**2. Biodiesel and current feedstocks**

#### **2.1 Biodiesel production**

Another approach for alternative biofuels is biodiesel. The most common type of biodiesel is the methyl esters of fatty acid (FAME), obtained by transesterification of lipid with methanol or ethanol. It can be used in pure form (B100) or may be blended with fossil diesel at any rate. The commonly used biodiesel is B99 because 1% of fossil fuel is applied to

Microbial Biodiesel Production - Oil Feedstocks Produced from Microbial Cell Cultivations 95

source of sugars for biofuel and bioproducts. This potential has been gaining much attention recently as it is been defined as "second generation of bioenergy", compared to current technologies that primarily are limited to food materials. Oil accumulation with microalgae and other similar technologies started to show some potential to produce biofuel products

Algae oil is being seriously considered because of the large oil yields it shows compared with other oilseeds. The Office of Fuels Development, a division of the Department of Energy, funded a program from 1978 through 1996 under the National Renewable Energy Laboratory known as the "Aquatic Species Program". The focus of this program was to investigate high-oil algaes that could be grown specifically for the purpose of widescale biodiesel production. NREL's research showed that one quad (7.5 billion gallons) of biodiesel could be produced from 200,000 hectares of desert land (200,000 hectares is equivalent to 780 square miles, roughly 500,000 acres). It would be preferable to spread the algae production around the country, to lessen the cost and energy used in transporting the feedstocks. Algae farms could also be constructed to use waste streams (either human waste or animal waste from animal farms) as a food source, which would spread algae production around the country. Nutrients can also be extracted from the algae for the production of a fertilizer high in nitrogen and phosphorous. By using waste streams (agricultural, farm animal waste, and human sewage) as the nutrient source, these farms essentially also provide a means of recycling nutrients from fertilizer to food to waste and back to fertilizer. Microalgae are generally autotrophic eukaryotic cells, and contain one or more types of chlorophyll plus additional pigments known as carotenoids and biloproteins (also called phycobilins). Carotenoids are yellow, orange, or red water-insoluble linear hydrocarbons; biloproteins are blue or red water-soluble pigment-protein complexes. The color of the different groups of algae depends on the ratio of these pigments. Almost all the microalgae cells contain chlorophyll, but the green color can be masked by the carotenoids, giving them a brown or red color. Microalgae cells are mostly unicellular, but some species are colonial or filamentous. They can grow autotrophically and/or heterotrophically, with a wide range of tolerance to different temperature, salinity, pH and nutrient availabilities. Cyanobacteria, although sometimes also referred as algae, specifically as blue-green algae, are by definition prokaryotic bacteria, instead of microalgae. The eukaryotes microalgae we are referring to include green algae, diatoms, yellow–green algae, golden algae, red algae, brown algae, dinoflagellates and others; and only limited species have the capability to accumulate high content of lipids in their cell biomass. Microbial species that can accumulate over 20% of

with massive scales (Benemann, 1996; Chisti, 2007; Jarvis et al., 1994).

**3. Microorganisms for microbial lipid production** 

lipids in their cell biomass are considered oleaginous species.

One of the characteristics of algae that separate them from other oilseeds is the quantity of lipids and fatty acids algae have as membrane components; some algae have been found to contain over 80% of lipids, which is of great interest for a sustainable feedstock for biodiesel production. Different microalgae species are listed in Table 2 as examples of lipid accumulation research. For example, *Chlorella vulgaris* is a commercially important green microalgae because it has the potential to serve as a food and energy source due to its high photosynthetic efficiency, which can, in theory, reach 8%. It can grow with both autotrophic and heterotrophic modes, and its mixotrophic growth rate is the sum of its autotrophic

**3.1 Microalgae** 

inhibit mold growth, which shortens its shelf life. Biodiesel is around 5-8% less efficient than conventional fossil diesel; yet, biodiesel has potential as a total or partial (in the cold regions) replacement to the fossil diesel, compatible to the current diesel engine. There are numerous environmental benefits to replace fossil diesel to biodiesel since combustion of biodiesel emits far less pollutants comparing to fossil diesel (except the NOx emission) and the entire process is close to carbon neutral considering that the plant oil used to produce biodiesel is synthesized in the agriculture from CO2 in the air. The production and utilization of biodiesel are significant in many aspects, for instance, increasing oilseed crop market, providing domestic job opportunities to the rural community, and decreasing the dependence on imported oil; therefore biodiesel has been commercialized around the globe. Another type of biodiesel is the hydrocarbon generated with direct decarboxylation of fatty acid or lipid. Although it shows superior features than current FAME biodiesel in many aspects, the chemical decarboxylation process to produce diesel still needs further development compared to the transesterification process for biodiesel production.

