An Overview of Current Pretreatment Methods Used to Improve Lipid Extraction from Oleaginous Microorganisms

Microbial oils, obtained from oleaginous microorganisms are an emerging source of commercially valuable chemicals ranging from pharmaceuticals to the petroleum industry. In petroleum biorefineries, the microbial biomass has become a sustainable source of renewable biofuels. Biodiesel is mainly produced from oils obtained from oleaginous microorganisms involving various upstream and downstream processes, such as cultivation, harvesting, lipid extraction, and transesterification. Among them, lipid extraction is a crucial step for the process and it represents an important bottleneck for the commercial scale production of biodiesel. Lipids are synthesized in the cellular compartment of oleaginous microorganisms in the form of lipid droplets, so it is necessary to disrupt the cells prior to lipid extraction in order to improve the extraction yields. Various mechanical, chemical and physicochemical pretreatment methods are employed to disintegrate the cellular membrane of oleaginous microorganisms. The objective of the present review article is to evaluate the various pretreatment methods for efficient lipid extraction from the oleaginous cellular biomass available to date, as well as to discuss their advantages and disadvantages, including their effect on the lipid yield. The discussed mechanical pretreatment methods are oil expeller, bead milling, ultrasonication, microwave, high-speed and high-pressure homogenizer, laser, autoclaving, pulsed electric field, and non-mechanical methods, such as enzymatic treatment, including various emerging cell disruption techniques.


Introduction
Over the last decades, the production of biofuels from renewable sources has gained more attention due to critical environmental issues, such as greenhouse gas emission, rapid depletion of fossil fuel supplies, and high energy cost [1]. Microbial oils are found to be a good option to produce biofuels, as many microorganisms, such as microalgae, yeast, bacteria, and fungi have the ability to accumulate oils under special cultivation conditions [2]. Moreover, microbial sources of lipids have many advantages over other sources, including higher lipid productivity in terms of g/L/day, being unaffected by any seasonal climate changes, low labor intensiveness, and easily scale-up [3,4]. The production costs are the major limiting factor to utilize microbial oils for biodiesel production, since the feedstocks to cultivate microorganisms account for 60% to 80% of the overall production cost [5]. To enable the commercial production of microbial lipids, these costs must be reduced by using low-cost feedstocks [5]. Oleaginous microorganisms can utilize various types of organic carbons, regardless of their origin, to accumulate oils in their cytoplasm. They have the specific

Folch Method
After the Bligh & Dyer method, the Folch method is the second most used method for lipid extraction from oleaginous microbial biomass [34]. It was initially developed to extract and purify the lipids from brain tissue in a two-step process. In the first step, the lipids are extracted from the homogenized tissue with 2:1 chloroform-methanol (v/v) and in the second step, the non-lipid substances are removed by phase separation after adding at least five-fold volumes of water to the filtrate from step one [34]. Researchers are using this method with minor modification, e.g., Cheirsilp and Kitcha (2015) used the Folch method for the extraction of lipids from the oleaginous fungus Aspergillus tubingensis TSIP9. They applied sonication on the mixture of dried biomass and chloroform:methanol (2:1) for 30 min, followed by filtration [35]. Kumar et al. (2015) used a similar method to extract lipids from homogenized biomass of the oleaginous bacteria Rhodococcus opacus with chloroform and methanol (2:1, v/v), followed by 15-20 min of shaking in an orbital shaker at ambient room temperature [36]. A modification of the Folch method for the extraction and purification of lipids from oleaginous yeast Cryptococcus terricolus was described by T. A. Pedersen, where the extracted crude lipids were dissolved in chloroform and methanol (2:1, v/v) and distilled water, followed by a separation step by centrifugation [37]. Hara and Radin (1978) also tried to extract lipids from the tissues with non-toxic solvents, such as hexane:isopropanol, followed by a washing step with aqueous sodium sulfate to remove non-lipid contaminants from the extract [38].

