A Look At Microalgae Biology Essay

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Microalgae are prokaryotic or eukaryotic photosynthetic microorganism unified primarily by their lack of roots, leaves, and stems that characterize higher plants. They can be found almost anywhere with water and sunlight as fundamental requirement including lakes, soils, rivers, hotsprings, and the ocean. Marine algae are responsible for creating the majority of the oxygen, where 40%-50% of the photosynthesis among them occurs on earth each year despite their photosynthetic biomass represents only about 0.2% of that on land (Parker et al., 2008). According to Mata el al. (2009), microalgae with its unicellular or simple multicellular structure have high growth rate and are able to live in harsh condition.

Microalgae contain the high value compounds like fatty acids (γ- linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acids (DHA), and etc), pigments (chlorophyll and carotenoids), vitamin such as biotin, vitamin C and E, and others (Converti et al., 2009). Owing to their high quality nutrient components, microalgae are able to enhance the nutritional content of conventional food preparation, and are generally applied in the field of human and animal nutrition. Nowadays, it can also be seen that microalgae for human nutrition are commonly sold in the market in the form of tablets, capsules, and liquids as food supplements and health foods. Indeed, the world's largest producer Hainan Simai Enterprising has an annual production of 200t of algal powder, which accounts for almost 10% of the world output (Spolaore et al., 2005). For animal nutrition, microalgae are primary food source for rotifers, brine shrimp, late larvae, juvenile fishes, and crustaceans in aquaculture (Pratoomyot et al., 2005).

Microalgae rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) mainly EPA (20:5, n-3) and DHA( 22:6, n-3) are always the area of interest in the human nutrition because they are believed to be essential for optimizing human health. DHA is important for brain and eyes development in pre-term and young infants as well as support cardiovascular health in adults. While, EPA is essential for the human metabolism, and is involved in the blood lipid equilibrium, prevent hypertriglyceridemia, anti-inflammatory, and so forth (Kroes et al., 2003; Ward & Singh, 2005; Farjardo et al., 2007). Commercially, production of n-3 PUFA-rich microalgal oils has grown and microalgal oils are already used in 84% of US infant formulas over the last few years (van Beelen et al., 2009).

Apart from that, microalgae contain a multitude of pigments particularly chlorophylls and carotenoids are also of particular interest. In addition to chlorophylls as the primary photosynthetic pigment in microalgae, they are also being used in the dyeing of foodstuffs such as ice cream and cold drinks (Macias-Sanchez et al., 2005; Pulz and Gross, 2004). On the other hands, carotenoids which protect microalgae from excessive solar radiation are also found to be essential to human health and important in commercial application (Felti et al., 2005). For example, -carotene act as pro- vitamin A has been proven to prevent xeropthlamia (Puah et al., 2005), astaxanthin act as natural colorant for muscle in marine animals like fish, lutein, zeaxantin and canthaxantin for chicken skin coloration, or for pharmaceutical purposes (Pulz and Gross, 2004), act as natural food additive (Del Campo et al., 2000& Vilchez et al., 1997), and etc.

1.2. Statement of problems

Chronic diseases including cancer and cardiovascular diseases are the main contributor to death in the world (Abd El-Baky et al., 2007). Due to the substantial increase in the evidence base about the health benefits of n-3 PUFA particularly cardiovascular diseases, it is crucial to ensure its sufficient intake from diet (Kris-Etherton et al., 2009). Virtually, animals and humans lack the ability to synthesize the n-3 PUFA because efficiency of metabolic conversion from α- linolenic acid to EPA and DHA appear to be low (Napier and Sayanova, 2005). Thus, majority of these PUFAs must be obtained through diet. Based on National Health and Nutrition Examination Survey (1999-2000), many of the US population (including all ages and both genders) are actually not meeting current recommendations for omega-3 fatty acid (500mg/day), with the mean intake of EPA and DHA is only about 100mg/day (Erwin et al., 2004; Kris-Etherton et al., 2009). Also, the n-3 PUFA intake of the average Malaysian is currently low, estimated to be 0.3% kcal (recommended intake is 0.3 - 1.2% kcal), with 200 mg from EPA and DHA on fish (Ng, 1995).

Previously, fish and fish oil is the principal dietary source of DHA and EPA. However, declining sources of marine fish stocks and fish oil related to the serious environmental consequences and continuous exploitation, which limiting the protective role of PUFA in human health has prompted the research into new sources of PUFA( Burja et al., 2007). In addition, certain disadvantages of fish oils such as unpleasant odor, possible pollutants, and mixed fatty acid properties also encouraging the discovery of the alternative sources of PUFA (Pulz and Gross, 2004). As PUFA found in fish originating from microalgae consumed, it is reasonable to consider microalgae as potential source of PUFA.

