3.12 Biofuel from Microalgae Z Wen, Iowa State University, Ames, IA, USA J Liu, The University of Hong Kong, Hong Kong, China F Chen, The University of Hong Kong, Hong Kong, China and Peking University, Beijing, China

© 2011 Elsevier B.V. All rights reserved.

3.12.1 Introduction and Scope 127 3.12.2 Major Algal Composition 128 3.12.3 Different Types of Biofuels from Microalgae 128 3.12.3.1 Biogas 128 3.12.3.2 Ethanol 129 3.12.3.3 Biodiesel 129 3.12.3.4 Bio-Oil and Syngas 129 3.12.4 Algal Biodiesel Production Pipeline 129 3.12.4.1 Algal Physiology and Genetic Engineering 129 3.12.4.2 Mass Algal Culture 130 3.12.4.3 Algae Harvesting and Dewatering 131 3.12.4.4 Biomass Processing for Oil Extraction 132 3.12.4.5 Conversion of Algal Oil into Biodiesel 132 3.12.5 Conclusion and Perspectives 133 References 133

Glossary biogas A gas mixture containing carbon dioxide and methane as major components that are generated through breakdown of organic matters by bacteria and/or archaea without oxygen. bio-oil A synthetic liquid fuel that is extracted by treating biomass in a reactor at temperature of about 500 °C without oxygen.

microalgae Commonly photosynthetic organisms that primarily use water, carbon dioxide, and sunlight to produce biomass and oxygen. syngas A gas mixture that contains varying amounts of carbon monoxide and hydrogen that are generated by gasification of coal or biomass. thermochemical conversion A process by which biomass is treated at high temperature with various catalysts to produce various liquid and/or gaseous fuels.

3.12.1 Introduction and Scope

Microalgae are mostly photosynthetic organisms that primarily use water, CO2, and sunlight to produce biomass and O2. The nutrients required for growing algae are nitrogen, phosphorus, mineral salts, trace elements, and silicon (for diatom). Most of those nutrients are available from municipal, industrial, and agricultural wastewater. Compared with terrestrial plants, microalgae have a high oil content and growth rate. Algal cells generally contain 4–12% oil (dry basis) but can be as high as 77% depending on species and growing conditions [1, 2]. Mass cultivation of microalgae can be performed on unexploited lands using saline water in arid regions, thus avoiding competition for limited arable lands. Due to these merits, microalgae have long been considered a promising alternative and renewable feedstock for biofuel production.

Depending on the biomass composition, microalgae can be processed into various types of biofuels including biogas, alcohol, biodiesel, and jet fuels. However, current algal biofuel production is still far from economical due to several major challenges such as low oil yield, high harvest cost, and the contamination of the native species [3]. Developing an economic algal biofuel production requires a collaborative effort between algal biologists and bioprocess engineers. This article provides an overview of the current status of algal biofuel production. The algal biomass composition and the various types of biofuels that can be produced from algae are discussed. At last, we use algal biodiesel production as an example to illustrate the production chain elements of algal biodiesel production.

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3.12.2 Major Algal Composition

Microalgal biomass contains carbohydrate, proteins, and lipids as major compositions. In general, proteins account for 40–60% of dry biomass, followed by carbohydrate (20–30%), and lipids (10–20%) [1]. Syntheses of these components are highly regulated by the culture conditions. For example, under nitrogen-limitation and high light conditions, microalgal cells tend to accumulate lipid instead of the starch [4]. In addition to these three major components, algal cells also contain small amounts (1–5%) of nucleic acids [1], and various pigments such as carotenoids. From the biofuel production point of view, lipids (oils) are the most interesting group of components.

