**1. Introduction**

132 Biodiesel – Feedstocks and Processing Technologies

Wang, B.; Li, Y.; Wu, N.; Lan, CQ. (2008). CO2 bio-mitigation using microalgae. Applied

Fossil-based fuels including oil, coal and gas play a pivotal role in modern world energy market. These fossil fuels, according to world energy outlook 2007, will remain the major sources of energy and are expected to meet about 84% of energy demand in 2030. However, fossil fuels are non-renewable and will be finally diminished. It has been recently estimated that the global oil, coal and gas last only approximately for 35, 100 and 37 years respectively, based on a modified Klass model (Shafiee & Topal, 2009). In order to sustain a stable energy supply in the future, it is necessary to develop other sources of energy, e.g., renewable energy. Renewable energy is derived from natural processes that are replenished constantly, including hydropower, wind power, solar energy, geothermal energy, biodiesel, etc. An estimated \$150 billion was invested in renewable energy worldwide in 2009, around 2.5 times of the 2006 investment (Figure 1).

It is well known that transport is almost totally dependent on petroleum-based fuels, which will be depleted within 40 years. An alternative fuel to petrodiesel must be technically feasible, easily available, economically competitive, and environmentally acceptable (Demirbas, 2008). Biodiesel is such a candidate fuel for powering the transport vehicles. Biodiesel refers to a biomass-based diesel fuel consisting of long-chain alkyl (methyl, propyl or ethyl) esters. In addition to being comparable to petrodiesel in most technical aspects, biodiesel has the following distinct advantages over petrodiesel (Knothe, 2005a):


Like petrodiesel, biodiesel operates in compression ignition engines. Biodiesel is miscible with petrodiesel in all ratios. Currently, the blends of biodiesel and petrodiesel instead of net biodiesel have been widely used in many countries and no engine modification is

Biodiesel can be produced from a variety of feedstocks, including plant oils, animal fats and waste oils as well as microalgae (Demiras, 2008). Each feedstock has its advantages and disadvantages in terms of oil content, fatty acid composition, biomass yield and geographic distribution. Depending on the origin and quality of feedstocks, changes may be required

The use of plant oils as biodiesel feedstocks has been long recognized and well documented in numerous studies (Abdullah et al., 2009; de Oliveira et al., 2005; Graef et al., 2009; Hawash et al., 2009; Hill et al., 2006; Jain & Sharma, 2010; Nakpong & Wootthikanokkhan, 2010; Patil & Deng, 2009; Rashid & Anwar, 2008; Sahoo & Das, 2009; Saka & Kusdiana, 2001). These feedstocks include the oils from soybean, rapeseed, palm, canola, peanut, cottonseed, sunflower and safflower. Based on the geographic distribution, soybean is the primary source for biodiesel in USA, palm oil is used as a significant biodiesel feedstock in Malaysia and Indonesia, and rapeseed is the most common base oil used in Europe for biodiesel production (Demiras, 2008). The vast majority of these plants are also used for food and feed production, which means that possible food versus fuel conflicts are present. Thus, the use of these plant oils as feedstocks for biodiesel seems insignificant for the developing countries which are importers of edible oils (Meher et al., 2008). In addition to these edible oils, various non-edible, tree-borne oils from jatropha, karanja, jojoba and neem are the potential biodiesel feedstocks (Jain & Sharma, 2009; Meher et al., 2008; Sahoo & Das, 2009). Jatropha and karanja are two oilseed plants that are not widely exploited due to the presence of toxic components in the oils.

In addition to the plant oils, animal fats and waste oils are the potential sources for commercial biodiesel production (Thompson et al., 2010). Among these feedstocks, tallow, lard, yellow grease and waste cooking oils have received most interest (Banerjee et al., 2009; Canakci, 2007; da Cunha et al., 2009; Dias et al., 2009; Diaz-Felix et al., 2009; Oner & Altun, 2009; Phan & Phan, 2008). However, animal fats and waste oils usually contain large amounts of free fatty acids, which can be as high as 41.8% (Canakci, 2007). Free fatty acids cannot be directly converted to biodiesel in alkali-catalyzed transesterificatoin but react with alkali to form soaps that inhibit the separation of biodiesel from glycerin and wash water fraction (Huang et al., 2010). A two-step process was developed for these high fatty acid feedstocks: acid-catalyzed pretreatment and alkali-catalyzed transesterificaton. Because animal fats and waste oils have relatively high level of saturation (Canakci, 2007), the

Microalgae represent a wide variety of aquatic photosynthetic organisms with the potential of producing high biomass and accumulating high level of oil. The production of biodiesel from microalgal oil has long been recognized and been evaluated in response to the United States Department of Energy for research in alternative renewable energy (Sheehan et al., 1998). Currently, the commercialization of algae-derived biodiesel is still in its infancy stage. Using microalgae as biodiesel feedstocks has received unprecedentedly increasing interest, including but not restricted to microalgal strain selection and genetic engineering, mass cultivation for biomass production, lipid extraction and analysis, transesterification technologies, fuel properties and engine tests (Abou-Shanab et al., 2011; Brennan & Owende, 2010; Demirbas, 2009; Greenwell et al., 2010; Miao & Wu, 2006; Pruvost et al., 2011; Radakovits et al., 2010; Rodolfi et al., 2009; Ross et al., 2008; Sydney et al., 2011). Considering their unique characteristics, microalgae have been considered as the most promising feedstock of biodiesel that has the potential to displace fossil diesel (Chisti, 2007). This review mainly focuses on the potential of using microalgae as biodiesel feedstocks, biodiesel production pipeline, and

possibility of employing genetic engineering for improving microalgal productivity.

for the production process of biodiesel.

In India, they are popularly used as biodiesel feedstocks.

biodiesel from these sources exhibits poor cold flow properties.

required (Singhania et al., 2008). These blends of biodiesel with petrodiesel are usually denoted by acronyms, for example B20 which indicates a blend of 20% biodiesel with petrodiesel (Knothe, 2005a).

