Evaluation of extraction methods for recovery of fatty acids from Botryococcus braunii LB 572 and Synechocystis sp. PCC 6803

Microalgae are a very diverse group of organisms that consist in both prokaryotic and eukaryotic forms. Some species of microalgae can be induced to overproduce particular fatty acids through simple manipulations of the physical and chemical properties of the culture medium. In this paper, the effect of different extraction techniques on the recovery of fatty acids from the freeze-dried biomass from two lipid-producing microalgal strains: Botryococcus braunii LB 572 (green algae) and Synechocystis sp. PCC 6803 (cyanobacteria) was examined. Five procedures were used: after conversion of the lipid material into the corresponding fatty acid methyl esters (FAMEs) via suitable derivatization reactions (extraction-transesterification) and direct transesterification of biomass to produce FAMEs (without the initial extraction step) that used differential types of catalysts and processing conditions. This study has shown that procedure 3, a one step practical procedure for lipid extraction and in situ methyl ester derivation could be used successfully for the determination of the fatty acid compositions of microalgae and cyanobacteria.     

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.     

Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae

A comparative evaluation of Bligh and Dyer, Folch, and Cequier-Sánchez methods for quantitative determination of total lipids (TLs) and fatty acids (FAs) was accomplished in selective green (Ulva fasciata), red (Gracilaria corticata), and brown algae (Sargassum tenerrimum) using a full factorial categorical design. Applications of sonication and buffer individually on lipid extraction solvent systems were also evaluated. The FA recoveries obtained from the aforementioned methods were compared with those of direct transesterification (DT) methods to identify the best extraction methods. The experimental design showed that macroalgal matrix, extraction method, and buffer were key determinants for TL and FA recoveries (P ? 0.05), exhibiting significant interactions. But sonication gave erratic results with no interaction with any of the factors investigated. The buffered solvent system of Folch rendered the highest TL yield in U. fasciata and G. corticata while the buffered system of Bligh and Dyer gave the highest yield in S. tenerrimum. DT methods were more convenient and accurate for FA quantification and rendered 1.5–2 times higher yields when compared with the best conventional method, minimizing the use of chlorinated solvents, their cost of analysis, and disposal. The buffered solvent system was found to be the most appropriate for lipid research in macroalgae.     

Zero-waste biorefinery of oleaginous microalgae as promising sources of biofuels and biochemicals through direct transesterification and acid hydrolysis

Oleaginous microalgae are considered as promising sources of biofuels and biochemicals due to their high lipid content and other high-value components such as pigments, carbohydrate and protein. This study aimed to develop an efficient biorefinery process for utilizing all of the components in oleaginous microalgae. Acetone extraction was used to recover microalgal pigments prior to processes for the other products. Microalgal lipids were converted into biodiesel (fatty acid methyl ester, FAME) through a conventional two-step process of lipid extraction followed by transesterification, and alternatively a one-step direct transesterification. The comparable FAME yields from both methods indicate the effectiveness of direct transesterification. The operating parameters for direct transesterification were optimized through response surface methodology (RSM). The maximum FAME yield of 256 g/kg-biomass was achieved when using chloroform:methanol as co-solvents for extracting and reacting reagents at 1.35:1 volumetric ratio, 70 °C reaction temperature, and 120 min reaction time. The carbohydrate content in lipid-free microalgal biomass residues (LMBRs) was subsequently acid hydrolyzed into sugars under optimized conditions from RSM. The maximum sugar yield obtained was 44.8 g/kg-LMBRs and the protein residues were recovered after hydrolysis. This biorefinery process may contribute greatly to zero-waste industrialization of microalgae based biofuels and biochemicals.     

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.     