#### **2.2 Agricultural feedstocks: Vegetable oil, animal fat and recycled grease**

The biodiesel industry suffered due to limited raw materials such as soybean oil or vegetable oil. Although there are numerous potential renewable and carbon neutral feedstocks for the production of biodiesel, none seems capable of displacing fossil diesel. Table 1 shows a comparison of the oil yields of some sources of biodiesel, the land area for their cultivation, and the percentage of existing United States (U.S.) cropping area to meet half of the transport fuel needs. Using soybean as feedstock for the production of biodiesel requires 326% of the U.S. cropping area to meet the 50% of all U.S. transport fuel needs. Based on this rough estimation, none of the terrestrial crops is able to completely substitute crude oil.


a For meeting 50% of all transport fuel needs of the U.S.

b 70% oil (by wt) in biomass.

c 30% oil (by wt) in biomass.

Table 1. Comparison of some sources of biodiesel (Chisti 2007)

Various raw materials are proposed to produce biodiesel, including waste cooking oil and various oil-accumulating plants, which could help solve the shortage for raw materials but are not sufficiently available. New approaches need to be developed to use an alternative biomass as the substrate for biofuel production, for example, biomass leftover from agricultural harvest or the biomass produced in non-traditional agricultural land as the source of sugars for biofuel and bioproducts. This potential has been gaining much attention recently as it is been defined as "second generation of bioenergy", compared to current technologies that primarily are limited to food materials. Oil accumulation with microalgae and other similar technologies started to show some potential to produce biofuel products with massive scales (Benemann, 1996; Chisti, 2007; Jarvis et al., 1994).

#### **3. Microorganisms for microbial lipid production**

#### **3.1 Microalgae**

94 Biodiesel - Feedstocks and Processing Technologies

inhibit mold growth, which shortens its shelf life. Biodiesel is around 5-8% less efficient than conventional fossil diesel; yet, biodiesel has potential as a total or partial (in the cold regions) replacement to the fossil diesel, compatible to the current diesel engine. There are numerous environmental benefits to replace fossil diesel to biodiesel since combustion of biodiesel emits far less pollutants comparing to fossil diesel (except the NOx emission) and the entire process is close to carbon neutral considering that the plant oil used to produce biodiesel is synthesized in the agriculture from CO2 in the air. The production and utilization of biodiesel are significant in many aspects, for instance, increasing oilseed crop market, providing domestic job opportunities to the rural community, and decreasing the dependence on imported oil; therefore biodiesel has been commercialized around the globe. Another type of biodiesel is the hydrocarbon generated with direct decarboxylation of fatty acid or lipid. Although it shows superior features than current FAME biodiesel in many aspects, the chemical decarboxylation process to produce diesel still needs further

development compared to the transesterification process for biodiesel production.

The biodiesel industry suffered due to limited raw materials such as soybean oil or vegetable oil. Although there are numerous potential renewable and carbon neutral feedstocks for the production of biodiesel, none seems capable of displacing fossil diesel. Table 1 shows a comparison of the oil yields of some sources of biodiesel, the land area for their cultivation, and the percentage of existing United States (U.S.) cropping area to meet half of the transport fuel needs. Using soybean as feedstock for the production of biodiesel requires 326% of the U.S. cropping area to meet the 50% of all U.S. transport fuel needs. Based on this rough estimation, none of the terrestrial crops is able to completely substitute

Corn 172 1540 846 Soybean 446 594 326 Canola 1190 223 122 Jatropha 1892 140 77 Coconut 2689 99 54 Oil palm 5950 45 24 Microalgaea 136,900 2 1.1 Microalgaec 58,700 4.5 2.5

Various raw materials are proposed to produce biodiesel, including waste cooking oil and various oil-accumulating plants, which could help solve the shortage for raw materials but are not sufficiently available. New approaches need to be developed to use an alternative biomass as the substrate for biofuel production, for example, biomass leftover from agricultural harvest or the biomass produced in non-traditional agricultural land as the

**Land area needed** [Mha]a

**Percent of existing US cropping area**<sup>a</sup>

**2.2 Agricultural feedstocks: Vegetable oil, animal fat and recycled grease** 

crude oil.

**Crop Oil yield**

[Liters per hectare]

a For meeting 50% of all transport fuel needs of the U.S.

