**3. Biodiesel production from microalgae**

The biodiesel production from microalgal oil shares the same processes and technologies as those used for other feedstocks derived oils. However, microalgae are microorganisms living essentially in liquid environments and thus have particular cultivation, harvesting, and downstream processing techniques for efficient biodiesel production. The microalgal biodiesel production pipeline is schematically presented in Figure 5, including strain selection, mass culture, biomass harvesting and processing, oil extraction and transesterification.

Fig. 5. Microalgal biodiesel production pipeline

nutrient that affects lipid metabolism of diatoms, and can promote the accumulation of neutral lipids as well as of saturated and monounsaturated fatty acids when depleted from culture medium (Roessler, 1988). Other types of nutrient deficiency include phosphorus and sulfur limitations are also able to enhance lipid accumulation in algae (Khozin-Goldberg & Cohen, 2006; Li et al., 2010b; Sato et al., 2000). These types of nutrient deficiency, however, do not always lead to elevated overall lipid production, because they at the same time exert negative effect on algal growth and contribute to the reduced algal biomass production that compromises the enhanced lipid yield resulting from increased lipid content. Therefore, the manipulation of these nutrients needs to be optimized to induce lipid accumulation while maintaining algal growth for maximal production of lipids. Iron is a micro-nutrient required in a tiny amount for ensuring algal growth. Within a certain range of concentrations, high concentrations of iron benefit algal growth as well as cellular lipid accumulation and thus the overall lipid yield in the green alga

Among the environmental factors, light is an important one that has a marked effect on the lipid production and fatty acid composition in algae (Brown et al., 1996; Damiani et al., 2010; Khotimchenko & Yakovleva, 2005; Napolitano, 1994; Sukenik et al., 1989; Zhekisheva et al., 2002, 2005). Generally, low light intensity favors the formation of polar lipids such as the membrane lipids associated with the chloroplast; whereas high light intensity benefits the accumulation of neutral storage lipids in particular TAGs. In *H. pluvialis,* for example, high light intensity resulted in a great increase of both neutral and polar lipids, but the increase extent of neutral lipids was much greater than that of polar lipids, leading to the dominant proportion of neutral lipids in the total lipids (Zhekisheva et al., 2002, 2005). Although the effect of light intensity on fatty acid composition differs among the algal species and/or strains, there is a general trend that the increase of light intensity contributes to the enhanced proportions of saturated and monounsaturated fatty acids and the concurrently the reduced proportion of polyunsaturated fatty acids (Damiani et al., 2010; Sukenik et al., 1989; Zhekisheva et al., 2002, 2005). Temperature is another important environmental factor that affects profiles of algal lipids and fatty acids. In response to temperature shift, algae commonly alter the physical properties and thermal responses of membrane lipids to maintain fluidity and function of membranes (Somerville, 1995). In general, increased temperature causes increased fatty acid saturation and at the same time decreased fatty acid unsaturation. For example, C14:0, C16:0, C18:0 and C18:2 increased and C18:3 (n-3), C18:4, C20:5 and C22:6 decreased in *Rhodomonas* sp., and C16:0 increased and C18:4 decreased in *Cryptomonas* sp. when temperature increased (Renaud et al., 2002). As for the effect of temperature on cellular lipid content, it differs in a species-dependent manner. In response to increased temperature, algae may show an increase (Boussiba et al., 1987), no significant change or even a decrease (Renaud et al., 2002) in lipid contents. Other environmental factors such as salinity, pH and dissolved O2 are also important and able to affect algal lipid

In addition to the nutritional and environmental factors, growth phase and aging of the culture affect algal lipids and fatty acids. Commonly, algae accumulate more lipids at stationary phase than at logarithmic phase (Bigogno et al., 2002; Mansour et al., 2003). Associated with the growth phase transition from logarithmic to stationary phase, increased proportions of C16:0 and C18:1 and decreased proportions of PUFAs are often observed. Besides, it is suggested that algal lipids and fatty acids can be greatly affected by cultivation modes. Algae growing under heterotrophic mode usually produce more

*Chlorella vulgaris* (Liu et al., 2008).

metabolism.