Direct transesterification of all classes of lipids in a one-step reaction

Conventional techniques for the determination of fatty acid composition of lipids require solvent extraction, purification, hydrolysis, and derivatization procedures that are both lengthy and cumbersome. A 1-hr direct transesterification procedure carried out in methanol-benzene 4:1 with acetyl chloride circumvented all these steps and was applicable for analysis of both simple (triglycerides) and complex lipids (cholesteryl esters, phospholipids, and sphingomyelin). Recoveries (greater than 95%) of standards unaffected by the presence of 5% water and 200 mg of silica suggested that the technique could be used for the quantitative analysis of total fatty acids as well as of fatty acids in classes of lipids separated on silica from biological samples. When compared to the Folch procedure, the technique led to a 20.1% increase in total fatty acids for plasma, 3.9% for feces, 7.4% for bile, and 9.7% for rat liver. We therefore conclude that this one-step direct transesterification procedure is superior to currently used methods, not only because of its simplicity and speed, but also because of its added precision.     

Review of methods used in lunar organic analysis: Extraction and hydrolysis techniques

Extraction, hydrolysis and crushing procedures have proven useful in analyzing for some of the carbon-containing compounds which are present in Apollo 11 and 12 lunar samples. Three main extraction methods employed with aqueous and nonaqueous solvents were refluxing in open and closed systems, Soxhlet extraction, and sonication. With water and acids, refluxing was the method of choice. Of the various nonaqueous solvent systems used, benzene: methanol mixtures were most often selected, and sonication was favored over Soxhlet extraction. Extraction of lunar samples with water followed by acid hydrolysis of the water extract was found to be superior to direct acid hydrolysis of lunar material in the search for amino acids or their precursors. Direct acid hydrolysis of lunar materials did demonstrate however, that carbides or carbide-like compounds are present on the moon. Hydrolysis with deuterated acids and bases showed that lunar samples contain indigenous methane and ethane and confirmed the presence of carbide-like materials. Crushing experiments also showed that gaseous hydrocarbons can be released from lunar samples.     

Lipid Extraction from <Emphasis Type="Italic">Tetraselmis</Emphasis> sp. Microalgae for Biodiesel Production Using Hexane-based Solvent Mixtures

Lipid extraction is a critical step in the downstream processing of biodiesel production from microalgae. Solvent extraction using mixtures of non-polar and polar solvents is one of the most well-known processes for this purpose. Hexane is the most common solvent of choice for large-scale lipid extractions due to its technical and economic advantages, especially its high selectivity toward lipids and low cost. In this study, extractions using mixtures of hexane and polar solvents were evaluated for their performance in order to develop a more efficient method for large-scale lipid extraction from microalgae. The combination of hexane and methanol resulted in the highest fatty acid methyl ester (FAME) yield for lipids from Tetraselmis sp. The effects of extraction conditions, including proportions of methanol to hexane, ratios of total solvent volume to dry biomass, and extraction time, on extraction yields were evaluated to determine optimum conditions providing higher lipid and FAME yields. The optimal conditions were as follows: proportion of hexane to methanol of 1:1, ratio of total solvent volume to dry biomass of 10 mL/g, and extraction time of 120 min. Finally, the selected solvent mixture and optimal conditions were applied to larger scale extraction experiments with scale-up factors of 10, 50, and 100. FAME yields of large-scale extractions were almost completely consistent with increasing scale-up factors. The results of this study suggest that a hexane and methanol mixture is a promising solvent for large-scale lipid extraction from microalgae.     

LIBERATION OF 5,8,11,14,17-EICOSAPENTAENOIC ACID AND OTHER POLYUNSATURATED FATTY ACIDS FROM LIPIDS AS A GRAZER DEFENSE REACTION IN EPILITHIC DIATOM BIOFILMS

