**6.1 Lipid extraction and biodiesel formation from microalgal biomass**

Conventional methodologies for lipid extraction involve the use of toxic organic solvents such as chloroform, methanol and hexane. While the solvent extraction process is effective it is difficult to adopt on a large scale. Novel methods for lipid extraction involve technologies such as acoustics, sonication, the use of mesoporous nanomaterials, and amphiphilic solvents (Cheng & Ogden 2011). Super critical fluid extraction has been reported to be safer and faster than the conventional solvent extractions (Andrich et al., 2005). Another technique of "milking" microalgae manipulates the hydrophobicity of the solvent system, which allows the extraction of lipids from living algal cells. A flat panel two-phase bioreactor designed by Hejazi & Wijffels (2004) was used in the milking process for *Dunaliella salina* production. In this process, microalgal cells grown under optimal growth conditions are stressed by excess light to stimulate the production of β-carotene, which is then extracted from the cells using lipophilic compounds. Important considerations for application of this "milking" process includes: a) cell wall and membrane properties of the microalgal strain; b) location and accumulation of the product inside the cell; and c) biocompatibility and chemical properties of the solvent used for the "milking" process (Hejazi & Wijffels 2004).

Lipid conversion to biodiesel can easily be achieved by chemical trans-esterification, enzymatic conversion, and catalytic cracking. Chemically, biodiesel is comprised of monoalkyl esters of fatty acids that are derived from triacylglycerols. These triacylglycerides can be produced from crop or microalgae oils, animal fats, and waste cooking oils. Such biodiesel is miscible with petroleum diesel and thus suitable blends of biodiesel-diesel can be obtained. These are denoted as BXX, where XX is the percent of biodiesel in the blend. For example, B40 is 40% biodiesel in a diesel-biodiesel blend (Tat et al., 2007).

### **6.2 Fuel properties of microalgal biodiesel**

There are several properties which determine the suitability of biodiesel as a biofuel including cetane number, kinematic viscosity, cold flow and oxidative stability (Ramos et al, 2009). These properties are greatly influenced by the fatty acid compositions of the feedstock oils (Figure 3, 4). Therefore, to determine the best composition of biodiesel, it is necessary to study the lipid profile of potential biomass feedstocks. The most common fatty acid methyl esters present in most biodiesel are palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) (Knothe, 2008). Biodiesel obtained upon trans-esterification of these common fatty acids has many advantages over petroleum-derived diesel fuel. However, there are several performance problems with biodiesel, notably poor cold flow properties, lower cetane number and insufficient oxidative stability (Knothe, 2009). Ignition delay time and combustion quality of a diesel fuel is determined by the cetane number. An adequate cetane number is required for better engine performance and a high cetane number is also associated with

Eco-Physiological Barriers and Technological Advances for Biodiesel Production from Microalgae 171

number depends on the microalgal feedstock biomass from which the oil is derived and the alcohol that is used during the trans-esterification process. The cetane number decreases with increasing unsaturation and increases with increasing chain length without branching of the CH2 moities. However, the straight chain fatty acid methyl esters with carbon numbers of 6, 10, 12, 14, 16 and 18 show a non-linear increase with carbon number. Esters of saturated fatty acids (such as palmitic and stearic acids) give high cetane numbers. In summary, cetane number increases with chain length, decreases with unsaturation (or the number of double bonds) and decreases as double bonds and carbonyl group move toward the centre of the chain (Graboski & McCormik, 1998). Oils from different microalgal feedstocks will have different fatty acid compositions and such fatty acids are different with respect to the chain length, degree of unsaturation, or presence of other chemical moieties. While esters of long chain saturated fatty acid show higher cetane number, they can have a high cloud point that results in nozzle clogging (see below). On the other hand, esters of unsaturated fatty acid show low cetane number and are prone to oxidation. Among the non-algal biodiesel feedstocks, palm oil is reported to have the highest cetane number (61), whereas peanut, sunflower and corn oils

To improve the properties of biodiesel, it is necessary to enrich the biodiesel with fatty esters with desirable properties. Genetic engineering can provide important opportunities in this regard. For example, a soybean transgenic crop variety designated 335-13 has been genetically altered to increase concentration of oleic acid by more than 85%, with a corresponding reduction of palmitic acid content to less than 4% (Tat et al., 2007). Saturated and long chain fatty acids gave a high cetane number, which increases with increasing saturation and the chain length (Knothe et al*.*, 2003; Bajpai & Tyagi, 2006; Knothe, 2009). Esters of highly unsaturated fatty acids such as linoleic (18:2) and linolenic (18:3) acids lower the cetane number (Knothe et al., 2003). According to Knothe et al. (2003) high cetane numbers were observed for esters of saturated fatty acids such as palmitic (C16:0) and stearic (C18:0) acids. Feedstock oils rich in these fatty acid

