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Municipal wastewater refer to the undesired wastewater collected from homes, businesses and also storm drainage which contain a broad variety of contaminants and nutrients. Those contaminants are necessary to be treated and removed in order to avoiding contamination.
In Hong Kong, wastewater treatment is divided in to two stages that is primary and secondary wastewater treatment respectively. Primary treatment contributes a majority of wastewater treatment in Hong Kong, therefore this type of treatment should be considered with first priority.
A stoichiometric formula for common nutrients in algal cell is found as C106H181O45N16P, this combination of nutrient can theoretically achieve an optimum growth for algae. (Oswald, 1988)
In primary wastewater, a certain type of contaminants is prior to be concerned which is Nitrogenous and Phosphorous chemicals because of its amount and necessity.
Nitrogen is one of the various contaminants that can be found in wastewater. Those nitrogenous compounds are produced from metabolism of human body and can be simply categorized into two forms that are organic and inorganic respectively. (Sedlak, 1991). In organic form, bacterial decomposition of protein and hydrolysis of urea can accelerate the process of conversion of organic matter into ammonium from. (Sedlak, 1991) The oxidized from of nitrogen, NO2, NO3 has a less contribution in the fresh water.
With the results from Drainage Services Department of Hong Kong, the average total nitrogen in the primary wastewater is about 39mg/L and those nitrogen compounds occur in various forms commonly in ammonium (NH4 +) and nitrate (NO3-) and is assimilated by microalgae (Oliver and Ganf, 2000). Ammonium is prior for the microalgae uptake but an ammonia toxic effect may occur while the ammonium concentration is higher than 20 mg per litre. (Borowitzka, 1998). In spite of these two nitrogen compounds, urea (CO(NH2)2) and nitrite (NO2 -) can also be used as nitrogen sources.
For the formation of nitrogen compounds, some mechanism involved that are nitrogen fixation, nitrification, denitrification, synthesis and ammonification. (Selecky, 2005)
Nitrogen fixation involves transforming the gaseous nitrogen into nitrogen compounds which can be assimilated by plants. (Selecky, 2005)
Nitrification is the process of oxidizing the ammonium ion to nitrate by bacteria. (Selecky, 2005)
Denitrification is the conversion of nitrate to nitrogen gas under anoxic conditions by heterotrophic bacteria. (Selecky, 2005)
Synthesis is the biochemical process in converting the ammonium ion or nitrate into organic nitrogen (plant protein). (Selecky, 2005)
Ammonification is the process of degrading the organic nitrogen into ammonia or ammonium ion by heterotrophic bacteria. (Selecky, 2005)
Phosphorus in the wastewater is generally in the form of phosphate and is essential for algal growth. Regarding to the source of phosphorus, there are three interconnected cycles involved which is inorganic cycle and two organic cycles respectively. (Cornel, 2009) In municipal wastewater, the major source of phosphorus is from the organic cycle that involved digestion of the food which containing phosphorus. (Cornel, 2009) With the results from Drainage Services Department of Hong Kong, the average total phosphorous in the primary wastewater is about 4.4mg/L.
With physical and chemical characteristics, phosphate is divided in to orthophosphate, condensed phosphate and organic phosphate. (Sedlak, 1991)
Orthophosphates are transformed into organic phosphates by phosphatases in active process requiring energy especially inorganic phosphate is insufficient. (Sedlak, 1991)
2.2 Introduction to algae
2.2.1 Division of algae
Algae is a oldest, simplest and large group of organism in the world, the first algae cyanobacteria appeared more than 3 billion year ago. Algae ranging in size from microscopic single cells to complex, multicellular seaweeds many meters long. (Philip 1993) In general it is a plant-like organisms. Most algae is aquatic and photosynthetic, it do not have true roots, leaves, stems and vascular tissue. According to the book " Freshwater algae identification and use as bioindicators ", The botanist divided the major freshwater algae in to ten phylum, phylum of Cyanophyta, phylum of Chlorophyta, phylum of Euglenophyta, phylum of Xanthophyta, phylum of Dinophyta, phylum of Cryptophyta, phylum of Chrysophyta, phylum of Bacillariophyta, phylum of Rhoophyta, phylum of Phaeophyta.