In general, algal lipids are divided into two classes: neutral lipids and polar lipids. Triacylglycerol (TAG) is the major neutral lipid found in algae. In addition to TAG, algae also contain small amounts of other neutral lipids such as monoacylglycerol, diacylgly­cerol, and sterols. Polar lipids are more complex than neutral lipids, of which glycolipids and phospholipids are the two most important and popular groups. Lipid composition and content are important factors to assess the potential of algae as biodiesel feedstock. Over the past few decades, numerous algal species have been screened and characterized for their lipid production potentials. The lipid composition and content of these oleaginous algae are species- and/or strain-dependent and may vary greatly. Under optimal growth conditions, algae generally synthesize a small amount of lipids with polar lipids being the main compo­nents; whereas under unfavorable environmental or stress conditions, algae may accumulate large quantities of lipids with neutral lipids, particularly TAG as the major components. This might be due to the shift of lipid metabolism from membrane polar lipids to storage neutral lipids. Algae can produce lipids up to 77% of dry weight, with TAG accounting for as much as 80% of total lipids. The synthesized TAGs are deposited in lipid bodies located in cytoplasm of algal cells. 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. From a biodiesel production point of view, TAGs are preferred to phospholipids or glycolipids because of their high proportions of fatty acids and lack of phosphate.

Algal fatty acids are in either saturated or unsaturated form, and the unsaturated fatty acids may vary in the number and position of double bonds on the acyl chain. Based on the number of double bonds, unsaturated fatty acids are classified into monounsa­turated fatty acids and polyunsaturated fatty acids. Many algae have been investigated for their fatty acid profiles. The fatty acids of algae are commonly in medium length, ranging from 16 to 18 carbons, although composition of those fatty acids varies greatly. In general, 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. However, it should be noted that these data are obtained from algal species under specific conditions that may vary greatly when the algal cells are exposed to different environmental or nutritional conditions such as light intensity, temperature, and nitrogen concentration.

3.12.3 Different Types of Biofuels from Microalgae

3.12.3.1 Biogas

Anaerobic digestion is widely used for treating various waste streams such as municipal sludge or animal waste. For treating microalgae using anaerobic digestion, the digesting materials can be either raw algal biomass or the residue after oil extracted from the biomass. Methane produced from anaerobic digestion can be used as a heat source or for electricity generation. Anaerobic digestion process can also mineralize the organic nitrogen and phosphorus contained in the algal biomass, resulting in a flux of ammonium and phosphate that can be used as a substrate for microalgae, thus reducing the use of fertilizer in the microalgal culture. The use of raw algal biomass for methane production can avoid the biomass-harvest and oil-extraction processes used in algal biodiesel production, and significantly reduce the production cost and energy debt. Anaerobic digestion of the cell residues after lipid extraction is strongly recommended for balancing both the energy and economy of the algal biodiesel production.

There are disadvantages of using anaerobic digestion for treating algal biomass. In general, algal cell contains a ‘tough’ cell wall that is difficult to be digested. The proteins contained in the biomass will release ammonia when degraded; a high level of ammonia can inhibit the microorganism in the digesters. The inhibition will become more severe when digesting the lipid extracted algal residues because the protein content is even higher. In addition, some marine algal species require high levels of sodium ions for growth. Nevertheless, sodium ions at high concentrations are strongly inhibitory to the anaerobic microflora [5]. All these factors will reduce the methane yield when raw microalgal biomass is being digested. Indeed, it has been reported that the degradation rates of Chlorella and Scenedesmus species are only 60–70% of that in active sludge digestions [6]. In another study on anaerobic digestion of Chlorella vulgaris, it is found that 50% of the biomass does not undergo anaerobic digestion even at a long retention time of 28 days [6].