Pretreatment of Oleaginous Microbial Biomass to Extract Lipids
The effect of microbial biomass pretreatment on the lipid extraction process has not yet been discussed extensively. The efficiency of lipid extraction varies depending on the pretreatment process that is used to disrupt the cellular integrity as it increases with an increasing degree of cell disruption. However, other parameters, such as residual water content in the case of wet biomass and particulate size in the case of dry biomass, may also affect the pre-treatment process [2,14]. The choice of pretreatment depends on the cellular structure of the microbial biomass. It can be a single-step or a multistep process depending on the physical condition of the biomass (dry or wet). Various pretreatment methods for the disintegration of cellular membranes are currently in use and they can be divided into two main groups of (i) mechanical and (ii) non-mechanical methods ( Figure 1). Some researchers divide the cellular disintegration into thermal treatment methods and non-thermal treatment methods, whereas others explain the pretreatment methods in combination with lipid extraction and categorize them as mechanical methods, such as oil expeller, ultrasonication, and microwave-assisted extraction or chemical methods, such as Soxhlet extraction, supercritical fluid extraction, and accelerated solvent extraction [20]. All of these pretreatment methods have been extensively utilized for the efficient lipid extraction from various oleaginous microorganisms, such as yeast, microalgae, fungi, and bacteria (Table 1).
However, other parameters, such as residual water content in the case of wet biomass and particulate size in the case of dry biomass, may also affect the pre-treatment process [2,14]. The choice of pretreatment depends on the cellular structure of the microbial biomass. It can be a single-step or a multistep process depending on the physical condition of the biomass (dry or wet). Various pretreatment methods for the disintegration of cellular membranes are currently in use and they can be divided into two main groups of (i) mechanical and (ii) non-mechanical methods ( Figure 1).

Mechanical Pretreatment Methods
Mechanical pretreatment for the disintegration of cellular structure is usually carried out by applying mechanical forces or energy transfer through conventional heat, waves, and electric currents. Mechanical forces can be divided into two forms, i.e., solid-shear forces (e.g., bead mill, high-speed homogenization) and liquid-shear forces (e.g., high-pressure homogenization, micro fluidization). The direct energy transfer to the cells can be achieved by waves (laser, ultrasonication, and microwave treatment), conventional heat (autoclave and water bath), or by applying a pulse electric field [2].

Oil or Expeller Pressing
Expeller pressing is the simplest method to extract oils by mechanical crushing. It has many advantages, like smooth and hands-free operational conditions and low needs for maintenance. The lipids are extracted from the dry biomass by applying mechanical pressure to squeeze out the oils from the broken cells. However, this method is relatively slow and requires large amounts of biomass [65]. The applied pressure must be optimal, otherwise it will result in excessive heat generation and blockage problems due to high pressure [66]. Although this method is usually used to extract oils from seeds, some microalgal lipids have also been extracted with this method. For example, filamentous algae were explored for lipid extraction by using the screw expeller press and almost 75% total lipids were extracted from algae by this method [67]. Depending on the type of biomass, various press configurations, such as screw, expeller, piston, etc., can be used. Some researchers suggest that this method is an expensive and slow process when applied on microbial biomass [24,68]. Johnson and Wen (2009) stated that it is a suitable method for feedstocks, like soybean or canola seeds, where lipids can be extracted from the crushed biomass with solvent, while the extraction process may not be suitable for microalgal cells (both mud-like form and dry powder form of algae), where rigid cell walls hinder the extraction process [69]. Topare et al. (2011) suggested that the solvent extraction by Soxhlet apparatus is effective and extracted more than 98% of the lipids from microalgal cells. But, since it is not a cost-effective method, they tried the expeller press method to extract lipids and this method could recover 75% of the oil from algae [67].