On the other hand, carotenoids are also receiving considerable public attention today. The nutritional and therapeutic relevance of certain carotenoids is always related to the potential pro-vitamin A activity, which can be converted into the vitamin A in the human body (Gouveia & Empis, 2003) as well as other properties like antioxidants and anti-inflammatory. According to Ishida & Chapman (2009), human must obtain carotenoids from dietary sources as they cannot synthesize it.

In most communities in developing countries, carotenoids are the major source of vitamin A (Tee, 1995).Unfortunately, vitamin A deficiency (VAD) remains a significant public health problem globally with an estimated 190 million children and 19 million pregnant women being affected (WHO, 2009). VAD is the leading cause of blindness in children and is also a major contributor to morbidity and mortality from infections, especially in children and pregnant women from low and middle income countries. Prior to the 1950's, vitamin A deficiency in Malaysia has also been reported to be a crucial disorder affecting mainly young children who taking unbalanced diet and from low socio-economic population (National Coordinating Committee on Food and Nutrition, 2005).

Many of the commercially available carotenoids, mainly -carotene supplements (more than 90%) are actually produced by synthetic mean (Leon et al., 2003). Unlike natural carotenoids which is mixture of carotenoids compound, synthetic form of carotenoids may not be readily metabolized rapidly (Puah et al., 2005). Besides, some studies have also shown that the natural carotenoids have antioxidant activity that the synthetic form lacks (Ben-Amotz & Levy, 1996). Accordingly, it has driven the research and development on the production and use of carotenoids from natural origin of microalgae to meet the increasing demand for natural products and act as substitute to the synthetic form of the carotenoids (Abd El-Baky, 2007; Del Campo et al., 2000).

Although microalgae contain important biochemical composition (particularly PUFA and carotenoids) , the extraction methods and efficiencies for plant material, especially for algae, are less well established where there are actually no standard extraction methods for determination of fatty acids contents and carotenoids in microalgae (Wiltshire et al., 2000). Felti et al. (2005) pointed out that absence of standard extraction method for carotenoids are actually attributed to the wide spectrum of analysed materials (foodstuffs, plant, animal, and human samples) and a wide range of carotenoid present. In fact, substantial problem associated with extraction methods may complicate the analysis of pigment and fatty acids composition of microalgae (Wiltshire et al., 1998).

1.3. Significance of study

Since n-3 PUFA and carotenoids play a major role in human health, it is necessary to find out appropriate alternative food sources to meet the requirement of the public today. At present, microalgae offer great possibilities for the isolation of these substances has made it with a great deal of added value (Macias-Sanchez et al., 2009). In addition, there is also a rising trend that people are going for natural products. Thus, functional ingredients such as n-3 PUFA, carotenoids, (e.g. lutein, lycopene, astaxanthin), and other pigments which are extracted from natural sources as like microalgae are preferable (Herrero, Cifuentes & Ibanez, 2005; Cohen & Vonshak, 1991; Mahajan & Kamat, 1995). These functional ingredients have potential commercial applications as nutraceuticals, pharmaceuticals, and important feed ingredient in marine culture has also greatly attracted attention from food industries and agriculture due to the economic and social demands.

Due to high nutritive value of the biochemical compositions of algae cells, mainly fatty acids compositions and carotenoids, research on quantitative determination of these biochemical compositions from microalgae is thus extremely important (Deventer & Heckman, 1996). The data can provide some insights to those researchers interested in this field of study. Meanwhile, it can also raise the awareness of public on the beneficial use of microalgae. Besides, the data may also beneficial to food manufacturers to enhance the nutritional value of their food products. In Malaysia, they are only limited research on determination of fatty acids composition and carotenoids in microalgae. Thus, it is hoped that the data are beneficial in constructing nutrition database in the future.

In order to determine the amount of biochemical composition of interest accurately, it is particularly important to find out efficient extraction methods, which can extract the maximum yield of biochemical composition from microalgae. Previously, various methods for the extraction of fatty acids from microbial biomass have been reported in many studies (Burja et al., 2007; Lewis et al., 2000; Grima et al., 1994; Tran et al., 2009; Lee et al., 2009). However, the different cell structure of microalgae may require further investigation on use of different organic solvents to extract the fatty acids. In addition, there is also lack of studies on the evaluation of extraction methods using different organic solvents for fatty acids from marine microalgae. On the other hand, there is only scarce studies reporting the extractability of carotenoids include an evaluation of extraction methods using different organic solvents (Armenta et al., 2006).

1.4. Objectives

1.4.1General objective

To study the various extraction methods on fatty acids composition and carotenoids from marine microalgae, Nannochloropsis oculata and Chaetoceros gracilis.

1.4.2 Specific objectives

To determine fatty acids composition in microalgae of different solvents extraction by using gas chromatography (GC).

To determine carotenoids (b-carotene, α-carotene, and lutein) in microalgae of different solvents extraction by using high performance liquid chromatography (HPLC).

To compare the fatty acids composition between microalgae Nannochloropsis oculata and Chaetoceros gracilis using different solvents extraction methods.

