**6.1 Factors affecting separation**

184 Biodiesel – Feedstocks and Processing Technologies

(Chen, 1996). However, several species (e.g. *Chlorella*, *Chlamydomonas, Phaeodactylum, Nitzschia, Tetraselmis* and *Crypthecodinium*) are also capable of heterotrophic growth (utilizing organic carbon such as glucose, acetate or glycerol as the sole source or carbon and energy) or mixotrophic growth (photoautotrophic growth supplemented by an organic

The advantages of using an organic carbon substrate are that it decreases dependence on light provision, allowing growth in conventional fermenters in the dark. Optimal growth conditions can be maintained, allowing higher cell concentrations and hence increased volumetric productivities to be reached (Chen, 1996). Higher productivities of both biomass and lipid have been reported under heterotrophic growth compared to autotrophic (Ceron Garcia, 2000; Miao & Wu, 2006). Disadvantages of feeding an organic carbon source include the fact that there are a limited number of algal species that can utilize organic carbon sources, the risk of bacterial contamination is greatly increased and the carbon substrate adds an additional cost, along with the environmental burden of its production. The use of a substrate such as glucose, commonly sourced from crop plants, adds a trophic level to the process, thereby removing the simplicity of the concept of microalgae as cellular factories

The optimal cultivation strategy (e.g. batch, fed-batch or continuous cultivation mode) is determined by the kinetics of growth, product accumulation and substrate uptake (Shuler & Kargi, 2005). For production of a primary product such as protein or biomass for food or feed, optimisation of biomass productivity is the main objective. In this case, batch or continuous systems are generally used. For production of a secondary product such as carotenoids or storage lipids, the use of two or more production stages to enhance yield has been proposed (Ben-Amotz, 1995; Huntley & Redalje, 2006; Richmond, 2004). The first stage is designed to optimize growth, while the second stage provides conditions that retard growth and encourage product synthesis, usually by applying some form of stress, e.g. nutrient deprivation in the case of lipid accumulation. Another potential two-stage strategy that could enhance lipid productivity is an initial photosynthetic stage, followed by a second heterotrophic phase, where feeding with an organic carbon source such as glucose may

The economic recovery of microalgal biomass remains a major challenge. Microalgae for biofuel are a low value product suspended in large volumes of water. Harvesting contributes 20 to 40% of the total cost of biomass production (Gudin & Therpenier, 1986; Molina Grima et al., 2003). The difficulty in separation can be attributed to the small size of the cells (3 to 300 m, Henderson et al., 2008), their neutral buoyancy and the fact that photoautotrophic microalgal cultures are relatively dilute, achieving concentrations in the order of 1 to 8 g.L-1 (Pulz, 2001). Each algal species presents unique challenges due to the array of sizes, shapes, densities and cell surface properties encountered. A low-cost, energy efficient method with a high recovery efficiency and concentration is required, minimizing

cell damage and allowing for water and nutrient recycle (Fig. 1).

carbon source).

producing liquid fuel from pure sunlight and CO2.

**5.2.6 Cultivation strategy** 

boost lipid content.

**6. Harvesting and processing** 

Several natural properties of microalgal cells affect the choice and efficiency of harvesting methods. Factors relevant to separation include density, surface charge, size, shape, hydrophobicity, salinity of the medium, adhesion and cohesion properties and settling or floating velocities. Table 3 highlights the variability in some of these parameters between species, indicating that species-specific solutions may be required. Algal cell characteristics can vary with culture age and growth conditions. For example, changes in biochemical composition such as lipid content could affect the buoyancy of algal cells. Surface charge, the chemical structure of the cell wall and the amount and composition of the extracellular organic matrix (EOM) can vary with growth phase and greatly influence the degree to which cells repel or stick to one another (Bernhardt & Clasen, 1994; Henderson et al., 2008). Danquah et al. (2009) found a strong correlation between growth phase and settling efficiency, with improved filtration, flocculation and sedimentation rates during the stationary phase.


Table 3. Characteristics relevant to harvesting of some microalgae (Henderson et al., 2008). Zeta potential is a measure of the degree of repulsion between adjacent particles due to surface charge.