#### **3.1 Microalgae selection**

There are more than 50,000 microalgal species around the world. Selection of an ideal species is of fundamental importance to the success of algal biodiesel production. Theoretically, an ideal species should own the following desirable characteristics: rapid growth rate, high oil content, wide tolerance of environmental conditions, CO2 tolerance and uptake, large cell size, easy of disruption, etc. However, it is unlikely for a single species to excel in all above mentioned characteristics. Thus, prioritization is required. Commonly, fast-growing strains with high oil content are placed on the priority list for biodiesel production. Fast growth makes sure the high biomass productivity and reduces the contamination risk owing to out-competition of slower growers. High oil content helps increase the process yield coefficient and reduce the cost of downstream extraction and purification. The selected species should be suitable for mass cultivation under local geographic and climatic conditions, for example, the inland prefers freshwater algae while the coastal place desires marine algal species. Ease of harvesting is an often-overlooked criterion and should be taken into account. Algal biomass harvest requires significant capital and accounts for up to 30% of total biomass production cost (Molina Grima et al., 2003). Therefore, it is desirable to choose algal species with properties that simplify harvesting, including large cell size, high specific gravity and autofloculation potential (Griffiths & Harrison, 2009). These properties can greatly influence the process economics for biodiesel production from algae. An additional algal characteristic is the suitability of lipids for biodiesel production; for example, neutral lipids in particular TAG are superior to polar lipids (phospholipids and glycolipids) for biodiesel and C18:1 has advantages over other fatty acids for improving biodiesel quality (Knothe, 2009).

#### **3.2 Microalgae cultivation**

#### **3.2.1 Factors affecting algal lipids and fatty acids**

Microalgae require several things to grow, including a light source, carbon dioxide, water, and inorganic salts. The lipid content and fatty acid composition are species/strainspecific and can be greatly affected by a variety of medium nutrients and environmental factors. Carbon is the main component of algal biomass and accounts for *ca* 50% of dry weight. CO2 is the common carbon source for algal growth. But some algal species are also able to utilize organic carbon sources, for example sugars and glycerol (Easterling et al., 2009; Liu et al., 2010). Sugars particularly glucose are preferred and can be used to boost production of both algal biomass and lipids (Liu et al., 2010). Nitrogen is an important nutrient affecting lipid metabolism in algae. The influence of nitrogen concentration on lipid and fatty acid production has been investigated in numerous algal species. Nitrate was suggested to be superior to other nitrogen sources such as urea and ammonium for algal lipid production (Li et al., 2008). Generally, low concentration of nitrogen in the medium favors the accumulation of lipids particularly TAGs and total fatty acids. But in some cases, nitrogen starvation caused decreased synthesis of lipids and fatty acids (Saha et al., 2003). Nitrogen concentration also affects algal fatty acid composition. For example, in cyanobacteria, increased levels of C16:0 and C18:1 and decreased C18:2 levels were observed in response to nitrogen deprivation (Piorreck & Pohl, 1984). In the marine alga *Pavlova viridis*, nitrogen depletion resulted in an increase in saturated, monounsaturated fatty acids and C22:6 (n-3) contents (Li et al., 2005). Nitrogen starvation brought about a strong increase in the proportion of C20:4 (n-6) in the green algal *Parietochloris incisa* (Solovchenko et al., 2008). Similar to nitrogen, silicon is a key

There are more than 50,000 microalgal species around the world. Selection of an ideal species is of fundamental importance to the success of algal biodiesel production. Theoretically, an ideal species should own the following desirable characteristics: rapid growth rate, high oil content, wide tolerance of environmental conditions, CO2 tolerance and uptake, large cell size, easy of disruption, etc. However, it is unlikely for a single species to excel in all above mentioned characteristics. Thus, prioritization is required. Commonly, fast-growing strains with high oil content are placed on the priority list for biodiesel production. Fast growth makes sure the high biomass productivity and reduces the contamination risk owing to out-competition of slower growers. High oil content helps increase the process yield coefficient and reduce the cost of downstream extraction and purification. The selected species should be suitable for mass cultivation under local geographic and climatic conditions, for example, the inland prefers freshwater algae while the coastal place desires marine algal species. Ease of harvesting is an often-overlooked criterion and should be taken into account. Algal biomass harvest requires significant capital and accounts for up to 30% of total biomass production cost (Molina Grima et al., 2003). Therefore, it is desirable to choose algal species with properties that simplify harvesting, including large cell size, high specific gravity and autofloculation potential (Griffiths & Harrison, 2009). These properties can greatly influence the process economics for biodiesel production from algae. An additional algal characteristic is the suitability of lipids for biodiesel production; for example, neutral lipids in particular TAG are superior to polar lipids (phospholipids and glycolipids) for biodiesel and C18:1 has advantages over other