Bead Milling
The history of bead milling goes far back to when it was first applied in the manufacturing of cosmetics to reduce the particle size of paint or lacquer and to grind minerals. After proving its effectiveness in the chemical industry, bead milling was successfully applied for the disruption of microbial cells for the downstream processing of intracellular products [20,[70][71][72]. This method has many advantages, such as the need of only single-pass, continuous module of operation, high disruption efficiency, easy biomass loading, mild operating temperature, and applicability to various types of biomass from lab-scale to industrial scale [20,67,71,72]. The operating conditions for the efficient disintegration of cells depend on various factors such as agitator geometry, speed, biomass concentration, slurry flow rate, bead size, bead-to-substrate ratio, etc. Montalescot et al. (2015) reported that disruption of two microalgae, Nannochloropsis oculata and Porphyridium cruentum, was performed by continuous bead milling where the highest bead filling ratio of >55% v/v was found to be optimal [73]. The type and size of beads also strongly affect the disintegration of microalgal cells. Doucha and Lívanský (2008) suggested that zirconium oxide (ZrO 2 ) beads are more efficient than glass beads for cellular disintegration because of their higher specific density [74]. Postma et al. (2015) reported that kinetic rate constants can be increased by increasing the speed of the agitator as well as the biomass concentration [72]. They investigated the disintegration of Chlorella vulgaris by using zirconium oxide (ZrO 2 ) beads with a diameter of 1 mm, which gives a lower specific energy consumption, while the agitator speed and biomass loading were 6 m/s and 145 g DW /kg [72]. However, similar specific energy consumptions were also achieved by changing the size of beads with similar flow rate and agitator speed [74]. Balasundaram et al. (2012) investigated the optimal balance between shear forces and impact forces that are required for a differential recovery of intracellular products from the cyanobacteria C. fritschii (PCC6912) when a custom-made energy efficient ball mill was used for disintegration [70]. Although bead milling is suitable for disintegration of cells, its high energy consumption during operation and its inefficient energy transfer from rotating shaft to individual cells make it an unfavorable method [20]. Oleaginous yeast Y. lipolytica IFP29 (ATCC 20460) was disrupted by ultrasound, microwave irradiation, and bead milling and the lipid extraction efficiency was compared to pretreatment with freezing/defrosting, cold-drying, bead milling, and microwave irradiation before the conventional solvent extractions process [75]. It was suggested that bead milling was efficient for lipid extraction from oleaginous yeast biomass while cold-drying under pressure was the best pretreatment method, giving two times more yield when compared to conventional methods [75].

High-Pressure Homogenization
This method is suitable for the stabilization of emulsification processes in cosmetic, pharmaceutical, and food industries, however, it has also been extensively utilized for the microbial cell disruption of microalgae [76], bacteria [77], and yeast [78]. The cell disruption efficiency varies according to the valve seat configurations of the homogenizer [79]. High disruption efficiency is usually achieved through shear forces of highly pressurized fluids on the stationary valve surface and hydrodynamic cavitation from the shear stress induced by pressure drop [80]. High-pressure homogenization has many advantages as it is a simple continuous operating system and can be applied for wet biomass, where the processing fluid is pressurized in intensifiers and passed through a homogenization chamber. The energy is accumulated in the fluid by the pressure and released into the passage through an orifice valve, where the velocity of the fluid increases to up to 200-400 m/s [81,82]. Increased velocity generates mechanical stress, such as shear and elongational forces, turbulence, and cavitation, which are responsible for disruption of cells [83]. Coccaro et al. (2018) suggested that the most efficient disintegration of Lactococcus lactis cells was achieved with small orifice valve size, high operating pressure, and low fluid viscosity [77]. In another study, oleaginous microalgae Nannochloropsis sp. were disrupted by prior incubation at 37 • C for 15 h before treatment with high-pressure homogenization at 1200 ± 100 bar, followed by lipid extraction with organic solvents [84]. It was a low solvent, low temperature method for efficient lipid extraction from wet concentrated paste where the recovery was reported to be up to 70% w/w of the total lipids and 86% w/w of neutral lipids using hexane as solvent [84].

