Fatty Acids Extraction from Algae- Chlorella Vulgaris

Chlorella is a single-celled, spherical shaped, flagellate organism which is about 2 to 10um diameter. It contains green photosynthetic pigments viz. chlorophyll a and chlorophyll b in its chloroplast. Through photosynthesis, it multiplies rapidly by utilizing only carbon dioxide, water, sunlight, and a small number of minerals to reproduce. Chlorella transforms inorganic matter to organic matter by using natural light via photosynthesis, at suitable growing conditions. It contains expert proteins, chlorophyll, dietary fiber, vitamins, minerals, enzymes, nucleic acids and phytonutrients that are advantageous to the humans. Growth and harvest of chlorella vulgaris in the closed and open pond system was done for the production of fatty acids. Growth was optimized at different parameters like temperature, pH, light, intensity, aeration, and medium in both open and closed pond system. For closed pond system chlorella vulgaris was cultivated in three different media like Bg11, TAP, N8. In both open and closed pond system, lipids were extracted using lipid extraction methods and the fatty acids were extracted by using methylation or esterification method. Key wordsBiochemical test, Chlorella vulgaris, Fatty acids, Lipid extraction method, Methylation.


I. INTRODUCTION
Microalgae can produce various commercial byproducts such as fats, oils, sugars. and functional bioactive compounds. Some are heterotrophic organisms and the other are photosynthetic organisms where photosynthetic organisms can be considered as plants in the world 1 . The algae have chlorophyll that can manufacture their food through photosynthesis. But some are can be considered as cyanobacteria because they have a prokaryotic cell structure 2 .
Microalgae are an extremely important species. They produce more oxygen than compared to all other plants in the world. An alga is an important food source for many animals such as little shrimps and huge whales 3 . One interesting information is found out with recent research by using algae to produce biofuels they are Biodiesel, butanol, gasoline, methane, ethanol, jetfuel, vegetable oil and fatty acids.

A. Chlorella Vulgaris:
The name "Chlorella" was derived from the Latin words 'Chloro' for green and 'Ella' for small. It has a size of 2-8 microns which makes it possible to be observed under a microscope 4 . The chlorella cell is equal to human red blood cells but differs in shape, where chlorella is spherical and the human red blood cell is disc-shaped 5 . Every 17-24 hours, chlorella produces four new cells. There are 25,000 species of algae which are plain plants without roots, stems, branches, and leaves. Chlorella vulgaris contains a high content of chlorophyll. It can manufacture its own food through the process of photosynthesis. It produces more oxygen than compared to other plants in the world. High amount of lipids are present in chlorella 6 .

B. Lipids:
Lipids are the substance of biological origin that are soluble in organic solvents such as chloroform and ethanol and are only sparingly soluble, if at all, in water 7 . They are a heterogeneous group of compounds related to the fatty acids either actual or potential and chemically they are esters of fatty acids and some alcohols 8 . Hence they are easily separated from other biological materials by extraction into organic solvents and may be further fractionated by such techniques as, adsorption chromatography, thin layer chromatography, and reversed phase chromatography 9 .  A. Cultivation of algae in closed pond: 3ml of algae is inoculated to 500ml of (Bg11, N8, and TAP) media observe and measure the growth from alternative days by using UV-Visible Spectrophotometer 11 .
Cultivation of algae in open pond: 10ml of algae is inoculated to 1.75 lit of TAP media observe and measure the growth from alternative days by using UV-Vis-Spectrophotometer 12 .
• Physical and chemical conditions: Closed pond: Optimized PH is 7.1, aeration is 7hr, and light is 14hr, for Bg11, TAP, and N8 Medias.  Anthrones test for carbohydrates estimation: Algae: HCl is added in the ratio of 1:2.5, add 1 molar of sodium carbonate until the smell will disappears, centrifuge 10,000 rpm for 5 min, add 1ml of 5% phenol and 3ml of 96% sulfuric acid, kept in dark place for 20min and take OD values at 490nm by using UV-Vis-Spectrophotometer 22 .
C. Lowry's method for protein estimation: Add double amount of Lysis buffer to the algae growth and incubate at 45 0 C for half an hour add small amount of SDS and centrifuge at maximum rpm for 5min. to the supernatant add equal amount of Reagent-I [48:1:1 of 2% sodium carbonate in 0.1 N NaOH: 1% sodium potassium tatarate:0.5% Cuso4.5H2O] to the collected supernatant, add equal amount of Reagent-II to the above mixture and incubate in dark place for 20 min. take OD values at 660nm by using UV-Vis-Spectrophotometer 23 .