Table 1. Comparison of some sources of biodiesel (Chisti 2007)

b 70% oil (by wt) in biomass. c 30% oil (by wt) in biomass. Algae oil is being seriously considered because of the large oil yields it shows compared with other oilseeds. The Office of Fuels Development, a division of the Department of Energy, funded a program from 1978 through 1996 under the National Renewable Energy Laboratory known as the "Aquatic Species Program". The focus of this program was to investigate high-oil algaes that could be grown specifically for the purpose of widescale biodiesel production. NREL's research showed that one quad (7.5 billion gallons) of biodiesel could be produced from 200,000 hectares of desert land (200,000 hectares is equivalent to 780 square miles, roughly 500,000 acres). It would be preferable to spread the algae production around the country, to lessen the cost and energy used in transporting the feedstocks. Algae farms could also be constructed to use waste streams (either human waste or animal waste from animal farms) as a food source, which would spread algae production around the country. Nutrients can also be extracted from the algae for the production of a fertilizer high in nitrogen and phosphorous. By using waste streams (agricultural, farm animal waste, and human sewage) as the nutrient source, these farms essentially also provide a means of recycling nutrients from fertilizer to food to waste and back to fertilizer.

Microalgae are generally autotrophic eukaryotic cells, and contain one or more types of chlorophyll plus additional pigments known as carotenoids and biloproteins (also called phycobilins). Carotenoids are yellow, orange, or red water-insoluble linear hydrocarbons; biloproteins are blue or red water-soluble pigment-protein complexes. The color of the different groups of algae depends on the ratio of these pigments. Almost all the microalgae cells contain chlorophyll, but the green color can be masked by the carotenoids, giving them a brown or red color. Microalgae cells are mostly unicellular, but some species are colonial or filamentous. They can grow autotrophically and/or heterotrophically, with a wide range of tolerance to different temperature, salinity, pH and nutrient availabilities. Cyanobacteria, although sometimes also referred as algae, specifically as blue-green algae, are by definition prokaryotic bacteria, instead of microalgae. The eukaryotes microalgae we are referring to include green algae, diatoms, yellow–green algae, golden algae, red algae, brown algae, dinoflagellates and others; and only limited species have the capability to accumulate high content of lipids in their cell biomass. Microbial species that can accumulate over 20% of lipids in their cell biomass are considered oleaginous species.

One of the characteristics of algae that separate them from other oilseeds is the quantity of lipids and fatty acids algae have as membrane components; some algae have been found to contain over 80% of lipids, which is of great interest for a sustainable feedstock for biodiesel production. Different microalgae species are listed in Table 2 as examples of lipid accumulation research. For example, *Chlorella vulgaris* is a commercially important green microalgae because it has the potential to serve as a food and energy source due to its high photosynthetic efficiency, which can, in theory, reach 8%. It can grow with both autotrophic and heterotrophic modes, and its mixotrophic growth rate is the sum of its autotrophic

Microbial Biodiesel Production - Oil Feedstocks Produced from Microbial Cell Cultivations 97

heterotrophically cultured cells exhibited better capability for biomass and lipid production. Miao and Wu (2006) reported that the oil content of heterotrophically cultured *C. protothecoides* was approximately four times greater than that in the corresponding autotrophic culture. Liu et al. (2010) demonstrated that the heterotrophically cultured cells of *Chlorella zofingiensis* showed 411% and 900% increases in dry cell weight and lipid yield, respectively, compared to autotrophically cultured cells. Moreover, biodiesel produced from heterotrophically cultured algae oils had similar properties to diesel fuel in terms of density, viscosity, heating value, and H/C ratio (Xu, Miao et al. 2006). In addition to lipid production, high value byproducts can be obtained from heterotrophically cultured microalgae, including polyunsaturated fatty acids and carotenoids (Chen and Chen 2006).