To increase the anaerobic digestion efficiency, pretreatment of algal biomass is needed so the organic substrates in the algal cells are more accessible to anaerobic microflora and readily biodegraded. Various pretreatment methods that are developed for treating other waste materials such as animal waste and municipal sewage sludge can be applied to treating algal biomass. These include the physical treatment (mechanical maceration, ultrasonic lysis, and heat treatment), chemical treatment (acid, base, neutral detergent, and ozonation), and biological and enzymatic treatment. The high protein content of the algal biomass usually leads to a low C/N ratio, which is imbalanced for anaerobic digestion. For example, freshwater algae have an average C/N ratio of 10.2, while terrestrial plants have an average C/N ratio of 36. Co-digestion of algal biomass with other organic matters such as waste paper to ensure a balanced C/N ratio of the influent composition can increase the digestion performance [7].

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3.12.3.2 Ethanol

Certain microalgae are capable of producing high levels of carbohydrates such as starch or cellulose as reserve materials, which are ideal feedstocks for ethanol production. Compared to terrestrial plants, algae have a high photosynthetic efficiency and can synthesize and accumulate large quantities of carbohydrate biomass. When making ethanol from terrestrial biomass such as corn stover and switchgrass, a harsh pretreatment step is usually needed to break down the complex structure of those lignocellulosic materials, so that the cellulose can be converted via hydrolysis into fermentable sugars. Aquatic algal cells, however, are buoyant and do not contain those structural biopolymers such as hemicellulose and lignin. This greatly simplifies the algal bioethanol production process by eliminating the complex and expensive pretreatment steps [8].

Ethanol from microalgae can be produced through the conventional method, that is, extract the starch or cellulose from the algal biomass, hydrolyze the starch/cellulose to be sugars, and ferment the sugars to produce ethanol by appropriate ethanol producers. First, the harvested algal cells are treated through mechanical means such as ultrasonic, explosive disintegration, mechanical shear, or enzymatic dissolution of cell walls. The starch is then extracted with water or an organic solvent. Once the starch is extracted, it can be further fermented to ethanol using the technology similar to other starch-based feedstocks, that is, saccharification and fermentation. This can be done through either a sequential step or a single step (simultaneous saccharification and fermentation). The ethanol is then purified by distillation to remove water and other impurities in the diluted alcohol product (10–15% ethanol), and then condensed into concentrated form (95% ethanol).

In addition to the above-mentioned conventional methods, some algal species are capable of producing ethanol through a dark, anaerobic-based self-fermentation process. When microalgae grow in dark and in the presence of oxygen, the algal cells usually consume storage starch for their maintenance, with water and CO2 as the starch-decomposition products. Under anaerobic conditions, however, the decomposition is incomplete, and a variety of products such as hydrogen, CO2, ethanol, lactic acid, formic acid, and acetic acid can be produced. Based on this mechanism, dark anaerobic algal fermentation process is developed for ethanol production. For example, the green microalga Chlamydomonas reinhardtii produced around 1% (w/w) of ethanol with 30–40% of the theoretical yield of 0.56 g ethanol/g of starch conversion rate through dark anaerobic fermentation [9]. The alga Chlorococcum littorale is also reported to produce ethanol through dark anaerobic fermentation, and 27% of the cellular starch is consumed within 24 h at 25 °C [10].

3.12.3.3 Biodiesel

Algal oil is ideal for biodiesel production. Compared with plant-based oil, algal oil has relatively high carbon and hydrogen contents, and low oxygen content. These characteristics make algal oil attractive for biodiesel production because it may lead to high-energy content, low viscosity, and low density. The basic chemical reaction required to produce biodiesel is the esterification of lipids with alcohol. Glycerol is produced as byproduct.

High lipid-containing algae are most desirable for biodiesel production, and the neutral lipids (TAGs) contained in the algal cells are an ideal feedstock for producing biodiesel. It is noticeable that some microalgae are capable of producing high levels of TAGs but their growth rates [11] are relatively low. Many marine microalgal species may produce higher levels of phospholipids than TAGs. Phospholipids, however, are not desirable in the transesterification process. All these factors need to be carefully considered before a process for algal biodiesel production can be developed.