Fig. 1. Global investment in renewable energy, 2004-2009. Adapted from REN21 (2010)

The global markets for biodiesel are entering a period of rapid and transitional growth. In the year 2007, there were only 20 nations producing biodiesel for the needs of over 200 nations; by the year 2010, more than 200 nations become biodiesel producing nations and suppliers (Thurmond, 2008). Global biodiesel production has massively increased to 16.6 billion liters per year over the last nine years (Figure 2). Much of the growth is happening in just three countries: the United States, Brazil and Germany, which together account for over half of biodiesel (Checkbiotech, 2009). The International Energy Agency's report suggests that world production of biodiesel could top 25 million tons per year by 2012 if the recent trends continue.

Fig. 2. Global biodiesel production, 2001-2009. Adapted from REN21 (2010)

required (Singhania et al., 2008). These blends of biodiesel with petrodiesel are usually denoted by acronyms, for example B20 which indicates a blend of 20% biodiesel with

Fig. 1. Global investment in renewable energy, 2004-2009. Adapted from REN21 (2010)

Fig. 2. Global biodiesel production, 2001-2009. Adapted from REN21 (2010)

The global markets for biodiesel are entering a period of rapid and transitional growth. In the year 2007, there were only 20 nations producing biodiesel for the needs of over 200 nations; by the year 2010, more than 200 nations become biodiesel producing nations and suppliers (Thurmond, 2008). Global biodiesel production has massively increased to 16.6 billion liters per year over the last nine years (Figure 2). Much of the growth is happening in just three countries: the United States, Brazil and Germany, which together account for over half of biodiesel (Checkbiotech, 2009). The International Energy Agency's report suggests that world production of biodiesel could top 25 million tons per year by 2012 if the recent trends continue.

petrodiesel (Knothe, 2005a).

Biodiesel can be produced from a variety of feedstocks, including plant oils, animal fats and waste oils as well as microalgae (Demiras, 2008). Each feedstock has its advantages and disadvantages in terms of oil content, fatty acid composition, biomass yield and geographic distribution. Depending on the origin and quality of feedstocks, changes may be required for the production process of biodiesel.

The use of plant oils as biodiesel feedstocks has been long recognized and well documented in numerous studies (Abdullah et al., 2009; de Oliveira et al., 2005; Graef et al., 2009; Hawash et al., 2009; Hill et al., 2006; Jain & Sharma, 2010; Nakpong & Wootthikanokkhan, 2010; Patil & Deng, 2009; Rashid & Anwar, 2008; Sahoo & Das, 2009; Saka & Kusdiana, 2001). These feedstocks include the oils from soybean, rapeseed, palm, canola, peanut, cottonseed, sunflower and safflower. Based on the geographic distribution, soybean is the primary source for biodiesel in USA, palm oil is used as a significant biodiesel feedstock in Malaysia and Indonesia, and rapeseed is the most common base oil used in Europe for biodiesel production (Demiras, 2008). The vast majority of these plants are also used for food and feed production, which means that possible food versus fuel conflicts are present. Thus, the use of these plant oils as feedstocks for biodiesel seems insignificant for the developing countries which are importers of edible oils (Meher et al., 2008). In addition to these edible oils, various non-edible, tree-borne oils from jatropha, karanja, jojoba and neem are the potential biodiesel feedstocks (Jain & Sharma, 2009; Meher et al., 2008; Sahoo & Das, 2009). Jatropha and karanja are two oilseed plants that are not widely exploited due to the presence of toxic components in the oils. In India, they are popularly used as biodiesel feedstocks.

In addition to the plant oils, animal fats and waste oils are the potential sources for commercial biodiesel production (Thompson et al., 2010). Among these feedstocks, tallow, lard, yellow grease and waste cooking oils have received most interest (Banerjee et al., 2009; Canakci, 2007; da Cunha et al., 2009; Dias et al., 2009; Diaz-Felix et al., 2009; Oner & Altun, 2009; Phan & Phan, 2008). However, animal fats and waste oils usually contain large amounts of free fatty acids, which can be as high as 41.8% (Canakci, 2007). Free fatty acids cannot be directly converted to biodiesel in alkali-catalyzed transesterificatoin but react with alkali to form soaps that inhibit the separation of biodiesel from glycerin and wash water fraction (Huang et al., 2010). A two-step process was developed for these high fatty acid feedstocks: acid-catalyzed pretreatment and alkali-catalyzed transesterificaton. Because animal fats and waste oils have relatively high level of saturation (Canakci, 2007), the biodiesel from these sources exhibits poor cold flow properties.

Microalgae represent a wide variety of aquatic photosynthetic organisms with the potential of producing high biomass and accumulating high level of oil. The production of biodiesel from microalgal oil has long been recognized and been evaluated in response to the United States Department of Energy for research in alternative renewable energy (Sheehan et al., 1998). Currently, the commercialization of algae-derived biodiesel is still in its infancy stage. Using microalgae as biodiesel feedstocks has received unprecedentedly increasing interest, including but not restricted to microalgal strain selection and genetic engineering, mass cultivation for biomass production, lipid extraction and analysis, transesterification technologies, fuel properties and engine tests (Abou-Shanab et al., 2011; Brennan & Owende, 2010; Demirbas, 2009; Greenwell et al., 2010; Miao & Wu, 2006; Pruvost et al., 2011; Radakovits et al., 2010; Rodolfi et al., 2009; Ross et al., 2008; Sydney et al., 2011). Considering their unique characteristics, microalgae have been considered as the most promising feedstock of biodiesel that has the potential to displace fossil diesel (Chisti, 2007). This review mainly focuses on the potential of using microalgae as biodiesel feedstocks, biodiesel production pipeline, and possibility of employing genetic engineering for improving microalgal productivity.