To compare the carotenoids between microalgae Nannochloropsis oculata and Chaetoceros gracilis using different solvents extraction methods.

. Null hypothesis

H0: There is no significant difference in the mean of fatty acids amount between microalgae Nannochloropsis oculata and Chaetoceros gracilis using different solvents extraction methods.

2) H0: There is no significant difference in the mean of carotenoids amount between microalgae Nannochloropsis oculata and Chaetoceros gracilis using different solvents extraction methods.



2.1. Microalgae

Alga is referred to as any organism with chlorophyll a, and a thallus not differentiated into roots, stems, and leaves while the term microalgae refer to the microscopic algae, and the oxygenic photosynthetic bacteria, such as the cyanobacteria. Microalgae are present in all existing earth ecosystems, not just water but also found on the surface of all type of soils, representing a big variety of species living in a wide range of environmental conditions (Richmond, 2004).

During the past decades, microalgae have begun attracted attention from researchers all over the world where extensive collections of microalgae have been created by researchers in different countries. For example, University of Coimbra (Portugal) is considered as one of the world's largest research institute in the collection of freshwater microalgae, having more than 4000 strains and 1000 species. This collection has revealed the benefit of the large variety of microalgae in a broad diversity of commercial applications, such as value added products for pharmaceutical purposes, food crops for human consumption and as energy source (Mata et al., 2009).

The nutritive quality of microalgae is dependent on several factors including biochemical composition, particle size and shape, toxicity, and ease of digestibility (related to cell wall structure and composition). The biochemical composition of microalgae is influenced by environmental conditions, including light, salinity, temperature, nutrients and growth rate. Particularly, temperature has a major effect on the biochemical composition of some microalgae. It has been reported that some microalgae respond to a decrease in temperature by increasing the ratio of unsaturated fatty acid to saturated fatty acids. Besides, high growth temperature has also been related to significant decrease in protein content, together with increases in lipids and carbohydrates. However, several studies found that the response of microalgae chemical composition to growth temperatures as well as other environment conditions stated above is actually varies from species to species (Durmaz et al., 2009; Renaud et al., 1995; Renaud et al., 2002).

According to Ariyadej et al. (2004), microalgae serve as the important basis of the food chain in the open sea where they are primary food source for larvae of many species of mollusks, crustaceans and fish. On the other hand, microalgae can also help to stabilize and improve the quality of the culture medium for marine animals (green-water technique) attributed to its ability to improve water quality by stabilization of oxygen and pH (Chuntapa et al., 2003& Muller-Feuga, 2000). Recently, microalgae has also been suggested as very good candidates for fuel production attributed to their advantages of higher photosynthetic efficiency, higher biomass production, faster growth compared to other energy crops, and much less land areas requirement (Widjaja et al., 2009).

Today, microalgae biomass and extracts from microalgae have gained a firm position on the market because there is an increasing demand for high-value products from microalgae, such as polyunsaturated fatty acids (PUFA), carotenoids, heat-induced proteins, or immunologically effective compounds. In 2004, the microalgal biomass market produces about 5000t of dry matter/year and approximately US $ 1.25 x 109 /year of turnover is generated (Pulz & Gross, 2004).

2.1.1 Nannochloropsis oculata

Nannochloropsis oculata is a marine unicellular algae belonging to the Eustigmatophyceae class. Eustigmatophytes are unicellular, with coccoid cells and polysaccharide cell walls where the cells do not accumulate starch. Nannochloropsis are characterized by the absence of chlorophyll b and its carotenoids composition is relative simple, containing -carotene, violaxanthin, and a vaucheraxanthin-thin pigments (Rebolloso-Fuentes., 2001). Nannochloropsis are small and nonmotile spheres with its diameter of about 2-4µm (Figure 1). All of its species do not express any other distinct morphological features, and cannot be distinguished by either light or electron microscopy. Typically, they live in salt-water habitats (Hoek, Mann & Jahns, 1995; Richmond, 2004).

Figure 1: Microscopy view of Nannochloropsis sp. (Adapted from Wikipedia, 2009)

Currently, the eustigmatophyte Nannochloropsis is widely used in many aquaculture as the basis of an artificial food chain. In Japan, Nannochloropsis oculata play an important role as cultured feed for the rotifer Brachionus plicatilis, and its concentrated suspensions and frozen biomass are commercially available (Chini Zittelli et al., 1999). The advantage of Nannochloropsis over other unicellular algae is primarily its unique fatty acid composition, where it has been recognized as a good potential source of fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid( DHA), an important polyunsaturated fatty acid in human diet (Rodolfi et al., 2003). Previously, a study assessed the fatty acid composition of 15 species of marine microalgae found that Nannochloropsis oculata exhibited relative simple fatty acids profile, dominated by three components- 16:0, 16:1 (n-7), and 20:5 ( n-3) with a near total absence of C18 PUFAs ( Zhukova & Aizdaicher, 1995).