Acute grazer toxicity of freshwater diatom biofilms was determined using Thamnocephalus platyurus Packard, an anostracan grazer, as the bioassay organism. The diatoms exhibited toxicity when the cells of the biofilm were freeze–thawed before the assay. The toxicity could be extracted from the biofilms with methanol and acetone, and only minimal toxicity was left in the insoluble residue. Bioassay-guided HPLC separation of the methanolic extract was performed to trace the most toxic components. Analysis by UV spectrometry, gas chromatography, and mass spectrometry showed that 5,8,11,14,17-eicosapentaenoic acid was responsible for most grazer toxicity. The 24-h LC₅₀ of this polyunsaturated fatty acid was 34 μM in the Thamnocephalus platyurus bioassay. The concentrations of other free fatty acids were not high enough to contribute significantly to the toxicity. Procedures that affected the integrity of the cells (e.g. solvent extraction, freezing and thawing, osmotic stress by addition of 20% NaCl, or grinding the cells in a mortar) were taken as model reactions for grazing and had the common effect of resulting in a dramatic increase of free polyunsaturated and saturated fatty acids. Under these conditions, about 30% of the total fatty acids of the diatoms was transformed from the bound into the free form. The time necessary for liberation was very short. With the exception of 5,8,11,14,17-eicosapentaenoic acid, which continued to be liberated, the hydrolysis of the other fatty acids was terminated less than 1 min after initiating the reaction. The classical extraction procedures using methanol and other solvents led to the appearance of a high percentage of free fatty acids in live cells. Treatment of biofilms with these solvents did not stop the hydrolysis of lipids initiated by the disintegration of the cells. However, boiling acetone completely suppressed the hydrolytic reactions, and free polyunsaturated fatty acids were not detected in live biofilm organisms, although nontoxic saturated fatty acids were present in moderate concentrations. These results were interpreted as an indication that the frequently reported existence of free polyunsaturated fatty acids in live biomass is an analytical artifact.     

千年桐种子油提取工艺研究及其主要脂肪酸成分分析

本文以氯仿、石油醚和正己烷-异丙醇(3:2,v/v)三种不同溶剂对千年桐种子油进行提取,比较了不同溶剂对种子出油率的影响,结果表明以氯仿为溶剂时出油率最高,达到了35％;并考查了提取时间和提取溶剂体积对出油率的影响.最终优化的提取工艺为:以氯仿为溶剂,液料比为12:1(v/w),提取时间6h,出油率达到了37％.提取的种子油经转酯化后,GC-MS分析其主要脂肪酸组分,结果表明千年桐种子油中总脂肪酸占总油酯的90.55％,其中棕榈酸3.87％,硬脂酸4.11％,亚油酸12.15％,油酸13.31％,亚麻酸12.09％,共轭亚麻酸51.20％和EPA(二十碳五烯酸)3.30％.千年桐种子油中富含不饱和脂肪酸,是一种良好的干性油.     

In situ alkaline transesterification of cottonseed oil for production of biodiesel and nontoxic cottonseed meal

The production of fatty acid methyl ester (FAME) by direct in situ alkaline-catalyzed transesterification of the triglycerides (TG) in cottonseeds was examined. The experimental results showed that the amount of cottonseed oil dissolved in methanol was approximately 99% of the total oil and the conversion of this oil could achieve 98% under the following conditions: less than 2% moisture content in cottonseed flours, 0.3-0.335mm particle size, 0.1mol/L NaOH concentration in methanol, 135:1 methanol/oil mole ratio, 40 degrees C reaction temperature and 3h reaction time. Further, the effects of co-solvent petroleum ether and methanol recycling on the cottonseed oil extraction and conversion were also investigated. The use of alkaline methanol as extraction and reaction solvent, which would be useful for extraction oil and gossypol, would reduce the gossypol content in the cottonseed meal. The free and total gossypol contents in the cottonseed meal obtained from in situ alkaline transesterification were far below the FAO standard. And the nontoxic cottonseed meal could be used as animal protein feed resources.     

Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids

The objective of this study was to investigate the extraction of lipids, for example, mono‐ and polyunsaturated fatty acids (PUFA) as well as carotenoids, from wet microalgae biomass using pressurized subcritical extraction solvents, which meet the requirements of food and feed applications. To demonstrate the effect of the solvent and temperature on the lipid yield, we chose two microalgae species, viz. Chlorella vulgaris and Phaeodactylum tricornutum, differing in their biochemical composition fundamentally. In case of P. tricornutum, ethanol showed the highest fatty acid yield of 85.9% w/w. In addition to eicosapentaenoic acid (EPA), the ethanolic extracts contained exceptional amounts of fucoxanthin (up to 26.1 mg/g d. w.), which can be beneficial to protect unsaturated fatty acids from oxidation processes and in terms of human nutrition. For C. vulgaris, a fatty acid yield of 76.5% w/w was achieved from wet biomass using ethyl acetate at 150°C. In general, an increase in the extraction temperature up to 150°C was found to be important in terms of fatty acid yield when extracting wet microalgae biomass. The results suggest that it is possible to efficiently extract both fatty acids and carotenoids from wet microalgae by selecting suitable solvents and thus circumvent energy‐intensive drying of the biomass.     

Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography: BF3/methanol transesterification of lyophilized samples instead of Folch extraction gives higher yields.

The amount of individual fatty acids contained in the main human lipoproteins VLDL, LDL, lipoprotein (a), HDL2, and HDL3 were determined by two different methods. In Method I, the lipids were first extracted by the classical Folch procedure and then transesterified with BF3/methanol and separated by capillary GC. In Method II the lipoprotein solution was freeze dried prior to transesterification with BF3/methanol. In all lipoproteins except VLDL significantly more fatty acids were found with Method II as compared to Method I. For total fatty acids the increase was up to 17.5%, for polyunsaturated fatty acids up to 24.5%. The total fatty acid content determined by Method II resembled closely the content independently derived from the enzymatically determined lipid composition. The results indicate that in case of lipoproteins quantification of fatty acids should be made with freeze-dried samples rather than with Folch extracts.     

Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate

The study documented the potential of isolated filamentous fungus Aspergillus sp. as whole cell biocatalyst for biodiesel production using Sabourauds dextrose broth medium (SDBM) and corncob waste liquor (CWL) as substrates. SDBM showed improvement in both biomass production (13.6 g dry weight/1000 ml) and lipid productivity (23.3%) with time. Lipid extraction was performed by direct (DTE) and indirect (IDTE) transesterification methods. DTE showed higher transesterification efficiency with broad spectrum of fatty acids profile over IDTE. CWL as substrate showed good lipid productivity (22.1%; 2g dry biomass; 48 h) along with efficient substrate degradation. Lipids derived from both substrates depicted high fraction of saturated fatty acids than unsaturated ones. Physical characteristics of fungal based biodiesel correlated well with prescribed standards. CWL derived biodiesel showed relatively good fuel properties (acid number, 0.40 mg KOH/g of acid; iodine value, 11 g I?/100 g oil; density, 0.8342 g/cm3) than SDBM derived biodiesel.     

Effect of biomass pre-treatment and solvent extraction on beta-carotene and lycopene recovery from Blakeslea trispora cells

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being beta-carotene, lycopene, and gamma-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C(max) values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44-56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in beta-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

不同提取溶剂和方法组合从落叶松毛虫蛹中提取的脂肪酸成分和含量的比较

落叶松毛虫Dendrolimus superans (Butler)蛹个体较大, 具有很高利用价值。为明确东北落叶松毛虫蛹中脂肪酸成分, 探讨最佳提取溶剂和提取方法的组合, 分别以正己烷、 石油醚和乙醚为提取溶剂, 结合超声波振荡萃取法、 索氏萃取法及溶剂萃取方法热浸和冷浸4种提取方法提取落叶松毛虫蛹油, 并采用毛细管色谱-质谱法分析提取物的脂肪酸种类和相对含量。结果表明： 正己烷溶剂与4种提取方法的组合中, 溶剂萃取热浸法提取率最高, 为25.60%。索氏萃取及溶剂萃取方法热浸和冷浸均检测到10种脂肪酸, 正己烷-超声波振荡萃取组合检测到9种脂肪酸。石油醚溶剂与4种提取方法的组合中, 索氏萃取提取率最高, 为29.31%, 均检测到10种脂肪酸。乙醚溶剂与4种提取方法的组合中, 溶剂萃取冷浸法提取率最高, 为29.11%, 检测到的脂肪酸种类为溶剂萃取冷浸法（13种）＞索氏萃取法（12种）＞溶剂萃取热浸法（11种）＞超声波振荡萃取法（9种）。在检测到的总脂肪酸中, 63%以上为不饱和脂肪酸, 其含量受提取溶剂和方法的影响不大。因此, 适合东北落叶松毛虫蛹中脂肪酸提取的最佳组合为石油醚溶剂 索氏萃取法。     