In addition to growth rate, lipid content is another important factor to assess the potential of microalgae for biodiesel production. Over the past few decades, thousands of algae and cyanobacterial species have been screened for high lipid production, and numerous oleaginous species have been isolated and characterized. The lipid contents of these oleaginous algae are species- and/or strains-dependent, vary greatly, and may reach as high as 68% of dry weight, as shown in Table 1. Generally, microalgae synthesize a low content of lipids under nutrient replete conditions (Figure 4), with membrane lipids (e.g., phospholipids and glycolidips) being the main components; whereas under stress conditions such as nitrogen deficiency, a great increase in total lipids was observed (Figure 4) with neutral lipids in particular triacylglycerols (TAGs) being the dominant components (Hu, 2004). TAGs are considered to be superior to phospholipids or glycolipids for biodiesel feedstocks because of their higher percentage of fatty acids and lack of phosphate (Pruvost et al., 2009). Unlike higher plants in which individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, algae produce these different lipids in a single cell (Hu et al., 2008b). The synthesized TAGs are deposited in lipid bodies located in

cytoplasm of algal cells (Damiani et al., 2010; Rabbani et al., 1998).

Lipid content (%) biomass productivity (g/L/day)

*Botryococcus braunii* Phototrophic 9.5-13.5 0.02-0.04 2.6-4.5 Chinnasamy et al., 2010 *Botryococcus braunii* Phototrophic 17.85 0.346 Órpez et al., 2009 *Botryococcus braunii* Phototrophic 24 0.077 21 Yoo et al., 2010

*Botryococcus* sp.Phototrophic 15.8-35.9 0.14-0.22 21.3-46.9 Yeesang and Cheirsilp,

*reinhardtii* Mixotrophic 12.2-46 0.21-0.36 29-95 Li et al., 2010a *Chlorella ellipsoidea* Phototrophic 32 0.07 22.4 Abou-Shanab et al.,

*Chlorella ellipsoidea* Phototrophic 15-43 11.4 Yang et al., 2011 *Chlorella protothecoides* Heterotrophic 48.1-63.8 1.02-1.73 3432-6293 De la Hoz Siegler et al.,

*Chlorella protothecoides* Heterotrophic 49 1.2 586.8 Gao et al., 2010 *Chlorella saccharophila* Phototrophic 12.9-18.1 0.02 2.7-4.2 Chinnasamy et al., 2010 *Chlorella sorokiniana* Phototrophic 19.3 0.23 44.7 Rodolfi et al., 2009 *Chlorella* sp. Phototrophic 33.9 0.528 178.8 Chiu et al., 2008 *Chlorella* sp. Phototrophic 22.4-66.1 0.08-0.34 51-124 Hsieh and Wu., 2009 *Chlorella* sp. Phototrophic 34.1 a 0.053 22 Matsumoto et al., 2010 *Chlorella* sp. Phototrophic 18.7 0.23 42.1 Rodolfi et al., 2009 *Chlorella vulgaris* Phototrophic 20-42 0.21-0.35 44-147 Feng et al., 2011

*Chlorella vulgaris* Phototrophic 19.2 0.17 32.6 Rodolfi et al., 2009 *Chlorella vulgaris* Phototrophic 26-52 11.6-13.2 Widjaja et al., 2009 *Chlorella vulgaris* Phototrophic 35 0.117 41 Yeh et al., 2010 *Chlorella zofingiensis* Heterotrophic 52 0.72 374.4 Liu et al., 2010

Lipid productivity (mg/L/day)

21-38 0.01-0.26 4-54 Liang et al., 2009

References

2011

2011

2011

conditions

Phototrophic, Mixotrophic, heterotrophic

Algal species Culture

**Chlorophyta** 

*Chlamydomonas* 

*Chlorella vulgaris* 

#### **2. Potential of using microalgae as biodiesel feedstocks**

Microalgae represent a large and diverse group of prokaryotic or eukaryotic photosynthetic microorganisms that are in unicellular or multicellular form. Examples of prokaryotic microorganisms are cyanobacteria (commonly referred to as blue-green algae) that are closely related to Gram-negative bacteria and eukaryotic ones are for example green microalgae and diatoms (Graham et al., 2009). Microalgae can be found in a wide range of environmental conditions, including water, land, and even unusual environments such as snow and desert soils (Lee, 2008). It is estimated that there are more than 50,000 species around the world, among which only about 30,000 have been studied and analyzed (Mata et al., 2010). Extensive collections of microalgae have been established by researchers in different countries, including the Freshwater Microalgae Collection of University of Coimbra (Portugal), the Collection of the Goettingen University (Germany), the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP, USA), the University of Texas Algal Culture Collection (USA), the CSIRO collection of Living Microalgae (CCLM, Australia), the National Institute for Environmental Studies Collection (NIES, Japan), the American Type Culture Collection (ATCC, USA), and the Freshwater Algae Culture Collection of Institute of Hydrobiology (China). Together more than 10,000 microalgal strains are available to be selected for use in a broad range of applications, for example, as biodiesel feedstocks.

The use of microalgae for biodiesel production has long been recognized and its potential has been widely reported by many research studies recently (Abou-Shanab et al., 2011; Afify et al., 2010; Ahmad et al., 2011; Cheng et al., 2009; Damiani et al., 2010; Gouveia et al., 2009; Liu et al., 2010; Rodolfi et al., 2009; Yoo et al., 2010). Microalgae reproduce themselves autotrophically using CO2 from air and light through photosynthesis. Compared with higher plants, microalgae exhibit higher photosynthetic efficiency and grow much faster, finishing an entire growth cycle within a few days (Christi, 2007). Typical growth rates are presented in Figure 3 as the doubling time for each microalgal species. A low doubling time corresponds to a high specific growth rate. Microalgae double themselves with an average time of 26 h, and some can even reproduce within 8 h. Moreover, they can be adapted to grow in a broad range of environmental conditions, suggesting the possibility of finding species best suited to local environments which is not suitable for cultivating oil plants (e.g. palm, soybean and rapeseed).