2.2.2 Biology of algae
22.214.171.124 Metabolisms of microalgae
Mata et al(2010)mention that Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms, since their simple multi-cellular or unicellular structure make it can live under stress environment and grow rapidly. K. Chojnacka al(2004) base on the metabolisms of microalgae to divide different type of trophic for example, autotrophic, heterotrophic, mixotrophic, photoheterotrophic). Mata et al(2010) summaries the algae in different type in to table X
Using light as a sole energy source that is converted to chemical energy through photosynthetic reactions
Utilizing only organic compounds as carbon and energy source.
Performing photosynthesis as the main energy source, though both organic compounds and CO2 are essential. Amphitrophy, subtype of mixotrophy, means that organisms are able to live either autotrophically or heterotrophically, depending on the concentration of organic compounds and light intensity available.
Known as photoorganitrophy, photoassimilation, photometabolism, describes the metabolism in which light is required to use organic compounds as carbon source.
Table X: Metabolisms of microalgae(Mata et al 2010)
The microalgae need photosynthesis directly or indirectly. This process the microalgae using light energy and inorganic compounds to convert the organic matter. The organic matter is a sources of energy to the microalgae growth and their metabolism.
Photosynthesis can be expressed as a redox reaction. It driven by light energy harvested by chlorophyll molecules in microalgae, in which carbon dioxide and water are converted to carbohydrates and oxygen. The photosynthesis process is show as figure X
Figure 2: Major products of the light and dark reaction of photosynthesis(Masojidek et al,2006)
From figure X, the photosynthesis process is divided into two stage, light reactions and dark reactions. In the light reaction, which are bound on photosynthetic membranes, the light energy is converted to chemical energy providing a biochemical reductant Nicotinamide Adenine Dinucleotide Phosphate Hydrogen(NADPH2) and a high energy compound Adenosine triphosphate(ATP). In the dark reactions, which take place in the stroma, NADPH2 and ATP are utilized in the sequential biochemical reduction of carbon dioxide to carbohydrates.
The light reactions is to provide the biochemical reductant NADPH2 and the chemical energy ATP for the assimilation of inorganic carbon. The light energy is trapped in two photoreactions carried out by two pigment-protein complexes, photosystem I (PS I) and photosystem II (PS II). These two system form the reaction call photophosphorylation. Figure X is the photophosphorylation reaction.
Figure X is the photophosphorylation reaction (Masojidek et al, 2006)
The dark reactions mechanism of carbon fixation was worked out by Calvin cycle.
In the dark reaction, the carbon dioxide fixation is using the NADPH2 and ATP produced in the light reaction of photosynthesis. The dark reaction can be expressed in figure X.
Figure X is the reaction of dark reaction (Masojidek et al,2006)
2.2.3 Culture method
126.96.36.199 Batch culture
Batch culture is a common method for commercial to culture the algae. The algae is culture in a fix volume culture medium, during the culture period it will not add any substrate to the culture medium. Batch culture only harvest one time when the biomass concentration is suitable for harvesting.
Under the batch culture, the algae will have different phase due to the environment change, nutrient concentration and the growth rate. Normally, the microorganism growth can be divided into four phases, lag phase, exponential phase, stationary phase and death phase. Dhont(1996)
Lag phase: the algae need to adapt the new environment for their growth, the algae concentration will keep fix
Exponential phase: after adaptation, the algae growth exponentially
Stationary phase; algae cell division and population growth stops because nutrients are exhausted
Death phase: the algae death rate large than the growth, the nutrients are fully exhausted
188.8.131.52 Semi-Continuous Cultures
In a semi-continuous culture, use of large tank cultures and the nutrient is continuous add to the medium to achieve the original nutrient level when the algae partial periodic harvesting. After add nutrient to the culture, it grown up again then partially harvested. The step is keep continuous when the nutrient is used so the cultures keep growth.
184.108.40.206 Continuous Cultures
In continuous cultures, the nutrient never runs out. This method of culturing algae permits the maintenance of cultures very close to the maximum growth rate. The nutrient is add regularly avoid the nutrient exhaust. In practice, the supplement culture medium is added automatically at a rate proportional to the growth rate of the alga, while an equal volume of culture is removed.
2.2.4 Scenedesmus sp.
Scenedesmus is under green algae, division of chlorophyta, class of chlorophyceae, order of Chlorococcales, family of Scenedesmaceae and Genus of Scenedesmus.