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel 187

input. Microstraining (filtration by natural gravity using low speed rotating drum filters) is a promising method due to ease of operation and low energy consumption (Mohn, 1980). Another option is cross-flow membrane filtration (Zhang et al., 2010). Using a tangential, turbulent flow of liquid across the membrane prevents clogging of the filter with cells. The efficiency of the process is very dependent on cell morphology and the transmembrane pressure (Petrusevski et al., 1995). A more unconventional approach is magnetic filtration. Here addition of magnetic metals, either taken up by algal cells, or used to flocculate them,

Sedimentation is the process whereby solid particles suspended in a fluid are settled under the influence of gravity or some other force. In microalgae, it depends on coagulation or flocculation of cells to produce flocs with a large enough size (> 70 m) or high enough density to induce settling (Vlaski et al., 1997). Sedimentation is typically used in wastewater treatment. It is suitable for large throughput volumes and has low operational costs. Flocculation, using a dense substance such as calcium carbonate, can greatly reduce settling time. Ultrasound (acoustic energy) can be used to induce aggregation and facilitate sedimentation (Bosma et al., 2003), however the energy requirement may be too high for

Centrifugation is essentially sedimentation under a rotational force rather than gravity. The efficiency of centrifugation depends on the size and density of the particles, the speed of the rotor, the time of centrifugation and the volume and density of the liquid. Almost all microalgae can be harvested by centrifugation. It is a highly efficient and reliable method, can separate a mixture of cells of different densities and does not require the addition of chemicals, but has a high energy consumption (Chisti, 2007). It is routinely used for recovery of high value products, or for small scale research operations, although large, flow-though centrifuges can be used to process large volumes. Many algae require speeds of up to 13 000 g which results in high shear forces (Harun et al., 2010; Knuckey et al., 2006) and can

Flotation operates by passing bubbles through a solid-liquid mixture. The particles become attached to the bubble surface and are carried to the top of the liquid where they accumulate. The concentrated biomass can be skimmed off (Uduman et al., 2010). Flotation is considered to be faster and more efficient than sedimentation (Henderson et al., 2008). It is associated with low space requirements and moderate cost. Addition of chemical coagulants or flotation agents is often required to overcome the natural repulsion between the negatively charged algal particles and air bubbles. The pH and ionic strength of the medium

After harvesting, the major challenge is in releasing the lipids from their intracellular location in the most energy efficient and economical way possible. Algal lipids must be separated from the rest of the biomass (carbohydrates, proteins, nucleic acids, pigments) and water. Common harvesting methods generally produce a slurry or paste containing between 5 and 25% solids (Shelef et al., 1984). Removing the rest of the water is thought to be one of the most expensive steps with literature values ranging from 20 to 75% of the total processing cost (Uduman et al., 2010, Molina Grima et al., 2003). Shelef et al. (1984) highlight a number of possible techniques for drying biomass: flash drying, rotary driers, toroidal driers, spray drying, freeze-drying and sun drying. Because of the high water content, sun-drying is not an effective method and spray-drying is not economically

could allow capture using a magnetic field (Bitton et al., 1975).

are important factors to optimize this recovery technique.

large-scale use.

damage sensitive cells.

**6.3 Processing** 

Morphological characteristics that influence harvesting include cell motility, size, shape, cell wall elongations such as spines and flagella, colony formation, and the presence of extracellular mucilage layers or capsules (Petrusevski et al., 1995; Jarvis et al., 2009). Larger particles allow for easier separation due to increased surface area and mass. Filamentous morphology or appendages also allow for easier filtration as cells cannot pass through filter pores. Density affects sedimentation and flotation. Most algae have a specific gravity close to that of water, rendering them with a neutral buoyancy. Some cyanobacteria can adjust their density through gas vacuoles (Anderson, 2005), rendering sedimentation difficult but enabling potential surface collection.

Cell surface charges influence the electrostatic interactions between cells and between cells and surfaces or bubbles. This directly affects the adhesion, adsorption, flotation and flocculation properties of algal cells. Most algae have a negatively charged cell surface, leading to electrostatic repulsion between cell walls. Addition of positively charged ions to the solution can help to neutralize the negative surface charge and aid cell flocculation. Changing the pH of the solution can also cause flocculation (Chen et al., 1998). Hydrophobicity is another, non-electric property affecting interaction of algal cells with each other and external surfaces. Most algae are naturally hydrophilic (Fattom & Shilo, 1984), but this can be altered by surfactants and pH (Jameson, 1999). Increasing the hydrophobicity of cells could cause them to adhere to bubbles, filters or other separation catalysts.