Fig. 3. Doubling time for some microalgal species. The dash line indicates the average value. *T*d=ln(2)/*μ*, *T*d, doubling time, *μ*, specific growth rate.

Microalgae represent a large and diverse group of prokaryotic or eukaryotic photosynthetic microorganisms that are in unicellular or multicellular form. Examples of prokaryotic microorganisms are cyanobacteria (commonly referred to as blue-green algae) that are closely related to Gram-negative bacteria and eukaryotic ones are for example green microalgae and diatoms (Graham et al., 2009). Microalgae can be found in a wide range of environmental conditions, including water, land, and even unusual environments such as snow and desert soils (Lee, 2008). It is estimated that there are more than 50,000 species around the world, among which only about 30,000 have been studied and analyzed (Mata et al., 2010). Extensive collections of microalgae have been established by researchers in different countries, including the Freshwater Microalgae Collection of University of Coimbra (Portugal), the Collection of the Goettingen University (Germany), the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP, USA), the University of Texas Algal Culture Collection (USA), the CSIRO collection of Living Microalgae (CCLM, Australia), the National Institute for Environmental Studies Collection (NIES, Japan), the American Type Culture Collection (ATCC, USA), and the Freshwater Algae Culture Collection of Institute of Hydrobiology (China). Together more than 10,000 microalgal strains are available to be selected for use in a

The use of microalgae for biodiesel production has long been recognized and its potential has been widely reported by many research studies recently (Abou-Shanab et al., 2011; Afify et al., 2010; Ahmad et al., 2011; Cheng et al., 2009; Damiani et al., 2010; Gouveia et al., 2009; Liu et al., 2010; Rodolfi et al., 2009; Yoo et al., 2010). Microalgae reproduce themselves autotrophically using CO2 from air and light through photosynthesis. Compared with higher plants, microalgae exhibit higher photosynthetic efficiency and grow much faster, finishing an entire growth cycle within a few days (Christi, 2007). Typical growth rates are presented in Figure 3 as the doubling time for each microalgal species. A low doubling time corresponds to a high specific growth rate. Microalgae double themselves with an average time of 26 h, and some can even reproduce within 8 h. Moreover, they can be adapted to grow in a broad range of environmental conditions, suggesting the possibility of finding species best suited to local environments which is not suitable for cultivating oil plants (e.g.

Fig. 3. Doubling time for some microalgal species. The dash line indicates the average value.

*T*d=ln(2)/*μ*, *T*d, doubling time, *μ*, specific growth rate.

**2. Potential of using microalgae as biodiesel feedstocks** 

broad range of applications, for example, as biodiesel feedstocks.

palm, soybean and rapeseed).

In addition to growth rate, lipid content is another important factor to assess the potential of microalgae for biodiesel production. Over the past few decades, thousands of algae and cyanobacterial species have been screened for high lipid production, and numerous oleaginous species have been isolated and characterized. The lipid contents of these oleaginous algae are species- and/or strains-dependent, vary greatly, and may reach as high as 68% of dry weight, as shown in Table 1. Generally, microalgae synthesize a low content of lipids under nutrient replete conditions (Figure 4), with membrane lipids (e.g., phospholipids and glycolidips) being the main components; whereas under stress conditions such as nitrogen deficiency, a great increase in total lipids was observed (Figure 4) with neutral lipids in particular triacylglycerols (TAGs) being the dominant components (Hu, 2004). TAGs are considered to be superior to phospholipids or glycolipids for biodiesel feedstocks because of their higher percentage of fatty acids and lack of phosphate (Pruvost et al., 2009). Unlike higher plants in which individual classes of lipids may be synthesized and localized in a specific cell, tissue or organ, algae produce these different lipids in a single cell (Hu et al., 2008b). The synthesized TAGs are deposited in lipid bodies located in cytoplasm of algal cells (Damiani et al., 2010; Rabbani et al., 1998).


*Nitzschia* sp. Phototrophic 32 0.013 Moazami et al., 2011

*tricornutum* Phototrophic 18.7 0.24 44.8 Rodolfi et al., 2009 *Skeletonema costatum* Phototrophic 21.1 0.08 17.4 Rodolfi et al., 2009 *Skeletonema* sp.Phototrophic 31.8 0.09 27.3 Rodolfi et al., 2009

*pseudonana* Phototrophic 20.6 0.08 17.4 Rodolfi et al., 2009

*Ellipsoidion* sp.Phototrophic 27.4 0.17 47.3 Rodolfi et al., 2009 *Monodus subterraneus* Phototrophic 12.9-15 a 0.34-0.49 47.5-67.5 Khozin-Goldberg and

*Monodus subterraneus* Phototrophic 16.1 0.19 30.4 Rodolfi et al., 2009 *Nannochloropsis oculata* Phototrophic 22.8-23 2.4-3.4 547.2-782 Araujo et al., 2011 *Nannochloropsis oculata* Phototrophic 26.2-30.7 0.37-0.50 84-151 Chiu et al., 2009 *Nannochloropsis oculata* Phototrophic 7.9-15.9 0.06-0.13 9.1-16.4 Converti et al., 2009 *Nannochloropsis* sp. Phototrophic 52 0.0465 Moazami et al., 2011 *Nannochloropsis* sp.Phototrophic 23.1-37.8 0.06 20 Huerlimann et al., 2010 *Nannochloropsis* sp. Phototrophic 28.7 0.09 25.8 Gouveia and Oliveira,

*Nannochloropsis* sp. Phototrophic 21.6-35.7 0.17-0.21 37.6-61 Rodolfi et al., 2009

*microscopica* Heterotrophic 7.1-15.3 0.26-0.44 30-50 Queiroz et al., 2011 *Crypthecodinium Cohnii* Heterotrophic 19.9 2.24 444.9 Couto et al., 2010 *Isochrysis galbana* Phototrophic 24.6 0.057 14.02 Lin et al., 2007