Scenedesmus is one of the commonest genera of freshwater algae comprising of hundreds of named species. The cells usually are arranged in a row to for 4 or 8 celled colonies in Scenedesmus. 2 to 16 celled colonies can present and there are more than 16 cells per colony very rarely. It is especially found in eutrophic and hypertrophic waters. (Bellinger et al 2010) Figure X showed the Scenedesmus sp. under the electron microscope, it can find that there are 4 cell colonies and 2 cell colonies
Figure X: Scenedesmus sp. under the electron microscope
2.2.5 Chlorella sp.
Chlorella is under green algae, division of chlorophyta, class of chlorophyceae, order of Chlorococcales, family of Chlorelllaceae and Genus of Chlorella.
Chlorella is a genus of single-cell green algae. The shape of Chlorella is spherical, the diameter about 2 to 10 Î¼m, and is without flagella. According to Wu et al (2001) Chlorella sp. are widespread in fresh water and in the sea, air, and soil. Chlorella have been widely use for studied and applied in various practical applications in biotechnology and agriculture.
Figure X: Chlorella sp. under the electron microscope
2.3Factors for algal growth and lipid production
2.3.1Mechanism of nutrient absorption
There are multi forms of nitrogen presented in waste water like ammonium (NH4+), nitrate (NO3-) and urea. To uptake these forms of nitrogen, it is necessary to reduce NO3, NO2 and urea into NH4 enzymatically. However addition of energy is required by the reduction reaction so it is preferable for algae to uptake NH4 when it is present. Then NH4+ is transformed into amino acid for protein synthesis. Therefore, nitrogen supply is important for algal cellular structures. (Sze, 1998).
On the other hand, less or even no nitrate uptake occurs while ammonium concentration is above ca 1mM. Therefore ammonium can inhabit the nitrate uptake in algae (Dortch, 1990)
For assimilation of N, carbohydrates is recommend to make corporation.(Rhee,1978); (Eppley & Coatsworth, 1968)
In waste water, phosphate (PO4 3-) is merely absorbed out of all forms of Phosphorus. Phosphate will be stored as polyphosphate when it is available and used when the environment is insufficient of P. P is important for nucleic acid synthesis which leading direct effect on the protein synthesis. (Barsanti & Gualtieri, 2005; Sze, 1998)
220.127.116.11 Total inorganic carbon
Total inorganic carbon consists of carbon dioxide (CO2), carbonic acid (H2CO3) and bicarbonate ion (HCO3-) in water body(Philip 1993). The inorganic carbon is derived from the atmospheric CO2, CO2 produce by the bacterial respiration and from dissolution of carbonate rock. (Philip 1993).
According to Karin (2006) in the photosynthesis process the microalgae can assimilate inorganic carbon. Solar energy is converted to chemical energy with oxygen (O2) as a by-product, and then CO2 and sugars is assimilate and convert in chemical energy. The overall stoichiometric formula for photosynthesis is:
6 H2O + 6 CO2 + light â‡’ C6H12O6 + 6 O2
Michael (1998) mention that the CO2 and HCO3- utilize by the microalgae. HCO3 requiring the enzyme carbonic anhydrase to convert it to CO2
18.104.22.168 Trace elements
The use of micro element is described by (Sze, 1998) that only small concentration is required for algae often as the cofactors in enzyme system.
2.3.2 Environmental factors
The pH value will be increased since photosynthetic CO2 assimilation. Therefore, for high density culture, CO2 adjustment is required for the increased pH. ( Chevalier, Proulx, Lessard, Vincent, and de la Noüe (2000); (Sze, 1998). Also the optimum pH for algal growth is 8.2-8.7. (Barsanti & Gualtieri, 2005). Moreover, the high pH value causes precipitation of phosphate (Barsanti & Gualtieri, 2005; Voltolina, Cordero, Nieves, & Soto, 1999)
Light intensity-Lux (photosynthetic rate)
22.214.171.124 Effect on nutrient concentration to algae
Liebig's Law of Minimum
Rhee(1978) investigated the multiple and single nutrient to algae and find that algal growth is controlled by only single nutrient but not multiple. (Barsanti & Gualtieri, 2005) has described Liebig's law of minimum, which follows the Rhee's findings
Under N depletion, the production of amino acid is decreased and the translation of mRNA is restricted as well as the reduction protein synthesis. Moreover, during the N limitation, the energy transfer to the reaction centre where in PS II is decreased and the reaction centre surrounded by light harvesting antenna is energetically isolated and disconnected in the photochemical reactions. Therefore, the overall photosynthetic rate is limited. In addition, since the respiration rate is linear to photosynthetic rate, respiration rate will be reduced as well. (Barsanti & Gualtieri, 2005)
Moreover, the algae tend to reduce the uptake of nitrogen and start to utilize the cellular nitrogen while in N-limitation.( El-sayed, 2010)
Rhee (1978) has made an experiment on the effect of different N/P atomic ratios and nitrate limitation to algal growth cell composition and nitrate uptake. He found that the cell P was higher and cell carbon was lower in N-limited condition compared with P-limited. Also, he investigated that the growth rate controlled cell compositions, for cell N ; lipid N and protein N in N limited state( i.e. N/P<30), the concentration was remain constant. His experiment also shows that N in chlorophyll-a was shifted to other processes in N-limited condition but also hinder chlorophyll-a production.