High-Speed Shearing Homogenization
High-speed shearing homogenization (HSH) is usually utilized to prepare foams, emulsions, and suspensions [85]. It is a very effective method to disrupt cells, where a slurry of biomass is stirred in a specific device consisting of a stator-rotor assembly with a small gap (100-3000 µm) [86]. The cells are disintegrated due to hydrodynamic cavitation and the shear forces that are caused by stirring at high rpm, which creates high shear rates (20,000-100,000 s −1 ) [20]. High-speed shearing homogenization was used to prepare the extracts of Agaricus blazei murill for the extraction of α-glucan with a final carbohydrate content of 96% [86]. Kwak et al. (2018) used a high-shear mixer to disrupt the cells and extract the lipids from the wet biomass of the oleaginous microalgae Aurantiochytrium sp. KRS101 [52]. They suggested that the performance of the high-shear mixer was quite similar between wet and dry biomass of microalgae when extracted with different solvents, such as hexane, hexane-isopropanol, and ethanol. The mixtures developed a strong shear stress and cavitation effect when stirred at 15,000 rpm for 10 min, which was enough to extract all the esterifiable lipids from the microalgae [52]. The most important feature of this method is that it can be directly used for high moisture containing samples, thus reducing the water footprint and downstream process costs [86]. However, extensive heat generation and high energy consumption during the operation are the major drawbacks when it comes to scale-up processes [14].

Ultrasonication
The ultrasonication method was found to be the most applicable and efficient method for lipid extraction from oleaginous microbial biomass. Cavitation and acoustic streaming are two different phenomena that are created during the application of ultrasound to the cells. Cavitation creates pressure on the cells in the form of microbubbles, leading to the disruption of cell walls and membranes. Ultrasonication for the disruption of microalgal cells has been tested with various types of solvents, such as chloroform-methanol [87][88][89], n-hexane [90,91], diethyl ether [92], and other solvents [87].
Three freshwater-isolated microalgal species Chlorella sp., Nostoc sp., and Tolypothrix sp. were disrupted by different methods, such as bead beating, autoclave, microwave, sonication, and 10% sodium chloride solution treatment [93]. Among the tested methods, sonication was found to be the most effective method to disrupt the microalgal cells and the highest lipid content was obtained by disruption of Chlorella sp. [93]. The three microalgae species Phaeodactylum tricornutum, Nannochloropsis gaditana, and Chaetoceros calcitrans were tested for lipid extraction with the conventional Bligh & Dyer extraction method (1959) with prior ultrasonication treatment [94]. Trichosporon oleaginosus and an oleaginous fungal strain were treated with ultrasonication (520 kHz, 40 W, and 50 Hz, 2800 W) using various solvents, including water, hexane, methanol, and chloroform-methanol (1:1, v/v), followed by lipid extraction and comparison of the process efficiency to the conventional chloroform-methanol (2:1, v/v) extraction method [95]. The results suggested that almost all of the lipids (100%) were extracted from T. oleaginosus and the SKF-5 strain after a very short incubation (15 min) at a relatively low temperature (25 • C) with chloroform-methanol, followed by pretreatment with ultrasonication at 50 Hz and 2800 W [95]. Mecozzi et al. (2002) performed an experiment for lipid extraction from marine mucilage samples using an ultrasonic cleaning bath at 35 kHz [92]. They used two different solvent systems and suggested that diethyl ether was more suitable than methanol to assist the ultrasonication for lipid extraction. Moreover, the disruption due to the acoustic cavitation phenomena minimized the oxidative damage on the lipids [92]. Wu et al. (2012) investigated the ultrasonication treatment at low frequency (20 kHz) with high intensity (0.0403 W/cm 3 ) and found it to be effective for the disruption of Microcystis aeruginosa [96]. They suggested that the acoustic cavitation phenomenon at a low ultrasonic frequency is mainly responsible for the damage of cells due to sufficient shear forces being directly applied to cells. But, while the mechanical energy of cavitation is lower at high ultrasonic frequencies, the ultrasonic degradation of water generates free radicals that weaken the cell wall of cyanobacteria [96]. In another study, the lipids were extracted from microalgae Scenedesmus obliquus by using sun-, freeze-, and oven-dried biomass. The cells were disrupted by microwave-, sonication-, autoclaving-, and osmotic shock treatment [53]. The results suggested that the lipid yield of dried samples that were subjected to microwave treatment (20.73 ± 4.16%) was higher than for autoclaving and osmotic shock, while the results were comparable with sonication (19.49 ± 3.30%) [53]. Wang et al. (2014) treated two microalgae Scenedesmus dimorphus and Nannochloropsis oculate with a high frequency focused ultrasound (3.2 MHz, 40 W) and a low frequency non-focused ultrasound (20 kHz, 100 W). The results revealed that high frequency focused ultrasound was a more energy efficient process for maximal cell disintegration [97].