3.12.3.4 Bio-Oil and Syngas

The organic components present in algal biomass can also be converted into crude bio-oils or syngas fuels through thermochemical conversion processes. Depending on the temperature and the availability of oxygen, the thermochemical conversion process can be categorized as gasification, pyrolysis, and thermochemical liquefaction. The end products vary from gas, liquid, to solid fuel, depending on different processes used.

In the gasification process, the carbonaceous materials in the algal biomass are converted into synthetic gas (syngas) by means of partial oxidation at a temperature ranging from 800 to 900 °C. The major compositions in the syngas are CO2, CO, CH4, and H2. Ammonium is another major component of syngas for biomass with high nitrogen content [12]. Pyrolysis (particularly the fast pyrolysis) converts biomass into bio-oil, charcoal, and gaseous fraction by heating the algal biomass at around 500 °C in the absence of the oxygen. A drying process is usually needed prior to the pyrolysis in order to save the energy used. Liquefaction is usually performed in an aqueous solution of alkali or alkaline salt at around 300 °C and 10 MPa. The advantage of liquefaction is that wet biomass can be directly treated without involving a drying process. The major products of the liquefaction are bio-oils. The gaseous phase of the liquefaction contains CO2, but not H2 and CO [13].

3.12.4 Algal Biodiesel Production Pipeline

3.12.4.1 Algal Physiology and Genetic Engineering

Phototrophic microalgae require several things to grow, including a light source, carbon dioxide, water, and inorganic salts. Algal lipid production depends largely on the growth conditions including the nutrients, temperature, light intensity, growth phase, and physiological status. The growth medium must contain inorganic elements that help make up the algal cell, such as nitrogen,

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phosphorus, iron, and sometimes silicon. In general, oleaginous algae produce only small quantities of neutral lipids (TAG) under optimal growth conditions. Syntheses and accumulation of TAG are facilitated by placing the algae under stress conditions that are imposed by chemical (nutrient limitation, salinity, and pH) or physical (temperature and light intensity) stimuli.

Of those nutrients, nitrogen is the most crucial factor influencing lipid metabolisms. An increase in lipid/TAGs accumulation under nitrogen limitation conditions has been observed in numerous algal species. In diatoms, silicon is another important nutrient affecting lipid metabolism. It has been reported that silicon-deficient Cyclotella cryptic cells had a higher neutral lipid than silicon-replete cells [14]. Phosphate and sulfate limitation also promote lipid accumulation for certain algal species. Temperature influences algal lipid production through altering the fatty acid composition. A general trend is that low temperature tends to increase the unsaturation of the fatty acids and vice versa. By contrast, however, there is no general trend in the effects of temperature on the total lipid production by microalgae. Light intensity also significantly influences algal lipid production. Typically, low light intensity induces the formation of polar lipids, particularly the membrane polar lipids associated with chloroplast, whereas high light intensity decreases total polar lipids and increases the neutral storage lipids.

In addition to the use of traditional approaches such as algal physiology for lipid production, genetic engineering is another important means that may improve algal productivity and, thus, the economics of algal biodiesel production.

Understanding the lipid biosynthesis is of great help to genetically engineer algal lipid production. Theoretically, overexpressing the genes involved in fatty acid synthesis would be able to increase lipid accumulation as fatty acids are the precursors for lipid biosynthesis. Because neutral lipids (TAG) are the most preferred type of lipids for biodiesel production, increasing TAG/total lipids ratio or cellular TAG content through genetic engineering is the most focusing area. Overexpressing genes involved in TAG assembly has been found to significantly increase TAG production in higher plants. Such strategies may also be applied to microalgae. Commonly, microalgae produce large amounts of lipids under unfavorable conditions, which go beyond the log growth phase. The enhanced lipid biosynthesis through genetic engineering, therefore, is likely to reduce algal proliferation and biomass production. In such a case, the genes involved in lipid biosynthesis need to be overexpressed after the target algae have achieved a high cell density and entered the stationary phase.

