Marine Research in Indonesia

oculata and the antioxidant activity of N. oculata. This study consists of several procedures including; cultivation, growth observation (cell density and biomass), extraction, and antioxidant activity of N. oculata extracts. The highest growth (cell density and biomass) of N. oculata was found in media F/2 + 0.5 ml, while the lowest was in media F/2 - 0.5 ml. The strongest antioxidant activity of N. oculata was found in media F/2 + 1 ml, and the lowest was in the media F/2 - 0.5 ml. Media modification gave different results on cell density, biomass, and antioxidant activity of microalgae N. oculata.


Introduction
Microalgae are single-celled microscopic plants that live in both marine and freshwater, having 3 -30 µm in diameter, and classified as algae classes (Abdurrachman et al., 2013). Nannochloropsis oculata is common microalga that is found in marine water. Classified as Eustigmatophyceae classes, N. oculata has been utilized as food source in aquaculture. N. oculata has high protein, fatty acid, and antioxidants pigment.
The uses of microalgae for our life have been significantly growing, such as the source of functional foods, cosmetics, biogas/biodiesel, antioxidant, and antibacterial. Lately, antioxidant activities of various microalgae have been frequently analyzed (Anam et al., 2014;Bariyyah et al., 2013;Hafsa et al., 2017).
Antioxidants are compounds that prevent or slow the damage caused by free radicals, which happened by the oxidation reaction. Antioxidants could be natural compounds, or made by human (synthetic). Common antioxidants that are sold in public are made from synthetic source, which could be dangerous for human`s life (Bariyyah et al., 2013). Therefore, the natural source of antioxidants becomes important.
Cultivation of microalgae requires a constant supply of several inorganic nutrients, such as nitrogen (N), phosphorous (P), and potassium (K) to optimize microalgae yield. One of the most common media used in microalgae cultivation is F/2 media. These media consists of NaNO3, NaH2PO4, Na2SiO3, vitamin, and trace metal (Reyimu and Özçimen, 2017). However, the optimum F/2 media composition for the growth of N. oculata is rarely informed. Therefore, the aims of this study are to determine the effect of F/2 medium modification on the growth of N. oculata and determine the effect of F/2 medium modification to the antioxidant activity of N. oculata.
Article history: Submitted 16 August 2019 1 st Revision 21 January 2020 2 nd Revision 21 July 2020 Accepted 27 July 2020 Published online 31 July 2020 Nannochloropsis oculata is a common microalga that can be found in marine water. One of the most commonly used media for microalga culture is F/2 media; however, the optimum F/2 media composition for N. oculata growth is rarely informed. Therefore, the aims of this study are to determine the effect of F/2 medium modification on the growth of N. oculata and the antioxidant activity of N. oculata. This study consists of several procedures including; cultivation, growth observation (cell density and biomass), extraction, and antioxidant activity of N. oculata extracts. The highest growth (cell density and biomass) of N. oculata was found in media F/2 + 0.5 ml, while the lowest was in media F/2 -0.5 ml. The strongest antioxidant activity of N. oculata was found in media F/2 + 1 ml, and the lowest was in the media F/2 -0.5 ml. Media modification gave different results on cell density, Marine microalgae (N. oculata) used in this study were the collection of RCO LIPI, Jakarta. EtOH was purchased from Merck while 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid were purchased from Sigma Chemical Co. The other chemicals and reagents used were of analytical grade commercially available.

Cultivation of N. oculata
Sterilized seawater mixed with F/2 media which consists of NaNO3, NaH2PO4, Na2SiO3, vitamin (B1, H and B12), and trace metal consists of ZnSO4 and CuSO4 (one ml/components/a liter of seawater) (F/2). In this study, Na2SiO3 was not used because the microalgae are not classified as diatom anddiatom needs silicon (Si) to build the outer cell wall. Since N. oculata does not have any cell wall, silicon is not needed (Orefice et al., 2019). Three different modifications were applied to the other cultures; reduction and addition from each component of normal F/2 media, NaNO3, NaH2PO4, Na2SiO3, vitamin solution, and trace metal solution -0.5 ml, +0.5 ml, and +1 ml, respectively). Table 1 shows the total size of each component that was used for all treatments, Table 2 shows the components of the vitamin solutions, and Table 3 shows the components of the trace metal solutions. The salinity was controlled to 26 ppt, and the temperature was 25°C. The culture was kept for 15 days in a room which controlled to 12 hours of light in average (3.250 lux) and another 12 hours without light.