Microwave Irradiation
Microwave irradiation is another extensively used method for lipid extraction from oleaginous microorganisms, where electromagnetic waves are applied to the suspension of cells in an organic solvent. During the microwave treatment of polar compounds, the applied alternative current is converted into electromagnetic energy, and finally, in heating energy [98], as the polar compounds align themselves in the direction of the applied electric field and rotate at high speed when the microwave field alters. The process is accelerated when ions are present in the working system [75]. High heat is generated during the frictional movement of polar compounds or ions, not involving conventional radiant heat [99]. Microwave heating consumes almost two to three times less energy than that involves in the conventional heating [100]. Guerra et al. (2014) treated the oleaginous microalgae Chlorella sp. with microwaves and enhanced lipid yield was recorded as compared to the conventional Bligh & Dyer method, therefore it further boosts chemical and energy savings. Moreover, lipid extraction using a single-step microwave-assisted extraction was more convenient and effective than the multistep, time consuming traditional Bligh & Dyer method [101]. Furthermore, Lee et al. (2010) stated that the lipid extraction yield was higher for the microwave method when compared to autoclaving, bead beating, ultrasonication, and 10% NaCl solution extraction methods [89]. Microwaves were reported to be a useful tool for the extraction of plant oils and animal fats, and, in addition, their implementation has the advantage of easy scale-up [24]. Teo et al. (2014) performed trials on the extraction of lipids from the marine microalgae Nannochloropsis sp. and Tetraselmis sp. using four different solvent extraction methods (Hara & Radin, Folch, Chen, and Bligh & Dyer) along with conventional heating and microwave irradiation. The highest lipid yield was obtained when they used the Hara & Radin (8.19%) and the Folch (8.47%) method following the microwave irradiation [102]. Boldor et al. (2010) used microwave treatment for the extraction of oils from Chinese tallow tree in batch and continuous flow mode [24]. They suggested that the application of microwave-assisted solvent extraction to extract the lipids from seeds has many advantages over conventional methods, including short operating time and reduced energy consumption [24]. The cells of three freshwater-isolated microalgal species, Botryococcus sp., Chlorella vulgaris and Scenedesmus sp., were disrupted by different methods, such as bead beating, autoclave, microwave, sonication, and 10% NaCl treatment [89]. Among all of the tested methods, the microwave oven was found to be the simplest, easiest, and most effective method to disrupt the microalgal cells. The highest lipid content was obtained by the disruption of Botryococcus sp. [89]. Although microwave treatment is a suitable technique to extract lipids in a short amount of time, it has some drawbacks. Its use is limited to polar solvents and the method is unsuitable for volatile compounds. Moreover, the formation of free radicals and the increased temperature make microwave treatment less favorable [103].

Autoclaving
Autoclaving is usually utilized for the sterilization of laboratory equipment and media prior to the growth of microorganisms. Various microalgal species, such as Haematococcus pluvialis [104], Botryococcus sp., C. vulgaris, and Scenedesmus sp. [89] were disrupted by autoclaving at 121 • C and 1.5 MPa for 5 or 30 min [89,104]. Rakesh et al. (2015) treated four oleaginous microalgae, Chlorococcum sp. MCC30, Botryococcus sp. MCC31, Botryococcus sp. MCC32, and Chlorella sorokiniana MICG5, with various methods, such as autoclaving, microwave irradiation, osmotic shock treatment, and pasteurization, and reported that the highest amount of nutraceutically important unsaturated fatty acids was obtained when Botryococcus sp. was treated with autoclaving [105]. Similarly, Florentino de Souza Silva et al. (2014) suggested that autoclaving is a more efficient technique than ultrasonication but not as efficient as microwaving and electroflotation by alternating current (EFAC), when mixed cultures of microalgae were treated with different methods [106].