Besides microalgae, many yeast and fungi species (e.g., *Mucor circillenous* or *Mortierella isabellina*) also can accumulate a high content of lipids (Xia 2011; Heredia-Arroyo, Wei et al. 2011). Many oleaginous yeasts were studied for lipid accumulation on different substrates, such as industrial glycerol (Meesters, Huijberts et al. 1996; Papanikolaou and Aggelis 2002), sewage sludge (Angerbauer, Siebenhofer et al. 2008), whey permeate (Ykema, Verbree et al. 1988; Akhtar, Gray et al. 1998), sugar cane molasses (Alvarez, Rodriguez et al. 1992), and rice straw hydrolysate (Huang, Zong et al. 2009). The use of non-starch biomass is critical so that lignocelluloses can be used for organic carbon supply without concern of using food crops for fuel sources. Recent studies detailed conversion of hemicellulose hydrolysate into lipids by oleaginous yeast strains and their tolerance degrees to lignocellulose degradation compounds (Chen, Li et al. 2009; Hu, Zhao et al. 2009; Huang, Zong et al. 2009). However, these strains were unable to efficiently produce lipids in the presence of inhibitors in the hydrolysate, necessitating detoxification treatment prior to fermentation, which increases the cost of the process. Thus, using strains capable of growing in the non-detoxified hydrolysate is necessary for viable microbial lipid production in an industrial context. In addition, previous reports indicate that temperature is an key factor in regulating the fatty acid composition in fungi

Similarly, some oleaginous filamentous fungi can also produce lipids by utilizing glycerol, acetic acid, soluble starch, wheat straw, and wheat bran. Dey et al screening two endophytic oleaginous fungi *Colletotrichum sp.* and *Alternaria sp*. with lipid content 30% and 58% respectively (Dey, Banerjee et al. 2011). Fifteen eukaryotic microorganism were tested for waste glycerol assimilation to produce lipid. Fungi accumulated lipid inside their mycelia (lipid content ranging between 18.1 and 42.6% ) (Chatzifragkou, Makri et al. 2011). Such capabilities provide potential to utilize sugars in the pretreated lignocellulosic materials hydrolysate. Moreover, because the fatty acid profile of microbial oils is quite similar to that of conventional vegetable oils, oleaginous filamentous fungi are suggested as a favorable feedstock for a sustainable biodiesel industry (Peng and Chen 2008; Zhao,

Microalgae cells can generally utilize sunlight, carbon dioxide and nutrients from waste water for their cell growth (Brennan and Owende 2010). Lipid accumulation with microalgae cultivation is relatively efficient due to its high production efficiency and less

(Kendrick and Ratledge 1992; Weinstein, Montiel et al. 2000).

**4. Feedstocks for microbial lipid production** 

**3.2 Yeast and fungi** 

Hu et al. 2010).

**4.1 Light and carbon dioxide** 

growth rate and heterotrophic growth rate, separately. *Chlorella protothecoides* is another single-cell green microalgae, which has high potential for the energy and food production. Heterotrophic growth of *C. protothecoides* supplied with acetate, glucose, or other organic compounds as carbon source, results in high biomass and high content of lipid in cells.


AC: autotrophic cultures; MC: mixotrophic cultures; HC: heterotrophic cultures

Table 2. Some examples of microalgae cultivation for oil accumulation

Lipid accumulation occurs within the microalgae cells and it varies from strain and growth conditions. There are many nutritional and environmental factors controlling the cell growth and lipid contents, such as organic and inorganic carbon sources, nitrogen source, and other essential macro- and micro-nutrients like magnesium and copper, temperature, pH level, salinity, agitation speed (dissolved oxygen). Many microalgae species, for example, *C. protothecoides*, accumulated a higher content of lipids in cells and achieved higher growth rate when the culture was under heterotrophic mode. Like yeast and fungi, heterotrophic algae can accumulate biomass and lipids using organic carbon as its source instead of carbon dioxide and sunlight. Compared with autotrophic algae, the heterotrophic growth process has the advantages of no light limitation, a high degree of process control, higher productivity, and low costs for biomass harvesting (Barclay, Meager et al. 1994). Table 2 shows the oil production of autotrophically and heterotrophically cultured microalgae. Most heterotrophically cultured algae have greater than ten times the biomass concentration, while the lipid productivity is also significantly higher than the theoretical data for autotrophic cultivation. For certain algae strains, it was suggested the heterotrophically cultured cells exhibited better capability for biomass and lipid production. Miao and Wu (2006) reported that the oil content of heterotrophically cultured *C. protothecoides* was approximately four times greater than that in the corresponding autotrophic culture. Liu et al. (2010) demonstrated that the heterotrophically cultured cells of *Chlorella zofingiensis* showed 411% and 900% increases in dry cell weight and lipid yield, respectively, compared to autotrophically cultured cells. Moreover, biodiesel produced from heterotrophically cultured algae oils had similar properties to diesel fuel in terms of density, viscosity, heating value, and H/C ratio (Xu, Miao et al. 2006). In addition to lipid production, high value byproducts can be obtained from heterotrophically cultured microalgae, including polyunsaturated fatty acids and carotenoids (Chen and Chen 2006).