*Isochrysis* sp. Phototrophic 23.5-34.1 0.09 20.95 Huerlimann et al., 2010 *Isochrysis* sp. Phototrophic 22.4-27.4 0.14-0.17 37.8 Rodolfi et al., 2009 *Pavlova lutheri* Phototrophic 35.5 0.14 50.2 Rodolfi et al., 2009 *Pavlova salina* Phototrophic 30.9 0.16 49.4 Rodolfi et al., 2009 *Pavlova viridis* Phototrophic 24.8-32 Li et al., 2005

*Pleurochrysis carterae* Phototrophic 9.7-12 0.03-0.04 2.7-4.4 Chinnasamy et al., 2010 *Porphyridium cruentum* Phototrophic 9.5 0.37 34.8 Rodolfi et al., 2009 *Rhodomonas* sp.Phototrophic 9.5-20.5 0.06 6.19 Huerlimann et al., 2010

*limacinum* Heterotrophic 50.3 a 3.48 1750 Ethier et al., 2011

*mangrovei* Heterotrophic 68 a 2.44 1659 Fan et al., 2007

*Spirulina maxima* Phototrophic 4.1 0.21 8.6 Gouveia and Oliveira,

*Thalassiosira weissflogii* Phototrophic 6.3-13.2 0.5-1.5 31.5-198 Araujo et al., 2011

Table 1. Lipid content and productivity of various microalgal species.

biomass productivity (g/L/day)

Lipid productivity (mg/L/day)

References

Cohen, 2006

2009

2009

Lipid content (%)

Algal species Culture

*Phaeodactylum* 

*Thalassiosira* 

**Others**  *Aphanothece* 

*Schizochytrium* 

*Schizochytrium* 

a Total fatty acid content

**Eustigmatophyceae**

conditions


*Dunaliella tertiolecta* Phototrophic 12.2-15.2 0.03-0.04 4.0-4.6 Chinnasamy et al., 2010 *Dunaliella tertiolecta* Phototrophic 16.7 0.12 20 Gouveia and Oliveira,

*Haematococcus pluvialis* Phototrophic 15.6-34.9 Damiani et al., 2010 *Micractinium pusillum* Phototrophic 24 0.108 25.7 Abou-Shanab et al.,

*Neochloris oleabundans* Phototrophic 19-56 0.03-0.15 10.7-38.8 Gouveia et al., 2009 *Neochloris oleabundans* Phototrophic 7-40.3 0.31-0.63 38-133 Li et al., 2008 *Ourococcus multisporus* Phototrophic 52 0.045 23.3 Abou-Shanab et al.,

*Parietochloris incisa* Phototrophic 18-34 a 0.23-0.47 46-160 Solovchenko et al., 2008

*Scenedesmus obliquus* Phototrophic 17.7 0.09 15.9 Gouveia and Oliveira,

Mixotrophic 12.6-58.3 0.51 270 Mandal and Mallick,

*Pseudochlorococcum* sp. Phototrophic 24.6-52.1 0.234-0.76 53-350 Li et al., 2011a *Scenedesmus obliquus* Phototrophic 21-58 0.08-0.09 19-43.3 Abou-Shanab et al.,

*Scenedesmus obliquus* Phototrophic 12-38.9 0.20-0.29 35.1-78.7 Ho et al., 2010

*like* Phototrophic 11.3-27 a 0.44-0.54 108-133 Lin and Lin, 2011

*quadricauda* Phototrophic 18.4 0.19 35.1 Rodolfi et al., 2009 *Scenedesmus* sp.Phototrophic 22-53 0.08 20.3 Xin et al., 2010 *Scenedesmus* sp.Phototrophic 18 0.203 39 Yoo et al., 2010 *Scenedesmus* sp.Phototrophic 21.1 0.26 53.9 Rodolfi et al., 2009 *Tetraselmis chui* Phototrophic 17.3-23.5 1-2.6 235-450 Araujo et al., 2011 *Tetraselmis* sp. Phototrophic 8.7-33 0.21 22.86 Huerlimann et al., 2010 *Tetraselmis suecica* Phototrophic 8.5-12.9 0.28-0.32 27-36.4 Rodolfi et al., 2009 *Tetraselmis tetrathele* Phototrophic 29.2-30.3 3.1-4.4 905-1333 Araujo et al., 2011

*Chaetoceros calcitrans* Phototrophic 39.8 0.04 17.6 Rodolfi et al., 2009 *Chaetoceros gracilis* Phototrophic 15.5-60.3 3.4-3.7 530-2210 Araujo et al., 2011 *Chaetoceros muelleri* Phototrophic 11.7-25.3 1.2-2.7 1404-6831 Araujo et al., 2011 *Chaetoceros muelleri* Phototrophic 33.6 0.07 21.8 Rodolfi et al., 2009

*closterium* Phototrophic 17-30 Pruvost et al., 2011 *Navicula* sp.Phototrophic 47.6 a 0.055 26.4 Matsumoto et al., 2010 *Nitzschia cf. pusilla* Phototrophic 48 0.065 31.4 Abou-Shanab et al.,

*Nitzschia laevis* Heterotrophic 12.8 2.02 258.6 Chen et al., 2008

biomass productivity (g/L/day)

Lipid productivity (mg/L/day)

References

2010

2009

2011

2011

2011

2009

2009

2011

Lipid content (%)

*Chlorella zofingiensis* Phototrophic 25.8 0.136 35.1 Liu et al., 2011 *Chlorococcum* sp. Phototrophic 19.3 0.28 53.7 Rodolfi et al., 2009 *Choricystis minor* Phototrophic 21-59.3 0.35 82 Sobczuk and Chisti,

Algal species Culture

*Scenedesmus obliquus* Phototrophic,

*Scenedesmus rubescens* 

**Bacillariophyceae**

*Cylindrotheca* 

*Scenedesmus* 

conditions


a Total fatty acid content

Table 1. Lipid content and productivity of various microalgal species.

