Effects of Nitrate and Salinity on Fatty Acid Composition of Marine Tetraselmis sp.: Potential as Biodiesel

Identifying sources of renewable energy is extremel y important. The potential of Tetraselmis sp. in biodiesel production was investigated using a completely randomized desi gn with four treatments. Te traselmis sp. was cultured on media containing different concentrations of nitrate ther e a e 2 mM and 15 Mm and s 25 ‰ and 35 ‰ salinity. The treatments namely are (N2S25), (N2S35), N15S25) and (N15S35). Analysis of the fatty acid content using gas chr omatography-mass spectrometry (GC-MS). The results showed that the h ighest growth occurred in culture that contained 15 mM of nitrate and 35 ‰ salinity. The lowest growth occurred in cu lture containing 2 mM of nitrate and 25‰ of salinit y. The highest lipid content was found in cultures containing 2 mM of nitrate and 25‰ salinity, it is 34.83%. Seven f atty acids were detected in culture containing 2 mM of nitrate and 25‰ salinity namely myristic acid (4.02%), palmitic a id (40.59%), palmitoleic acid (29.06%), stearic acid (0.95%), ol eic acid (12.52%), gamma-linolenic acid (2.56%), an d rachidonic acid (9.38%). Four fatty acids palmitoleic acid (8. 99%), palmitic acid (37.34%), oleic acid(44.89%), a nd stearic acid (8.78%) were detected in 2 mM of nitrate with 35‰ s alinity. The fatty acids have potential to be used as raw material for biodiesel production.


Introduction
Based on data from the Ministry of Energy and Mineral Resources (ESDM) in 2006 [1], Indonesia is dependent on several resources for energy. These include oil (46.99%), natural gas (21.8%), coal (23.5%), hydropower (2.4%), and geothermal energy (5.5%). The nation's energy consumption is predicted to continue increasing with the growth of transportation and industry, both of which are major consumers of petroleum. All human life on earth requires energy. As oil is not renewable, we need to take to secure a domestic energy supply. Microbial biodiesel production is an alternative renewable energy to solar-based fuels. It can be derived from vegetable oil by transesterification and esterification.
In general, biodiesel is produced from palm oil, sunflower, soybean, and canola. However requires a vast land area and may cause damage and threaten food security [2]. Different with biodiesel from marine microalgae, it doesn't need crop land area. As an alternative to plant-based biodiesels, researchers are concentrating their efforts on the production of biodiesel from marine microorganisms, which do not cause environmental damage or use valuable crop land.
Marine microalgae they have several advantages for the examples, saline or marine water can be used for culture, thereby not depleting fresh water supplies. In addition, microalgae can absorb CO 2 for photosynthesis. Depending on its productivity, certain species of microalgae may produce more than 30,000 L of oil or about 200 barrels per acre compared with 100,000 L/ha produced by soybean cultivation [3].
The biochemical composition of microalgae depends on environmental conditions and macronutrients, such as nitrogen and phosphorus, and carbon sources [4]. Tetraselmis sp. which cultured in medium supplemented with phosphorus (20 mL/L) showed growth increasing, but the lipid yield was lowest (9.41 ± 0.27% dry weight). Higher lipid yields were observed at lower phosphorus concentrations. The study also showed that in CO 2 supplementation (10%) and nitrogen uptake (0.316 ± 0.08 NH-N /cell/day indicated increasing of lipid content (10. 95 ± 15% dry weight) [5].
The present study focused on the sea microalgae Tetraselmis sp. which has the potential to accumulate almost 50% of its dry weight as lipids [6]. Based on a previous study, modifying the composition of growth media can increase microbial growth and lipid levels [7]. The aim of the present study was to enhance the growth and lipid levels of Tetraselmis sp. by culturing the microalga in media containing different concentrations of nitrate and different levels of salinity.