Another feasible approach to increasing the cellular lipid content is inhibiting metabolic pathways that lead to other carbon storage compounds, such as starch. Starch synthesis shares common carbon precursors with lipid synthesis in algae. Blocking starch synthesis is able to redirect carbon flux to the lipid biosynthetic pathway, resulting in the overproduction of fatty acids and thus total lipids [15].

The important properties of biodiesel, such as cetane number, viscosity, cold flow, and oxidative stability, are largely determined by the characteristics of biodiesel feedstocks such as the carbon chain length and unsaturation degree of the fatty acids of the oil [16]. Thus, the genetic modification of algal fatty acid composition is of interest. Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially polyunsaturated, fatty esters have improved low-temperature properties [17]. 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 [17]. Oleic acid is converted to linoleic acid (C18:2) catalyzed by a Δ12 desaturase enzyme encoded by the FAD2 gene. Inactivation of this desaturation step can greatly increase the proportion of oleic acid in soybean and may represent a possible strategy for elevated accumulation of oleic acid in microalgae.

Genetic engineering can also be used potentially to improve tolerance of algae to stress factors such as temperature, salinity, and pH. These improved attributes will allow for the cost reduction in algal biomass production and be beneficial for growing selected algae under extreme conditions that limit the proliferation of invasive species. Photoinhibition could also be addressed by genetic engineering. The adoption of engineered algae having a higher inhibition light threshold will significantly improve biodiesel production economics.

Although the great potential of using engineered microalgae for biodiesel production has been proposed for some time, the research in genetic engineering of microalgae is still in its infancy. The lack of full or near-full genome sequences and robust transformation systems makes genetic engineering of algae lag much behind that of bacteria, fungi, and higher eukaryotes. Although certain algal species have been reported for efficient transformation, it proves to be difficult to produce stable transformants. Currently, sophisticated genetic engineering whereby several genes are concurrently overexpressed or downregulated is only realistically applicable to the green alga C. reinhardtii.

3.12.4.2 Mass Algal Culture

There are a variety of photoautotrophic-based microalgal culture systems. For example, in those culture systems, the algae may be grown in suspension culture or attached on solid surface. Each system has its own advantages and disadvantages. Currently, the suspension-based open ponds and enclosed photobioreactors are commonly used for algal biofuel production. An open pond culture system usually consists of a series of raceways-type of ponds placed outdoors, while a photobioreactor is a sophisticated reactor design that can be placed outdoors (in most cases) or indoors (e.g., in greenhouse).

Open ponds are the oldest and simplest systems for mass cultivation of microalgae. In this system, the shallow pond is usually about 1 ft. deep; algae are cultured under conditions identical to their natural environment. The pond is designed in a raceway configuration, in which a paddle wheel provides circulation and mixing of the algal cells and nutrients [1]. The raceways are typically made from poured concrete, or they are simply dug into the earth and lined with a plastic liner to prevent the ground from soaking up the liquid. Baffles in the channel guide the flow around bends in order to minimize space. The system is often operated in a continuous mode, that is, the fresh feed (containing nutrients including nitrogen, phosphorus, and inorganic salts) is added in

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front of the paddle wheel, and algal broth is harvested behind the paddle wheel after it has been circulated through the loop [1]. Depending on the nutrient requirements by algal species, a variety of wastewaters can be used for algal culture such as dairy/swine lagoon effluent and municipal wastewater. For some marine type microalgae, seawater or water with high salinity can be used.

Although open ponds cost less to build and operate than enclosed photobioreactors, the open pond system has its intrinsic disadvantages. Because of the open-air nature, the open pond often experiences a lot of water loss due to evaporation. Thus, in open ponds the microalgae fail to use carbon dioxide efficiently, and thus biomass production is limited [2]. Biomass productivity is also limited by contamination with unwanted algal species as well as organisms that feed on algae. In addition, optimal culture conditions are difficult to maintain in open ponds and recovering the biomass from such a dilute culture is expensive [18].

