**6.1 Cell harvest methods**

The algae cell harvest from pond water and the subsequent water reuse have been one of the major obstacles for the algae-to-fuel approach. Microalgae cell harvest is technically challenging, especially considering the low cell densities (typically in the range of 0.3–5 g/L) of autotrophic microalgae due to limited light penetration, the small size of the oleaginous algal cells (typically in the range of 2–40 um), and their similar density to water (Li, Horsman et al. 2008). Oleaginous microalgae cells are usually suspended in the water and are hard to settle by natural gravity force due to their negative charges. The recovery of microalgae biomass generally requires one or more solid–liquid separation steps, and usually accounts for 20–30% of the total costs of production, according to one source (Uduman, Qi et al. 2010).

How to harvest microalgae cells is dependent on the characteristics of the microalgae, such as size and density(Olaizola 2003). All of the available harvest approaches, which include flocculation, flotation, centrifugal sedimentation, and filtration, have limitations for effective, cost-efficient production of biofuel (Shelef, Sukenik et al. 1984). For instance, flotation methods, based on the trapping of algae cells using dispersed micro-air bubbles, is

Different fermentation processes are applied to obtain high productivity of lipids and high conversion ratio of substrate for the fermentation, such as batch cultivation, fed-batch cultivation, and continous cultivation. Fed-batch cultivatioin is a modified batch model that can reach high cell density and it has many applications in the fermentative lipid accumulation process. For example, *Rhorosporidum toruloides* reach much higher cell density with 48% lipid compared to its batch cultivation (Li, Zhao et al. 2007). The high productivity of fed-batch cultivation was conformed by *Phodotorula glutinis* (Xue, Miao et al. 2008), *C. curvatus* (Meesters, Huijberts et al. 1996), and *L. starkeyi* (Yamauchi, Mori et al. 1983). Continuous cultivation has advantages of easy maintenance and time-saving, although it is difficult to control the contamination. It has limited applicatioins in the fermentative lipid

Besides the commonly used submerged cultivations, solid state fermentation, as a compact process for lipid production, showed many advantages, such as low requirements to the raw materails; low capital cost; low energy expenditure; less expensive downstream processing; less water usage and low water output; potential higher volumetric productivity; less fermentation space; easy operation and maintenance. The research for *Aspergillus oryzae* growing on rice bran and wheat bran through solid state fermentation resulted to the lipid content of cell biomass at about 10-11% (Da Silveira, Oliveira et al. 2010). The lipid yield reached 62.87 mg/gds in solid state fermentation on the 6th day after Plackett-Burman design (PBD) by *A. oryzae* A-4 (Lin, Cheng et al. 2010) . Currently, the solid state fermentation research is still in its infancy and many barriers are hindering this process from commercilization. The lipid yield is relatively low compared to submerge cultivation. Modern biotechnological approaches, such as heterogenous expression of hydrolytic enzymes and UV radiation, are available to enhance the hydrolytic enzymes production (Li, Yang et al. 2010; Awan, Tabbasam et al. 2011). Semi-solid state fermentation is used to avoid high sugar concentration on the surface of lignocellulose. An oleaginous fungus *M. isabellina*  was cultured at semi-solid state fermentation with the results of 11g oil per 100g sweet

The algae cell harvest from pond water and the subsequent water reuse have been one of the major obstacles for the algae-to-fuel approach. Microalgae cell harvest is technically challenging, especially considering the low cell densities (typically in the range of 0.3–5 g/L) of autotrophic microalgae due to limited light penetration, the small size of the oleaginous algal cells (typically in the range of 2–40 um), and their similar density to water (Li, Horsman et al. 2008). Oleaginous microalgae cells are usually suspended in the water and are hard to settle by natural gravity force due to their negative charges. The recovery of microalgae biomass generally requires one or more solid–liquid separation steps, and usually accounts for 20–30% of the total costs of production, according to one source

How to harvest microalgae cells is dependent on the characteristics of the microalgae, such as size and density(Olaizola 2003). All of the available harvest approaches, which include flocculation, flotation, centrifugal sedimentation, and filtration, have limitations for effective, cost-efficient production of biofuel (Shelef, Sukenik et al. 1984). For instance, flotation methods, based on the trapping of algae cells using dispersed micro-air bubbles, is

accumulation.

sorghum (Economou, Makri et al. 2010) .