In addition, this microalga is also well known as a source of different valuable pigment like carotenoids (astaxanthin, zeaxanthin and canthaxanthin) and chlorophyll a,

which is essential for commercial use. (Marcillaa et al., 2009). Indeed, the use of Nannochloropsis has been recognized in human diets where several studies have been conducted to investigate its incorporation in foods such as noodles to improve nutritional profile ( Rebolloso- Fuentes, 2001).

2.1.2 Chaetoceros gracilis

Chaetoceros sp. is probably the largest genus of marine diatoms with approximately 400 species described. Diatoms are a major group of eukaryotic algae, and are one of the most common types of phytoplankton. Most diatoms are unicellular, although they can exist as colonies in the shape of filaments or ribbons. Commonly, diatoms are between 20-200 microns in diameter or length. However, some species of diatoms can reach up to 2 millimetres in length. Besides, most diatoms are non-motile, although some may move using flagella. A unique feature of these diatom cells is that they are encased within a cell wall made of silica (hydrated silicon dioxide) called frustule, which provide the diatom access to the external environment for process such as waste removal and mucilage secretion. Diatoms are belonging to group of heterokonts in which its yellowish-brown chloroplasts are the typical characteristic with four membranes and containing pigments such as the carotenoid (fucoxanthin) as shown in Figure 2 (Wikepedia, 2009).

They play an important role in oceans, where they contributes up to 45% of the total oceanic primary production. Diatoms, in particular, are regarded as useful neutral lipid sources. Indeed, some marine species of Chaetoceros are critical food sources for marine cultures. For example, Chaetoceros gracilis has been shown to be an adequate exclusive feed source for larvae and postlarvae of the shrimp Metapenaeus ensis (Chu, 1989).

Besides, Tsitsa-Tzardis et al. (1993) also reported that Chaetoceros sp. is the most effective phytoplankton in promoting juvenile oyster growth because its isolation contains cholesterol which is the major sterol in oysters. In addition to high lipid content, diatoms are also abundant of certain polyunsaturated fatty acids (PUFAs) such as large amounts of eicosapentaenoic acids (EPA). Thus, the ability to manipulate the quantity and quality of lipid in Chaetoceros species is effective for several applications such as renewable resource as fuel and food (McGinnis et al., 1997). A study has also shown that Chaetoceros gracilis contain primarily C14, C16, and C20 fatty acids composition, specifically 14:0, 16:0, 16:1, and 20:5 under the different condition of temperature, light intensity and silicate (Mortensen et al., 1988).

Figure 2: Microscopy view of Chaetoceros gracilis

(Adapted from www.reed-mariculture.com/microalgae/chgra.htm)

2.2. Carotenoids

Carotenoids are natural pigments that are uniquely synthesized in plants, algae, fungi, and bacteria (Sandmann, 2001). The pigments may vary in yellow, orange, and red color. The structure of the carotenoids is long, aliphatic, and conjugated double bond systems (Felti et al., 2005). They are isoprenoid polyenes formed by head-to-tail linkage of C5 isoprene unit, and only in the center of the molecule is a tail-to-tail lingkage that makes the molecules symmetrical (Nollet, 2000). Most of carotenoids contain 40 carbons atoms ( C40 carotenoids) and can be divided into two groups, which are hydrocarbon (made up of carbon and hydrogen atoms only) called as carotenes and oxygenated derivatives of carotenes (containing at least one oxygen function such as hydroxy, keto, epoxy, methoxy or carboxylic acid groups) called as xanthophylls (Rodriguez-Amaya, 2001). In nature, most carotenoids occur in the all-trans form. Cis isomers are frequently present in small amounts ( Nollet, 2000).

Some common structures of carotenoids are shown as below:















(Adapted from http://www.food-info.net/uk/caro/stru.htm)

2.2.1 Physiochemical properties of carotenoids

Carotenoids are lipophilic pigments, where they are insoluble in water and soluble in organic solvents, such as acetone, alcohol, ethyl ether, chloroform, and ethyl acetate. The solubility of the carotenoids depends on its specific structural. For example, carotenes are readily soluble in petroleum ether, hexane, and toluene; xanthophylls dissolve better in methanol and ethanol. Accordingly, the proper selection of extraction method and pre-concentration agent are especially required to obtain reliable result.

The presence of conjugated double-bond system which forms the light-absorbing chromophore gives carotenoids their intensely color and provides the visible absorption spectrum. This characteristic is important for their identification and quantification, where changes or loss of color during analysis may indicate degradation or structural modification of carotenoids. Indeed, the first diagnostic tool for the identification of carotenoids is the ultraviolet and visible spectrum. The greater the number of conjugated double bonds, the higher the λmax values.

