Extraction Review

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Eur. J. Lipid Sci. Technol. 2011, 000, 0000–0000

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Review Article Developments in oil extraction from microalgae*
Paula Mercer and Roberto E. Armenta
Ocean Nutrition Canada, Dartmouth, Nova Scotia, Canada

Microalgae are a diverse group of organisms with significant potential for industrial applications: as feedstock in aquaculture as well as in the production of valuable bioproducts such as lipids, carotenoids and enzymes. Lately, developments in molecular biology have improved production yields of algae bioproducts, thus increasing their industrial relevance. Additionally, variations in bioprocessing factors (i.e. temperature, pH, light, carbon source, salinity, nutrients, etc.) have been used to enhance both biomass and specific bioproducts’ productivities. Particularly, microalgae have increasingly gained research interest as a source of specialty lipids such as arachidonic, eicosapentaenoic and docosahexaenoic acids, which are often reported in literature to provide several health benefits. Moreover, there has been a recent resurgence in interest in microalgae as an oil producer for biofuel applications. Significant advances have also been made in upstream processing to generate cellular biomass and oil. However, extracting and purifying oil from algae continues to prove a significant challenge in producing both microalgae bioproducts and biofuel, as microbial oil extraction is relatively energy-intensive and costly. Thus, developing inexpensive and robust oil extraction and purification processes is a major challenge facing both the microalgae to bioproduct, and biofuel industries. This paper presents an overview, based on the last 10 years, of advances made in technologies for extracting and purifying microalgae oil. We compared solvent extraction technologies with extraction alternatives such as mechanical milling and pressing, enzymatic and supercritical fluid extraction. We also reviewed recent advances based on molecular engineering of microbes to aid oil extraction. Downstream processing for the potential commercial production of microalgae oil not only must consider economic costs, but should also consider minimizing environmental impacts in order to attain sustainable production processes.
Keywords: bioprocessing / downstream processing / microalgae oil / oil extraction methods / omega-3 fatty acids

Received: September 2, 2010 / Revised: December 8, 2010 / Accepted: January 10, 2011 DOI: 10.1002/ejlt.201000455

1 Introduction
Microalgae are a heterogeneous group of organisms with a host of characteristics that distinguish them, some of those being cell size, colour, inhabitation of aquatic environments, as well as being unicellular and quite often, photoautotrophic [1]. Microalgae can also be prokaryotic or eukaryotic, and in evolutionary terms, they can be either ancient species or recent ones. This diversity creates the capability for microalgae to be a valuable source of a multitude of products that, at some level, support the food (both human and animal nutrition), cosmetic, pharmaceutical and fuel industries [1]. In recent years, microalgae have gained attention as a possible solution to some imminently critical issues,
Correspondence: Dr. Roberto E. Armenta, Ocean Nutrition Canada, 101 Research Drive, Dartmouth, Nova Scotia, B2Y 4T6, Canada E-mail: [email protected] Fax: 1 902 480 3241

including the role of an alternative fuel source, and as a potential source of many bioactive compounds intended for human consumption (i.e. omega-3 fatty acids, which are commonly sourced from fish stocks). Due to potential fish sustainability concerns, microalgae have the potential to be a viable alternative for obtaining oils rich in omega-3 fatty acids. Microorganisms bear multiple advantages over traditional energy crops. They have a higher growth rate than other crops, a shorter maturity rate, a higher biomass production rate than other cash crops, as well as using far less land than conventional crops [2]. Furthermore, with microalgae there is no competition for land space that could be used for food crops. For example, using corn as a feedstock for making ethanol creates a negative competition between human and animal consumption or fuel production [3]. Microalgae also have the potential to counteract a portion
*This paper is based on an oral presentation given at the 2010 FEBS workshop of microbial lipids in Vienna.

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of the greenhouse effect and water pollution, because through photosynthesis, microalgae have the ability to fix carbon dioxide produced by industrial plants. Some microalgae also fix nitrogen and absorb other contaminants such as heavy metals and phosphorous [4, 5]. One of the main obstacles to fully taking advantage of lipid-producing microalgae, is the ability to successfully and efficiently extract oil from the cell biomass. Table 1 shows some of the more recent extraction methods and their abilities to recover oil, more specifically, valuable fatty acids. Additionally, there is the concern of extracting the oil in the safest and most environmentally sustainable manner,

therefore solvent extraction may not always be the best solution for recovering the oil from the microalgal biomass. There is an abundant amount of literature available on the topic of microalgae and its potential uses, the aim of this review is to focus on the most relevant extraction methods reported within the last decade. There are a few well-documented procedures for extracting oil from microalgae, those being mechanical pressing, homogenization, milling, solvent extraction, supercritical fluid extraction, enzymatic extractions, ultrasonic-assisted extraction and osmotic shock. All of these methods have their individual benefits and drawbacks (Table 2).