Therefore, form (Sze, 1998)and Rhee, cell protein can only remain constant or decrease but not increase.
The paper form Rhee(19780 also pointed out that Surplus P in cell is higher in N- limited condition.
Moreover, there are more papers stating the consequences of N-limited condition. Xin, Hong-ying, Ke, and Ying-xue (2010)stated the composition of thylakoid membrane is decreased in N-limited state. However, there are different result on lipid production compared with Rhee(1978).Takagi, Watanabe, Yamaberi, and Yoshida (2000); Xin et al. (2010)claimed the lipid production and triacylglycerol (TAG)[per cell] is enhanced, but Rhee find that Lipid N remained constant. (Takagi et al., 2000) had focused on the mechanism on lipid production that N-limitation activate of acyl hydrolase and stimulate of the phospholipids hydrolysis. Consequencly,the above reactions increase the intracellular content of fatty acid acyl-CoA which will be converted to TAG.
First, the rate of light utilization for carbon fixation is reduced by lmitating the synthesis and regeneration of substrate in Calvin Cycle. Second, the nucleic acid synthesis is reduced, in term of genome replication or RNA synthesis, which is responsible in transcriptional control, hence, the synthesis of proteins for photosynthetic apparatus is restricted. This effect makes cell metabolism and oxidative problems. Hence, the cell membrane phospholipids will be decreased and the insufficient of nucleic acid limits cell division. If all orthophosphate is used, autotrophic growth will stop. (Barsanti & Gualtieri, 2005)
However there are reverse findings in papers. In P limited condition i.e. N/P> 30, Rhee (1978) found that cell P is lower and carbon started to increase after reaching this N/P ratio. Also, he noted that the concentration cell P was determined by growth rate but not the amount of excess N. Besides, the excessive N is stored as protein fraction.Xin et al. (2010);(Khozin-Goldberg & Cohen, 2006);(Rodolfi et al., 2009) (Takagi et al., 2000) which is increase in the same manner with lipid N (Rhee,1978). Rhee explained with an example that physiological adaptation is the feedback of nutrient stress for growth, which is relied on turnover rate, and when the living condition is insufficient of P amino acid production is the outcome of protein turnover or breakdowns are recombined to synthesize certain kind of enzyme called alkaline phosphatase.(Hino, 1988);(Cao, Song, & Zhou, 2010) explained the use of alkaline phosphatase, which has the function of utilizing organic phosphorus under P deficiency.
N/P ratio to Scenedesmus
Regarding to the experiment from (Li, 2010), the appropriate N/P ratio for nutrient removal to Scenedesmus is proved around 5:1 to 20:1. (Li, 2010)
According to the result, the efficiency of nitrogen removal decrease when TP of 1.3 mg Lÿ1 and TN is more than 10 mg Lÿ1. The three ratios 2:1, 4:1 and 8:1 have a nearly hundred percent removal of nitrogen excluding 12:1 and 20:1. (Li, 2010) It means Scenedesmus will have a less efficient nutrient removal while N/P ratio larger than 8:1. (Li, 2010) On the other hand, the nutrient removal efficiency will also decrease when TN of 10 mg Lÿ1 and TP is lower than 0.5 mg Lÿ1. The three ratios 5:1, 10:1 and 20:1 also have a very high nitrogen removal but excluding 50:1 and 100:1. It shows that Scenedesmus will have a less efficient nutrient removal while N/P ratio larger than 20:1. (Li, 2010)
Considering with different N/P ratio to nutrient removal and biomass production, a range of optimum N/P ratio should be found for the following experiment. From the result of Li Xin, the mass production of Scenedesmus is generally proportional to the concentration and ratio of nitrogen and phosphorous. It means the Scenedesmus will have a higher population while concentration and ratio of nutrient increase. (Li, 2010)
N/P ratio to Chlorella
The most optimum inorganic N/P ratio to freshwater algae was about 6.8 - 10:1. (Darley, 1982) (Reynolds, 1984) (Martin, 1985).