The highly unsaturated carotenoids are susceptible to the isomerization and oxidation particularly in the presence of heat, light, acids, trace of metal ions and availability of oxygen. Isomerization of usual configuration of carotenoids, which shift from trans-cis form, may have effect on the overall shape of the carotenoids molecules and thus its properties like loss of color or provitamin A activity. On the other hand, extensive losses of carotenoids may arise from oxidation. Therefore, concern must be undertaken during carotenoids analysis. For example, carotenoids analysis carried out under vacuum and a nitrogen or argon atmosphere can assist to exclude oxygen. Meanwhile, antioxidants (e.g. butylated hydroxytoluene, BHT) can also be added to minimize oxidation especially during prolonged storage. Besides, carotenoids analysis must also be done in subdued light. For example, vessels containing carotenoids should be wrapped with aluminum foil to prevent from direct sunlight exposure. Due to thermolability of carotenoids, its extracts or solution should also be concentrated in a rotary evaporator at reduced pressure and at temperature below 40oC and the solvent used for extraction should be low boiling point. Yet, it is important to prevent the extracts from complete dryness in rotary evaporator as it may cause degradation of carotenoids especially lycopene (Felti et al., 2005; Rodriguez-Amaya, 2001; van den Berg et al., 2000).

2.2.2 Health benefits of carotenoids

Human must obtain carotenoids from the dietary sources since they cannot synthesize it. Usually, plants serve as the primary sources of carotenoids to human (Ishida & Chapman, 2009). These compounds are essential to the human health attributed to its physiological and biological functions. The essential role of carotenoids especially -carotene as provitamin A has been best documented for many years (Ishida & Chapman, 2009; Holden et al., 1999). With its potential provitamin A activity in human diet, they are well-known to be capable of preventing xeropthlamia, a night blindness disease (Takahashi et al., 2006). In addition, epidemiological studies have also shown an intake of carotenoids (lutein and zeaxanthin) correlated with decreased risk of cataract and age-related macular degeneration (AMD) (Basu et al., 2001).

Moreover, carotenoids in particular lycopene and -carotene have also been claimed as displaying anticancer activities. For example, overview of observational epidemiologic studies has shown that carotenoids are associated with reduced risk of cancer mainly lung and stomach cancer (van Poppel & Goldbohm, 1995). In the case of cardiovascular diseases (CVD) risk, several ecologic, cross-sectional, and cohort studies suggested a protective association between carotenoids (-carotene) and cardiovascular disease and its risk indicator (Kohlrneier & Hastings, 1995). Also, some research has shown the efficiency of carotenoids (lycopene and -carotene) in skin protection by providing protection against ultraviolet radiation sunlight ( Sies & Stahl, 2004 ) as well as having immunomodulatory effects such as the reduction in UV-induced immunosuppression ( Fuller et., 1992).

In fact, protective effects of carotenoids against certain diseases stated above are associated to its antioxidant properties, which are capable of inactivating reactive oxygen species (ROS), thereby help delay or prevent oxidative damage (O'Connell et al., 2007). In addition, carotenoids also act as free radical scavenger (Zanfini et al., 2009). Free radical can cause damage to the structure and function of cell membranes, DNA, and protein (Ishida & Chapman, 2009).

Owing to functional chemical properties of carotenoids, they are increasingly used in foods, pharmaceutical and cosmetics industries. According to Lyn Patrick (2006), more than 600 carotenoids have been isolated in nature. However, only a small number of carotenoids are used commercially, including -carotene, lycopene, astaxanthin, canthaxanthin, and lutein (Del Campo et al., 2000). They play vital role as natural food colorants (e.g orange juice), food additives, and feed additives in agriculture (poultry, fish) (Spolaore et al., 2006). For example, astaxanthin is best known for creating pinkish red color in flesh of salmonids, shrimp, lobsters, and crayfish (Jin & Melis, 2003). Annual worldwide sales of this particular compound are estimated at US$200 million with its market value of US$2500/kg (Hejazi & Wijffels, 2004; Spolaore et al., 2006).

Until the early 1980s, commercial production of -carotene was all synthetic form. During the 1970s, natural -carotene derived from microalgae (i.e. Dunaliella) has been discovered by researchers and this discovery is currently a substantial and growing industry (Richmond, 2004).

2.3. Fatty acid

The structure of fatty acid comprised of hydrocarbon chain (carbon and hydrogen atoms) with a carboxyl group (COOH) at one end and a methyl group (CH3) at the other end (Figure 3). Usually, fatty acid chain contains even number of carbons and lengths vary from 2 to 80 with either presence or absence of double bonds.

Figure 3: Structure of saturated fatty acid

(Adapted from http:// chemistryinmydailylife.blogspot.com/)

Fatty acids without double bonds are known as saturated fatty acid while fatty acids containing double bonds are known as unsaturated fatty acids. If there is only one double bond present in an unsaturated fatty acid, it is said to be a monounsaturated fatty acid (MUFA) while if there is two or more double bonds present in an unsaturated fatty acid, it is called as polyunsaturated fatty acid (PUFA). Unsaturated fatty acids are named by identifying the number of double bonds and the position of the first double bond counted from the methyl terminus. For example, 18-carbon fatty acid with two double bonds in the acyl chain and with the first double bond on carbon number six from the methyl terminus is notated as 18:2, n-6. The common name of this fatty acid is linoleic acid (Lunn & Theobald, 2006).