Materials and Methods
This research was divided into three phases. In the first phase, Tetraselmis sp. from Balai Besar Pengembangan Budidaya Laut, Lampung was grown in growth medium containing 1 L of marine water collected from Sea World Indonesia in Jakarta and Conwy/Walne medium consisting of 1 mL of macronutrients, 1 mL of micronutrients, and 0.1 mL of vitamins. The laboratory equipment and marine water were sterilized by digital autoclaving at 121 °C, 2 atm for 15 min. Tetraselmis sp. was cultured with a stone aerator in 100 mL, 250 mL, 500 mL, 1 L, 2 L, and 5 L of growth medium. An 18watt fluorescent lamp was used as a source of light.
In the second phase, the microalga was cultured in several concentrations of nitrate and salinity in 10 L of growth medium. Concentration of nitrate were 2 mM and 15 mM, and salinity were 25‰ and 35‰. In the third phase, the microalgae was exposed to four different treatments: medium consisting of 2 mM of nitrate and 25‰ salinity, 2 mM of nitrate and 35‰ salinity, 15 mM of nitrate and 25‰ salinity, and 15 mM of nitrate and 35‰ salinity. The growth, lipid concentration, and fatty acid lipid composition were then analyzed.
The research used a completely randomized design, and ANOVA test was used to analyze the data. If the factors of treatment were significantly different according to the ANOVA test, Least Significance Difference (LSD) test was conducted. A value of α = 5% was considered a significant difference between the standard treatment and its interaction.
Culture of Tetraselmis sp. Before culturing in different saline and nitrate concentration Tetraselmis sp. were derived from pure isolates. It increased by using 100 mL, 250 mL, 500 mL, 1 L, 2 L, up to 10 L of growth medium. The cell density was increased for six to seven days. After the seventh day of culture, it was moved to larger media. To maintain a supply of pure isolates, its liquid form was kept in refrigerator. Thus, its frequent agitation was preserved to prevent precipitation, and it was useful for air circulation.
After the amount of Tetraselmis sp. cells were enough for a treatment, the cultures will be taken with salinity and nitrate treatments. The aquarium was filled with seawater which has been assorted by Conwy/Walne and nitrate in a volume of 28 × 22 × 17.5 cm 3 a capacity of 10 L. The inoculation may reserve microalgae for 30% (v/v) due to the used medium. Then, the culture, which was ready to be paired on airstone and hose, was connected to the aerator. The culture was placed in a culture chest, with an 18-watt fluorescent lamp. Meanwhile, the culture was used to calculate the growth curve to determine the period of time in undertaking subcultures and gaining microalgae cells to compute lipid extraction and its dry weight. The population of microalgae was calculated with a haemocytometer under a microscope.
The biomass of microalgae was produced by filtering the cells using microfilter paper. The accumulated biomass was transferred to a wide petri dish and dried with fluorescent light radiation.
Lipid extraction with the Soxhlet method. Samples dry biomass 1-3 g were wrapped in filter paper. Then, The filter paper was placed in the Soxhlet. The condenser was mounted upon the top and underneath the fat. Hexane was placed in a flask with a volume of fat Soxhlet at one quarter. The minimum period of time to use reflux was 5 h due to the solvent was clear. The hexane solvent in the flask was distilled to obtain lipids from fat pumpkin. The fat pumpkin containing the lipid extract was heated in an oven at 80 °C for 1 h to remove residual water and any remaining solvent. Once it was dried to keep its cool and weight in a desiccator, lipids were also be pondered. The weight of lipids was calculated as follows: Analysis of fatty acids with gas chromatographymass spectrometry (GC-MS). The extracted lipid samples were analyzed with GC-MS to determine the components of fatty acid. Before it was injected into the GC-MS, a sample of transesterification reaction was conducted with 4.5 mL of 0.5N NaOH in methanol, vortex, the heating of water bath within 5 min, and kept cool. Then, a 14% solution of BF3 was added to 3 mL of methanol, vortexed, heated, and cooled. The addition of n-hexane (grade chromatography) in samples, vortex, heat it and keep it cool to perform the centrifugation at 3000 rpm. The samples (5 µL) were transferred into GC tubes ready for injection into the GC column. The samples were injected into a 30 m column of dimethyl polysiloxane with a thickness of 0.25 µm df at a temperature of 80 °C. The GC was ran with the hexane solvent. The injector temperature was 250 °C, and types of split injector. The starting temperature was 80 °C. This was maintained for 2 min and increased to 210 °C at 10 °C per min at intervals of 1 min. The temperature was increased again to 280 °C, with an increase of 6 °C per min and held for 10 min. The last injection phase was at a temperature of 280 °C.