A higher content of polyunsaturated fatty esters in oil derived from the feedstock can reduce the quality of biodiesel upon storage due to oxidation in the presence of air, light, heat, peroxides, trace metals, or even the structural features of the fatty acids themselves. Thus, oxidation stability is another major issue affecting the use of biodiesel fuels (Ramos et al., 2009; Knothe, 2009). The number and position of double bonds in the chains of unsaturated fatty esters are both factors affecting the susceptible to autoxidation reaction (Bajpai & Tyagi, 2006; Frankel, 2005). Most biodiesel fuels contain significant amounts of alkyl esters of oleic, linoleic and linolenic acids, which influence the oxidation stability of the fuels. The relative rates of autoxidation for oleic acid methyl ester, linoleic acid methyl ester and linolenic acid methyl ester have been reported to be 1, 41 and 98 respectively (Knothe et al.,

The high viscosity of non-esterified vegetable oils leads to operational problems in diesel engines by increasing the level of engine deposits. Reduction of the high viscosity of vegetable oils is facilitated by the production of alkyl esters from the oil by transesterification. Since viscosity increases with decreasing temperature, handling of fuels in lower temperatures is facilitated by this lower viscosity as well (Knothe, 2009). This can be achieved by increasing the length and degree of saturation of the carbon chains (Knothe et al., 2005). Wax settling and plugging of filters and fuel lines are typical

typically have a cetane values of 53 (Ramos et al., 2009).

compounds would have higher cetane values.

2005; Frankel, 2005; Ramos et al., 2009; Knothe, 2009).

biodiesel with good cold start properties and reduced NOx exhaust emissions (Ramos et al., 2009; Knothe, 2009; Ladommatos et al., 1996).

Fig. 3. Fatty acid composition of various vegetable oil crop feedstocks. (Data from Ramos et al 2009). HOSME (high oleic sunflower methyl ester), PME (palm methyl ester), RME (rape methyl ester), SME (soy methyl ester), CME (corn methyl ester), OME (olive methyl ester).

Fig. 4. Fatty acid composition of different microalgal species (Huerlimann et al., 2010). Data for *Chlorella* sp and *Scenedesmus* sp obtained from the author's investigations.

The cetane number of biodiesel can be determined using established standards such as ASTM D975 in the USA. Cetane is the common name for a long straight-chain hydrocarbon, hexadecane (C16H34), which is the high quality standard on the cetane scale with an assigned Cetane number of 100. In contrast, the low quality standard is a highly branched compound heptamethylnonane (C16H34) which exhibits poor ignition quality and has an assigned cetane number of 15 (Gopinath et al 2010). According to ASTM D975, conventional diesel fuel requires a minimum cetane number of 40 whereas a minimum of 47 has been prescribed for biodiesel (ASTM D6751) or (EN14214). For biodiesel, the cetane

biodiesel with good cold start properties and reduced NOx exhaust emissions (Ramos et

Fig. 3. Fatty acid composition of various vegetable oil crop feedstocks. (Data from Ramos et al 2009). HOSME (high oleic sunflower methyl ester), PME (palm methyl ester), RME (rape methyl ester), SME (soy methyl ester), CME (corn methyl ester), OME (olive methyl ester).

Fig. 4. Fatty acid composition of different microalgal species (Huerlimann et al., 2010). Data

The cetane number of biodiesel can be determined using established standards such as ASTM D975 in the USA. Cetane is the common name for a long straight-chain hydrocarbon, hexadecane (C16H34), which is the high quality standard on the cetane scale with an assigned Cetane number of 100. In contrast, the low quality standard is a highly branched compound heptamethylnonane (C16H34) which exhibits poor ignition quality and has an assigned cetane number of 15 (Gopinath et al 2010). According to ASTM D975, conventional diesel fuel requires a minimum cetane number of 40 whereas a minimum of 47 has been prescribed for biodiesel (ASTM D6751) or (EN14214). For biodiesel, the cetane

for *Chlorella* sp and *Scenedesmus* sp obtained from the author's investigations.

al., 2009; Knothe, 2009; Ladommatos et al., 1996).