Microalgae require several things to grow, including a light source, carbon dioxide, water, and inorganic salts. The lipid content and fatty acid composition are species/strainspecific and can be greatly affected by a variety of medium nutrients and environmental factors. Carbon is the main component of algal biomass and accounts for *ca* 50% of dry weight. CO2 is the common carbon source for algal growth. But some algal species are also able to utilize organic carbon sources, for example sugars and glycerol (Easterling et al., 2009; Liu et al., 2010). Sugars particularly glucose are preferred and can be used to boost production of both algal biomass and lipids (Liu et al., 2010). Nitrogen is an important nutrient affecting lipid metabolism in algae. The influence of nitrogen concentration on lipid and fatty acid production has been investigated in numerous algal species. Nitrate was suggested to be superior to other nitrogen sources such as urea and ammonium for algal lipid production (Li et al., 2008). Generally, low concentration of nitrogen in the medium favors the accumulation of lipids particularly TAGs and total fatty acids. But in some cases, nitrogen starvation caused decreased synthesis of lipids and fatty acids (Saha et al., 2003). Nitrogen concentration also affects algal fatty acid composition. For example, in cyanobacteria, increased levels of C16:0 and C18:1 and decreased C18:2 levels were observed in response to nitrogen deprivation (Piorreck & Pohl, 1984). In the marine alga *Pavlova viridis*, nitrogen depletion resulted in an increase in saturated, monounsaturated fatty acids and C22:6 (n-3) contents (Li et al., 2005). Nitrogen starvation brought about a strong increase in the proportion of C20:4 (n-6) in the green algal *Parietochloris incisa* (Solovchenko et al., 2008). Similar to nitrogen, silicon is a key

**3.1 Microalgae selection** 

**3.2 Microalgae cultivation** 

fatty acids for improving biodiesel quality (Knothe, 2009).

**3.2.1 Factors affecting algal lipids and fatty acids** 

nutrient that affects lipid metabolism of diatoms, and can promote the accumulation of neutral lipids as well as of saturated and monounsaturated fatty acids when depleted from culture medium (Roessler, 1988). Other types of nutrient deficiency include phosphorus and sulfur limitations are also able to enhance lipid accumulation in algae (Khozin-Goldberg & Cohen, 2006; Li et al., 2010b; Sato et al., 2000). These types of nutrient deficiency, however, do not always lead to elevated overall lipid production, because they at the same time exert negative effect on algal growth and contribute to the reduced algal biomass production that compromises the enhanced lipid yield resulting from increased lipid content. Therefore, the manipulation of these nutrients needs to be optimized to induce lipid accumulation while maintaining algal growth for maximal production of lipids. Iron is a micro-nutrient required in a tiny amount for ensuring algal growth. Within a certain range of concentrations, high concentrations of iron benefit algal growth as well as cellular lipid accumulation and thus the overall lipid yield in the green alga *Chlorella vulgaris* (Liu et al., 2008).

