Comparison of Cell Disruption Methods for Improving Lipid Extraction from Thraustochytrid Strains

Lipid extraction is an integral part of biodiesel production, as it facilitates the release of fatty acids from algal cells. To utilise thraustochytrids as a potential source for lipid production. We evaluated the extraction efficiency of various solvents and solvent combinations for lipid extraction from Schizochytrium sp. S31 and Thraustochytrium sp. AMCQS5-5. The maximum lipid extraction yield was 22% using a chloroform:methanol ratio of 2:1. We compared various cell disruption methods to improve lipid extraction yields, including grinding with liquid nitrogen, bead vortexing, osmotic shock, water bath, sonication and shake mill. The highest lipid extraction yields were obtained using osmotic shock and 48.7% from Schizochytrium sp. S31 and 29.1% from Thraustochytrium sp. AMCQS5-5. Saturated and monounsaturated fatty acid contents were more than 60% in Schizochytrium sp. S31 which suggests their suitability for biodiesel production.     

Biobased Solvents for Pressurized Liquid Extraction of Nannochloropsis gaditana Omega-3 Lipids

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.     

Profiling of Polar Lipids in Marine Oleaginous Diatom Fistulifera solaris JPCC DA0580: Prediction of the Potential Mechanism for Eicosapentaenoic Acid-Incorporation into Triacylglycerol

The marine oleaginous diatom Fistulifera solaris JPCC DA0580 is a candidate for biodiesel production because of its high lipid productivity. However, the substantial eicosapentaenoic acid (EPA) content in this strain would affect the biodiesel quality. On the other hand, EPA is also known as the essential health supplement for humans. EPAs are mainly incorporated into glycerolipids in the microalgal cell instead of the presence as free fatty acids. Therefore, the understanding of the EPA biosynthesis including the incorporation of the EPA into glycerolipids especially triacylglycerol (TAG) is fundamental for regulating EPA content for different purposes. In this study, in order to identify the biosynthesis pathway for the EPA-containing TAG species, a lipidomic characterization of the EPA-enriched polar lipids was performed by using direct infusion electrospray ionization (ESI)-Q-TRAP-MS and MS/MS analyses. The determination of the fatty acid positional distribution showed that the sn-2 position of all the chloroplast lipids and part of phosphatidylcholine (PC) species was occupied by C16 fatty acids. This result suggested the critical role of the chloroplast on the lipid synthesis in F. solaris. Furthermore, the exclusive presence of C18 fatty acids in PC highly indicated the biosynthesis of EPA on PC. Finally, the PC-based acyl-editing and head group exchange processes were proposed to be essential for the incorporation of EPA into TAG and chloroplast lipids.     

LC-PUFA-Enriched Oil Production by Microalgae: Accumulation of Lipid and Triacylglycerols Containing n-3 LC-PUFA Is Triggered by Nitrogen Limitation and Inorganic Carbon Availability in the Marine Haptophyte Pavlova lutheri

In most microalgal species, triacyglycerols (TAG) contain mostly saturated and monounsaturated fatty acids, rather than PUFA, while PUFA-enriched oil is the form most desirable for dietary intake. The ability of some species to produce LC-PUFA-enriched oil is currently of specific interest. In this work, we investigated the role of sodium bicarbonate availability on lipid accumulation and n-3 LC-PUFA partitioning into TAG during batch cultivation of Pavlova lutheri. Maximum growth and nitrate uptake exhibit an optimum concentration and threshold tolerance to bicarbonate addition (~9 mM) above which both parameters decreased. Nonetheless, the transient highest cellular lipid and TAG contents were obtained at 18 mM bicarbonate, immediately after combined alkaline pH stress and nitrate depletion (day nine), while oil body and TAG accumulation were highly repressed with low carbon supply (2 mM). Despite decreases in the proportions of EPA and DHA, maximum volumetric and cellular EPA and DHA contents were obtained at this stage due to accumulation of TAG containing EPA/DHA. TAG accounted for 74% of the total fatty acid per cell, containing 55% and 67% of the overall cellular EPA and DHA contents, respectively. These results clearly demonstrate that inorganic carbon availability and elevated pH represent two limiting factors for lipid and TAG accumulation, as well as n-3 LC-PUFA partitioning into TAG, under nutrient-depleted P. lutheri cultures.     