Fatty acids Algal species C12:0 C14:0 C15:0 C16:0 C16:1 C16:2 C16:3 C16:4 C17:0 C18:0 C18:1 C18:2 C18:3 C18:4 C20:0 C20:4 C20:5 C22:5 C22:6 Refs

*Botryococcus braunii* 29.5 3.4 1 44.9 21.2 Yoo et al., 2010 *Botryococcus* sp*.* 3.95 1.56 30.04 0.94 1.54 12.02 37.68 5.01 7.35 0.63 Yeesang and

*reinhardtii* 30.7 3 1.8 1.6 2.7 3.2 27.2 18.3 11 0.5 James et al., 2011 *Chlorella ellipsoidea* 2 26 4 40 23 5 Abou-Shanab et

*protothecoides* 14.3 1 0.32 2.7 71.6 9.7 Cheng et al 2009 *Chlorella pyrenoidosa* 0.7 17.3 0.8 7 9.3 1.2 3.3 18.5 41.8 D'Oca et al 2011 *Chlorella sorokiniana* 25.4 3.1 10.7 4.1 1.4 12.4 34.4 7.1 Chen and Johns,

*Chlorella* sp. 3.78 5.24 16.1 10.88 9.79 4.74 4.35 8.45 14.36 18.79 Li et al., 2011b *Chlorella vulgaris* 24 2.1 1.3 24.8 47.8 Yoo et al., 2010 *Chlorella zofingiensis* 22.62 1.97 7.38 1.94 0.22 2.09 35.68 18.46 7.75 0.49 Liu et al., 2010 *Chlorocuccum littorale* 20.9 5.6 14.4 29.7 7.2 22.2 Ota et al., 2009 *Choricystis minor* 36 0.4 12.3 31.2 9.9 3.8 1.9 Sobczuk and

*splendida* 13.88 69.59 1.21 0.38 1.11 12.14 0.42 Afify et al 2010 *Dunaliella tertiolecta* 26.4 2.3 1.27 0.6 16.8 13.1 39.6 Chen et al 2011

*pluvialis* 0.21 1.25 22.5 0.64 0.19 3.15 19.36 26.9 17.04 0.2 0.89 0.57 Damiani et al

*pusillum* 33 1 31 17 18 Abou-Shanab et

*oleabundans* 23.3 0.6 1.6 2.4 0.2 4.5 43 17.8 5.8 Levine et al., 2011 *Neochloris* sp. 5.22 29.4 5.2 6.6 17.5 23.6 12.6 Moazami et al.,

*multisporus* 2 19 1 5 26 11 36 Abou-Shanab et

*Parietochloris incise* 9.1 0.7 0.6 2.1 15.1 9.3 1.6 1.2 58.9 Khozin-Goldberg

*Scenedesmus obliquus* 1.48 21.8 5.95 3.96 0.68 0.43 0.45 17.93 21.74 3.76 0.21 Gouveia and

*Scenedesmus* sp*.* 36.3 4 2.7 25.9 31.1 Yoo et al., 2010 *Tetraselmis* sp.0.6 27.8 0.9 28.2 9.3 23.9 3.7 0.9 3.4 Huerlimann et

*Chaetoceros* sp. 23.6 9.2 36.5 6.9 2.6 2 3 1.4 0.6 4.1 8 1 Renaud et al.,

*Cyclotella cryptica* 1.4 15.2 10.7 3.9 1.2 3.5 9.7 1.7 Pahl et al., 2010 *Navicula* sp.45 52.7 0.6 1.1 0.6 Matsumoto et al.,

*Nitzschia cf. pusilla* 6 31 57 0.27 6 Abou-Shanab et

*Nitzschia laevis* 16.9 28.5 23.9 0.7 5.1 3.4 4.1 5 11.7 Chen et al 2008 *Nitzschia* sp. 9 3.5 37.4 4.6 5.3 16.9 11.6 Moazami et al.,

*Nostoc commune* 23.5 22.5 5.6 21.1 14.1 Pushparaj et al.,

*Nostoc flagelliforme* 0.65 21.27 14.91 6.2 22.59 15.03 19.35 Liu et al., 2005 *Spirulina* 49.2 5.9 1.7 2.9 22.7 17.5 Chaiklahan et al

*Spirulina maxima* 0.34 40.16 9.19 0.42 0.16 1.18 5.43 17.89 18.32 0.08 0.06 Gouveia and

*PCC6803* 52 3 1 3 9 29 3 Wada and

*Monodus subterraneus* 3.3 19.8 34.3 9.7 9 0.8 0.7 2.8 15.5 Khozin-Goldberg

*oculata* 62 11 5 8 15 Converti et al

*Nannochloropsis* sp*.* 23.4 7.14 45.4 11.7 12.2 Moazami et al.,

Cheirsilp, 2011

al 2011

1991

Chisti, 2010

2010

al 2011

2011

al 2011

et al., 2002

al., 2010

2002

2010

al 2011

2011

2008

2008

2009

2011

Oliveira, 2009

Murata, 1990

and Cohen, 2006

Oliveira, 2009

*Chlorophyta* 

*Chlamydomonas* 

*Dictyochloropsis* 

*Haematococcus* 

*Micractinium* 

*Neochloris* 

*Ourococcus* 

*Bacillariophyceae* 

*Cyanobacteria* 

*Synechocystis* 

*Eustigmatophyceae* 

*Nannochloropsis* 

*Chlorella* 

Fig. 4. Lipid content under nitrogen replete (open squares) and nitrogen deficient (filled circles) conditions for *Chlorophyta*. *B*. sp., *Botryococcus* sp. (Yeesang and Cheirsilp, 2011); *C. reinhardtii*, *Chlamydomonas reinhardtii* (Li et al., 2010); *C. littorale, Chlorocuccum littorale* (Ota et al., 2009); *C.* sp., *Chlorella* sp. (Hsieh and Wu, 2009); *C. vulgaris*, *Chlorella vulgaris* (Feng et al., 2011); *C. zofingiensis*, *Chlorella zofingiensis* (Liu et al., 2010); *H. pluvialis*, *Haematococcus pluvialis* (Damiani et al 2010); *N. oleabundans*, *Neochloris oleabundans* (Gouveia et al., 2009); *P. incisa, Parietochloris incisa* (Solovchenko et al., 2010); *P*. sp., *Pseudochlorococcum* sp. (Li et al., 2011); *S. obliquus*, *Scenedesmus obliquus* (Mandal and Mallick, 2009); *S. rubescens*, *Scenedesmus rubescens* (Mandal and Mallick, 2009); *T. suecica*, *Tetraselmis suecica* (Rodolfi et al., 2009).