Among the environmental factors, light is an important one that has a marked effect on the lipid production and fatty acid composition in algae (Brown et al., 1996; Damiani et al., 2010; Khotimchenko & Yakovleva, 2005; Napolitano, 1994; Sukenik et al., 1989; Zhekisheva et al., 2002, 2005). Generally, low light intensity favors the formation of polar lipids such as the membrane lipids associated with the chloroplast; whereas high light intensity benefits the accumulation of neutral storage lipids in particular TAGs. In *H. pluvialis,* for example, high light intensity resulted in a great increase of both neutral and polar lipids, but the increase extent of neutral lipids was much greater than that of polar lipids, leading to the dominant proportion of neutral lipids in the total lipids (Zhekisheva et al., 2002, 2005). Although the effect of light intensity on fatty acid composition differs among the algal species and/or strains, there is a general trend that the increase of light intensity contributes to the enhanced proportions of saturated and monounsaturated fatty acids and the concurrently the reduced proportion of polyunsaturated fatty acids (Damiani et al., 2010; Sukenik et al., 1989; Zhekisheva et al., 2002, 2005). Temperature is another important environmental factor that affects profiles of algal lipids and fatty acids. In response to temperature shift, algae commonly alter the physical properties and thermal responses of membrane lipids to maintain fluidity and function of membranes (Somerville, 1995). In general, increased temperature causes increased fatty acid saturation and at the same time decreased fatty acid unsaturation. For example, C14:0, C16:0, C18:0 and C18:2 increased and C18:3 (n-3), C18:4, C20:5 and C22:6 decreased in *Rhodomonas* sp., and C16:0 increased and C18:4 decreased in *Cryptomonas* sp. when temperature increased (Renaud et al., 2002). As for the effect of temperature on cellular lipid content, it differs in a species-dependent manner. In response to increased temperature, algae may show an increase (Boussiba et al., 1987), no significant change or even a decrease (Renaud et al., 2002) in lipid contents. Other environmental factors such as salinity, pH and dissolved O2 are also important and able to affect algal lipid metabolism.

In addition to the nutritional and environmental factors, growth phase and aging of the culture affect algal lipids and fatty acids. Commonly, algae accumulate more lipids at stationary phase than at logarithmic phase (Bigogno et al., 2002; Mansour et al., 2003). Associated with the growth phase transition from logarithmic to stationary phase, increased proportions of C16:0 and C18:1 and decreased proportions of PUFAs are often observed. Besides, it is suggested that algal lipids and fatty acids can be greatly affected by cultivation modes. Algae growing under heterotrophic mode usually produce more

**Culture systems Open ponds Enclosed bioreactors** 

Operation regime Batch or semi-continuous Batch or semi-continuous

Temperature control difficult More uniform temperature

Table 4. Comparison of open ponds and photobioreactors for microalgae cultivation (Mata

Algal harvesting is the concentration of diluted algal suspension into a thick algal paste, with the aim of obtaining slurry with at least 2–7% algal suspension on dry matter basis. Biomass harvest is a very challenging process and may contribute to 20-30% of the total biomass production cost (Molina Grima et al., 2003). The most common harvesting methods include sedimentation, filtration, centrifugation, sometimes with a pre-step of flocculation or flocculation-flotation. Flocculation is employed to aggregate the microalgal cells into larger clumps to enhance the harvest efficiency by gravity sedimentation, filtration, or centrifugation (Molina Grima et al., 2003). The selection of a harvesting process for a particular strain depends on size and properties of the algal strain. The selected harvest

Filtration is the most commonly used method for harvesting algal biomass. The process can range from micro-strainers to pressure filtration and ultra-filtration systems. Vacuum filtration is feasible for harvesting large microalgae such as *Coelastrum proboscideum* and *Spirulina platensis* but unsuitable for recovering small size algal cells such as *Scenedesmus, Dunaliella*, or *Chlorella* (Molina Grima et al., 2003). Membrane-based microfiltration and ultrafiltration have also been used for harvesting algal cells for some specific application purposes, but overall, they are more expensive. Centrifugation is an accelerated sedimentation process for algae harvesting. Generally, centrifugation has high capital and operation costs, but its efficiency is much higher than natural sedimentation. Because of its high cost, centrifugation as an algae harvesting method is usually considered only feasible

After harvesting, chemicals in the biomass may be subject to degradation induced by the process itself and also by internal enzyme in the algal cells. For example, lipase contained in

Contamination control Difficult Easy Contamination risk High Reduced Sterility None Achievable Process control Difficult Easy Species control Difficult Easy Mixing Very poor Uniform

Area/volume ration Low High Algal cell density Low High Investment Low Hight Operation cost Low High Light utilization efficiency Poor High

Productivity Low High Hydrodynamic stress on algae Very low Low-high Evaporation of growth medium High Low Gas transfer control Low High

method must be able to handle a large volume of algal culture broth.