In general, fatty acids are critical for energy storage as triacylglycerol (3 fatty acids attached to a glycerol backbone), for cell membrane formation with a phospholipids bilayer (2 fatty acids attached to a phospholipid polar head group), and for the formation of cholesteryl esters (one fatty acid attached to free cholesterol). In human plasma, the most abundant fatty acids are the saturated fatty acids: palmitic acid (16:0) and stearic acid (18:0), the monounsaturated fatty acid: oleic acid (18:1n-9), and the polyunsaturated fatty acid: linoleic acid (18:2n- 6) (Johnson & Schaefer, 2006).

2.3.1 Physiochemical properties of fatty acids

Double bonds in unsaturated fatty acids can be arranged in one of two ways with either cis- or trans- configurations. Double bonds with cis- configuration are mostly found in foods, where both hydrogen atoms are found on the same side of the fatty acid. This fatty acid exists as liquid at room temperature since the presence of a cis- bond in a fatty acid lowers the melting point of the fatty acid. On the other hand, trans-fatty acids, where the hydrogen atoms are situated on opposite sides of the fatty acid, are less common in nature. Commonly, they are produced during the hydrogenation (hardening) of unsaturated oils as was traditionally used in margarine manufacture (Lunn & Theobald, 2006).

The susceptible of different fats to oxidative rancidity is also varies, depending upon the degree of unsaturation of the component of fatty acids besides the availability of antioxidants, and the presence of transition metal such as iron and copper. In other words, the greater the number of double bond in fatty acid, the more prone it is to peroxidation. Thus, PUFA are more susceptible to oxidation as compared to MUFA (Allen & Hamilton, 1994).

On the other hand, the physical properties of fatty acids such as melting point, water solubility, viscosity, and refractive index are also depending on the length chain of fatty acids as well as its degree of unsaturation. Melting point of fatty acids will increase with carbon number while the presence of double bond in the fatty acids (unsaturated fatty acids) will have lower melting point as compared to saturated fatty acids. Then, an increase in the chain length and degree of unsaturation will decrease the water solubility of fatty acids, yet increasing its refractive index. Unlike other physical properties, viscosities of fatty acid will increase with the chain length, but decrease with an increasing degree of unsaturation (Allen & Hamilton, 1994).

2.3.2 Health benefits of PUFA

PUFA are always claimed to be most beneficial to human health among the various type of fatty acids. The predominant PUFA are the omega-3 and omega-6 fatty acid. In human body, there are two PUFA namely omega-6 linoleic acid (18:2, n-6) and omega-3 α-linolenic acid (18:3, n-3) cannot be synthesized because they lack the desaturase enzymes necessary to synthesize these components. As a result, they must be obtained from plant material in the diet and are known as essential fatty acids (Tapiero et al., 2002). Once consumed, the body can metabolize them by the introduction of further double bonds (desaturation) and by lengthening the acyl chain (elongation) to form other PUFAs such as arachidonic acid (AA, 20:4, n-6), eicosapentaenoic acid (EPA; 20:5,n-3) and docosahexaenoic acid (DHA; 22:6, n-3) that are more readily used in the body ( Ng, 2006). The pathway of its conversion is shown in Figure 4 (Calder, 2004).

Figure 4: Pathway of the conversion of linoleic and α-linolenic acids into their longer chain derivatives

PUFA particularly n-3 fatty acids ( EPA and DHA), which supplied mostly from fish or fish oil are believed to have beneficial effect in prevention and treatment of a wide range of human diseases and disorders although their underlying mechanism of action are still unclear (Zittelli et al., 1999; GISSI-Prevenzione Investigators, 1999). They are considered as pivotal component in the human diet (Napier & Sayanova, 2005).

According to Ward & Singh (2005), health benefits of PUFAs has began drawing attention from researchers when it was noted that populations with high consumption of fish had a much lower incidence of heart disease. Indeed, there is substantial evidence from epidemiological and case control studies shown the protective effect of n-3 fatty acids in reducing the risk of cardiovascular mortality such as myocardial infarction (Calder, 2004). A study also suggested that consumption of fish (good source of n-3 fatty acids) at least once per week may reduce the risk of sudden cardiac death in men (Albert et al., 1998). As information, high MUFA intake has also been shown associated with reduced cardiovascular disease risk (Kris-Etherton, 1999).