Table 1. Some common extraction methods explored in the last decade, and their effectiveness at recovering lipids and lipid products
Extraction method Solvent/saponification Bligh and dyer (wet) Bligh and dyer (dry) SC-CO2 Bligh and dyer SC-CO2 SC-CO2 SC-CO2 Solvent UAE Soxhlet Organism Porphyridium cruentum Mortierella alpina Recovered oil (%) 59.5 27.6 41.1 2.1 5.5 25 40 77.9 96.1 25.9 4.8 Fatty acid (% in recovered oil) EPA – 79.5 ARA – 73.2 N/A Oleic – 49.3 Palmitic – 15.3 GLA – 31.3 GLA – 73.0 EPA – 32.1 Palmitic – 17.8 GLA – 13.0 GLA – 20.2 Palmitic – 40.0 EPA – 23.7 Palmitoleic – 19.2 DHA – 39.3 Palmitic – 37.9 DHA – 39.5 Palmitic – 38.0 N/A Oleic – 56.3 Linolenic – 19.0 Palmitic – 59.2 Oleic – 16.7 References

[22] [19]

Arthrospira (spirulina) maxima Nannochloropsis sp. Arthrospira (spirulina) maxima Spirulina (arthrospira) platensis Phaeodactylum tricornutum Crypthecodinium cohnii

[26] [48] [30] [29] [49]

[6]

Bligh and dyer (dry) Solvent/transesterification

Chlorella vulgaris Botryococcus braunii Synechocystis sp.

52.5 12.1 7.3 25.3 21.2 21.0 20.5 6.3 18.8 14.9 14.4 10.7 5.6 8.6 19.9

[50] [51]

Wet milling French press Sonnication Bead-beater Soxhlet Bead-beater French press Wet milling Sonnication Soxhlet SC-CO2 Bligh and dyer

Scenedesmus dimorphus

Chlorella protothecoides

N/A

[16]

Crypthecodinium cohnii

DHA – 42.7 Palmitic – 25.3 DHA – 49.5 Palmitic – 22.9

[52]

SC-CO2 ¼ supercritical carbon dioxide extraction, UAE ¼ ultrasonic assisted extraction EPA ¼ eicosapentaenoic acid, C20:5(v-3), ARA ¼ arachidonic acid, C20:4(v-6), oleic ¼ oleic acid (C18:1), palmitic ¼ palmitic acid (C16:0), GLA ¼ g-linolenic acid, C18:3(v-6), DHA ¼ docosahexaenoic acid, C22:6(v-3), linolenic ¼ linolenic acid (C18:3).
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Table 2. Advantages and disadvantages of popular extraction methods for recovering oil from microalgae
Extraction method Pressing Solvent extraction Advantages Easy to use, no solvent involved Solvents used are relatively inexpensive; results are reproducible Limitations Large amount of sample required, slow process Most organic solvents are highly flammable and/or toxic; solvent recovery is expensive and energy intensive; large volume of solvent is required High power consumption; expensive/difficult to scale up at this time High power consumption; difficult to scale up References

[53]
[54, 55]

Supercritical fluid extraction

Non-toxic (no organic solvent residue in extracts); ‘green solvent’; non-flammable and simple operation Reduced extraction time; reduced solvent consumption; greater penetration of solvent into cellular materials; improved release of cell contents into bulk medium

[56, 57]

Ultrasonic-assisted

[58, 59]