Enclosed photobioreactors have been employed to overcome the contamination and evaporation problems encountered in open ponds [18]. These systems are made of transparent materials and generally placed outdoors for illumination by natural light. The cultivation vessels have a large surface-area-to-volume ratio.

The most widely used photobioreactor is a tubular design, which has a number of clear transparent tubes, usually aligned with the Sun’s rays [2]. The tubes are generally less than 10 cm in diameter to maximize sunlight penetration. The medium broth is circulated through a pump to the tubes, where it is exposed to light for photosynthesis, and then back to a reservoir. A portion of algal cells is usually harvested after the solar collection tubes. In some photobioreactors, the tubes are coiled spirals to form what is known as a helical tubular photobioreactor, but these sometimes require artificial illumination, which adds to the production cost, so this technology is only used for high-value products, not for biodiesel feedstock. The algal biomass is prevented from settling by maintaining a highly turbulent flow within the reactor using either a mechanical pump or an airlift pump [2].

The result of photosynthesis will generate oxygen. In an open raceway system, this is not a problem as the oxygen is simply returned to the atmosphere. However, in the closed photobioreactor, the oxygen levels will build up until they inhibit and poison the algae. The culture must periodically be returned to a degassing zone, an area where the algal broth is bubbled with air to remove the excess oxygen. In addition, the algae use carbon dioxide, which can cause carbon starvation and an increase in pH. Therefore, carbon dioxide must be fed into the system in order to successfully cultivate the microalgae on a large scale. Photobioreactors require cooling during daylight hours, and the temperature must be regulated in night hours as well. This may be done through heat exchangers located either in the tubes themselves or in the degassing column.

The advantages of the enclosed photobioreactors are obvious. It can overcome the problems of contamination and evaporation encountered in open ponds [18]. The biomass productivity of photobioreactors can be 13 times more than that of a traditional raceway pond on average [2]. Harvest of biomass from photobioreactors is less expensive than that from a raceway pond, since the typical algal biomass is about 30 times as concentrated as the biomass found in raceways [2]. However, enclosed photobioreactors also have some disadvantages. For example, the reactors are difficult to scale-up. Moreover, light limitation cannot be entirely overcome because light penetration is inversely proportional to the cell concentration. Attachment of cells to the tube walls may also prevent light penetration. Although the enclosed photobioreactor systems can enhance the biomass concentration, the growth of microalgae is still suboptimal due to variations in temperature and light intensity.

3.12.4.3 Algae Harvesting and Dewatering

Algal harvesting is the concentration of diluted algal suspension into a thick algal paste, with the aim of obtaining slurry with at least 2–7% algal suspension on dry matter basis. In general, algal biomass harvest is a very challenging step in the algal biofuel production chain. Because the size of the algal cells is very small (3–30 µm diameter) and cell concentration is very dilute (∼1 g l−1 for open pond system and <5 g l−1 for photobioreactor culture), a large volume of suspension needs to be treated. As a result, algal biomass harvesting is a very costly process; it is estimated that the recovery of biomass from the culture suspension accounts for 20–30% of the total cost of producing biomass.

Microalgae can be harvested by sedimentation, filtration, or centrifugation. The selection of a harvesting process for a particular strain depends on the size and properties of the algal strain. The sedimentation and flotation harvesting techniques mainly apply to open pond cultivation systems, while filtration and centrifugation apply to photobioreactors. The selected harvest method must be able to handle a large volume of algal culture broth.

A gravity sedimentation system is suitable for microalgae that have naturally high sedimentation rates. This is performed in thickeners or clarifiers, in standard processes in water-treatment plants. The capital and operation costs are low. If the strain has poor sedimentation properties, a flocculation agent can be used.