Apart from that, the n -3 PUFAs such as EPA and DHA have also been suggested to reduce risk for metabolic syndrome, which is currently represent major public health problem in industrialized societies by lowering plasma triglycerides, reducing hypertension, improve lipoprotein profile and insulin sensitivity (Graham et al., 2004). Metabolic syndrome is described as 'clustering' of several risk factors for cardiovascular disease including obesity (particularly abdominal obesity), dyslipidaemia, insulin resistance and hypertension. (Nugent, 2004).

There is also increasing evidence from studies shown that n -3 fatty acids play a key role in brain and retina development in preterm infants and also for the prevention or delaying the process of dementia and age-related macular degeneration (Dangour & Uauy, 2008; Johnson & Schaefer, 2006; Hoffman et al, 1993). Furthermore, n-3 fatty acids are also important component of cell membrane phospholipids from some tissues like retina, brain, and myocardium (Surette, 2008). Membranes containing high proportions of the long chain PUFAs can ensure efficient intracellular metabolism as they are most permeable to water (Lunn & Theobald, 2006).

Recent studies have revealed the importance of omega-6/omega-3 fatty acids ratio, whereby a very high omega-6/omega-3 ratio (excessive amounts of omega-6 fatty acids) as is found in today's Western diets, promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases. On the contrary, lower omega-6/ omega-3 ratio (increased levels of omega-3 fatty acids) exert suppressive effects. This implies the importance of small ratio of omega-6/omega-3 fatty acids in reducing the risk of many of the chronic diseases ( Simpoulos, 2001).

Although there is increasing awareness of general public regarding health-beneficial properties of dietary consumption of n-3 PUFA such as EPA and DHA, the natural resources that provide these oils are in danger of being exhausted and may inadequate for supplying the expanding market to meet its high demand (Opsahl-Ferstad et al. 2003; Sijtsma & de Swaaf, 2004). Recently, microalgae has been exploited and studied to be the alternative processes for PUFA-production (Sijtsma and de Swaaf, 2004). However, DHA is the only current algal PUFA commercially available (Spolaore et al., 2006).

2.4. Mechanism of extraction

Extraction is the process by which analytes from sample matrix are selectively separated from other, often undesired compounds. Extraction of analytes from sample matrix is an important step prior the HPLC or GC analysis. One of the most common extraction methods is two-phase system for liquid-liquid extraction (also called biphasic aqueous/organic system), which depend on differential solvent solubility and solvent immiscibility. Virtually, the use of two- phase aqueous organic systems has been proved to be an effective way to separate the poorly water-soluble compounds (Leon et al., 2003).

A polar, aqueous solutions are often paired with non-polar organic solvents such as chloroform which is volatile, non reactive, immiscible with and denser than water to form a two-phase system for liquid-liquid extraction as shown in Figure 5. This allows the separation of analytes in either polar or non-polar solvents according to its relative solubility, and for subsequent chromatographic separation and analysis (Sana & Fischer, 2007).


Figure 5: Mechanism of two-phase system for liquid-liquid extraction

( Adapted from Sana & Fischer, 2007)

Basically, the sampled material matrix plays an important role in extraction process as analyte may be strongly bound to the matrix, which affects the extraction efficiency (Anklam & von Holst, 2005). In whole-cell systems, organic solvent might be incorporated within membrane lipids causing the disruption or alteration of membrane functions, inactivation or denaturation of membrane bound enzymes, collapse of transport mechanisms, and even cell lysis when a cell is exposed to biocompatible solvent (Cruz et al., 2001). In general, the cells will carry out the bioconversion when a biocompatible organic solvent is in contact with the aqueous phase, where the biochemical compounds or analytes are continuously extracted into the organic phase due to a permeability effect of the solvent on cell membrane (Xu et al., 2004).

On the other hand, using mixed solvent has also gained the interest of researchers. Indeed, the use of mixed solvent compared to single solvent will increase the permeability effect of solvents and concentration of the solvent accumulated in the cell membrane. Mojaat et al. (2008) reported that the mixture dichloromethane-decane is able to extract -carotene six times more efficiently than the pure decane. Accordingly, the selection of the solvent is particular important in order to ensure extraction efficiency, where it must satisfy certain requirements such as biocompatibility, maximum solubility and important extraction ability.

2.4.1 Fatty acids extraction in microalgae

Fatty acids exist in a great variety of molecules which differ in chemical and physical characteristics poses a challenge with respect to the application of optimal extraction methods for quantitative extraction of the fatty acids from different matrices (Jensen, 2008). Indeed, different methods can actually lead to a three-fold difference in lipid contents (Smedes, 1999).

In spite of many extraction methods developed recently including supercritical or subcritical fluid extraction, microwave and ultrasound assisted extraction, the involvement of organic solvent in lipid extraction from microalgae including chloroform/methanol mixture, was still found superior in comparison to these techniques (Mendes et al., 2006). The extraction yields of lipids/fatty acids using solvents dependent upon the nature of the microbial cell structures, the sample-solvent ratio, temperature, time of extraction and the extraction techniques used (Chaiklahana et al., 2008).