The important properties of biodiesel such as cetane number, viscosity, cold flow, oxidative stability, are largely determined by the composition and structure of fatty acid esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstocks, for exmaple carbon chain length and unsaturation degree (Knothe, 2005b). Fatty acids are either in saturated or unsaturated form, and the unsaturated fatty acids may vary in the number and position of double bones on the acyl chain. Based on the number of double bones, unsaturated fatty acids are clarified into monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). The fatty acid profile of a great many algal species has been investigated and is shown in Table 2. The synthesized fatty acids in algae are commonly in medium length, ranging from 16 to 18 carbons, despite the great variation in fatty acid composition. Specifically, the major fatty acids are C16:0, C18:1 and C18:2 or C18:3 in green algae, C16:0 and C16:1 in diatoms and C16:0, C16:1, C18:1 and C18:2 in cyanobacteria. It is worthy to note that these data are obtained from algal species under specific conditions and vary greatly when algal cells are exposed to different environmental or nutritional conditions such as temperature, pH, light intensity, or nitrogen concentration (Guedes et al 2010; James et al., 2011; Sobczuk & Chisti, 2010; Tatsuzawa et al., 1996). Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially

140 Biodiesel – Feedstocks and Processing Technologies

Fig. 4. Lipid content under nitrogen replete (open squares) and nitrogen deficient (filled circles) conditions for *Chlorophyta*. *B*. sp., *Botryococcus* sp. (Yeesang and Cheirsilp, 2011); *C. reinhardtii*, *Chlamydomonas reinhardtii* (Li et al., 2010); *C. littorale, Chlorocuccum littorale* (Ota et al., 2009); *C.* sp., *Chlorella* sp. (Hsieh and Wu, 2009); *C. vulgaris*, *Chlorella vulgaris* (Feng et al., 2011); *C. zofingiensis*, *Chlorella zofingiensis* (Liu et al., 2010); *H. pluvialis*, *Haematococcus pluvialis* (Damiani et al 2010); *N. oleabundans*, *Neochloris oleabundans* (Gouveia et al., 2009); *P. incisa, Parietochloris incisa* (Solovchenko et al., 2010); *P*. sp.,

*Pseudochlorococcum* sp. (Li et al., 2011); *S. obliquus*, *Scenedesmus obliquus* (Mandal and Mallick,

The important properties of biodiesel such as cetane number, viscosity, cold flow, oxidative stability, are largely determined by the composition and structure of fatty acid esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstocks, for exmaple carbon chain length and unsaturation degree (Knothe, 2005b). Fatty acids are either in saturated or unsaturated form, and the unsaturated fatty acids may vary in the number and position of double bones on the acyl chain. Based on the number of double bones, unsaturated fatty acids are clarified into monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs). The fatty acid profile of a great many algal species has been investigated and is shown in Table 2. The synthesized fatty acids in algae are commonly in medium length, ranging from 16 to 18 carbons, despite the great variation in fatty acid composition. Specifically, the major fatty acids are C16:0, C18:1 and C18:2 or C18:3 in green algae, C16:0 and C16:1 in diatoms and C16:0, C16:1, C18:1 and C18:2 in cyanobacteria. It is worthy to note that these data are obtained from algal species under specific conditions and vary greatly when algal cells are exposed to different environmental or nutritional conditions such as temperature, pH, light intensity, or nitrogen concentration (Guedes et al 2010; James et al., 2011; Sobczuk & Chisti, 2010; Tatsuzawa et al., 1996). Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially

2009); *S. rubescens*, *Scenedesmus rubescens* (Mandal and Mallick, 2009); *T. suecica*,

*Tetraselmis suecica* (Rodolfi et al., 2009).


also be used for sequestration of carbon dioxide from industrial flue gases and wastewater treatment by removal of nutrients (Chinnasamy et al 2010; Fulke et al., 2010; Levine et al., 2011; Yang et al., 2011). Coupled with these environment-beneficial approaches, the production

> Oil yeild (L/ha year)

Land area needed (M ha)a Percentage of existing US cropping areaa

(% dry weight)

Table 3. Comparison of microalgae with other biodiesel feedstocks.

**3. Biodiesel production from microalgae** 

Fig. 5. Microalgal biodiesel production pipeline

Corn 44 172 3480 1912 Hemp 33 363 1650 906 Soybean 18 636 940 516 Jatropha 28 741 807 443 Camelina 42 915 650 357 Canola 41 974 610 335 Sunflower 40 1070 560 307 Castor 48 1307 450 247 Palm oil 36 5366 110 60.4 Microalgae (low oil content) 30 58,700 10.2 5.6 Microalgae (medium oil content) 50 97,800 6.1 3.4 Microalgae (high oil content) 70 136,900 4.4 2.4

a For meeting all transport fuel needs of the United States. Adapted from Chisti, 2007 and Mata et al., 2010.

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.

potential of microalgae derived biodiesel is desirable.

Feedstocks Oil content


Table 2. Fatty acid composition of various algal species (% of total fatty acids)

polyunsaturated, fatty esters have improved low-temperature properties (Knothe, 2008). In this regard, it is suggested that the modification of fatty esters, for example the enhanced proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote the quality of biodiesel (Knothe, 2009). Thus, microalgae with high oleic acid are suitable for biodiesel production.