Pressing and homogenization essentially involve using pressures to rupture cell walls, in order to recover the oil from within the cells. Milling on the other hand, uses grinding media (consisting of small beads) and agitation to disrupt cells. These methods are usually used in combination with some kind of solvent extraction. Solvent extraction entails extracting oil from microalgae by repeated washing or percolation with an organic solvent. Hexane is a popular choice due to its relatively low cost and high extraction efficiency. Supercritical fluid extraction involves the use of substances that have properties of both liquids and gases (i.e. CO2) when exposed to increased temperatures and pressures. This property allows them to act as an extracting solvent, leaving no residues behind when the system is brought back to atmospheric pressure and RT. Enzymes can also be used to facilitate the hydrolysis of cell walls to release oil into a suitable solvent. The use of enzymes alone, or in combination with a physical disruption method such as sonnication, has the potential to make extractions faster and with higher yields. The use of sonnication alone can also enhance the extraction process immensely due to a process called cavitation. Ultrasonic waves create bubbles in the solvent, the bubbles burst near the cell walls of microalgae, which produce shock waves, causing the contents (i.e. lipids) to be released into the solvent [6, 7]. Osmotic shock, a less-employed procedure, makes use of an abrupt lowering of osmotic pressure that causes cells to burst and release their contents [8]. Figure 1 outlines steps of the downstream processing used in extracting oil from microorganisms.

the microalgal biomass (or more commonly, seeds or nuts) to high-pressure, which ruptures cell walls and releases the oil. Alternatively, homogenization is the process of forcing biomass through an orifice, which results in a prompt pressure change as well as high shearing action. Bead milling (Figure 2) entails vessels packed with very small beads that are agitated at high speeds. Biomass agitation within the grinding media (beads) results in damaged cells, where the degree of disruption depends mostly on contact between biomass and beads; also size, shape and composition of the beads, and strength of the microalgal cell walls [10]. Bead milling is generally used in conjunction with solvents to recover oil, and are most effective and economical when cell concentrations are significant and when extracted products are easily separated after disruption. Typically, this type of cell disruption is most effective and energy-wise when biomass concentrations of 100 to 200 g/L are used [9]. In terms of finding an effective and efficient mode of disrupting cells, there are multiple options when it comes to using this kind of technology. Some of which include the identification of biological features of the organism that make it possible to weaken the cell wall prior to mechanical disruption, such as pre-treatments (i.e. acid/alkali, and enzymatic), thus potentially minimizing the use of solvents.

3 Solvent extraction methods
Organic solvents, such as benzene, cyclohexane, hexane, acetone and chloroform have shown to be effective when used on microalgae paste; they degrade microalgal cell walls and extract the oil because oil has a high solubility in organic solvents [4]. However, it is feasible (at least using Botryococcus braunii) to extract oil from microalgal biomass without damaging cell walls, as long as the solvent is not toxic to the cells [11]. This is possible if the species of microalgae is one that actively secretes oil, which is then recovered by a non-toxic, biocompatible solvent such as decane [12]. Instances of such
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2 Mechanical cell disruption
Mechanical disruption, which includes pressing, bead milling (Figure 2) and homogenization, is an approach that minimizes contamination from external sources, while maintaining the chemical integrity of the substance(s) originally contained within the cells [9]. Pressing involves subjecting
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Figure 1. Extraction/downstream processing of microbial oils. PEF ¼ pulsed electric field, MAE ¼ microwave-assisted extraction.