#### **3.2 Yeast and fungi**

96 Biodiesel - Feedstocks and Processing Technologies

growth rate and heterotrophic growth rate, separately. *Chlorella protothecoides* is another single-cell green microalgae, which has high potential for the energy and food production. Heterotrophic growth of *C. protothecoides* supplied with acetate, glucose, or other organic compounds as carbon source, results in high biomass and high content of lipid in cells.

Microalgae Cultures Substrates Growth Rate Lipid Content AC MC HC

3.74g/L - 144h 55.2%

the first 20 Hr 21%

14-22%

0.098/h

double in 14h after adaptation

exponential during

CO2

lactate / CO2

*cohnii* X glucose/ CO2 40g/L - 60-90h 15-30%

*Gracilis* X CO2 14-20%

*sp* glycerol/ CO2 130 -140h (28°C) 55%

*Braunii* X CO2 low growth rate 20 - 86%

*salina* X x CO2 ~70%

Lipid accumulation occurs within the microalgae cells and it varies from strain and growth conditions. There are many nutritional and environmental factors controlling the cell growth and lipid contents, such as organic and inorganic carbon sources, nitrogen source, and other essential macro- and micro-nutrients like magnesium and copper, temperature, pH level, salinity, agitation speed (dissolved oxygen). Many microalgae species, for example, *C. protothecoides*, accumulated a higher content of lipids in cells and achieved higher growth rate when the culture was under heterotrophic mode. Like yeast and fungi, heterotrophic algae can accumulate biomass and lipids using organic carbon as its source instead of carbon dioxide and sunlight. Compared with autotrophic algae, the heterotrophic growth process has the advantages of no light limitation, a high degree of process control, higher productivity, and low costs for biomass harvesting (Barclay, Meager et al. 1994). Table 2 shows the oil production of autotrophically and heterotrophically cultured microalgae. Most heterotrophically cultured algae have greater than ten times the biomass concentration, while the lipid productivity is also significantly higher than the theoretical data for autotrophic cultivation. For certain algae strains, it was suggested the

*pusillum* x X CO2 0.94g/L - 24h

*platensis* X X glucose/ CO2 0.008 /h

AC: autotrophic cultures; MC: mixotrophic cultures; HC: heterotrophic cultures Table 2. Some examples of microalgae cultivation for oil accumulation

*protothecoides* X X glucose, acetate /

*vulgaris* X X X glucose, acetate,

*obliquus* X X glucose/ CO2

*reinhardtii* X x X acetate/ CO2

*Chlorella* 

*Chlorella* 

*Crypthecodinium* 

*Chlamydomonas* 

*Micractinium* 

*Schizochytrium* 

*Euglena* 

*Spirulina* 

*Botryococcus* 

*Dunaliella* 

*Scenedesmus* 

Besides microalgae, many yeast and fungi species (e.g., *Mucor circillenous* or *Mortierella isabellina*) also can accumulate a high content of lipids (Xia 2011; Heredia-Arroyo, Wei et al. 2011). Many oleaginous yeasts were studied for lipid accumulation on different substrates, such as industrial glycerol (Meesters, Huijberts et al. 1996; Papanikolaou and Aggelis 2002), sewage sludge (Angerbauer, Siebenhofer et al. 2008), whey permeate (Ykema, Verbree et al. 1988; Akhtar, Gray et al. 1998), sugar cane molasses (Alvarez, Rodriguez et al. 1992), and rice straw hydrolysate (Huang, Zong et al. 2009). The use of non-starch biomass is critical so that lignocelluloses can be used for organic carbon supply without concern of using food crops for fuel sources. Recent studies detailed conversion of hemicellulose hydrolysate into lipids by oleaginous yeast strains and their tolerance degrees to lignocellulose degradation compounds (Chen, Li et al. 2009; Hu, Zhao et al. 2009; Huang, Zong et al. 2009). However, these strains were unable to efficiently produce lipids in the presence of inhibitors in the hydrolysate, necessitating detoxification treatment prior to fermentation, which increases the cost of the process. Thus, using strains capable of growing in the non-detoxified hydrolysate is necessary for viable microbial lipid production in an industrial context. In addition, previous reports indicate that temperature is an key factor in regulating the fatty acid composition in fungi (Kendrick and Ratledge 1992; Weinstein, Montiel et al. 2000).