#### **6.2 Harvesting methods**

Harvesting requires one or more solid-liquid separation techniques (Molina Grima et al., 2003). In order to achieve the levels of concentration required, various chemical, biological and physical separation steps may be necessary. Common methods of cell harvesting include flocculation, filtration, sedimentation, centrifugation and flotation (Mata et al., 2010). The small cell size of microalgae makes them difficult to dewater. Flocculation is used to 'clump' the cells, grouping them together to form larger particle sizes. This is often suggested as a pretreatment step prior to filtration, sedimentation or flotation. Flocculation occurs when the repulsion between cells is reduced, allowing them to either aggregate directly onto each other or through an intermediate bridging surface. The extent of flocculation is dependent on pH, temperature, density, hydrophobicity, surface charge and culture age (Lee et al., 1998). Flocculation can be induced by addition of positively charged ions or polymers, e.g. minerals such as lime, calcium and salts, metal salts such as aluminium sulphate and ferric chloride, and naturally occurring flocculants such as starch derivatives and tannins. Drawbacks to the use of chemical flocculants are the high dosages required, the need for pH correction (Pushparaj et al., 1993) and the contamination of the biomass and media with the flocculant, meaning that media cannot be recycled without removal of the chemical. Autoflocculation can be induced through pH change (Csordas & Wang, 2004), nutrient limitation (Schenk et al., 2008), excretion of macromolecules (Benemann et al., 1980) or aggregation between microalgae and bacteria (Lee et al., 2009). Conventional filtration is only effective for larger (> 70 m) or filamentous species such as *Coelastrum* and *Spirulina* (Brennan & Owende, 2010, Lee et al., 2009). For smaller cells, microfiltration, ultra-filtration and membrane-filtration can be used, though usually only for small volumes (Brennan & Owende, 2010, Petrusevski et al., 1995, Borowitzka, 1997). Fouling (accumulation of material on the surface of the membrane, slowing filtration) is a major problem. If filtration were to be considered for mass production, a high driving force for

separation (high pressure or suction) would be required, which necessitates a high energy

input. Microstraining (filtration by natural gravity using low speed rotating drum filters) is a promising method due to ease of operation and low energy consumption (Mohn, 1980). Another option is cross-flow membrane filtration (Zhang et al., 2010). Using a tangential, turbulent flow of liquid across the membrane prevents clogging of the filter with cells. The efficiency of the process is very dependent on cell morphology and the transmembrane pressure (Petrusevski et al., 1995). A more unconventional approach is magnetic filtration. Here addition of magnetic metals, either taken up by algal cells, or used to flocculate them, could allow capture using a magnetic field (Bitton et al., 1975).

Sedimentation is the process whereby solid particles suspended in a fluid are settled under the influence of gravity or some other force. In microalgae, it depends on coagulation or flocculation of cells to produce flocs with a large enough size (> 70 m) or high enough density to induce settling (Vlaski et al., 1997). Sedimentation is typically used in wastewater treatment. It is suitable for large throughput volumes and has low operational costs. Flocculation, using a dense substance such as calcium carbonate, can greatly reduce settling time. Ultrasound (acoustic energy) can be used to induce aggregation and facilitate sedimentation (Bosma et al., 2003), however the energy requirement may be too high for large-scale use.

Centrifugation is essentially sedimentation under a rotational force rather than gravity. The efficiency of centrifugation depends on the size and density of the particles, the speed of the rotor, the time of centrifugation and the volume and density of the liquid. Almost all microalgae can be harvested by centrifugation. It is a highly efficient and reliable method, can separate a mixture of cells of different densities and does not require the addition of chemicals, but has a high energy consumption (Chisti, 2007). It is routinely used for recovery of high value products, or for small scale research operations, although large, flow-though centrifuges can be used to process large volumes. Many algae require speeds of up to 13 000 g which results in high shear forces (Harun et al., 2010; Knuckey et al., 2006) and can damage sensitive cells.

Flotation operates by passing bubbles through a solid-liquid mixture. The particles become attached to the bubble surface and are carried to the top of the liquid where they accumulate. The concentrated biomass can be skimmed off (Uduman et al., 2010). Flotation is considered to be faster and more efficient than sedimentation (Henderson et al., 2008). It is associated with low space requirements and moderate cost. Addition of chemical coagulants or flotation agents is often required to overcome the natural repulsion between the negatively charged algal particles and air bubbles. The pH and ionic strength of the medium are important factors to optimize this recovery technique.