For Chlorella, the optimum N/P ratio is found around 8:1 (Kapdan and Aslan, 2008)
The most efficient N/P ratio to nutrient removal is around 5:1 - 8:1 in a study. (Li, 2010)
Another study showed that chlorella will have a steady state of growth while N/P ratio increases up to 30:1 and this ratio can be confirmed as an optimum ratio for the growth of Chlorella. (Rhee, 1978)(Rhee, 1978)
3.1 Composition of synthetic waste water
There were 4 sets of synthetic waste water used in the experiment, namely 3:1 SWW, 9:1 SWW, 16:1 SWW and 30:1 SWW. The 9:1 SWW was formulated by DSD 2011 yearly averaged sewage N/P data. Also the other sets of SWW are to simulate the N-limited and P limited conditions. The detailed composition was attached in appendix
3.2 Experiment Set up
Scenedesmus quadricauda and chlorella vulgaris was cultivated in 9:1 SWW. The ideal cell number for the experiment was 1x105. 1L of algae is combined with 6L of SWW and triplicate method was performed.
Samples were collected in day 0, 1, 3, 5, 7, 10 and 14. 60ml sample were collected from each tank.1ml sample for cell count, 4ml sample for optical density, 20ml sample for total suspended solid and 30ml filtered sample were collected by 50ml centrifuge tube for nutrient analyses which are ammonia, nitrite, nitrate and orthophosphate. Another 20ml sample with cell is collected for other nutrient analyses which are TN, TKN, TP, TOC and COD. 300ml sample were collected from each tank for lipid content analysis in day 0, 3, 5, 7, 10 and 14.
10ml auto pipette and autoclaved pipette tube were used to collect the sample from the tank and two 50ml centrifuge tube were used to storage the sample. 600ml autoclaved beakers were used to collect 300ml sample from the tank for lipid content analysis and storage in 500ml plastic bottle.
3.3 Physical Parameter
3.3.2 pH & temperature
3.4 Chemical parameters
HACH 60 Low Range (0 to 0.350 mg/L NO2--N) (Method 8507) (Hach Company, 2009)
The analysis of NO2 by HACH Method (8507) performs through chemical reaction by adding NitriVer 3 Nitrite Reagent Powder Pillow into the sample and is analyzing by colorimeter.
Mechanism of analysis- To measure the amount of nitrite present in the water, sulfanilic acid is added and is combined with nitrite to form diazonium salt. During the reaction of nitrite and that chemical, a pink colored compound is formed with chromotropic acid and which is directly proportional to the amount of nitrite. Therefore the readings of measurement can be obtained and quantified by colorimeter.
Ultraviolet Spectrophotometric Screening Method (SM 4500-NO3- B) (Lenore S. et al, )
This method is used only when organic matter content inside the sample is low. Moreover, acidification with 1N HCl may used to avoid interference from hydroxide or carbonate concentrations up to 1000 mgCaCO3/L. Wavelengths used in this method are 220nm and 275nm. The wavelength, 220nm, is able to determine the NO3 - rapidly. However, the sample may also contain other dissolved organic matter and 220nm is also absorbed by those matters. Therefore, the wavelength, 275nm, is also measured for the reason that NO3 - does not absorb at 275nm. Consequently, after the empirical correction, the concentration of NO3 - can be recorded.
0.7218 g KNO3 is prepared and dissolved in 100 ml milli-q water which calls stock solution. Then 10ml stock solution is transferred into 100ml volumetric flask and is diluted by milli-q water which becomes intermediate nitrate solution. After that 1.00, 2.00, 4.00, 7.00 and 35.00ml intermediate nitrate solution are transferred into 50ml volumetric flasks.
Salicylate method (Verdouw, H. et al, 1977)
The ammonia in the sample reacts with phenol, hypochlorite and sodium salicylate, therefore, indophenol compound which is blue in color is formed. Sodiumnitroprusside and potassiumferrocyanide are used and act as catalysts in this method. Sodium hydroxide may need to be added to increase the sensitivity when the pH value is less than 12. Less indophenol is dissociated; therefore, less color will be developed when the pH is less than 12. The wavelength, 660 nm, is used to determine concentration of ammonia.