Similarly, some oleaginous filamentous fungi can also produce lipids by utilizing glycerol, acetic acid, soluble starch, wheat straw, and wheat bran. Dey et al screening two endophytic oleaginous fungi *Colletotrichum sp.* and *Alternaria sp*. with lipid content 30% and 58% respectively (Dey, Banerjee et al. 2011). Fifteen eukaryotic microorganism were tested for waste glycerol assimilation to produce lipid. Fungi accumulated lipid inside their mycelia (lipid content ranging between 18.1 and 42.6% ) (Chatzifragkou, Makri et al. 2011). Such capabilities provide potential to utilize sugars in the pretreated lignocellulosic materials hydrolysate. Moreover, because the fatty acid profile of microbial oils is quite similar to that of conventional vegetable oils, oleaginous filamentous fungi are suggested as a favorable feedstock for a sustainable biodiesel industry (Peng and Chen 2008; Zhao, Hu et al. 2010).

#### **4. Feedstocks for microbial lipid production**

#### **4.1 Light and carbon dioxide**

Microalgae cells can generally utilize sunlight, carbon dioxide and nutrients from waste water for their cell growth (Brennan and Owende 2010). Lipid accumulation with microalgae cultivation is relatively efficient due to its high production efficiency and less

Microbial Biodiesel Production - Oil Feedstocks Produced from Microbial Cell Cultivations 99

attributed to chemical precipitation and biological assimilation (around 50% each). In removing ammonia, the HRAP is superior to the traditional bacterial nitrification– denitrification process, which requires the assimilation of extra-organic carbon as a carbon source. Phosphorus removal by microalgae is largely thought to be due to its uptake for normal growth, as an essential element required for making cellular constituents such as phospholipids, nucleotides, and nucleic acids. Under certain conditions microalgae can be triggered to uptake more phosphorus than is necessary for survival, in the form of polyphosphate (Powell, Shilton et al. 2011). Phosphorus removal by luxury uptake (amount of P uptake more than growth required) was confirmed to occur in the microalgae growing in the wastewater treatment facility; further research is needed in this prosperous field

Several research studies revealed that organic carbon sources, if the algae have the capability to grow on heterotrophic mode, can significantly increase the cell growth rate and dramatically enhance the lipid content of the biomass. For example, *Chlorella protothecoides* can only accumulate 18-25% lipid in the autotrophic culture, while the lipid content can reach to 55.2% dry cell biomass if cultured with addition of organic carbons (Xiong, Li et al. 2008). However, the heterotrophic cultivation of microalgae mostly request pure monosugars, which are usually costly and limited. Alternative materials with relative abundance and zero or negative valued organic materials, such as lignocellulosic biomass from agriculture, are the only choice for this route. Agricultural feedstocks contribute a large part of renewable resource for biodiesel production, which are the target of biodiesel resource of cost reduction of biodiesel and non-human food resource discovery. Most of these substrates are locally available and thus are expected to support mainly small production facilities. Lignocellulosic materials are mainly composed of cellulose, hemicellulose, and lignin, which make up approximately 90% of the dry weight of most plant materials (Kumar, Barrett et al. 2009). Cellulose and hemicellulose can be converted to fermentable sugars for microbial lipid production. However, the direct enzymatic hydrolysis of cellulose and hemicellulose to sugars is impeded by the cell wall physico-chemical and structural composition. Thus, biomass pretreatment prior to enzymatic hydrolysis is essential to enhance enzymatic digestibility. Distinctly different from autotrophic microalgae cultures, this process is more similar to cellulosic ethanol production, where hydrolysis and

Three cultivation processes were designed to culture microalgae and other oleaginous cells, including open-pond system, photobioreactor, and fermentation. The open-pond system is typically a closed loop with a pump to create microalgae flow in the channel. The channel is 0.2-0.5 meter deep, and the pump keeps the microalgae cell well mixed for continuous growth. This type of open-pond system has been used for several years because it is easy to operate and inexpensive to maintain. The open-pond system also can be upgraded to large-scale microalgae production. On the other hand, a high cell density of microalgae cannot be reached because of limited capability to assimilate the sun light and low carbon dioxide concentration of air. Increasing the CO2 concentration by using

about the detailed mechanism and applications.

**4.3 Lignocellulosic biomass** 

fermentation are needed for conversion.