**3.3 Biomass harvesting and concentration** 

for high value products rather than biofuels.

**3.4 Biomass processing for oil extraction** 

et al., 2010)

O2 inhibition < bioreactors Great problem Scale-up Difficult Difficult

lipids in particular TAG and higher proportion of C18:1 than under photoautotrophic mode (Liu et al., 2011).

#### **3.2.2 Raceway ponds and photobioreactors**

Currently, the commonly used culture systems for large-scale production of algal biomass are open ponds and enclosed photobioreactors. An open pond culture system usually consists of a series of raceways-type of ponds placed outdoors. In this system, the shallow pond is usually about one foot deep; algae are cultured under conditions identical to their natural environment. The pond is designed in a raceway configuration, in which a paddle wheel provides circulation and mixing of the algal cells and nutrients (Chisti, 2007). The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid. Compared with photobioreactors, open ponds cost less to build and operate, and are more durable with a large production capacity. However, the open pond system has its intrinsic disadvantages including rapid water loss due to evaporation, contamination with unwanted algal species as well as organisms that feed on algae, and low biomass productivity. In addition, optimal culture conditions are difficult to maintain in open ponds and recovering the biomass from such a dilute culture is expensive.

Unlike open ponds, enclosed photobioreactors are flexible systems that can be employed to overcome the problems of evaporation, contamination and low biomass productivity encountered in open ponds (Mata et al., 2010). These systems are made of transparent materials with a large surface area-to-volume ratio, and generally placed outdoors using natural light for illumination. The tubular photobioreactor is the most widely used one, which consists of an array of straight transparent tubes aligned with the sun's rays (Chisti, 2007). The tubes are generally no more than 10 cm in diameter to maximize sunlight penetration. The medium broth is circulated through a pump to the tubes, where it is exposed to light for photosynthesis, and then back to a reservoir. In some photobioreactors, the tubes are coiled to form what is known as a helical tubular photobioreactor. Artificial illumination can be used for photobioreactor. But it adds to the production cost and thus is used for the production of high value products instead of biodiesel feedstock. The algal biomass is prevented from settling by maintaining a highly turbulent flow within the reactor using either a mechanical pump or an airlift pump (Chisti, 2007). The result of photosynthesis will generate oxygen. The oxygen levels will accumulate in the closed photobioreactor and inhibit the growth of algae. Therefore, the culture must periodically be returned to a degassing zone, an area where the algal broth is bubbled with air to remove the excess oxygen. In addition, carbon dioxide must be fed into the system to provide carbon source and maintain culture pH for algal growth. Photobioreactors require cooling during daylight hours and temperature regulation in night hours. This may be done through heat exchangers located either in the tubes themselves or in the degassing column.

Table 4 shows the comparison between open ponds and photobioreactors for microalgae cultivation.

Photobioreactors have obvious advantages over open ponds: offer better control, prevent contamination and evaporation, reduce carbon dioxide losses and allow to achieve higher biomass productivities. However, enclosed photobioreactors cost high to build and operate and the scale-up is difficult, limiting the number of large-scale commercial systems operating globally to high-value production runs (Greenwell et al., 2010). In this context, a hybrid photobioreactor-open pond system is proposed: using photobioreactors to produce contaminant-free inoculants for large open ponds.

lipids in particular TAG and higher proportion of C18:1 than under photoautotrophic

Currently, the commonly used culture systems for large-scale production of algal biomass are open ponds and enclosed photobioreactors. An open pond culture system usually consists of a series of raceways-type of ponds placed outdoors. In this system, the shallow pond is usually about one foot deep; algae are cultured under conditions identical to their natural environment. The pond is designed in a raceway configuration, in which a paddle wheel provides circulation and mixing of the algal cells and nutrients (Chisti, 2007). The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid. Compared with photobioreactors, open ponds cost less to build and operate, and are more durable with a large production capacity. However, the open pond system has its intrinsic disadvantages including rapid water loss due to evaporation, contamination with unwanted algal species as well as organisms that feed on algae, and low biomass productivity. In addition, optimal culture conditions are difficult to maintain in open ponds and recovering the biomass from