In literature, Bligh & Dyer method and Folch method are the one most common used to determine the fatty acids in various food sources including microalgae, where mixtures of chloroform and methanol were used to extract lipids/fatty acids. (Lewis, Nichols, & McMeekin, 2000; Molina Grima et al., 1994; Wiltshire et al, 2000). Other solvents may include hexane, ethanol, diethyl ether, methanol, acetone, petroleum ether, and so forth.

In fact, solvent extraction methods have been evaluated for the GC analysis of fatty acids/lipids from different food products (Sahasrabudhe & Smallbone, 1983; Somashekar et al., 2001), but it does not include marine microalgae. Many studies regarding the evaluation of extraction methods for fatty acids analysis from microalgae are focused on the different extraction techniques (e.g. direct saponification, microwave-assistant extraction, supercritical fluid extraction, and others) instead of evaluation of solvent extraction methods (Burja et al., 2007; Grima, E.M., 1994).

2.4.2 Carotenoids extraction

During carotenoids analysis of foods, significant errors are typically arising from extraction procedures and the error associated with chromatography is only minor. However, it is difficult to standardize the variety of published methods available between laboratories (Rodriguez-Amaya et al., 2006).

In literature, the most widely accepted methods involve extraction of carotenoids with one or mixture organic solvents including hexanes, chloroform, dichloromethane, tetrahydrofuran, methanol, ethanol, ethyl acetate, n-butyl-alcohol, diethyl ether, 2-propanol, and petroleum ether. Many extraction procedures require freeze-dried material, involve saponification to remove lipids and chlorophylls and antioxidants utilization (e.g., butylated hydroxytoluene (BHT) or pyrogallol) (Howe & Tanumihardjo, 2006). Although numerous extraction procedures of carotenoids from foods involved various types of solvent as well as solvent combinations have been proposed previously, the relative efficacies of these existing methods have not yet been evaluated, especially on microalgae (Taungbodhitham et al., 1998). In any case, proper selection of the solvent for a particular carotenoid extraction is of principally importance (Feltl et al., 2005).

2.5. High Performance Liquid Chromatography (HPLC)

2.5.1 Principle

The underlying principle of chromatography is that molecules not only dissolve in liquids but can also absorb (dissolve) on to or interact with the surface of solids. If a molecule dissolved in a liquid (mobile phase) is passed down a column of solid particles (stationary phase) with which it interacts, it will take longer time to pass down the column as it spends some time dissolve in the mobile phase and some on the stationary phase. In contrast, to those molecules that do not interact with stationary phase, they will elute first from the column. The separation of molecules by chromatography varies, depending upon physical properties of the molecules in samples such as solubility in water, solubility in organic solvents, net positive or negative charge, and size. In sum, HPLC is a physical method of the separation of one type of molecule from others and thus can be used to identify and simultaneously quantify many components of a complex mixture (Bird, 1989).

2.5.2 Application of reverse-phase HPLC (RP-HPLC)

Among all different separation modes, RP-HPLC is the most widely used in modern column liquid chromatography (more than 70%) which utilizes a non-polar stationary phase and a polar mobile phase. In RP-HPLC, solutes are retained due to hydrophobic interactions with the non-polar stationary phase. Thus, the higher the polarity of the solutes, the more likely it is being eluted from column. Recently, RP-HPLC has become the method of choice for both qualitative and quantitative analysis of carotenoids besides determination of water and fat soluble vitamins, food dyes, phenolic flavor compounds (such as vanillin), and other pigments like chlorophyll ( Nielsen, 1998 ; Nollet, 2000).

2.6. Gas chromatography (GC)

2.6.1 Principle

GC is a powerful and widely used tool for the separation, identification and quantification of components in a mixture. In this technique, a sample is converted to the vapor state and a flowing stream of inert carrier gas (usually helium or hydrogen) sweeps the sample into a thermally-controlled column. The column is usually packed with solid particles that are coated with a non-volatile liquid, referred to as the stationary phase. As the sample mixture moves through the column, sample components that interact strongly with the stationary phase will spend more time in the stationary phase than the moving carrier gas. Accordingly, more time is required for sample components to move through the column (Truman State University CHEM 222 Lab Manual, 2008).

2.6.2 Application of GC

GC is used broadly in various analyses, including determination of fatty acids, triglycerides, cholesterol, and other sterols, gases, solvent analysis, water, alcohols, and simple sugars, as well as oligosaccharides, amino acids, vitamins, food additives, antioxidants, and so forth. However, GC is ideally suited only to the analysis of thermally stable volatile substances. According to Seppänen-Laakso et al. (2002), GC is a highly applicable tool in micro-scale analytical work in a number of research areas of fatty acids. Normally, GC requires an esterified sample of fatty acids such as the methyl ester (FAME), and this increase the volatility lipids components, thus providing better separation ( Tran et al., 2009).