Currently the commercial production of biodiesel is mainly from plant oils and animal fats. However, the plant oil derived biodiesel cannot realistically meet the demand of transport fuels because large arable lands are required for cultivation of oil plants, as demonstrated in Table 3. Based on the oil yield of different plants, the cropping area needed is calculated and expressed as a percentage of the total U.S. cropping area. If soybean, the popular oil crop in United States is used for biodiesel production to meet the existing transport fuel need, 5.2 times of U.S. cropland will need to be employed. Even the high-yielding oil plant palm is planted as the biodiesel feedstock, more than 50% of current U.S. arable lands have to be occupied. The requirement of huge arable lands and the resulted conflicts between food and oil make the biodiesel from plant oils unrealistic to completely replace the petroleum derived diesel in the foreseeable future. It is another case, however, if microalgae are used to produce biodiesel. As compared with the conventional oil plants, microalgae possess significant advantages in biomass production and oil yield and therefore the biodiesel productivity. In terms of land use, microalgae need much less than oil plants, thus eliminating the competition with food for arable lands (Table 3).

In addition to biodiesel, microalgae can serve as sources of other renewable fuels such as biogas, bioethanol, bio-oil and syngas (Chisti, 2008; Demirbas, 2010; Mussgnug et al., 2010). Moreover, microalgal biomass contains significant amounts of proteins, carbohydrates and other high-value compounds that can be potentially used as feeds, foods and pharmaceuticals (Chisti, 2007). Thus, integrating the production of such co-products with biofuels will provide new insight into improving the production economics of microalgal biodiesel. Microalgae can

Fatty acids Algal species C12:0 C14:0 C15:0 C16:0 C16:1 C16:2 C16:3 C16:4 C17:0 C18:0 C18:1 C18:2 C18:3 C18:4 C20:0 C20:4 C20:5 C22:5 C22:6 Refs

*galbanan* 19.3 18.1 29.5 2.6 3.6 13.8 4.1 7.5 Lin et al., 2007 *Isochrysis* sp. 8.9 0.4 13.7 5.1 0.2 22.8 2.3 4.8 22.5 0.1 0.6 1.7 12.7 Huerlimann et

*Pavlova lutheri* 5.54 19 31.46 1.11 2.55 4.46 5.37 6.63 16.07 7.8 Guedes et al 2010 *Pavlova viridis* 19.9 13.9 16.1 21.2 8.7 Hu et al 2008a *Pavlova viridis* 10.34 17.3 17.87 3.16 1.33 2.48 2.23 10.46 14.78 Li et al., 2005

*cruentum* 14.5 8.5 10.5 14 10.8 6.1 10.5 Oh et al., 2009

*cohnii* 2.9 13.4 22.9 0.4 2.6 7.6 0.5 49.5 Couto et al., 2010

*chrysoplasta* 22 4.4 4 6.6 3.9 5.5 39.2 13.3 Kawachi et al.,

*Rhodomonas* sp*.* 7.8 0.4 19.7 1.5 3 8.4 3 29.8 11.7 0.6 8.6 1.7 3 Huerlimann et

*limacinum* 3.96 54.61 3.86 6.47 31.09 Ethier et al 2011

polyunsaturated, fatty esters have improved low-temperature properties (Knothe, 2008). In this regard, it is suggested that the modification of fatty esters, for example the enhanced proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote the quality of biodiesel (Knothe, 2009). Thus, microalgae with high oleic acid are suitable for

Currently the commercial production of biodiesel is mainly from plant oils and animal fats. However, the plant oil derived biodiesel cannot realistically meet the demand of transport fuels because large arable lands are required for cultivation of oil plants, as demonstrated in Table 3. Based on the oil yield of different plants, the cropping area needed is calculated and expressed as a percentage of the total U.S. cropping area. If soybean, the popular oil crop in United States is used for biodiesel production to meet the existing transport fuel need, 5.2 times of U.S. cropland will need to be employed. Even the high-yielding oil plant palm is planted as the biodiesel feedstock, more than 50% of current U.S. arable lands have to be occupied. The requirement of huge arable lands and the resulted conflicts between food and oil make the biodiesel from plant oils unrealistic to completely replace the petroleum derived diesel in the foreseeable future. It is another case, however, if microalgae are used to produce biodiesel. As compared with the conventional oil plants, microalgae possess significant advantages in biomass production and oil yield and therefore the biodiesel productivity. In terms of land use, microalgae need much less than oil plants, thus

In addition to biodiesel, microalgae can serve as sources of other renewable fuels such as biogas, bioethanol, bio-oil and syngas (Chisti, 2008; Demirbas, 2010; Mussgnug et al., 2010). Moreover, microalgal biomass contains significant amounts of proteins, carbohydrates and other high-value compounds that can be potentially used as feeds, foods and pharmaceuticals (Chisti, 2007). Thus, integrating the production of such co-products with biofuels will provide new insight into improving the production economics of microalgal biodiesel. Microalgae can

Table 2. Fatty acid composition of various algal species (% of total fatty acids)

eliminating the competition with food for arable lands (Table 3).

al., 2010

2002

al., 2010

*Prymnesiophyceae Isochrysis* 

*Rhodophyta Porphyridium* 

*Glossomastrix* 

*Schizochytrium* 

biodiesel production.

**Others**  *Crypthecodinium*  also be used for sequestration of carbon dioxide from industrial flue gases and wastewater treatment by removal of nutrients (Chinnasamy et al 2010; Fulke et al., 2010; Levine et al., 2011; Yang et al., 2011). Coupled with these environment-beneficial approaches, the production potential of microalgae derived biodiesel is desirable.


a For meeting all transport fuel needs of the United States. Adapted from Chisti, 2007 and Mata et al., 2010.

Table 3. Comparison of microalgae with other biodiesel feedstocks.