Pulsed Electric Field
Pulsed electric field (PEF) treatment works based on electroporation phenomena, including electromechanical compression and electric field-induced tension, where an external electric field is used to induce the critical electrical potential across the cell membrane [107,108]. The increase in membrane porosity is directly proportional to the strength of the applied electric field and pulses and the pore formation in the membrane can be reversible or irreversible, depending on the size and number of pores in comparison to the total surface area of the membrane or cell wall [107]. Eing et al. (2013) treated the oleaginous microalgae Auxenochlorella protothecoides with a PEF at 35 kV/cm and the pulse duration was set to 1 µs. They suggested that the lipid yield after PEF treatment and extraction with ethanol was four times higher than it was for untreated cells [109]. Similarly, the oleaginous microalga Synechocystis PCC 6803 was treated with a pulsed electric field (intensity > 35 kWh/m 3 ) and isopropanol as solvent [110].
In another study, oleaginous microalgae Ankistrodesmus falcatus wet biomass was treated with PEF using the green solvent ethyl acetate and the results demonstrated that the lipid yield was 83-88% higher when compared to the untreated cells [111].

Laser
Laser treatment is a well-known technique to disintegrate the cellular membrane without damaging the compartments of the cell factory or other interior compounds. Most importantly, laser treatment is free of the use of any organic solvent, fast, and requires no laborious effort [112,113]. Previously, researchers have studied the efficiency and mechanism of this method of cell lysis in static mode for different microorganisms, like Escherichia coli, Saccharomyces cerevisiae, and microalgae at various wavelengths and energy inputs. In a study, oleaginous microalgae N. oculata cells were disrupted by using various pretreatment methods, such as microwave, water bath, blender, ultrasonic, and laser treatment, and it was revealed that the highest disruption efficiency was achieved with laser treatment (96.53%), followed by microwave treatment (94.92%) [114]. However, the number and scope of these studies are limited and further investigations, especially in the continuous system, are required in order to examine the potential applications of this cell disruption method.

Acid-Catalyzed Hot-Water
Hot water treatment is a well-known technique to disintegrate the crystalline nature of cellulosic biomass [115]. Hot water requires high pressure at an elevated temperature in order to remain in liquid form. It is to be noted that this pretreatment under acidic conditions is applicable for the extraction of lipids from biomass in wet condition ensuring the cost-effectiveness since no extra energy input is required for dewatering processes. Lipids with high free fatty acid content were extracted from C. vulgaris by using acid-catalyzed hot-water treatment and the anionic surfactant sodium dodecyl benzene sulfonate (SDBS) [116]. The lipid extraction yield was 266.0 mg/g of cell weight from a total fatty acid content of 296.0 mg/g of cell weight, when the concentration of sulfuric acid and SDBS were 2.0% and 0.2%, respectively [116]. C. vulgaris cells were disrupted for efficient lipid extraction using acid-catalyzed hot-water treatment [117]. The lipid extraction yield was 337.4 mg/g of cell weight from a total fatty acid content of 381.6 mg/g of cell weight, given a 1% sulphuric acid concentration and heating at 120 • C for 60 min, when compared to 83.2 mg/g of cell weight lipid yield with no heating and no catalyst [117]. This method is also suitable to extract lipids rich in docosahexaenoic acid (DHA) from Aurantiochytrium sp. [118]. During acid-catalyzed hot-water treatment, cells are disrupted along with the degradation of other cellular components, leading to excess acid loading and devaluation of co-products.