number depends on the microalgal feedstock biomass from which the oil is derived and the alcohol that is used during the trans-esterification process. The cetane number decreases with increasing unsaturation and increases with increasing chain length without branching of the CH2 moities. However, the straight chain fatty acid methyl esters with carbon numbers of 6, 10, 12, 14, 16 and 18 show a non-linear increase with carbon number. Esters of saturated fatty acids (such as palmitic and stearic acids) give high cetane numbers. In summary, cetane number increases with chain length, decreases with unsaturation (or the number of double bonds) and decreases as double bonds and carbonyl group move toward the centre of the chain (Graboski & McCormik, 1998). Oils from different microalgal feedstocks will have different fatty acid compositions and such fatty acids are different with respect to the chain length, degree of unsaturation, or presence of other chemical moieties. While esters of long chain saturated fatty acid show higher cetane number, they can have a high cloud point that results in nozzle clogging (see below). On the other hand, esters of unsaturated fatty acid show low cetane number and are prone to oxidation. Among the non-algal biodiesel feedstocks, palm oil is reported to have the highest cetane number (61), whereas peanut, sunflower and corn oils typically have a cetane values of 53 (Ramos et al., 2009).

To improve the properties of biodiesel, it is necessary to enrich the biodiesel with fatty esters with desirable properties. Genetic engineering can provide important opportunities in this regard. For example, a soybean transgenic crop variety designated 335-13 has been genetically altered to increase concentration of oleic acid by more than 85%, with a corresponding reduction of palmitic acid content to less than 4% (Tat et al., 2007). Saturated and long chain fatty acids gave a high cetane number, which increases with increasing saturation and the chain length (Knothe et al*.*, 2003; Bajpai & Tyagi, 2006; Knothe, 2009). Esters of highly unsaturated fatty acids such as linoleic (18:2) and linolenic (18:3) acids lower the cetane number (Knothe et al., 2003). According to Knothe et al. (2003) high cetane numbers were observed for esters of saturated fatty acids such as palmitic (C16:0) and stearic (C18:0) acids. Feedstock oils rich in these fatty acid compounds would have higher cetane values.

A higher content of polyunsaturated fatty esters in oil derived from the feedstock can reduce the quality of biodiesel upon storage due to oxidation in the presence of air, light, heat, peroxides, trace metals, or even the structural features of the fatty acids themselves. Thus, oxidation stability is another major issue affecting the use of biodiesel fuels (Ramos et al., 2009; Knothe, 2009). The number and position of double bonds in the chains of unsaturated fatty esters are both factors affecting the susceptible to autoxidation reaction (Bajpai & Tyagi, 2006; Frankel, 2005). Most biodiesel fuels contain significant amounts of alkyl esters of oleic, linoleic and linolenic acids, which influence the oxidation stability of the fuels. The relative rates of autoxidation for oleic acid methyl ester, linoleic acid methyl ester and linolenic acid methyl ester have been reported to be 1, 41 and 98 respectively (Knothe et al., 2005; Frankel, 2005; Ramos et al., 2009; Knothe, 2009).

The high viscosity of non-esterified vegetable oils leads to operational problems in diesel engines by increasing the level of engine deposits. Reduction of the high viscosity of vegetable oils is facilitated by the production of alkyl esters from the oil by transesterification. Since viscosity increases with decreasing temperature, handling of fuels in lower temperatures is facilitated by this lower viscosity as well (Knothe, 2009). This can be achieved by increasing the length and degree of saturation of the carbon chains (Knothe et al., 2005). Wax settling and plugging of filters and fuel lines are typical

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**9. References** 

problems associated with biodiesel fuels at low temperatures. The maximum temperature at which the first solids appear for a particular fuel is known as the cloud point (CP) and such solids can lead to fuel filter plugging. The pour point (PP) is typically a few degrees below the cloud point and represents the temperature at which the fuel can no longer be poured (Dunn & Bagby, 1996). Key properties of biodiesel fuels at low-temperature are determined by the cold filter plugging point (CFPP) and low-temperature flow test (LTFT) (Dunn & Bagby, 1996; Knothe, 2009). The cloud point (CP) and CFPP are included in the biodiesel standards but as "soft" specifications (Knothe, 2009). For instance, The cloud point in ATSM D6751 requires a report, while the CFPP in UNE-EN 14214 varies with time of year and geographic location.

The properties of biodiesel at low-temperature are correlated with the properties of individual fatty acids, which mostly depend on the saturated ester content. In contrast, the effect of unsaturated fatty acids is considered negligible (Imahara et al., 2006; Ramos et al., 2009). Saturated fatty acids have significantly higher melting points than unsaturated fatty acids and in a mixture saturated fatty acids will crystallize at higher temperature. Therefore, biodiesel fuels derived from fats or oils with significant amounts of saturated fatty compounds display higher values of CP and CFPP (Knothe, 2003).