Figure 2. Bead-milling process for recovering oil.
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milking of components from microbial cells (i.e. carotenoids) using so-called biocompatible solvents are described in the work of Hejazi et al. [13, 14] using Dunaliella salina. In addition, a suitable solvent should be insoluble in water, preferentially solubilize the compound of interest, have a low boiling point to facilitate its removal after extraction, and have a considerably different density than water. Also, for process cost-effectiveness, it should be easily sourced, as well as inexpensive and reusable [11]. Due to these qualities, hexane is typically the solvent of choice for large scale extractions. An efficient extraction requires the solvent to fully penetrate the biomass and to match the polarity of the targeted compound(s) (i.e. non-polar solvent such as hexane for extracting non-polar lipids). This, in conjunction with the ability to make physical contact with the lipid material and solvate the lipid, makes for a successful extraction solvent. One way to facilitate this is to mechanically disrupt cells prior to exposing them to the solvent [15]. Shen et al. [16] illustrated some differences between cell disruption techniques and solvent extraction systems. In their experiments, they found that for Scenedesmus dimorphus (an autotrophic algae), wet milling followed by hexane extraction gave the best recovery, with 25.3%, compared to 6.3% using soxhlet extraction. They also showed that for Chlorella protothecoides (a heterotrophic algae), using a bead-beater followed by hexane extraction, was most effective at recovering the oil; 18.8%, compared to 5.6% using soxhlet extraction. Differences in oil recovery effectiveness could be due to dissimilarity in cell shape and structure of the two species. Alternatively, solvent extraction can be enhanced by using organic solvents at temperatures and pressures above their boiling point; this is called accelerated solvent extraction (ASE), and is applicable to solid and semi-solid matrices, thus requiring samples to be dried prior to extraction [15]. When this procedure is used with water as the solvent, it is referred to as pressurized low polarity water extraction. Increasing the pressure decreases polarity of water, thus modifying its solvent power, allowing the extraction of compounds not normally soluble in water. Traditionally, lipids have been extracted from biological matrices using a combination of chloroform, methanol and water [17]. This procedure, known as the Bligh and Dyer method, originally designed to extract lipids from fish tissue, has been used as a benchmark for comparison of solvent extraction methods. A disadvantage of using this method is that at large scale, significant quantities of waste solvent are generated, making solvent recycling costly, as well as raising safety concerns due to handling of large amounts of organic solvents [18]. Although the Bligh and Dyer method has been proven effective when used on wet material, there is an example that contradicts it. Zhu et al. [19] extracted significantly more oil from dried microbial biomass compared with oil extraction yield from wet biomass (41.1 and 27.6%, respectively). A potential explanation is that their work was done using Mortierella alpina, an oleaginous fungus, showing
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the Bligh and Dyer method may not be applicable to certain kinds of wet biomaterials, such as the mycelium of fungus. In addition, organic solvents can lead to contamination in the form of solvent residues being present in the final product. This is why most organic solvents may have limited application in food processing. Similar to other extraction procedures, moisture can prove troublesome with solvents as well, restricting solvent access to cells by acting as a barrier between solvent and cells. Several methods involve simultaneous extraction and transesterification of microalgal biomass to recover fatty acids [20, 21]. Direct transesterification of the microalgal biomass has been shown to be more efficient (at least 15–20% over extraction–transesterification experiments) and to produce better oil yields when solvents are added in order of increasing polarity, and the reaction is allowed to progress for an optimized length of time. For example, it was shown there were significant increases in saturated, monounsaturated and polyunsaturated fatty acid concentrations from 15 to 120 min of reaction time, but not beyond 120 min [21]. In addition, fatty acids can also be recovered through concurrent solvent extraction and saponification [22]. In experiments by GuilGuerrero et al. [22], solvent extraction using 96% v/v ethanol and simultaneous saponification with KOH, the fatty acid profile of extracts were similar to the fatty acid profile of the initial biomass after direct methylation. A similar experiment was performed using mechanical cell disruption via a bead mill prior to subsequent saponification with KOH and hexane extraction [23]. This experiment showed that mechanical cell disruption was indeed necessary to achieve acceptable yields of oil, as well as being a scalable process with the potential to be applied at a industrial level.

4 Supercritical fluid extraction
An extraction method that has gained acceptance in recent years is the use of supercritical fluids to extract high-value products from microalgae. This is because it produces highly purified extracts that are free of potentially harmful solvent residues, extraction and separation are quick, as well as safe for thermally sensitive products [18, 24, 25]. Also, fractionation of specific compounds is feasible, which may reduce separation costs, as well as possibly counteracting greenhouse gas effects by using CO2 waste from industry [24–27]. Table 3, based on the work of Sahena et al. [18], compares traditional solvent extraction methods with supercritical fluid extraction. Supercritical fluid extraction takes advantage of the fact that some chemicals behave as both a liquid and a gas, and have increased solvating power when they are raised above their critical temperature and pressure points. Carbon dioxide is favoured because of its relatively low critical temperature (31.18C) and pressure (72.9 atm) [15]. Supercritical CO2 extraction efficiency is affected by four main factors: pressure, temperature, CO2 flow rate and extraction time
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Table 3. Comparison of supercritical CO2 and solvent extractions
Solvent extraction Presence of solvent is inevitable; residual solvent concentration (usually in the order of ppm) depends on the solvent used Heavy metal contamination is also unavoidable, and depends on the solvent, solvent recycling procedure, source of raw material and what the machinery parts are made from Inorganic salt content is also difficult to avoid (same reasons as above) Solvents have poor selectivity; during solvent removal, polar substances form polymers which lead to discolouration of the extract and poor flow characteristics Both polar and non-polar colours are extracted Solvent removal requires extra unit operations, which results in higher cost and lower recoveries Supercritical CO2 extraction Procedure is completely free of solvents and thus extracts are very pure Free of heavy metals; not extracted, even if they’re present in the raw material. There are no heavy metals present in CO2 or the equipment Free of inorganic salts (same reasons as above) CO2 is highly selective, so there is no chance of polar substances forming polymers Only non-polar colours get extracted No extra unit operations required, and yield is very high