Filtration is most commonly used to harvest algal biomass. The process can range from simple screening or microstrainers to complex vacuum or pressure filtration systems. Microstrainers, rotating screen filters with a backwash, are widely used for collecting algae such as Spirulina but it is unlikely that this would be economical for collecting algae for nonfood products [11]. Filtration under pressure or vacuum has been successfully used for recovering large microalgae such as Coelastrum proboscideum and Spirulina platensis [19]. The main limitation of filtration is plugging, particularly for harvesting small-size algal cells such as Scenedesmus, Dunaliella, or Chlorella [19]. This can be solved by vibrating screens or tangential filtrations. Deep-bed filtration is also commonly used to avoid plugging, but it requires mixing the solution with sand. Some combined systems use pressing and screening belts, having the advantage of continuous operation [20]. The efficiency of the filtration algal cells also depends on the filter materials selected. For example, sand filter and cellulose fiber have proved to be impractical for harvesting Dunaliella cells [21], while diatomaceous earth can successfully harvest this type of algal cells [22]. Membrane-based microfiltration and ultrafiltration have also been used for harvesting algal cells for some specific application purposes, but, overall, they are more

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commonly used for harvesting algal biomass for biofuel production as the membrane replacement and pumps are the major cost factors. In general, these membrane-based filtration processes can only be used for treating small volume of the algal culture broth.

Centrifugation is an accelerated sedimentation process for algae harvesting. Capital and operation costs of centrifugation are usually high, but its efficiency is much higher than natural (gravity) sedimentation. Due to its high cost, centrifugation as an algae-harvesting method has only been feasible for relatively high-value products [23], although continuous centrifugation has been explored that might be more economical on a large scale. Among various centrifugation processes, hydrocyclone may be the most promising technology that can be used in large-scale economical application due to its simplicity and few moving parts. In hydrocyclone system, the walls of the hydrocyclone chamber are fixed, while algal cells in the chamber move in a spiral fashion creating centripetal forces that result in the denser particles to be spun out of the traversing liquid. Although the technique has been widely used in petroleum and mining industries such as removing dense particles from liquid streams or separating oil from water, their application to soft algal cells is experimental [11].

To enhance the harvest efficiency by gravity sedimentation, filtration, or centrifugation, various flocculation methods can be used to aggregate the microalgal cells into larger clumps that are more easily filtered and/or settled. Cell flocculation can be achieved through either chemical flocculants or culture autoflocculation. Microalgal cells carry a negative charge that prevents aggregation in suspension. The surface charge can be neutralized or reduced by adding flocculants such as multivalent cations or cationic polymers (polyelectrolytes). The ideal flocculants should be inexpensive, nontoxic, and effective at low concentrations. The commonly used multivalent metal salts include ferric chloride, aluminum sulfate, and ferric sulfate. The effectiveness of polyelectrolytes depends on many factors such as molecular weight, the charge density, the dosage of the polymers, as well as the biomass concentration, the broth pH and ionic strength, and the mixing of the fluid. In addition to chemical flocculation, autoflocculation is another way for aggregating algal cells. For example, with the photosynthetic CO2 consumption, the elevation of pH may result in the precipitation of carbonate salt with algal cells [24]. Prolonged culture under sunlight with limited CO2 supply assists autoflocculation [25]. Elevating pH of algal culture broth can also stimulate autoflocculation [23].

3.12.4.4 Biomass Processing for Oil Extraction

The harvested algal biomass slurry usually results in 50–200-fold concentration. After harvesting, chemicals in the biomass may be subject to degradation induced by the process itself and also by internal enzyme in the algal cells [20]. For example, after cells die, lipase contained in the cells can rapidly hydrolyze cellular lipids into free fatty acids (FFAs) so the content of the lipid suitable for biodiesel production can be significantly reduced. Therefore, the biomass slurry must be processed rapidly or it will spoil within a few hours. Drying is a major step to keep the quality of the oil. In addition, the solvent-based oil extraction can be difficult when wet biomass is used. Various drying methods such as sun drying, spray drying, freeze drying, and drum drying can be used for drying algal biomass. Spray drying and freeze drying are normally expensive, and thus are not suitable for biofuel production purpose. In addition, spray drying can cause significant deterioration of the cellular components. Due to the high-energy requirement, drying is the economical bottleneck of the entire process that can account for 70% of the total cost. In general, evaporating 1 kg of water will always require at least 800 kcal of energy [1].