Results and Discussion
Indicating of Tetraselmis sp. growth is increasing of cell density and the green color of the culture became stronger. The exponential phase occurred from the third to the fifth days. The stationary or death phase started on the third or fifth day.
Growth of Tetraselmis sp. The culture medium affects the growth of microalgae [8]. Figure 1 shows the growth curve of Tetraselmis sp. in the lag phase, exponential phase (logarithmic), stationary phase, and death phase. The lag phase of Tetraselmis sp. had a short duration of two days. This may be related to the initial inoculation (v/v) of the cell growth media with 30% microalgae. The length of the lag phase depends on the growth media and environmental conditions, as well as the age and number of inoculums. The metabolism when lag phase which occurred in the cells Tetraselmis sp. but was not involved the activity of cell division so that the cell density was not increased. The cell density of Tetraselmis sp. increased in the exponential phase (logarithmic) due the growth of the cells. In this phase, photosynthesis produces carbohydrate, and protein synthesis provides proteins for growth and increased cell division [9]. In the stationary phase, the limiting factor and the growth rate were balanced, which resulted in a relatively constant cell density [10]. The interaction between the effects of nitrate and the influence of salinity significantly affected the number of cells (Fig  1). According to the ANOVA test, the interactive effects of nitrate and salinity affected cell growth from the third to the ninth day but not on the sixth and seventh day. On the third day, the highest and lowest numbers of cells were found in the N 15 S 25 (1.51 × 10 6 cells/mL ± 0.02 × 10 6 and N 2 S 25 treatments (1.07 × 10 6 cells/mL ± 0.02 × 10 6 ). However, on the fifth day, the highest and lowest number of cells were found in the N 15 S 25 and N 2 S 25 treatments (1.95 × 10 6 cells/mL ± 0.03 × 10 6 and 0.66 × 10 6 cells/mL ± 0.01 × 10 6 , At the end of the experiment on the ninth day, the highest numbers of cells were observed in N 15 S 35 (0.92 × 10 6 cells/mL± 0.03 × 10 6 ) treatment and the lowest numbers in the N 2 S 25 treatment ( 0.31 × 10 6 cells/mL ± 0.02 x 10 6 ), Nitrate and salinity influence the growth Tetraselmis sp., with previous research reporting that a low level of nitrogen in macronutrients may limit the growth of cells [8]. Another study reported that a high concentration of carbon and nitrogen affected the growth of Chlorella sp. 227 [11].
Therefore, the stationary phase may easily to achieve within its cultures with low nitrogen. A low level of nitrate may also have an adverse effect on the metabolism of Tetraselmis sp., cell division and thereby reducing cell population levels. Previous research demonstrated that low levels of nitrogen in media reduce the productivity of microalgae [12]. Nitrate is a source of nitrogen in carbon As shown in an earlier study, the amount of chlorophyll decreases in accordance with a reduction in the concentration of nitrogen in culture media [13].
The reduction in chlorophyll also hampers the processes of photosynthesis and assimilation, which, in turn, inhibits growth.
Salinity affects the growth and the productivity of microalgae, with research showing the growth rate of microalgae varies according to the level of salinity in the surrounding environment [9]. As demonstrated previously, Tetraselmis sp. from Penang National Park coastal water can be grown under 16 g/L salinity [14].
In the present study, Tetraselmis sp. grew at salinity levels of 25‰ and 35‰. Based on research that the optimum growth of Tetraselmis sp. may applies to 20-35 ppm salinity [15].

Dry weight (biomass).
The dry weight was determined at the end of the growth phase and on the ninth day (death phase) in 10 L medium culture. The biomass of Tetraselmis sp. was determined by estimating the dry weight of 4.02 mg/10 6 cells [16].
On the third day at the end of the exponential phase, the highest biomass dry weight in the N 15 S 25 treatment in 10 L was 6.07 g. In the N 2 S 25 treatment, it was 4.30g. On the fifth day, the highest biomass dry weight was 7.83g in the N 15 S 35 treatment. The lowest in theN 2 S 25 medium was 2.65 g. In the death phase (ninth day), the highest biomass dry weight in the N 15 S 35 treatment was 3.69 g, and the lowest biomass dry weight in the N 2 S 25 treatment was 1.24 g. On the ninth day, the highest dry weight of Tetraselmis sp. was 3.68 g in the N 15 S 35. The data shows that increasing the nitrate concentration to 15 mM and the salinity 25-35‰ can increase the biomass dry weight of Tetraselmis sp.

