatty acids (PUFAs). Ahlgren et al. (1990) showed that the long-chained PUFAs produced by cryptophytes enhance reproduction in zooplankton, including Daphnia. Many studies of algal biochemistry have been undertaken in the past on cells that were either growing at the maximal rate (µmax) or at the stationary phase. Growth rates of algae measured in lakes are frequently found to be at some intermediate growth rate (Lehman and Sandgren, 1985; Sommer, 1989), rather than at stationary phase or µmax. Algae grown in continuous or semi-continuous culture have reduced variation in physiological conditions, can be easily replicated, and are more representative of the in-situ condition. Immediate effects of phosphorus limitation include a reduction in the synthesis and regeneration of substrates in the Calvin-Benson cycle and a consequential reduction in the rate of light utilization required for carbon fixation (Reitan Barsanti and Gualtieri, 2005). Phosphate limitation also reduces the synthesis of n-3 PUFA (Reitan Barsanti and Gualtieri, 2005). Salinity and pH (T6) shocks together on day 15 and combination of pH and salinity and starvation (T2) shocks on day 18 induced DHA production in N. oculata at the end of dark period, However, pH shock alone did influence the cell EPA level to more than 47% of FAME on day 15 and 63% of FAME on day 18 at the end of the dark period. 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