N. oculata growth measurements
Two variables of growth were measured in this study:cell density and biomass. Measurement was conducted every day from day one until day fifteen. Cell density (cell/ml) was measured with hemocytometer Neuerbauer, and biomass was measured with Whatman filter paper GF/A that weighed on analytical scales (every 30 ml of samples were filtered on Whatman filter paper every day). N. oculata has diameter in average ranged from 2.52 to 2.63 µm (Kandilian et al., 2013), which can be filtered by Whatman filter paper GF/A that has filtration size of 1.6 µm.
2.4 Extraction of N. oculata culture N. oculata culture was centrifuged at 3000 RPM for 7 min to get the pellet of the culture. Afterward, the pellet was macerated with two different solvents; 50% acetone, and 70% ethanol. The result of maceration is a dry extract of N. oculata which will be used for antioxidants assessment with DPPH method (Goh et al., 2010).
2.5 Antioxidant measurement of N. oculata DPPH (α, α-diphenyl-β-picrylhydrazyl) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources (Kedare and Singh, 2011) and it was used in this study. 10 mg of dry N. oculata extract was prepared for the assessment. The extract was then diluted into several concentrations (5, 2, 1, 0.5, 0.2, 0.1, and 0.05 mg/ml), and mixed with methanol as their solvent and also the DPPH. The antioxidant value of N. oculata was measured by microplate reader at 515 nm two times for each culture (F/2 normal, F/2 -0.5 ml, F/2 + 0.5 ml, and F/2 + 1 ml). From that, inhibitions were obtained and could be used to determine antioxidant activity at IC50 (Inhibitory Concentration 50%) unit. IC50 value was determined using a linear regression equation (Y=bX+a) from inhibition graph of each treatment. Y is the dependent variable (on this case, 50 to determine the IC50 value), a is the constant (the concentration) while b is the regression slope (the inhibition value).

Results
3.1 Effects of media differentiation on N. oculata growth (density and biomass) From the cultivation of N. oculata for 15 days, the highest cell density and biomass results were obtained in the treatment of F/2 + 0.5 ml (cell density 19.68 x 10 6 cells/ml and biomass 0.0012433 gr/ml). The treatment of F/2 -0.5 ml produced the lowest value for cell and biomass density (6.58 x 10 6 cells/ml and 0.0009233 gr/ml). Normal F/2 media produced cell density of 15.18 x 10 6 cells/ml and 0.0011733 gr/ml biomass, while F/2 + 1 ml media  produced cell density and biomass higher than normal F/2 but lower than F/2 + 0.5 ml (19.45 x 10 6 cells/ml and 0.00122 gr/ml). Graphs of cell density and biomass respectively can be seen in Figures 1 and Figure 2. One way ANOVA was conducted to determine the effect of media differences in cell density and biomass. The results of statistical tests show that media differences do not give a significant effect on cell density and biomass of microalgae N. oculata (P ≥ 0.05).

Effects of media differentiation on antioxidant activities of N. oculata
From all of the treatments, the F/2 + 1 ml medium had the strongest antioxidant activity (inhibition of 55.03% and IC50 4.53 mg / ml). Reduction of F/2 (F/2 -0.5 ml) media had the lowest antioxidant activity with inhibition of 18.94% and IC50 14.99 mg/ml. Normal F/2 media had an inhibition value of 23.66% and IC50 14.46 mg/ml, and for F/2 + 0.5 ml the inhibition and IC50 values were 21.26% and 13.88 mg/ml, respectively. All inhibition percentage values were obtained from the highest dilution concentration (5 mg/ml). Figure 3 and Figure 4 show the antioxidant activities of N. oculata from different concentration of F/2 media.
Factorial ANOVA was conducted to determine the effect of media differences on the antioxidant activity of N. oculata. The results showed that the media differences had no significant effect on the antioxidant activity of N. oculata (P ≥ 0.05). It can be concluded that the differences in F/2 media concentration on the antioxidant activity of N. oculata is still normal (not significant).