Level of lipids.
Lipid levels were measured at the end of each growth phase, the end of the exponential phase (third and fifth days), and the end of the final death phase (ninth day). The results showed that the lipid levels were highest (11.83-34.83%) at the end of the death phase and lowest (4.22-26.89%) at the end of the exponential phase. The ANOVA test results revealed significant effects (p<0.01) of the concentration of nitrate and salinity levels on the content of Tetraselmis sp. Based on the results of the LSD, every combination of nitrate and salinity treatments were significantly differ from each other (Figure 2). The highest and lowest lipid levels were found on the third day in the N 2 S 25 and N 15 S 35 treatments (26.89% ± 0.38% and 4.22% ± 1.02%, respectively). The highest and lowest levels of lipids were observed in the N 2 S 25 and N 15 S 35 treatments (32.5% ± 0.50% and 7.83% ± 0.76%, respectively) on the on the fifth day. On the ninth day at the start of the death phase, the highest and lowest levels of lipids were produced in the N2S25 and N 15 S 35 treatments (34.83% ± 0.29% and11.83% ± 0.58%, respectively).
The productivity of Tetraselmis sp. is affected by environmental factors and the composition of the culture media. Based on this study, nitrate increased the amount of biomass but not the yield lipid. Lipids are synthesized by the formation of gliseraldehide in the process of glycolysis and Co-A acetyl in the decarboxylation of pyruvic acid. Glycerol and fatty acids are converted to lipids [3]. The high level of lipids in the N 2 S 25 treatment in the stationary phase may be caused by the process of synthesizing, which the biomass in logarithmic phase was quick due to the limited availability of nitrogen. In addition, the metabolism of the cells changed, switching from the formation of cell structure to lipid synthesis.
The biochemical composition in the exponential phase is different from that in the stationary phase [17]. In this study, the types of fatty acids produced in the N 2 S 25 and N 2 S 35 cultures differed because of the effect of the different nitrate and salinity treatments. As shown in a previous study, the content of fatty acid in microalgae may vary based on its growth conditions [18].  Table 2). The highest concentrations of fatty acids were found in the N 2 S 25 treatment. Palmitic acid (C 16:0 ) and palmitoleic acid (C 16:1 ) of 40.59% and 29.06%, respectively. Palmitic acid (C 16:0 ) and palmitoleic acid (C 16:1 ) was produced in the N 2 S 25 treatment more than in the N 2 S 35 treatment.

Test of gas chromatography (GC
The type of fatty acid in N 2 S 35 treatment is oleic acid (C 18:1 ) and palmitic acid (C 16:0 ) respectively 44.89% and 37.34%. Both of fatty acids have potential as biodiesel.
As reported previously, the use of saturated fatty acids in biodiesel production results in better oxidation and fewer emissions than unsaturated fatty acids [19].
Research has also shown that palmitoleic acid (C16:1) has a greater advantage as biodiesel than oleic acid [20] because high levels of oleic acid increase the resistance to oxidation [18].
The highest level of oleic acid (44.89%) was produced by Tetraselmis sp. in the N 2 S 35 treatment. The level of acids produced was better than that found with other sources of crude oils, such as oil palm, soybeans, sunflowers, and corn ( Table 3). The palmitic acid content was 37.34% and in N 2 S 25 was 40.59%. Linoleic acid was the dominant fatty acid component (23.06%) when Tetraselmis sp. was cultured in the marine water and Conway medium (4:1). Based on these data, the type of fatty acids produced by Tetraselmis sp. is dependent on the composition of the culture medium [21].
The best optimum treatment to increase the growth of Tetraselmis sp.was a concentration of 15 mM nitrate and a salinity level of 35%. The treatment also showed the highest biomass dry weight was 7.83 g. The best optimal treatment to increase lipid levels of Tetraselmis sp. was a concentration of 2 mM nitrate and a salinity level of 25% (p<0.05), it is 34.83% Seven types of fatty acids there are myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, gamma-linolenic acid, and arachidonic acid were detected in the treatment with 2 mM nitrate and salinity of 25%. Four types of fatty acids (palmitoleic acid, palmitic acid, oleic acid, and stearic acid) were detected in the 2 mM nitrate and 35% salinity treatment. The different concentrations of nitrate and salinity levels exerted a significant effect (p<0.05) on the growth of Tetraselmis sp. and the kinds of fatty acids produced.