#### **6.3 Processing**

186 Biodiesel – Feedstocks and Processing Technologies

Morphological characteristics that influence harvesting include cell motility, size, shape, cell wall elongations such as spines and flagella, colony formation, and the presence of extracellular mucilage layers or capsules (Petrusevski et al., 1995; Jarvis et al., 2009). Larger particles allow for easier separation due to increased surface area and mass. Filamentous morphology or appendages also allow for easier filtration as cells cannot pass through filter pores. Density affects sedimentation and flotation. Most algae have a specific gravity close to that of water, rendering them with a neutral buoyancy. Some cyanobacteria can adjust their density through gas vacuoles (Anderson, 2005), rendering sedimentation difficult but

Cell surface charges influence the electrostatic interactions between cells and between cells and surfaces or bubbles. This directly affects the adhesion, adsorption, flotation and flocculation properties of algal cells. Most algae have a negatively charged cell surface, leading to electrostatic repulsion between cell walls. Addition of positively charged ions to the solution can help to neutralize the negative surface charge and aid cell flocculation. Changing the pH of the solution can also cause flocculation (Chen et al., 1998). Hydrophobicity is another, non-electric property affecting interaction of algal cells with each other and external surfaces. Most algae are naturally hydrophilic (Fattom & Shilo, 1984), but this can be altered by surfactants and pH (Jameson, 1999). Increasing the hydrophobicity of

Harvesting requires one or more solid-liquid separation techniques (Molina Grima et al., 2003). In order to achieve the levels of concentration required, various chemical, biological and physical separation steps may be necessary. Common methods of cell harvesting include flocculation, filtration, sedimentation, centrifugation and flotation (Mata et al., 2010). The small cell size of microalgae makes them difficult to dewater. Flocculation is used to 'clump' the cells, grouping them together to form larger particle sizes. This is often suggested as a pretreatment step prior to filtration, sedimentation or flotation. Flocculation occurs when the repulsion between cells is reduced, allowing them to either aggregate directly onto each other or through an intermediate bridging surface. The extent of flocculation is dependent on pH, temperature, density, hydrophobicity, surface charge and culture age (Lee et al., 1998). Flocculation can be induced by addition of positively charged ions or polymers, e.g. minerals such as lime, calcium and salts, metal salts such as aluminium sulphate and ferric chloride, and naturally occurring flocculants such as starch derivatives and tannins. Drawbacks to the use of chemical flocculants are the high dosages required, the need for pH correction (Pushparaj et al., 1993) and the contamination of the biomass and media with the flocculant, meaning that media cannot be recycled without removal of the chemical. Autoflocculation can be induced through pH change (Csordas & Wang, 2004), nutrient limitation (Schenk et al., 2008), excretion of macromolecules (Benemann et al., 1980) or aggregation between microalgae and bacteria (Lee et al., 2009). Conventional filtration is only effective for larger (> 70 m) or filamentous species such as *Coelastrum* and *Spirulina* (Brennan & Owende, 2010, Lee et al., 2009). For smaller cells, microfiltration, ultra-filtration and membrane-filtration can be used, though usually only for small volumes (Brennan & Owende, 2010, Petrusevski et al., 1995, Borowitzka, 1997). Fouling (accumulation of material on the surface of the membrane, slowing filtration) is a major problem. If filtration were to be considered for mass production, a high driving force for separation (high pressure or suction) would be required, which necessitates a high energy

cells could cause them to adhere to bubbles, filters or other separation catalysts.

enabling potential surface collection.