**5. Conversion processes for microbial lipid production 5.1 Autotrophic microalgae cultivation and lipid accumulation** 

demand of agricultural land. Most microalgal ponds have a solar energy conversion efficiency of 1–4% under normal operating conditions and higher efficiencies can be achieved with closed photo-bioreactor systems. There is a considerable margin for improvement, which is being targeted through accelerated breeding programs and genetic modification. Autotrophic microalgae cells also absorb CO2 as their carbon source to support their cell growth, which makes the microalgae an attractive option for the biological CO2 fixation. Atmospheric CO2 accumulation, derived mainly from fossil fuel combustion, is proved as the leading cause of global warming. Current mitigation methods such as physicochemical adsorption, injection into deep oceans and geological formations are not economically feasible due to the high cost of implementing these methods and possible CO2 leakage. After CO2 is fixed via microalgae assimilation, the cell biomass can be utilized to generate methane gas, biochar, or the oil can be extracted to generate biodiesel. Microalgae can fix not only atmospheric CO2, but also the CO2 from industrial exhaust gases and from the carbonate salt, which are chemically fixed. Atmospheric CO2 levels (0.0387%) are not sufficient to support the high microalgae growth rates and productivities needed for commercial biofuel production, adding CO2 into autotrophic microalgae culture is an effective method to accelerate the microalgae growth rate. Utilization of CO2, for example, flue gas from electrical plant, by means of microalgae alleviates the impact of CO2 on the environment (greenhouse effect) and renders algal biomass production less expensive. It was also reported that the CO2 content was reduced from 44–48% to 2.5–11% when the microalgae cultivation was integrated with anaerobic digestion to remove impurities of biogas produced from anaerobic digestion. Microalgae also assimilated other impurities such as ammonia and hydrogen sulfide; and the gas leaving the algae pond had 88–97% by volume of methane 23. Although there are conflicting results from different references about the toxicity of ammonia, hydrogen sulfide and other impurities on the growth of microalgae cells, integration of microalgae cultivation together with this industrial processes can be more sustainable and economically feasible than the individual microalgae cultivation process to generate oil for biodiesel production.

#### **4.2 Wastewater**

Culturing microalgae with nutrients from wastewater, such as nitrogen and phosphate, can decrease the cost of the raw materials and also provide some environmental benefits. Agricultural effluent and municipal wastewater, even after treated with anaerobic digestion (AD), cannot be disposed directly because of their high nutrient level (Levine, Costanza-Robinson et al. 2011). In contrast, these wastewater can be considered as a cost-effective candidate of raw materials for biodiesel production (Siddiquee and Rohani 2011). Cultivation of microalgae, yeast, or fungi can be integrated with AD system to reduce the remaining COD, phosphate, and ammonia. Microalgae were also studied to absorb metal ion, waste pharmaceutical chemicals and dye into their cell biomass in order to remove the pollutants from wastewater once their cell biomass were stabilized and harvested. A typical example is the recently developed high-rate algae pond (HRAP) in the tertiary wastewater treatment facility (El Hamouri 2009). HRAP functions behind a two-step upflow anaerobic reactor (pre-treatment) and was followed by one maturation pond (MP) for polishing. The HRAP was revealed to have no activity for removing the COD from the wastewater; however, it removed 85% of total N and 63% of total P. Nitrogen removal was discovered due to the assimilation of microalgae for their growth, and denitrification did not play any role in removing the nitrogen in this process. Phosphorus removal in this process was attributed to chemical precipitation and biological assimilation (around 50% each). In removing ammonia, the HRAP is superior to the traditional bacterial nitrification– denitrification process, which requires the assimilation of extra-organic carbon as a carbon source. Phosphorus removal by microalgae is largely thought to be due to its uptake for normal growth, as an essential element required for making cellular constituents such as phospholipids, nucleotides, and nucleic acids. Under certain conditions microalgae can be triggered to uptake more phosphorus than is necessary for survival, in the form of polyphosphate (Powell, Shilton et al. 2011). Phosphorus removal by luxury uptake (amount of P uptake more than growth required) was confirmed to occur in the microalgae growing in the wastewater treatment facility; further research is needed in this prosperous field about the detailed mechanism and applications.