**6. Cell harvest and lipid extraction** 

**6.1 Cell harvest methods** 

(Uduman, Qi et al. 2010).

very limited in its technical and economic viability. Most conventional and economical separation methods such as filtration and gravitational sedimentation are widely applied in wastewater treatment facilities to harvest relatively large (>70 µm) microalgae such as *Coelastrum* and *Spirulina*. However, they cannot be used to harvest algae species approaching bacterial dimensions (<30 µm) like *Scenedesmus*, *Dunaliella*, and *Chlorella* (Brennan and Owende 2010), to which most oleaginous microalgae species belong. Centrifugation is a method widely used to recover microalgae biomass, especially smallsized algae cells; however, its application is restricted to algae cultures for high-value metabolites due to intensive energy needs and high equipment maintenance requirements. While flocculation is used to harvest small-sized microalgae cells, it is a preparatory step to aggregate the microalgae cells and increase the particle size so that other harvesting methods such as filtration, centrifugation, or gravity sedimentation can be applied (Molina Grima, Belarbi et al. 2003). Several flocculants have been developed to facilitate the aggregation of microalgae cells, including multivalent metal salts like ferric chloride (FeCl3), aluminium sulphate (Al2(SO4)3), and ferric sulphate (Fe2(SO4)3), and organic polymers such as Chitosan (Li, Horsman et al. 2008). Chemical flocculation can be reliably used to remove small algae cells from pond water by forming large-sized (1–5 mm) flocs (Sharma, Dhuldhoya et al. 2006). However, the chemical reactions are highly sensitive to pH and the high doses of flocculants required produce large amounts of sludge and may leave a residue in the treated effluent. In summary, most technologies including chemical and mechanical methods greatly increase operational costs for algal production and are only economically feasible for production of high-value products (Park, Craggs et al. 2011).

Besides traditional methods mentioned above, there are several new technology developments in this field. DOE-ARPA-E recently funded a research project for Algae Venture Systems (AVS) to develop a Harvesting, Dewatering, and Drying (AVS-HDD) technology by using the principles of liquid adhesion and capillary action to extract water from dilute microalgae solutions. Attached algal culture systems have been developed for growing microalgae on the surface of polystyrene foam (Wilkie and Mulbry 2002) (Johnson and Wen 2010) to simplify the cell harvest. New bioflocculants, which are more environmentally friendly, are also proposed to address the cost and environmental concerns for current flocculation method (Uduman, Qi et al. 2010). All these methods are innovative and will decrease the harvest cost to some extent if developed successfully, but heavy investments on equipment and chemical supplies are still needed.

Dr. Bo Hu's research group at University of Minnesota developed an innovative approach to enhance natural algae aggregation and to encourage simple gravity settling or filtration by co-culturing filamentous fungal cells at the end of the microalgae cultures. Instead of suspended culture, this approach uses pelletized or granulized culture where cells form pellets in culture medium. In submerged cultures, many filamentous microorganisms tend to aggregate and grow as pellets/granules. They are spherical or ellipsoidal masses of hyphae with variable internal structure, ranging from loosely packed hyphae, forming "fluffy" pellets, to tightly packed, compact, dense granules (Hu and Chen 2007; Hu and Chen 2008; Hu, Zhou et al. 2009; Chunjie Xia 2011). Besides merits from the cell immobilization, there are several other advantages, especially for the micro-oil production: a). easy to harvest cells, and b). easy to re-use pond water (Johnson and Wen 2010; Xia 2011). As the first research group to introduce pelletized liquid fermentation (PLF) into biofuel production, this research group at University of Minnesota found key operational conditions that induce the fungal pelletization. They discovered that changing conditions

Microbial Biodiesel Production - Oil Feedstocks Produced from Microbial Cell Cultivations 105

Supercritical fluid extraction takes advantage that some chemicals behave as both a liquid and a gas, and have increasing solvating power when they are raised above their critical temperature and pressure points. Carbon dioxide is the most commonly used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol. Critical temperature and critical pressure of carbon dioxide is at 31°C and 74 bar, respectively (Cooney, Young et al. 2009). Supercritical fluids produce highly purified extracts without using toxic solvent; and the process is fast and safe for thermally sensitive products. Supercritical CO2 extraction efficiency is affected by four main factors: pressure, temperature, CO2 flow rate, and extraction time. Ethanol (10 -15%), co-solvent, lead to similar results of Bligh and Hyer method at extracting oil from *Arthrospira maxima* and *Spirulina platensis* (Mendes, Reis et al. 2006; Sajilata, Singhal et al. 2008). The limitation of supercritical fluid extraction is high

Besides the methods mentioned above, numerous technologies are being tested at different labs to harvest lipids from cells. Genetic engineering has been applied to improve the porosity of the cell membranes in order to increase the release of lipids directly from the cells (Greenwell, Laurens et al. 2010). Enzymes treatment and pulsed electric field technology are other effective methods to break the cell wall and membrane and enhance the mass transfer across the cell membrane for oil extraction (Shah, Sharma et al. 2004; Guderjan, Elez-Martinez et al. 2007). Microwave technology is a portential pretreatment method, which heats the cell components in order to increase the release of oil. Oil yield increased from 4.8% to 17.7% from microalgae *Crypthecodinium chnii* when microwave was applied (Cravotto, Boffa et al. 2008). Microwave technology is featured for its time-savings, but its disadvantages include the oxidative damage to products and its intense energy need. Sonnication is a timely and efficient method, free of toxic materials. Cavitation occurs when high voltage is applied into cell lipids. Vapour bubbles form with negative pressure and cause a violent collapse when compressed under positive pressure while growing; then the cell contents are released (Wei, Gao et al. 2008). The sonnication is, however, difficult to