3.4.4 Total nitrogen
HACH 58 Test 'N Tube (0.0 to 25.0 mg/L N) (Method 10071) (Hach Company, 2009)
All forms of nitrogen in the sample converts into nitrate by alkaline persulfate digestion. After the digestion, sodium metabisulfite is added to eliminate halogen oxide interferences that exist in the sample. Under strongly acidic conditions, nitrate reacts with chromotrophic acid to form a yellow complex and be measured with an absorbance near 420 nm.
Ascorbic Acid Method (SM 4500-P E) (Lenore S. et al, )
Orthophosphate in the sample react with ammonium molybdate and antimony potassium in acid medium to form heteropoly acid-phosphomolybdic acid. Blue molybdenum color is formed by reduction of heteropoly acid-phosphomolybdic acid which ascorbic acid is added. The wavelength, 880nm, is used to determine the concentration of orthophosphate in the sample.
3.4.6 Total phosphorous
HACH 82 (0.00 to 3.50 mg/LPO43-) (Method 8190) (Hach Company, 2009)
Phosphate can be analyzed after it is converted to reactive orthophosphate. Therefore, pretreatment is needed to be carried out. For inorganic phosphates, it will convert to orthophosphate with existence of acid and heat which create a conditions for hydrolysis. For organic phosphate, heat and persulfate is needed for conversion of orthophosphate. After orthophosphate is formed, it will react with molybdate in an acid medium and, therefore, produce phosphomolybdate complex. Blue color can be observed while ascorbic acid react with and reduce the complex. The concentration of total phosphate can be known by DR 890 colorimeter.
3.4.7 Chemical oxygen demand
HACH 16 (0 to 150 mg/L COD) (Method 8000) (Hach Company, 2009)
The sample and strong oxidizing agent, potassium dichromate, are heated for two hours. Then dichromate ion (Cr2O7 2- ) is reduced to green chromic ion (Cr3+). After that the amount of Cr6+ remaining in the sample is measured as the 0- 150 mg/L colorimetric method is used. The concentration of COD is determined from the mg of O2 that is consumed per liter of sample. However, silver which is a catalyst and mercury ions which is used to complex the chloride interference, are appeared in COD reagent and caused interference. Therefore, dilute the sample, which has high chloride concentrations, to reduce the chloride concentration.
3.5 Biological parameters
3.5.1 Optical density
A Shimadzu® UV-1800 UV-Visible Spectrophotometer was used to analyze growth rates of algal species by measuring optical density. The culture was use to find the spectrum and find the maximum absorbance wavelength. 683nm wavelength were use for all optical density measurements to Scenedesmus sp. and Chlorella sp. Standard 10-mm pathlength quartz cuvettes with a sample holding capacity of 3 ml were used to hold the samples in the spectrophotometer. The absorbance was set to zero by blank UV cuvette. Then the sample UV cuvette was inserted and the data was recorded.
3.5.3 Total suspended solid
3.5.4 Chlorophyll-a ,
3.6 Lipid Extraction
Bligh and Dyer method is a low-toxicity, low interference method (Atsushi Hara and Norman S.Radin,1978) and it is a rapid method in lipid extraction .Also,it is a high efficiency method.less than 1% lipid loss in methanol layer(Bligh,E.G. and Dyer,W.J. 1959)
This method, it is important to measure the total lipid content of biological samples accurately. The Bligh and Dyer method of extraction was developed as a rapid but effective method for determining total lipid content(Sara J. Iverson*, Shelley L.C. Lang, and Margaret H. Cooper,2001).First, it need freeze-drying algae cells. The water molecules sublimate directly during the drying process, the sample structure remains intact. Second, 25mg dry cells was added into 3mL solvent (chloroform-methanol) (1/1) and Mix for 30 second. Third, add 1mL solvent (chloroform-methanol)(1/1)and 1 mL 0.75% NaCl water mix again and centrifuge 2000rpm for 10min. They have a reaction and obtain a 2-phase mixture. The upper layer need to remove and collect the lower layer(chloroform layer).Most of the lipid was dissolve in lower layer(chloroform layer). Fourth, combined solvent layers pass through anhydrous sodium sulfate to drying and use gravimetrically to weight. Anhydrous sodium sulfate will absorb the water and get a purify total lipid.