#### **4.3 Lignocellulosic biomass**

98 Biodiesel - Feedstocks and Processing Technologies

demand of agricultural land. Most microalgal ponds have a solar energy conversion efficiency of 1–4% under normal operating conditions and higher efficiencies can be achieved with closed photo-bioreactor systems. There is a considerable margin for improvement, which is being targeted through accelerated breeding programs and genetic modification. Autotrophic microalgae cells also absorb CO2 as their carbon source to support their cell growth, which makes the microalgae an attractive option for the biological CO2 fixation. Atmospheric CO2 accumulation, derived mainly from fossil fuel combustion, is proved as the leading cause of global warming. Current mitigation methods such as physicochemical adsorption, injection into deep oceans and geological formations are not economically feasible due to the high cost of implementing these methods and possible CO2 leakage. After CO2 is fixed via microalgae assimilation, the cell biomass can be utilized to generate methane gas, biochar, or the oil can be extracted to generate biodiesel. Microalgae can fix not only atmospheric CO2, but also the CO2 from industrial exhaust gases and from the carbonate salt, which are chemically fixed. Atmospheric CO2 levels (0.0387%) are not sufficient to support the high microalgae growth rates and productivities needed for commercial biofuel production, adding CO2 into autotrophic microalgae culture is an effective method to accelerate the microalgae growth rate. Utilization of CO2, for example, flue gas from electrical plant, by means of microalgae alleviates the impact of CO2 on the environment (greenhouse effect) and renders algal biomass production less expensive. It was also reported that the CO2 content was reduced from 44–48% to 2.5–11% when the microalgae cultivation was integrated with anaerobic digestion to remove impurities of biogas produced from anaerobic digestion. Microalgae also assimilated other impurities such as ammonia and hydrogen sulfide; and the gas leaving the algae pond had 88–97% by volume of methane 23. Although there are conflicting results from different references about the toxicity of ammonia, hydrogen sulfide and other impurities on the growth of microalgae cells, integration of microalgae cultivation together with this industrial processes can be more sustainable and economically feasible than the individual microalgae cultivation

Culturing microalgae with nutrients from wastewater, such as nitrogen and phosphate, can decrease the cost of the raw materials and also provide some environmental benefits. Agricultural effluent and municipal wastewater, even after treated with anaerobic digestion (AD), cannot be disposed directly because of their high nutrient level (Levine, Costanza-Robinson et al. 2011). In contrast, these wastewater can be considered as a cost-effective candidate of raw materials for biodiesel production (Siddiquee and Rohani 2011). Cultivation of microalgae, yeast, or fungi can be integrated with AD system to reduce the remaining COD, phosphate, and ammonia. Microalgae were also studied to absorb metal ion, waste pharmaceutical chemicals and dye into their cell biomass in order to remove the pollutants from wastewater once their cell biomass were stabilized and harvested. A typical example is the recently developed high-rate algae pond (HRAP) in the tertiary wastewater treatment facility (El Hamouri 2009). HRAP functions behind a two-step upflow anaerobic reactor (pre-treatment) and was followed by one maturation pond (MP) for polishing. The HRAP was revealed to have no activity for removing the COD from the wastewater; however, it removed 85% of total N and 63% of total P. Nitrogen removal was discovered due to the assimilation of microalgae for their growth, and denitrification did not play any role in removing the nitrogen in this process. Phosphorus removal in this process was

process to generate oil for biodiesel production.

**4.2 Wastewater** 

Several research studies revealed that organic carbon sources, if the algae have the capability to grow on heterotrophic mode, can significantly increase the cell growth rate and dramatically enhance the lipid content of the biomass. For example, *Chlorella protothecoides* can only accumulate 18-25% lipid in the autotrophic culture, while the lipid content can reach to 55.2% dry cell biomass if cultured with addition of organic carbons (Xiong, Li et al. 2008). However, the heterotrophic cultivation of microalgae mostly request pure monosugars, which are usually costly and limited. Alternative materials with relative abundance and zero or negative valued organic materials, such as lignocellulosic biomass from agriculture, are the only choice for this route. Agricultural feedstocks contribute a large part of renewable resource for biodiesel production, which are the target of biodiesel resource of cost reduction of biodiesel and non-human food resource discovery. Most of these substrates are locally available and thus are expected to support mainly small production facilities. Lignocellulosic materials are mainly composed of cellulose, hemicellulose, and lignin, which make up approximately 90% of the dry weight of most plant materials (Kumar, Barrett et al. 2009). Cellulose and hemicellulose can be converted to fermentable sugars for microbial lipid production. However, the direct enzymatic hydrolysis of cellulose and hemicellulose to sugars is impeded by the cell wall physico-chemical and structural composition. Thus, biomass pretreatment prior to enzymatic hydrolysis is essential to enhance enzymatic digestibility. Distinctly different from autotrophic microalgae cultures, this process is more similar to cellulosic ethanol production, where hydrolysis and fermentation are needed for conversion.