A complete techno-econoic analysis for the microbial biodiesel production is difficult, especially considering that most of the technologies are still in the early research stage. Initial investment into microalgal biofuels has mostly failed and several early start-up companies have closed. Different versions of economic analysis for microalgae biofuel production have been published recently, and Table 4 lists an analysis conducted by Seed

Table 4 shows that although photobioreactor has a higher cell concentration and utilizes CO2, its cost to produce lipid is the highest of all methods. Heterotrophic fermentation, however, appears to be the most economically feasible route to produce microbial biodiesel. Techno-economic analyses may vary from different research group, but their conclusions are similar. The biomass and oil generated from heterotrophic fermentation are more close to current fossile fuel cost. Heterotrophic fermentation relies less on local climate conditions and can be carried out in close fermentors, which may facilitate their commercialization.

**7. Techno-economic analysis and life cycle assessment** 

Science Ltd, sporsored by the British Columbia Innovation Council in Canada.

**6.2.3 Supercritical fluid extraction** 

capital cost and high cost for maintainence.

**6.2.4 Other methods** 

scale-up.

during cell cultivation can force fungal cells to aggregate and form pellets. This method avoids traditional approaches that use CaCO3 powder to induce the fungal pelletization (Liao, Liu et al. 2007; Liu, Liao et al. 2008), which are costly and cause solid waste disposal issues. Self aggregated pelletization/granulation dramatically improves mass transfer and cell cultivation performance and facilitates cell harvest and separation. A simple filtration can be used to separate the cell biomass from the fermentation broth. This approach brings tremendous advantages to decrease the harvest cost of biofuel production, especially when the raw materials only contain very diluted sugar, (which are the cases for many agricultural waste). This would appear to be the most promising option to achieve both a high-quality treated effluent in terms of total suspended solids and economically recovering algal biomass for biofuel use (Uduman, Qi et al. 2010). It will also be more environmentally sound than current procedures which may need chemical addition.

#### **6.2 Lipid extraction methods**

Oil extraction also contributes a large part of the cost in the process to generate microbial biodiesel. Several oil extraction technologies are currently available to process the microbial biomass in order to meet the requirement of being low cost, easy and safe to operate, and environmentally friendly.

#### **6.2.1 Mechanical methods**

Mechanical methods include pressing, bead milling, and homogenization. Pressing is a technology to harvest lipids out of cells by high pressure. Bead milling works in a container to destruct the cell wall by high speed small beads. Homogenization provides a sudden pressure change when cells go through an orifice. The mechanical technologies are often used in combination with solvent methods to separate the lipid from the cell biomass. The mechanical methods are energy intensive and better operated at the high cell density condition; in addition, pretreatments are necessary to obtain high recovery ratio (Greenwell, Laurens et al. 2010).

#### **6.2.2 Solvent extraction methods**

Solvent extraction is a commonly used method for soybean processing, and it is also used to extract lipids from microbial cells. Organic solvents should be insoluble in water, be easy to obtain, have a low boiling point, and be reusable. Current industrial solvents for microlipids accumulation include hexane, chloroform, acetone, benzene, and cyclohexane, can dissolve lipid without residual cell. The extraction process is significantly affected by operation condition, such as temperature and pressure. Accelerated solvent extraction (ASE) is named when the operation temperature is higher than that of solvent boiling point, which can be used for oil extraction from dry biomass (Cooney, Young et al. 2009). Mixture chloroform and methanol (Bligh and Hyer method) is the most common organic solvent to extract oil from biomass. This organic mixture can extract oil not only from dry biomass but also from wet biomass. However, the efficiency is different at certain condition (Zhu, Zhou et al. 2002). The efficiency of oil extraction was not working well at wet *Mortierella alpina* biomass. The process generated large amounts of wastewater and solvent often contaminated the final products. Simultaneous extraction and tranesterification is more efficient (15-20%) than the separate process (Belarbi, Molina et al. 2000); however, the important point of the simultaneous process is to balance the reaction time for the best components of product (Lewis, Nichols et al. 2000).