Lipids and Composition of Fatty Acids of Saccharina latissima Cultivated Year-Round in Integrated Multi-Trophic Aquaculture

This study is evaluating the seasonal lipid and fatty acid composition of the brown seaweed Saccharina latissima. Biomass was sampled throughout the year (bi-monthly) at the commercial cultivation site near a fish farm in an integrated multi-trophic aquaculture (IMTA) and at a reference site in Denmark (2013–2014). Generally, there was no difference in the biomass composition between sites; however, significant seasonal changes were found. The lipid concentration varied from 0.62%–0.88% dry weight (DW) in July to 3.33%–3.35% DW in November (p < 0.05) in both sites. The fatty acid composition in January was significantly different from all the other sampling months. The dissimilarities were mainly explained by changes in the relative abundance of 20:5n-3 (13.12%–33.35%), 14:0 (11.07%–29.37%) and 18:1n-9 (10.15%–16.94%). Polyunsaturated fatty acids (PUFA’s) made up more than half of the fatty acids with a maximum in July (52.3%–54.0% fatty acid methyl esters; FAME). This including the most appreciated health beneficial PUFA’s, eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), but also arachidonic (ARA) and stearidonic acid (SDA), which are not found in land vegetables such as cabbage and lettuce. Compared to fat (salmon) and lean fish (cod) this seaweed species contains higher proportions of ARA and SDA, but lower EPA (only cod) and DHA. Conclusively, the season of harvest is important for the choice of lipid quantity and quality, but the marine vegetables provide better sources of EPA, DHA and long-chain (LC)-PUFA’s in general compared to traditional vegetables.     

Rice Bran Oil: Emerging Trends in Extraction,Health Benefit,and Its Industrial Application

Rice bran oil(RBO) is unique among edible vegetable oils because of its unique fatty acid composition, phenolic compound(γ-oryzanol, ferulic acid) and vitamin E(tocopherol and tocotrienol). It has become a great choice of cooking oil because of its very high burning point, neutral taste and delicate flavour. Non-conventional methods of RBO extraction are more efficient and environmentally friendly than conventional extraction methods. Advances in RBO extraction using innovative extraction strategies like super/sub-critical CO_2, microwave-assisted, subcritical H_2 O, enzyme-assisted aqueous and ultrasoundassisted aqueous extraction methods have proven to significantly improve the yields along with improved nutritional profile of RBO. The compositions and strategies for stabilization of RBO are well discussed. The constituents are present in the RBO contribute to antioxidative, anti-inflammatory, antimicrobial, antidiabetic and anti-cancerous properties to RBO. This has helped RBO to become an important substrate for the application in food(cooking oil, milk product and meat product) and non-food industries(polymer, lubricant, biofuel, structural lipid and cosmetic). This review provided comprehensive information on RBO extraction methods, oil stabilization, existing applications and health benefits.     

Ethanolic fermentation of phosphoric acid hydrolysates from olive tree pruning

The objective of this work was to study the feasibility of using phosphoric acid to hydrolyze the hemicellulosic fraction of olive tree pruning, as a step in the bioconversion process to produce ethanol. Milled olive tree pruning was submitted to hydrolysis at 90 °C by phosphoric acid in a concentration range 0.3–8N for 240 min. The hydrolysates were then fermented by Pachysolen tannophilus. The hydrolysis stage was evaluated by the evolution of glucose and reducing sugars generated and by the conversion of hemicellulose fraction. The main parameters determined in the fermentation were: maximum specific growth rate, specific substrate consumption rate, specific ethanol production rate and ethanol yield. The maximum ethanol yield (0.38 kg/kg, equivalent to 74.5% of the theoretical yield) was obtained when hydrolysing with 0.5N phosphoric acid. Hemicellulose conversion is however incomplete at these operational conditions. Higher acid concentrations lead to higher hydrolysis of hemicellulose, but the ethanol yields resulting from the fermentation are lower.     

Evaluation of Ultrasound,Microwave, Ultrasound–Microwave,Hydrothermal and High Pressure Assisted Extraction Technologies for the Recovery of Phytochemicals and Antioxidants from Brown Macroalgae