[4, 18, 28, 29]. These factors, along with the use of modifiers (most commonly ethanol as a co-solvent), can be altered and adjusted to optimize extractions. When ethanol is used as a co-solvent, polarity of the extracting solvent (in this case, CO2) is increased and the viscosity of the fluid is subsequently altered. The resulting effect is an increase in the solvating power of the CO2, and the extraction requires lower temperature and pressure, making it more efficient [24, 26, 27, 30]. Supercritical CO2 extraction, in combination with a small concentration (usually 10–15%) of ethanol as a co-solvent, has shown to give comparable lipid yields to the benchmark Bligh and Dyer solvent system of chloroform, methanol and water when extracting oil from Arthrospira maxima, as well as Spirulina platensis [17, 30, 31]. In addition, supercritical fluid extraction has produced equivalent fatty acid profiles to those obtained with traditional solvent extraction of dried biomass, ¨ when applied to both Spirulina platensis and Hippophae thamnoides L. [28, 29]. Since CO2 is a gas at room temperature, it is easily removed when extraction is completed, thus it is safe for food applications [18, 30], and it can safely be recycled, which is an environmental benefit [18]. One restriction to supercritical CO2 extraction is the level of moisture in the sample. High moisture content can reduce contact time between the solvent and sample. This is because microalgal samples tend to acquire a thick consistency and moisture acts as a barrier against diffusion of CO2 into the sample, and diffusion of lipids out of the cells. This is why samples are dried prior to supercritical fluid extraction [18]. Further downstream processing or refining is often necessary depending on what the final product is and its intended usage (i.e. food grade oils that require bleaching and deodorization).

the liquid is lower than its vapour pressure. These bubbles grow when pressure is negative and compress under positive pressure, which causes a violent collapse of the bubbles. If bubbles collapse near cell walls, damage can occur and the cell contents are released [4, 7]. One company (OriginOil), has taken advantage of this property of ultrasonics, by developing a process in which a combination of ultrasound and electromagnetic pulses are used to disrupt algal cell walls. Carbon dioxide is then injected into the resulted slurry of algae biomass to lower pH, which facilitates separation as cell debris sinks to the bottom, and oil rises to the top of the aqueous layer [32]. Both ultrasound and microwave-assisted (described later) methods improve extractions of microalgae significantly, with higher efficiency, reduced extraction times and increased yields, as well as low to moderate costs and negligible added toxicity. An example of this is illustrated by experiments performed using Crypthecodinium cohnii, where ultrasound was most effective at disruption of cell walls, increasing oil yield to 25.9%, from 4.8% yield obtained using a soxhlet extraction with hexane [6]. It is worth noting that to date it has not been determined if ultrasonic methods may negatively impact oil quality and/or stability of polyunsaturated fatty acid rich oils. Finally, this technology may be difficult to scale up.

6 Metabolic engineering/genetic methods
More recently, research has been focused on the application of metabolic engineering to create microalgae with optimized productivity of specialty oil products as well as biofuels. Several known species of microalgae are able to accumulate significant amounts of lipids [33], which in theory may be optimized through metabolic engineering to enhance oil-producing microalgae strains. The average microalgae produces somewhere between 1 and 70% lipid (relative to dry cell weight), while other species, when exposed to certain conditions, can produce up to 90% of their dry weight as lipids
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5 Ultrasound/sonnication
Through cavitation, ultrasonic-assisted extractions can recover oils from microalgal cells. Cavitation occurs when vapour bubbles of a liquid form in an area where pressure of
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[34–37]. Steen et al. [38] engineered Escherichia coli to produce fatty acids. This was achieved through expression of plant derived genes that encoded several thioesterases, which have previously been shown to prefer varied chain length fatty acyl-ACP’s (acyl carrier proteins) [39]. Also, Ribeiro et al. [40] demonstrated a similar concept of free fatty acid excretion in Saccharomyces cerevisiae. This genetic tool makes custom free fatty acid production, for the purpose of fuel or chemical products, a possibility. This proved the concept of excretion of fatty acids through bacterial and yeast membranes. Although yields were low and thus not yet economically feasible, this finding is significant as it could be the basis for future improvements in the excretion of microbial free fatty acids. Microorganims excrete free fatty acids when these compounds are in high concentrations intracellularly as they can be detrimental to microbial cells due to lipotoxicity [9]. The ability of microbial cells to secrete oil could facilitate downstream processing of oil due to a reduced energy requirement for cell lysing. This may allow the use of much simpler technologies, such as centrifugation, to recover the oil.