Non-Mechanical Pretreatment Methods
Conventional mechanical techniques have several drawbacks, including insufficient extraction yields, the use of toxic solvents, and long processing time. Hence, there is a need for rapid, less energy intensive methods for the lipid extraction from wet biomass. Non-mechanical disruption methods, such as enzymatic and chemical cell lysis, are mainly used in lab-scale processes for bioanalytical purposes. The energy consumption of mechanical methods is always higher when compared to non-mechanical methods. Lee et al. (2012) compared the energy consumed by lipid extraction from microalgae with the energy levels that are required for other methods [119]. The advantages and disadvantages along with process parameters of various cell disruption methods are summarized and compared in Table 2.

Enzymatic Pretreatment
The extraction of lipids using enzymatic pretreatment completely depends on the cell wall characteristics of the subjected oleaginous microorganism [130]. This technique includes various cell wall degrading enzymes, such as xylanase, cellulase, amylase, papain, pectinase, and hemicellulase [131]. Enzymatic pretreatment is a well-known technique in the vegetable oil industry to degrade the structural polysaccharides of the cell wall of oily seeds [134,135]. It constitutes a favourable cell disintegration method due to its specificity and mild operating temperature, as well as its low time and energy requirements. Furthermore, the method is devoid of harmful solvents and harsh physical conditions, such as shear forces [132]. It has been reported that enzymatic pretreatment is suitable for extracting lipids from the oleaginous yeast. For example, the oleaginous yeast Rhodosporidium toruloides was treated with the recombinant β-1,3-glucomannanase plMAN5C, and almost 96.6% of the total lipid content was extracted directly from the culture with ethyl acetate at room temperature and atmospheric pressure without dewatering [25]. Moreover, this method has also been applied to extract lipids from oleaginous microalgae. The microalga C. vulgaris was treated with cellulases for 72 h and the hydrolysis efficiency of the cell wall carbohydrates was 85.3%. After enzymatic hydrolysis, the lipid extraction efficiency by solvent extraction was higher than without hydrolysis [136]. Bonturi et al. (2015) extracted lipids from intact and pretreated cells of oleaginous yeasts R. toruloides and L. starkeyi using various methods such as Folch, Pedersen, hexane, and Bligh & Dyer methods involving acid and enzyme pretreatment. They suggested that enzymatic pretreatment is not an efficient technique for L. starkeyi due to the sulfide bonds in its cell wall, which increase the strength and rigidity of its organelles [47].
In another study, the oleaginous microalga Scenedesmus sp. was treated with various enzymes, such as cellulase, xylanase, and pectinase under varying conditions, including enzyme concentration, temperature, pH, and incubation time [50]. The results demonstrated that the combination of cellulase, xylanase, and pectinase for 190 min improved the lipid extraction yields by 96.4% when compared to the untreated microalga [50]. Another oleaginous marine microalga, Nannochloropsis sp., was treated with cellulase and mannanase and the results revealed the improvement of lipid extraction yields from 40.8% to over 73% [137]. Treatment of the same microalga with similar enzymes under different conditions significantly improved the recovery of lipids from Nannochloropsis sp. biomass [133]. The enzymatic hydrolysis of Chloroccum sp. by using cellulase obtained from Trichoderma reesei, ATCC 26921, was an effective method to enhance the saccharification process of microalgal biomass for bioethanol production [138]. Hence, enzymatic treatment can improve the lipid extraction from various oleaginous microorganisms and scaling up the process is relatively easy. However, long processing times and high capital costs hinder the scale-up of enzymatic pretreatment for lipid extraction in the biorefineries [138].