This study aims to explore novel extraction technologies (ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), ultrasound–microwave-assisted extraction (UMAE), hydrothermal-assisted extraction (HAE) and high-pressure-assisted extraction (HPAE)) and extraction time post-treatment (0 and 24 h) for the recovery of phytochemicals and associated antioxidant properties from Fucus vesiculosus and Pelvetia canaliculata. When using fixed extraction conditions (solvent: 50% ethanol; extraction time: 10 min; algae/solvent ratio: 1/10) for all the novel technologies, UAE generated extracts with the highest phytochemical contents from both macroalgae. The highest yields of compounds extracted from F. vesiculosus using UAE were: total phenolic content (445.0 ± 4.6 mg gallic acid equivalents/g), total phlorotannin content (362.9 ± 3.7 mg phloroglucinol equivalents/g), total flavonoid content (286.3 ± 7.8 mg quercetin equivalents/g) and total tannin content (189.1 ± 4.4 mg catechin equivalents/g). In the case of the antioxidant activities, the highest DPPH activities were achieved by UAE and UMAE from both macroalgae, while no clear pattern was recorded in the case of FRAP activities. The highest DPPH scavenging activities (112.5 ± 0.7 mg trolox equivalents/g) and FRAP activities (284.8 ± 2.2 mg trolox equivalents/g) were achieved from F. vesiculosus. Following the extraction treatment, an additional storage post-extraction (24 h) did not improve the yields of phytochemicals or antioxidant properties of the extracts.     

Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.     

Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility and fermentability of bran protein and dietary fibre complex

Bran is a good source of dietary fibre, phytochemicals, and also protein, but highly insoluble and recalcitrant structure of bran hinders accessibility of these components for gastrointestinal digestion. In the present work, influence of bioprocessing on the microstructure and chemical properties of rye bran and wheat bread fortified with the rye bran were studied. In vitro protein digestibility, and release of short chain fatty acids (SCFA) and ferulic acid in a gut model were studied. Bioprocessing of rye bran was performed with subsequent treatments with cell-wall hydrolysing enzymes (40 °C, 4 h) and yeast fermentation (20 °C, 20 h). Bioprocessing of rye bran resulted in reduced total dietary fibre content, caused mainly by degradation of fructan and β-glucan, and increased soluble fibre content, caused mainly by solubilisation of arabinoxylans. Microscopic analysis revealed degradation of aleurone cell wall structure of the bioprocessed rye bran. Bioprocessing caused release of protein from aleurone cells, assessed as a larger content of soluble protein in bran and a higher hydrolysis rate in vitro. Bioprocessed bran had also faster SCFA formation and ferulic acid release in the colon fermentation in vitro as compared to native bran.     

Unusual Ether Lipids and Branched Chain Fatty Acids in Sea Cucumber (Cucumaria frondosa) Viscera and Their Seasonal Variation

The sea cucumber, Cucumaria frondosa, is harvested primarily for its muscular bands and body wall. Development of a nutraceutical product based on lipid recovered from its viscera would give commercial value to the entire organism; however, such development requires knowledge of the lipid and fatty acid (FA) profiles of the viscera. Here, we describe the lipid and FA composition of viscera recovered from C. frondosa harvested in coastal waters in the northwest Atlantic, taking into account variation due to harvest season. We found highest lipid content at ~29% in winter, with diacylglyceryl ethers (DAGE) comprising ~55% of the total lipid mass and triacylglycerols (TAG), phospholipids (PL) and monoacylglycerol ethers (MAGE) at 5–25% each. The branched chain FA, 12-methyltetradecanoic acid (12-MTA), represented 42% of total FA mass in DAGE. In summer, lipid content was lower at 24% and TAG was the dominate lipid, with proportions more than double that found in winter (45% vs. 20%); DAGE in summer dropped to ~30% of total lipids. In TAG, 12-MTA was much lower than found in DAGE in winter, at only 10% but eicosapentaenoic acid (EPA) content was ~20%, which brought the total EPA% to 28% of total FA—the highest among all three seasons. There was little effect of season on MAGE or PL proportions. These data can help harvesters maximize catch efforts in terms of lipid yield and profile.     

Exploring the Potential of Nannochloropsis sp. Extract for Cosmeceutical Applications

Recently, there has been emerging interest in various natural products with skin protective effects as they are recognized as safe and efficient. Microalgae have developed chemical defense systems to protect themselves against oxidative stress caused by UV radiation by producing various bioactive compounds including a number of secondary metabolites, which have potential for cosmeceutical applications. In addition, microalgae have various advantages as a sustainable source for bioactive compounds with diverse functions due to their rapid growth rate, high productivity, and use of non-arable land. In this study, we aimed to investigate the cosmeceutical potential of ethanol extract from Nannochloropsis sp. G1-5 (NG15) isolated from the southern West Sea of the Republic of Korea. It contained PUFAs (including EPA), carotenoids (astaxanthin, canthaxanthin, β-carotene, zeaxanthin, violaxanthin), and phenolic compounds, which are known to have various skin protective functions. We confirmed that the NG15 extract showed various skin protective functions with low cytotoxicity, specifically anti-melanogenic, antioxidant, skin-moisturizing, anti-inflammatory, anti-wrinkling, and UV protective function, by measuring tyrosinase inhibition activity; melanin content; DPPH radical scavenging activity; expression of HAS-2, MMP-1, and Col1A1 genes; and elastase inhibition activity as well as cell viability after UV exposure. Our results indicated that the NG15 extract has the potential to be used for the development of natural cosmetics with a broad range of skin protective functions.     