**6.2 Harvesting methods** 

After harvesting, the major challenge is in releasing the lipids from their intracellular location in the most energy efficient and economical way possible. Algal lipids must be separated from the rest of the biomass (carbohydrates, proteins, nucleic acids, pigments) and water. Common harvesting methods generally produce a slurry or paste containing between 5 and 25% solids (Shelef et al., 1984). Removing the rest of the water is thought to be one of the most expensive steps with literature values ranging from 20 to 75% of the total processing cost (Uduman et al., 2010, Molina Grima et al., 2003). Shelef et al. (1984) highlight a number of possible techniques for drying biomass: flash drying, rotary driers, toroidal driers, spray drying, freeze-drying and sun drying. Because of the high water content, sun-drying is not an effective method and spray-drying is not economically

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel 189

Fig. 2. Lipid contents and biomass productivities required for economic feasibility in (a) large-scale, outdoor raceway ponds and (b) large-scale, outdoor photobioreactors. Dark grey region: productivities economically feasible at US\$ 50 per barrel crude oil (cost of algal oil per L lower than cost of regular diesel per L). Additional region for crude oil price US\$ 90

Based on this model, the results for raceway ponds show that algal biodiesel will not be economically feasible, either in ponds or photobioreactors, at current costs below a biomass productivity of 1 g.L-1.day-1. Assuming a maximum realistically achievable lipid content of 50% DW, algal biodiesel becomes economically feasible at biomass productivities of 1.5 g.L-1.day-1 (US\$ 130 per barrel crude oil), close to 2 g.L-1.day-1 (US\$ 90), and 2.5 g.L-1.day-1 (US\$ 50) in raceway ponds. At lower lipid contents, higher biomass productivity is required, e.g. at a lipid content of 25% DW, algal biodiesel only becomes cost effective at 2 g.L-1.day-1 for US\$ 130 per barrel. The model for photobioreactors is based on a lower cost per kg algal biomass than raceway ponds, hence economic feasibility is reached at slightly lower biomass productivities and lipid contents, e.g. at a biomass productivity of 2 g.L-1.day-1, a

lipid content of only 20% DW is required to be viable at US\$ 130 per barrel crude oil.

Currently reported biomass productivities in outdoor raceway ponds average around 0.17 g.L-1.day-1, with a lipid content of 26% DW (Griffiths and Harrison, 2009), which is far from being economically feasible. Biomass productivities for closed photobioreactors (1.33 g.L-1.day-1) are closer to being within the economically viable range, if they can be maintained in the long term, concurrent with sufficiently high lipid content. As a reflection of this, there are currently no industrial facilities producing biodiesel from microalgae (Lardon, 2009). For cultivation to be economically viable, productivities must be increased, costs lowered, or additional income streams developed. The economics of algal biofuel production could be greatly improved through the production of co-products. For example, high value compounds such as pigments could be produced along with lipid. The residual biomass after lipid extraction could be sold as animal feed, fertilizer or soil conditioner, anaerobically digested to produce biogas, gasified or merely burned to provide some of the

In addition to economic feasibility, algal biodiesel must be environmentally desirable. It is critical that the energy embodied in the fuel produced is greater than the energy input required to produce it. Net energy analysis and life cycle analysis (LCA) are tools used to quantify the environmental burdens at every stage of production, from growth of the

per barrel = mid-grey and US\$ 130 = light grey

heat or electricity required in the process.

feasible for low value products (Mata et al., 2010). The selection of drying technique is dependent on the scale of operation, the speed required and the downstream extraction process (Mohn, 1980).

Lipid extraction can be done in a number of ways. Solvent extraction techniques are popular, but the cost and toxicity of the solvent (e.g. hexane) is of concern and solvent recovery requires significant energy input. Other methods involve disruption of the cell wall, usually by enzymatic, chemical or physical means (e.g. homogenization, bead milling, sonication (Mata et al., 2010)), allowing the released oil to float to the top of the solution. Ultrasound and microwave assisted extraction methods have been investigated (Cravotto et al., 2008). Supercritical CO2 extraction is an efficient process, but is too expensive and energy intensive for anything but lab-scale production. Direct transesterification (production of biodiesel directly from algal biomass) is also possible. Some of these techniques do not require dry biomass, but the larger the water content of the algal slurry, the greater the energy and solvent input required.

Once the algal oil is extracted, it can be treated as conventional vegetable oil in biodiesel production. Direct pyrolysis, liquefaction or gasification of algal biomass have also been suggested as means of producing fuel molecules. One of the concerns for biodiesel production through transesterification, shared with any biodiesel feedstock, is the quality of the biodiesel produced. Biodiesel must meet certain international regulations, for example, the ASTM international standards or the EN14214 in Europe. It has been calculated that the fatty acid profile of certain microalgal species will produce biodiesel that does not meet these specification, therefore blending or additives may be required (Stansell, 2011).