Other Emerging Methods for the Extraction of Lipids from Oleaginous Microorganisms
Researchers have used a limited number of other pretreatment methods, including chemical treatments to disrupt the microbial cells. Supercritical fluid extraction is an extensively used method to extract lipids from oleaginous microbial biomass [9,127,130,[139][140][141][142]. Bai et al. (2014) used free nitrous acid (FNA) as an effective and low cost pretreatment method to extract lipids from microalgae [143]. Boyd et al. (2012) used switchable hydrophilicity solvents, such as N,N-dimethylcyclohexylamine for the lipid extraction from freeze-dried samples of Botryococcus braunii microalgae [144]. Two microalgae species, N. oculata and Dunaliella salina, were treated with a novel simultaneous distillation and extraction process (SDEP) for lipid extraction under wet conditions by using d-limonene, a-pinene and p-cymene as solvents [145]. Du et al. (2013) tried to use switchable solvents such as secondary amines for the extraction of lipids from wet and non-broken algae [146]. Kim SO 3 ] and methanol for the extraction of lipids from C. vulgaris [147].  treated Chlorella sp. biomass with organic nanoclays, such as Mg-APTES clay, Al-APTES clay, Ca-APTES clay, and Mg-N3 clay [148]. In other reports, researchers used H 2 O 2 with or without FeSO 4 to disrupt the cell walls of C. vulgaris [149]. Jo et al. (2014) reported a quick method for dimethyl carbonate-mediated lipid extraction from Chlorella sp. [150]. Hydrothermal liquefaction (HTL) is the most emerging technique to convert high moisture algal biomass to crude biooil, producing more lipids per mass of microalgae than the other extraction method [151]. This technique is more advantageous than the traditional thermochemical conversion processes, such as pyrolysis, where high energy is required for drying the biomass [152]. In this process, biomolecules are decomposed in hot compressed water via the combined action of elevated temperature, elevated pressure, and hydrolytic attack. Reaction temperature and catalysts are two important factors in hydrothermal liquefaction that decide the fractionation of water-soluble and water-insoluble biocrude from algae [153]. Sheehan and Savage (2017) developed a kinetic model for hydrothermal liquefaction to predict the biocrude yield from protein, lipid, and carbohydrate rich microalgae, and suggested that feedstocks containing more proteins or lipids give higher biocrude yields than those that are abundant in carbohydrates [154]. For example, Hietala et al. (2016) performed isothermal and non-isothermal hydrothermal liquefaction with microalgae Nannochloropsis sp. and predicted that up to 46% w/w biocrude yields are achievable with short holding time of 1 min at 400 • C [155].

Conclusions and Recommendations
This review article summarizes the various techniques that are available for total lipid extraction from the biomass of various oleaginous microorganisms using different mechanical and non-mechanical pretreatment methods. Since all mechanical pretreatment methods are followed by the solvent extraction of lipids, overall costs of downstream processing can be increased. Some researcher use green solvents instead of potentially harmful chloroform and methanol to make the extraction process more feasible. Supercritical fluid extraction techniques appear to be a good option to avoid toxic solvents, but the initial capital cost for equipment is high. Another alternative, involving the use of solvent-free extractions, is offering an environment-friendly and cost-effective option on a laboratory scale. However, more research is required in order to scale up the process. Drying of biomass prior to pretreatment is another energy-intensive step, so the target should be to extract lipids from wet cellular biomass to avoid extra cost for drying. Several pretreatment methods allow for the extraction process in wet conditions. For example, hydrothermal liquefaction is an emerging method in which wet microalgae are converted into crude biooil and this technique is also suitable for scale-up of process. However, using only one single pretreatment method may not be sufficient to reach a maximal lipid extraction yield from lysed biomass, hence it may be advantageous to apply multiple different pretreatment methods on both lab-and large-scale. For example, if ultrasonication and microwave irradiation are used in combination for the cell disruption under wet conditions, four different physical phenomena would work together to easily break the cells and release the lipids to the external environment. During ultrasonication, the transmission of sonic waves causes cavitation where microbubbles form during the rarefaction phase of the sound wave and collapse during the compression phase. Disintegrated microbubbles release shock waves in the form of mechanical energy, which causes irreparable shearing in the cell wall of oleaginous microorganisms. After the ultrasonication step, microwave treatment causes the alignment of polar compounds in the cellular compartments in the direction of the applied electric field followed high-speed rotation when the microwave field alters. With this method combination, both the duration of the process as well as the solvent consumption could be decreased.
Author Contributions: All the authors participated in the collection of data from the literature, analysis of data and drafting the manuscript. The final publication was prepared with contributions from all authors.

Funding:
The authors will like to thank Bio4Energy, a strategic research environment appointed by the Swedish government, for supporting this work.

Conflicts of Interest:
The authors declare no conflict of interest.