High-EPA Biomass from Nannochloropsis salina Cultivated in a Flat-Panel Photo-Bioreactor on a Process Water-Enriched Growth Medium

Nannochloropsis salina was grown on a mixture of standard growth media and pre-gasified industrial process water representing effluent from a local biogas plant. The study aimed to investigate the effects of enriched growth media and cultivation time on nutritional composition of Nannochloropsis salina biomass, with a focus on eicosapentaenoic acid (EPA). Variations in fatty acid composition, lipids, protein, amino acids, tocopherols and pigments were studied and results compared to algae cultivated on F/2 media as reference. Mixed growth media and process water enhanced the nutritional quality of Nannochloropsis salina in laboratory scale when compared to algae cultivated in standard F/2 medium. Data from laboratory scale translated to the large scale using a 4000 L flat panel photo-bioreactor system. The algae growth rate in winter conditions in Denmark was slow, but results revealed that large-scale cultivation of Nannochloropsis salina at these conditions could improve the nutritional properties such as EPA, tocopherol, protein and carotenoids compared to laboratory-scale cultivated microalgae. EPA reached 44.2% ± 2.30% of total fatty acids, and α-tocopherol reached 431 ± 28 µg/g of biomass dry weight after 21 days of cultivation. Variations in chemical compositions of Nannochloropsis salina were studied during the course of cultivation. Nannochloropsis salina can be presented as a good candidate for winter time cultivation in Denmark. The resulting biomass is a rich source of EPA and also a good source of protein (amino acids), tocopherols and carotenoids for potential use in aquaculture feed industry.     

Changes in some biochemical components of wheat grain that was infected with Fusarium graminearum

Wheat kernels that were lightly and moderately infected by Fusarium graminearum were analyzed in terms of their carbohydrate, lipid, and protein contents to determine any compositional changes. The significant compositional changes in lightly infected wheat were increases in reducing sugars (24%), non-starch lipids (5%), and decreases in cellulose (17%) and hemicellulose (20%) components. In moderately infected wheat, the increases in protein (6%), total sugars (26%), reducing sugars (14%), non-starch (20%) and starch (8%) lipids, and decreases in apparent and total amylose (11–20%), cellulose (43%) and hemicellulose (37%) components were also significant. Furthermore, infection with F. graminearum decreased the proportions of water-extractable protein (albumin) and storage protein (glutenin) by 33 and 80%, respectively, in moderately infected wheat.     

Anti-Obesity and Gut Microbiota Regulation Effects of Phospholipids from the Eggs of Crab,Portunus Trituberculatus,in High Fat Diet-Fed Mice

There are resourceful phospholipids in the eggs of the crab, Portunus trituberculatus (Pt-PL). However, their components and bioactivities regarding obesity were unclear. Here, we investigated the composition of Pt-PL and their fatty acids. Moreover, its effects on obesity and gut microbiota were also evaluated in high fat diet (HFD)-fed mice. The results showed that Pt-PL contained 12 kinds of phospholipids, mainly including phosphatidylcholine (PC, 32.28%), phosphatidylserine (PS, 26.51%), phosphatidic acid (PA, 19.61%), phosphatidylethanolamine (PE, 8.81%), and phosphatidylinositol (PI, 7.96%). Polyunsaturated fatty acids (PUFAs) predominated in the fatty acids components of Pt-PL, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Animal experiments demonstrated that Pt-PL significantly alleviated body weight gain, adipose gain, hepatic gain, fasting blood glucose, serum insulin, lipid levels in serum and the liver, and systematic inflammation in HFD-fed mice. Furthermore, Pt-PL regulated gut microbiota, especially in a dramatic reduction in the ratio of Firmicutes to Bacteroidetes at phylum level, as well as significant amelioration in their subordinate categories. Pt-PL reduced fecal lipopolysaccharide and total bile acids, and elevated fecal short chain fatty acid (SCFA) concentrations, particularly acetate and butyrate. These findings suggest that Pt-PL possesses anti-obesity effects and can alter gut microbiota owing to the abundance of PUFAs. Therefore, Pt-PL may be developed as an effective food supplement for anti-obesity and regulation of human gut health.     