Unlike open ponds, enclosed photobioreactors are flexible systems that can be employed to overcome the problems of evaporation, contamination and low biomass productivity encountered in open ponds (Mata et al., 2010). These systems are made of transparent materials with a large surface area-to-volume ratio, and generally placed outdoors using natural light for illumination. The tubular photobioreactor is the most widely used one, which consists of an array of straight transparent tubes aligned with the sun's rays (Chisti, 2007). The tubes are generally no more than 10 cm in diameter to maximize sunlight penetration. The medium broth is circulated through a pump to the tubes, where it is exposed to light for photosynthesis, and then back to a reservoir. In some photobioreactors, the tubes are coiled to form what is known as a helical tubular photobioreactor. Artificial illumination can be used for photobioreactor. But it adds to the production cost and thus is used for the production of high value products instead of biodiesel feedstock. The algal biomass is prevented from settling by maintaining a highly turbulent flow within the reactor using either a mechanical pump or an airlift pump (Chisti, 2007). The result of photosynthesis will generate oxygen. The oxygen levels will accumulate in the closed photobioreactor and inhibit the growth of algae. Therefore, the culture must periodically be returned to a degassing zone, an area where the algal broth is bubbled with air to remove the excess oxygen. In addition, carbon dioxide must be fed into the system to provide carbon source and maintain culture pH for algal growth. Photobioreactors require cooling during daylight hours and temperature regulation in night hours. This may be done through heat exchangers located either in the tubes themselves or in the degassing

Table 4 shows the comparison between open ponds and photobioreactors for microalgae

Photobioreactors have obvious advantages over open ponds: offer better control, prevent contamination and evaporation, reduce carbon dioxide losses and allow to achieve higher biomass productivities. However, enclosed photobioreactors cost high to build and operate and the scale-up is difficult, limiting the number of large-scale commercial systems operating globally to high-value production runs (Greenwell et al., 2010). In this context, a hybrid photobioreactor-open pond system is proposed: using photobioreactors to produce

mode (Liu et al., 2011).

**3.2.2 Raceway ponds and photobioreactors** 

such a dilute culture is expensive.

column.

cultivation.

contaminant-free inoculants for large open ponds.


Table 4. Comparison of open ponds and photobioreactors for microalgae cultivation (Mata et al., 2010)

#### **3.3 Biomass harvesting and concentration**

Algal harvesting is the concentration of diluted algal suspension into a thick algal paste, with the aim of obtaining slurry with at least 2–7% algal suspension on dry matter basis. Biomass harvest is a very challenging process and may contribute to 20-30% of the total biomass production cost (Molina Grima et al., 2003). The most common harvesting methods include sedimentation, filtration, centrifugation, sometimes with a pre-step of flocculation or flocculation-flotation. Flocculation is employed to aggregate the microalgal cells into larger clumps to enhance the harvest efficiency by gravity sedimentation, filtration, or centrifugation (Molina Grima et al., 2003). The selection of a harvesting process for a particular strain depends on size and properties of the algal strain. The selected harvest method must be able to handle a large volume of algal culture broth.

Filtration is the most commonly used method for harvesting algal biomass. The process can range from micro-strainers to pressure filtration and ultra-filtration systems. Vacuum filtration is feasible for harvesting large microalgae such as *Coelastrum proboscideum* and *Spirulina platensis* but unsuitable for recovering small size algal cells such as *Scenedesmus, Dunaliella*, or *Chlorella* (Molina Grima et al., 2003). Membrane-based microfiltration and ultrafiltration have also been used for harvesting algal cells for some specific application purposes, but overall, they are more expensive. Centrifugation is an accelerated sedimentation process for algae harvesting. Generally, centrifugation has high capital and operation costs, but its efficiency is much higher than natural sedimentation. Because of its high cost, centrifugation as an algae harvesting method is usually considered only feasible for high value products rather than biofuels.