#### **6.2.3 Supercritical fluid extraction**

104 Biodiesel - Feedstocks and Processing Technologies

during cell cultivation can force fungal cells to aggregate and form pellets. This method avoids traditional approaches that use CaCO3 powder to induce the fungal pelletization (Liao, Liu et al. 2007; Liu, Liao et al. 2008), which are costly and cause solid waste disposal issues. Self aggregated pelletization/granulation dramatically improves mass transfer and cell cultivation performance and facilitates cell harvest and separation. A simple filtration can be used to separate the cell biomass from the fermentation broth. This approach brings tremendous advantages to decrease the harvest cost of biofuel production, especially when the raw materials only contain very diluted sugar, (which are the cases for many agricultural waste). This would appear to be the most promising option to achieve both a high-quality treated effluent in terms of total suspended solids and economically recovering algal biomass for biofuel use (Uduman, Qi et al. 2010). It will also be more environmentally

Oil extraction also contributes a large part of the cost in the process to generate microbial biodiesel. Several oil extraction technologies are currently available to process the microbial biomass in order to meet the requirement of being low cost, easy and safe to operate, and

Mechanical methods include pressing, bead milling, and homogenization. Pressing is a technology to harvest lipids out of cells by high pressure. Bead milling works in a container to destruct the cell wall by high speed small beads. Homogenization provides a sudden pressure change when cells go through an orifice. The mechanical technologies are often used in combination with solvent methods to separate the lipid from the cell biomass. The mechanical methods are energy intensive and better operated at the high cell density condition; in addition, pretreatments are necessary to obtain high recovery ratio (Greenwell,

Solvent extraction is a commonly used method for soybean processing, and it is also used to extract lipids from microbial cells. Organic solvents should be insoluble in water, be easy to obtain, have a low boiling point, and be reusable. Current industrial solvents for microlipids accumulation include hexane, chloroform, acetone, benzene, and cyclohexane, can dissolve lipid without residual cell. The extraction process is significantly affected by operation condition, such as temperature and pressure. Accelerated solvent extraction (ASE) is named when the operation temperature is higher than that of solvent boiling point, which can be used for oil extraction from dry biomass (Cooney, Young et al. 2009). Mixture chloroform and methanol (Bligh and Hyer method) is the most common organic solvent to extract oil from biomass. This organic mixture can extract oil not only from dry biomass but also from wet biomass. However, the efficiency is different at certain condition (Zhu, Zhou et al. 2002). The efficiency of oil extraction was not working well at wet *Mortierella alpina* biomass. The process generated large amounts of wastewater and solvent often contaminated the final products. Simultaneous extraction and tranesterification is more efficient (15-20%) than the separate process (Belarbi, Molina et al. 2000); however, the important point of the simultaneous process is to balance the reaction time for the best components of product

sound than current procedures which may need chemical addition.

**6.2 Lipid extraction methods** 

environmentally friendly.

**6.2.1 Mechanical methods** 

Laurens et al. 2010).

**6.2.2 Solvent extraction methods** 

(Lewis, Nichols et al. 2000).

Supercritical fluid extraction takes advantage that some chemicals behave as both a liquid and a gas, and have increasing solvating power when they are raised above their critical temperature and pressure points. Carbon dioxide is the most commonly used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol. Critical temperature and critical pressure of carbon dioxide is at 31°C and 74 bar, respectively (Cooney, Young et al. 2009). Supercritical fluids produce highly purified extracts without using toxic solvent; and the process is fast and safe for thermally sensitive products. Supercritical CO2 extraction efficiency is affected by four main factors: pressure, temperature, CO2 flow rate, and extraction time. Ethanol (10 -15%), co-solvent, lead to similar results of Bligh and Hyer method at extracting oil from *Arthrospira maxima* and *Spirulina platensis* (Mendes, Reis et al. 2006; Sajilata, Singhal et al. 2008). The limitation of supercritical fluid extraction is high capital cost and high cost for maintainence.

### **6.2.4 Other methods**

Besides the methods mentioned above, numerous technologies are being tested at different labs to harvest lipids from cells. Genetic engineering has been applied to improve the porosity of the cell membranes in order to increase the release of lipids directly from the cells (Greenwell, Laurens et al. 2010). Enzymes treatment and pulsed electric field technology are other effective methods to break the cell wall and membrane and enhance the mass transfer across the cell membrane for oil extraction (Shah, Sharma et al. 2004; Guderjan, Elez-Martinez et al. 2007). Microwave technology is a portential pretreatment method, which heats the cell components in order to increase the release of oil. Oil yield increased from 4.8% to 17.7% from microalgae *Crypthecodinium chnii* when microwave was applied (Cravotto, Boffa et al. 2008). Microwave technology is featured for its time-savings, but its disadvantages include the oxidative damage to products and its intense energy need. Sonnication is a timely and efficient method, free of toxic materials. Cavitation occurs when high voltage is applied into cell lipids. Vapour bubbles form with negative pressure and cause a violent collapse when compressed under positive pressure while growing; then the cell contents are released (Wei, Gao et al. 2008). The sonnication is, however, difficult to scale-up.