Once the algae biomass is dried, several approaches can be applied to extracting lipids from the biomass, including solvent extraction, osmotic shock, ultrasonic extraction, and supercritical CO2 extraction. Oil extraction from dried biomass can be performed in two steps, mechanical crushing followed by solvent extraction in which hexane is the main solvent used. For example, after the oil extraction using an expeller, the leftover pulp can be mixed with cyclohexane to extract the remaining oil. The oil dissolves in the cyclohexane and the pulp is filtered out from the solution. The oil and cyclohexane are separated by means of distillation. These two stages (cold press and hexane solvent) are able to derive more than 95% of the total oil present in the algae [11].

Oil extraction from algal cells can also be facilitated by osmotic shock or ultrasonic treatment to break the cells. Osmotic shock is a sudden reduction in osmotic pressure, causing cells to rupture and release cellular components including oil. The algae lacking the cell wall are suitable for this process. In the ultrasonic treatment, the collapsing cavitation bubbles near to the cell walls cause cell walls to break and release the oil into the solvent [11]. Supercritical CO2 is another way for efficiently extracting algal oil, but the high energy demand is a limitation for commercialization of this technology.

3.12.4.5 Conversion of Algal Oil into Biodiesel

Algal oil contained in algal cells can be converted into biodiesel through the transesterification process that can be catalyzed by enzymes, acid, or alkaline. After the reaction, the final products are separated into two phases. The upper phase contains fatty acid methyl ester, which is the major composition of the biodiesel, while the lower phase consists of crude glycerol, excess alcohol, water, and impurities inherent in the raw material. The crude biodiesel still contains contaminants such as soaps, excess methanol, residual catalyst, and glycerol. It can be purified by washing with warm water to remove residual catalyst or soaps. After washing, a clear amber-yellow liquid with a viscosity similar to petroleum diesel will be obtained. This product is fuel-grade biodiesel, but only if it meets the specifications outlined in ASTM D6751.

Algal cells usually contain a high level of FFAs (20–50% of total fatty acids) [26, 27] and, therefore, are not suitable for making biodiesel using the alkaline catalyst-based transesterification because of the formation of soap under alkaline conditions. Acid- and

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enzyme-based catalysts can avoid the soap formation problem, and are recommended for making algal biodiesel. However, these two types of the catalysts have inherent limitations. For example, the acid-based transesterification rates are very low, while the enzymes are expensive and unable to complete the transesterification that meet the ASTM standard [28].

3.12.5 Conclusion and Perspectives

Algal biofuel is an ideal biofuel candidate that eventually could replace petroleum-based fuel due to several advantages, such as high oil content, high-yield production, less land required, and environmentally friendly. Currently, algal-biofuel production is still too expensive to be commercialized. Due to the static cost associated with oil extraction and biodiesel processing and the variability of algal-biomass production, future cost-saving efforts for algal-biofuel production should focus on the production technology of the oil-rich algae itself. This needs to be approached through enhancing algal biology (in terms of biomass yield and oil content) and culture-system engineering. In addition, using all components of microalgae for producing value-added products besides algal fuels (e.g., in an integrated biorefinery) is an appealing way to lower the cost of algal-biofuel production. Indeed, microalgae contain a large amount of oil, with the remaining parts consisting of large quantities of proteins, carbohydrates, and other nutrients or bioactive compounds. This makes the residues after oil extraction attractive for use as animal feed or in other value-added products.

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