Lipid Indexes and Quality Evaluation of Omega-3 Rich Oil from the Waste of Japanese Spanish Mackerel Extracted by Supercritical CO2

Japanese Spanish mackerel (JSM) (Scomberomorus niphonius) is a marine fish species containing health-beneficial polyunsaturated fatty acids (PUFAs). In the present study, the quality of JSM by-products oils extracted by supercritical CO₂ (SC-CO₂) and organic solvent extraction was compared in terms of physico-chemical properties of the oils. Eicosapentaenoic acid (EPA) is one of the important polyunsaturated fatty acids present in SC-CO₂-extracted skin and muscle oil 5.81 ± 0.69% and 4.93 ± 0.06%, respectively. The amount of docosahexaenoic acid (DHA) in SC-CO₂-extracted skin and muscle oil was 12.56 ± 0.38% and 15.01 ± 0.28%, respectively. EPA and DHA are considered as important PUFAs for the development of brain function and the prevention of coronary heart diseases. Extracted oils showed considerable antioxidant activity. In the obtained oils, atherogenic index (AI) values varied from 0.72 to 0.93 and thrombogenic index (TI) ranged from 0.75 to 0.92, which is considered an acceptable level. Fatty acid composition, bio potentiality, thermogravimetric, and vitamin D analysis showed that oils extracted from JSM by-products can be a good source of oil for application in food, pharmaceutical and cosmetic industries. Therefore, the present research revealed the potentiality of green valorisation of S. niphonius by-products as a possible sustainable approach for targeting the era of zero waste.     

Polar Lipids Composition,Antioxidant and Anti-Inflammatory Activities of the Atlantic Red Seaweed Grateloupia turuturu

Grateloupia turuturu Yamada, 1941, is a red seaweed widely used for food in Japan and Korea which was recorded on the Atlantic Coast of Europe about twenty years ago. This seaweed presents eicosapentaenoic acid (EPA) and other polyunsaturated fatty acids (PUFAs) in its lipid fraction, a feature that sparked the interest on its potential applications. In seaweeds, PUFAs are mostly esterified to polar lipids, emerging as healthy phytochemicals. However, to date, these biomolecules are still unknown for G. turuturu. The present work aimed to identify the polar lipid profile of G. turuturu, using modern lipidomics approaches based on high performance liquid chromatography coupled to high resolution mass spectrometry (LC–MS) and gas chromatography coupled to mass spectrometry (GC–MS). The health benefits of polar lipids were identified by health lipid indices and the assessment of antioxidant and anti-inflammatory activities. The polar lipids profile identified from G. turuturu included 205 lipid species distributed over glycolipids, phospholipids, betaine lipids and phosphosphingolipids, which featured a high number of lipid species with EPA and PUFAs. The nutritional value of G. turuturu has been shown by its protein content, fatty acyl composition and health lipid indices, thus confirming G. turuturu as an alternative source of protein and lipids. Some of the lipid species assigned were associated to biological activity, as polar lipid extracts showed antioxidant activity evidenced by free radical scavenging potential for the 2,2′-azino-bis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS^●+) radical (IC₅₀ ca. 130.4 μg mL⁻¹) and for the 2,2-diphenyl-1-picrylhydrazyl (DPPH^●) radical (IC₂₅ ca. 129.1 μg mL⁻¹) and anti-inflammatory activity by inhibition of the COX-2 enzyme (IC₅₀ ca. 33 µg mL⁻¹). Both antioxidant and anti-inflammatory activities were detected using a low concentration of extracts. This integrative approach contributes to increase the knowledge of G. turuturu as a species capable of providing nutrients and bioactive molecules with potential applications in the nutraceutical, pharmaceutical and cosmeceutical industries.     

A Novel Lipid Extraction Method from Wet Microalga Picochlorum sp. at Room Temperature

A novel method using ethanol was proposed for extracting lipids from wet microalga Picochlorum sp. at room temperature and pressure. In this study, Central Composite design (CCD) was applied to investigate the optimum conditions of lipid extraction. The results revealed that the solvent to biomass ratio had the largest effect on lipid extraction efficiency, followed by extraction time and temperature. A high lipid extraction yield (33.04% of the dry weight) was obtained under the following extraction conditions: 5 mL solvents per gram of wet biomass for 37 min with gentle stirring at room temperature. The extraction yield was comparable to that obtained by the widely used Bligh-Dyer method. Furthermore, no significant differences in the distribution of lipid classes and fatty acid composition were observed according to different extraction methods. In conclusion, these results indicated that the proposed procedure using ethanol could extract lipids from wet biomass efficiently and had giant potential for lipid extraction at large scale.