#### **3.4 Biomass processing for oil extraction**

After harvesting, chemicals in the biomass may be subject to degradation induced by the process itself and also by internal enzyme in the algal cells. For example, lipase contained in

In addition to heat, a catalyst is needed to facilitate the transesterification. The transesterification of triglycerides can be catalyzed by acids, alkalis or enzymes. Acid transesterification is considered suitable for the conversion of feedstocks with high free fatty acids but its reaction rate is low (Gerpen, 2005). In contrast, alkali-catalyzed transesterification has a much higher reaction rate, approximately 4000 times faster than the acid-catalyzed one (Fukuda et al., 2001). In this context, alkalis (sodium hydroxide and potassium hydroxide) are preferred as catalysts for industrial production of biodiesel. The use of lipases as transesterification catalysts has also attracted much attention as it produces high purity product and enables easy separation from the byproduct glycerol (Ranganathan et al., 2008). However, the cost of enzyme is still relatively high and remains a barrier for its industrial implementation. In addition, it has been proposed that biodiesel can be prepared from oil via

Although lipid metabolism, in particular the biosynthesis of fatty acids and TAG, is poorly understood in algae, it is generally recognized that the basic pathways for fatty acid and

Algae synthesize the *de novo* fatty acids in the chloroplast using a single set of enzymes. A simplified schedule for saturated fatty acid biosynthesis is shown in Figure 7. Acetyl-CoA is the basic building block of the acyl chain and serves as a substrate for acetyl CoA

Fig. 7. Simplified overview of saturated fatty acid biosynthesis in algal chloroplast. ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; ENR, enoyl-ACP reductase; HD, 3-hydroxyacyl-ACP dehydratase; KAR, 3-ketoacyl-ACP reductase; KAS,

3-ketoacyl-ACP synthase; MAT, malonyl-CoA:ACP transacylase.

transesterification with supercritical methanol (Demirbas, 2002).

TAG biosynthesis are similar to those demonstrated in higher plants.

**4. Genetic engineering of microalgae** 

**4.1 Microalgal lipid biosynthesis** 

the cells can rapidly hydrolyze cellular lipids into free fatty acids that are not suitable for biodiesel production. Therefore, the harvested biomass need be processed rapidly. Drying is a major step to keep the quality of the oil. In addition, the solvent-based oil extraction can be difficult when wet biomass is used. Various drying methods such as sun drying, spray drying, freeze drying, and drum drying can be used for drying algal biomass (Mata et al., 2010). Due to the high water content of algal biomass, sun-drying is not a very effective method for algal powder production. Spray drying and freeze drying are rapid and effective, but also expensive and not economically feasible for biofuel production. Because of the high energy required, drying is considered as one of the main economical bottlenecks in the entire process.

There are several approaches for extracting oil from the dry algal biomass, including solvent extraction, osmotic shock, ultrasonic extraction and supercritical CO2 extraction. Oil extraction from dried biomass can be performed in two steps, mechanical crushing followed by solvent extraction in which hexane is the main solvent used. For example, after the oil extraction using an expeller, the leftover pulp can be mixed with cyclohexane to extract the remaining oil. The oil dissolves in the cyclohexane and the pulp is filtered out from the solution. These two stages are able to extract more than 95% of the total oil present in the algae. Oil extraction from algal cells can also be facilitated by osmotic shock or ultrasonic treatment to break the cells. Osmotic shock is a sudden reduction in osmotic pressure causing cells to rupture and release cellular components including oil. The algae lacking the cell wall are suitable for this process. In the ultrasonic treatment, the collapsing cavitation bubbles near to the cell walls cause cell walls to break and release the oil into the solvent. Supercritical CO2 is another way for efficient extraction of algal oil, but the high energy demand is a limitation for commercialization of this technology (Herrero et al., 2010).