D. Pigment estimation:
About 10 ml of the sample is taken and is centrifuged at 5000rpm for 5 min. The supernatant was discarded and washed with distilled H2O. The pellet is mixed with 10 ml of 90% ethanol and again it is centrifuged. The procedure from step1 to step4 is repeated until the pellet becomes colorless. The sample was measured at 663nm by using 90% ethanol as blank in UV-VIS-Spectrophotometer. The amount of chlorophyll is derived by using the following formula 24 F. Methylation /esterification of fatty acids: The lipids are dried under a water bath at 80°C for 10 min. 0.4gm NaOH in 20ml methanol is added into the lipids. Incubate in a water bath for 10 min at 80°C. 5ml of the extract is added into each one of the screw-capped test tubes. 1ml of boron trifluoride is added into the test tube. The extract is refluxed for 10 min in a water bath with stirring at 80°C. After cooling, 700ul of 1 bromo-tetra-decane and 300ul of n-heptane is added. 1 ml saturated NaCl solution is added into the test tube and shaken well. The upper layer is carefully collected with the help of a pipette. Finally, PUFA (polyunsaturated fatty acids) are collected.
F. Methylation /esterification of fatty acids: The lipids are dried under a water bath at 80°C for 10 min. 0.4gm NaOH in 20ml methanol is added into the lipids. Incubate in a water bath for 10 min at 80°C. 5ml of the extract is added into each one of the screw-capped test tubes. 1ml of boron trifluoride is added into the test tube. The extract is refluxed for 10 min in a water bath with stirring at 80°C. After cooling, 700ul of 1 bromo-tetra-decane and 300ul of n-heptane is added. 1 ml saturated NaCl solution is added into the test tube and shaken well. The upper layer is carefully collected with the help of a pipette. Finally, PUFA (polyunsaturated fatty acids) are collected.
G. Fatty Acid estimation by Gas Chromatography: The fatty acid composition of the oils was determined by gas chromatography (GC) as fatty acid methyl esters (FAME). FAME was prepared in accordance with the official method of the IUPAC (1987). Chromatographic analysis was performed in Shimadzu GC-2010 chromatograph using a DB-23 fused silica capillary column (30m, 0.25mm), 0.25 µm film thickness, Agilent j and W, USA). Helium is used as a carrier gas which is operated at a flow rate of 1.00ml/min. The column temperature was isothermal at 190⁰c where in the injector and detector temperatures were 230⁰C and 240⁰C, respectively. FAME was identified by comparison of their retention times with those of the reference standards.

III. RESULTS
A. Open pond:          In an open pond, the alga was inoculated and growth was observed after 19 days.

Closed pond
In an closed pond, the alga was inoculated and growth was observed after 19 days. It is explained Fig 7. Algae extract by using soxhlet The algae was collected from the conical flask and transferred into the falcon tubes. The pellet is obtained by centrifugation for 30min at 1500 rpm. 1.25ml of hexane and 1ml of chloroform was added to the pellet and again centrifuged. The algae supernatant was collected from the falcon tubes with the help of micropipette and was poured into the petri plates. The plates were then kept in the MS oven for 15 minutes at 50 0 C.
Finally the algae powder was collected and was utilized in Soxhlet to obtain algae extract 23 .
Phytochemical test for primary and secondary metabolites

Carbohydrates and protein estimation values in open pond
In open pond, sample readings were read on alternative days using UV-Vis-Spectrophotometer. From the result obtained, it is observed that the biomass and protein readings gradually increased and the carbohydrates gradually decreased. This can be interpreted as the carbohydrates present are utilized by the algae for its growth and development leading to the formation of biomass and protein. After 19 days, lipids and fatty acids content was estimated.

Carbohydrates and protein estimation values in closed pond
In closed pond, sample readings were read on alternative days using UV-Vis-Spectrophotometer. From the result obtained, it is observed that the biomass and protein readings gradually increased and the carbohydrates gradually decreased. This can be interpreted as the carbohydrates present are utilized by the algae for its growth and development leading to the formation of biomass and protein. After 19 days, lipids and fatty acids content was estimated.

Lipids and fatty acid values at final day in closed Pond
After 19 days lipids and fatty acids content was estimated in closed pond and highest percentage of lipids and fatty acids are obtained from Tap medium. i.e. 13.6% and 6.8% respectively.
Lipids are extracted from algae The algae is collected from the conical flask and transferred into falcon tubes. The pellet is obtained by centrifugation for 30min at 1500 rpm. 1.25ml of hexane and 1ml of chloroform was added to the pellet and again centrifuged. The algae pellet is transferred to a conical flask and incubated in a water bath for 10 min at 90 0 C. The Lipids which float in the conical flask were collected with the help of micropipette.

Extract the fatty acids from lipids
The lipid samples were taken in different test tubes and were kept in water bath at 90 0 C for 5 minutes. Then 0.3gm of sodium hydroxide pellets were added to 20 ml methanol and the mixture was added to each of the test tubes. The tubes were again kept in the water bath at 90 0 C for 5 minutes. From the sample, 5ml of extract were transferred to another set of test tubes. To these, add 1ml of boron trifluoride to each test tube. The tubes were again kept in water bath at 90 0 C for 5 minutes. Then to each tube 300ul of n-hexane and 700ul of 1bromotetradecane was added, finally 1ml saturated NaCl solution was added and the contents in the test tube were mixed thoroughly. After mixing, two layers were formed. The upper layer in the test tubes were collected as it contains fatty acids.

Fatty acid profile
The 1st batch is TAP media (closed pond

CONCLUSIONS:
Chlorella vulgaris can easily grow in any environment without any specific conditions. It can produce high lipid content when grown in nutrition rich media when compared to other species. It is very efficient in producing biofuels which helps the mankind to solve the problem of dependence on fossil fuels and hence save the non renewable resources present underneath the earth. It is also used for the production of unsaturated fatty acids. The daily intake recommended by American heart association is 13 grams per day on a 2,000 calorie daily diet. Omega-3 fatty acids are critical for brain