7 Other oil extraction technologies
Another potential extraction method worth noting is the use of pulsed electric field technology (PEF), in which cells are processed by exposing them to brief pulses of a strong electric field. Electric pulses permeabilize cell walls, enhancing mass transfers across cell membranes, which makes it a promising pre-treatment prior to solvent or mechanical extraction methods, or both, to recover lipids [41–43]. PEF is a relatively benign non-thermal process and has been used in applications such as oil extraction from rapeseed [41], and oil recovery from other plant products, such as maize, olives, soy beans and juice from alfalfa [43]. Guderjan et al. [41] illustrated an improvement in rapeseed oil yields of approximately 10% by pretreating hulled seeds with PEF, then pressing and hexane extracting the oil. This technology is relatively new for this type of application, thus there is definitely an opportunity for improvement and optimization. An alternative, relatively safe and environmentally conscientious method involves using enzymes to breakdown cell walls of microalgal species to release cell contents. Enzymatic treatment of microbial biomass has the potential to partially or fully disrupt cells with minimal damage to the inside product (i.e. oil). Enzymatic treatment has proven to be successful extracting oil from plant seeds, such as Jatropha curcas L. (in combination with sonnication) [44], or borage seed, using cold pressing as the physical means of oil recovery [45]. One distinct challenge with enzymes is that in order to design an effective enzymatic procedure for hydrolyzing microbial cells, it is necessary to determine composition of the cells being worked with. Hence, the most appropriate enzymes can be chosen to optimize extraction conditions and aim for improvements of oil yields.
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Microwave technology is based on the principle that microwave heating is very selective, and releases very little heat to the environment. Microwaves directly affect polar solvents and/or materials, so when they are used on even dried plant material, trace amounts of moisture in cells are affected. Moisture is evaporated, generating a significant amount of pressure that stresses the microorganism’s cell wall, eventually rupturing it and releasing desired contents from within [46]. This leads to potentially effective oil extraction procedures, however, to date, microwaves have a limited scope of application. Nonetheless, microwaves have facilitated the development of a quick, safe and potentially inexpensive method for lipid extraction that does not require initial dehydration of biomass. An example of this would be methods that use a combination of microwaves and smaller volumes of solvent (microwave-assisted solvent extractions) to extract oil from the microalgae Crypthecodinium cohnii, obtaining an oil yield of 17.8% compared to 4.8% obtained with soxhlet extraction [6]. One obvious drawback of using microwaves is the potential for oxidative damage to valuable lipid products [18]. In addition, microwave extraction of microbial oils could still prove difficult to scale up. A recent study by Balasubramanian et al. [47] was successful in demonstrating that microwave assisted extractions (MAE) of Scenedesmus obliquus, can give oil recoveries of 77% compared to 47% for the control soxhlet extraction. The major advantage of this type of extraction is the 20-fold reduction in processing time; 30 min, compared to the benchmark soxhlet extraction of 10 h. Additionally, the quality of the resulting oil was better than that of the control or the soxhlet extraction, containing more unsaturated fats, as well as more omega-3 and omega-6 fatty acids [47].

8 Conclusions
To date, extraction of microbial oils is mainly conducted with solvents such as hexane, coupled with mechanical disruption techniques. A mechanical cell disruption method that has been tested at both small and large scales is bead milling. Most recent developments on downstream processing for extracting microbial oils include promising non-solvent methods. Nonetheless, the effect of these methods on the chemical stability of valuable compounds prone to oxidation (i.e. omega-3 fatty acids) remains to be investigated. These non-solvent extraction technologies include the use of pulse electric field, enzymes, microwaves, ultrasonic energy and mechanical disruption. Some of them have shown significant oil extraction yields at the laboratory bench scale. However, little or no testing at pilot or large (production) scales have been performed. This represents an opportunity for research to define efficient technologies for oil extraction from microalgae as there is a need for these extraction methods to be tested at scales beyond the laboratory.
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