#### **3.5 Oil transesterification**

Algal oil contained in algal cells can be converted into biodiesel through transesterification. Transesterification is a chemical conversion process involving reacting triglycerides of vegetable oils or animal fats catalytically with a short-chain alcohol (typically methanol or ethanol) to form fatty acid esters and glycerol (Figure 6). This reaction occurs stepwise with the first conversion of triglycerides to diglycerides and then to monoglycerides and finally to glycerol. The complete transesterification of 1 mol of triglycerides requires 3 mol of alcohol, producing 1 mol of glycerol and 3 mol of fatty esters. Considering that the reaction is reversible, large excess of alcohol is used in industrial processes to ensure the direction of fatty acid esters. Methanol is the preferred alcohol for industrial use because of its low cost, although other alcohols like ethanol, propanol and butanol are also commonly used.


Fig. 6. Transesterification of oil to biodiesel. R1-3 indicates hydrocarbon groups.

the cells can rapidly hydrolyze cellular lipids into free fatty acids that are not suitable for biodiesel production. Therefore, the harvested biomass need be processed rapidly. Drying is a major step to keep the quality of the oil. In addition, the solvent-based oil extraction can be difficult when wet biomass is used. Various drying methods such as sun drying, spray drying, freeze drying, and drum drying can be used for drying algal biomass (Mata et al., 2010). Due to the high water content of algal biomass, sun-drying is not a very effective method for algal powder production. Spray drying and freeze drying are rapid and effective, but also expensive and not economically feasible for biofuel production. Because of the high energy required,

There are several approaches for extracting oil from the dry algal biomass, including solvent extraction, osmotic shock, ultrasonic extraction and supercritical CO2 extraction. Oil extraction from dried biomass can be performed in two steps, mechanical crushing followed by solvent extraction in which hexane is the main solvent used. For example, after the oil extraction using an expeller, the leftover pulp can be mixed with cyclohexane to extract the remaining oil. The oil dissolves in the cyclohexane and the pulp is filtered out from the solution. These two stages are able to extract more than 95% of the total oil present in the algae. Oil extraction from algal cells can also be facilitated by osmotic shock or ultrasonic treatment to break the cells. Osmotic shock is a sudden reduction in osmotic pressure causing cells to rupture and release cellular components including oil. The algae lacking the cell wall are suitable for this process. In the ultrasonic treatment, the collapsing cavitation bubbles near to the cell walls cause cell walls to break and release the oil into the solvent. Supercritical CO2 is another way for efficient extraction of algal oil, but the high energy demand is a limitation for commercialization of this technology

Algal oil contained in algal cells can be converted into biodiesel through transesterification. Transesterification is a chemical conversion process involving reacting triglycerides of vegetable oils or animal fats catalytically with a short-chain alcohol (typically methanol or ethanol) to form fatty acid esters and glycerol (Figure 6). This reaction occurs stepwise with the first conversion of triglycerides to diglycerides and then to monoglycerides and finally to glycerol. The complete transesterification of 1 mol of triglycerides requires 3 mol of alcohol, producing 1 mol of glycerol and 3 mol of fatty esters. Considering that the reaction is reversible, large excess of alcohol is used in industrial processes to ensure the direction of fatty acid esters. Methanol is the preferred alcohol for industrial use because of its low cost,

although other alcohols like ethanol, propanol and butanol are also commonly used.

Fig. 6. Transesterification of oil to biodiesel. R1-3 indicates hydrocarbon groups.

drying is considered as one of the main economical bottlenecks in the entire process.

(Herrero et al., 2010).

**3.5 Oil transesterification** 

In addition to heat, a catalyst is needed to facilitate the transesterification. The transesterification of triglycerides can be catalyzed by acids, alkalis or enzymes. Acid transesterification is considered suitable for the conversion of feedstocks with high free fatty acids but its reaction rate is low (Gerpen, 2005). In contrast, alkali-catalyzed transesterification has a much higher reaction rate, approximately 4000 times faster than the acid-catalyzed one (Fukuda et al., 2001). In this context, alkalis (sodium hydroxide and potassium hydroxide) are preferred as catalysts for industrial production of biodiesel. The use of lipases as transesterification catalysts has also attracted much attention as it produces high purity product and enables easy separation from the byproduct glycerol (Ranganathan et al., 2008). However, the cost of enzyme is still relatively high and remains a barrier for its industrial implementation. In addition, it has been proposed that biodiesel can be prepared from oil via transesterification with supercritical methanol (Demirbas, 2002).