Discussions
Microalgae cultivation requires a constant supply of several nutrients, such as nitrogen (N), phosphates (P), and potassium (K) to obtained optimum yields. (Brahmantara et al., 2015). Macronutrient such as nitrate is used for the metabolism of microalgae, while phosphate is used for reproduction and energy transfer. Not only macronutrients, microalgae also need other media constituents (i.e micronutrients) for their life nutrition (Sari et al., 2012). However, because the function of nitrate In this study, we found that the modification of nutrients in the media (reduced or added), affect the cell density and biomass of N. oculata. When the F/2 media is reduced to 0.5 ml per component per liter (F/2 -0.5 ml), the cell density and biomass of N. oculata were lower compared to the normal F/2 media (1 ml per component per L); however, the tdifference in the media concentration does not give a significant effect on the growth of microalgae N. oculata.
The addition of F/2 media (1.5 and 2 ml per component per L) showed higher cell density and biomass compared to the normal F/2 media. From this study, it can be concluded that the addition of F/2 + 0.5 ml per component per L is better than the addition of 1 ml per component per L due to the results of cell density and biomass after cultivation. The addition of 0.5 ml gives the highest cell density and biomass of N. oculata. The results of this studyindicated thatN. oculata have optimum nutrient levels for its growth (cell density and biomass).
There are no previous studies that examined the effect phosphate on the growth of N. oculata and Nannochloropsis sp., with various range of modification at the nitrate and phosphate composition. The studies show that different levels of nitrate and phosphate on the culture media will affect the growth of microalgae N. oculata and Nannochloropsis sp. Furthermore, using a linear regression correlation, Brahmantara et al. (2015) found the most optimum amount of nitrate and phosphate at F/2 media for the growth of Nannochloropsis sp., are 178.14 gr/l and 13.34 gr/l, respectively. Antioxidant ability of algae is affected by the molecular weight of polysaccharides, monosaccharide composition, and sulfate content (Hafsa et al., 2017). From this study, it can be confirmed that the sulfate content affects the antioxidant ability of N. oculata. The F/2 had sulfate content in forms of ZnSO4 and CuSO4 compounds which are the components of the trace metal solutions. The lowest antioxidant activity of N. oculata (both inhibition and IC50) was found in the reduction of F/2 media (0.5 ml per component per L) which had the lowest sulfate content compared to the other treatments. The highest antioxidant activity was found in the addition of F/2 media (2 ml per component per liter) where the sulfate content was also higher than the other treatments.
The strongest antioxidant activity in this study had an IC50 value of 4.53 mg/ml (4,530 µg/ml), and it can be concluded that antioxidant activity of N. oculata was very weak.
There are no previous studies that examined antioxidant activity of N. oculata with modified F/2 media treatments. From this study, it can be assumed that the optimal addition of nutrient levels can increase antioxidant activity of N. oculata, since the results of antioxidant assessment shows differences from each media used in this study.
Several studies had examined antioxidant activity of N. oculata with various treatments (as can be seen in Table  4.). Sangeetha et al., (2018) found a higher antioxidant activity of N. oculata compared to this study The results might be different due to the cultivation process and sample concentration. Sangeetha et al., (2018) did not cultivate the microalgae and used less sample concentration (500 µg/ml). Therefore, it can be assumed that cultivation process might affect the antioxidant activity of N. oculata.
Another study carried by Hafsa et al., (2017), who cultivate the N. oculata with F/2 media for 11 days and extracted with distilled water. Hafsa et al., (2017) found the antioxidant activity was 24.79 % (from 1 mg/ml extract) while antioxidant activity found in this study was 23.66 % (from 5 mg/ml extract). Hence, it can be assumed that the extraction process would effect the antioxidant activity of microalga.

Conclusions
The modification of F/2 media in this study did not give a significant effect on growth (cell density and biomass) and antioxidant activity of N. oculata. Therefore, further studies on the composition of F/2 media to obtain the optimum growth and antioxidant activity of N. oculata is required.