Sonication-assisted production of biodiesel using soybean oil and supercritical methanol

High temperature and pressure are generally required to produce biodiesel using supercritical methanol. We reduced the harsh reaction conditions by means of sonicating the reaction mixture prior to transesterification using supercritical methanol. Soybean oil was selected as the raw material for transesterification. As soybean oil contains more unsaturated fatty acid triglycerides, the biodiesel degraded more at high temperature. The reactants were sonicated for 60 min at 35 °C prior to transesterification to avoid degradation of the product and to enhance biodiesel yield at temperatures <300 °C. The process parameters were optimized using central composite design. The variables selected for optimization were temperature, time, and the oil to methanol molar ratio. The temperature and oil to methanol molar ratios were varied from 250 to 280 °C and 1:40–1:50, respectively. The reaction time was tested between 4 and 12 min. The biodiesel was analyzed for any possible degradation by gas chromatography–mass spectroscopy and for the wt% of fatty acid methyl esters (FAME) obtained. The maximum FAME yield (84.2 wt%) was obtained at a temperature of 265.7 °C, an oil to alcohol molar ratio of 1:44.7, and a time of 8.8 min. The optimum yield was obtained at a pressure of 1,500 psi. The pressure and optimum temperature used to obtain the maximum yield were the lowest reported so far without the use of a co-solvent. Thus, the severity of the supercritical reactions was reduced by adding sonication prior to the reaction.     

Optimization of direct conversion of wet algae to biodiesel under supercritical methanol conditions

This study demonstrated a one-step process for direct liquefaction and conversion of wet algal biomass containing about 90% of water to biodiesel under supercritical methanol conditions. This one-step process enables simultaneous extraction and transesterification of wet algal biomass. The process conditions are milder than those required for pyrolysis and prevent the formation of by-products. In the proposed process, fatty acid methyl esters (FAMEs) can be produced from polar phospholipids, free fatty acids, and triglycerides. A response surface methodology (RSM) was used to analyze the influence of the three process variables, namely, the wet algae to methanol (wt./vol.) ratio, the reaction temperature, and the reaction time, on the FAMEs conversion. Algal biodiesel samples were analyzed by ATR-FTIR and GC-MS. Based on the experimental analysis and RSM study, optimal conditions for this process are reported as: wet algae to methanol (wt./vol.) ratio of around 1:9, reaction temperature and time of about 255 °C, and 25 min respectively. This single-step process can potentially be an energy efficient and economical route for algal biodiesel production.     

Direct preparation of biodiesel from rapeseed oil leached by two-phase solvent extraction

A new method which coupled the two-phase solvent extraction (TSE) with the synthesis of biodiesel was studied. Investigations were carried out on transesterification of methanol with oil-hexane solution coming from TSE process in the presence of sodium hydroxide as the catalyst. Biodiesel (fatty acid methyl esters) were the products of transesterification. The influential factors of transesterification, such as reaction time, catalyst concentration, mole ratio of methanol to oil and reaction temperature were optimized. The results showed that the optimal reaction parameters were sodium hydroxide concentration 1.1% by weight of rapeseed oil, mole ratio of methanol to oil 9:1, reaction time 120 min, and reaction temperature 55-60 degrees C. Under these conditions, the TG conversion would rise up to 98.2%. Based on the new method, biodiesel production process could be simplified and the biodiesel cost could be reduced.     

Comparative Analysis of the Fatty Acid Composition of Microalgae Obtained by Different Oil Extraction Methods and Direct Biomass Transesterification

One of the main challenges for the successful production and use of microalgae for biodiesel production is to obtain a satisfactory level of fatty acid methyl esters (FAME). The aims of this study are to identify the best method of lipid extraction and provide high FAME levels and to evaluate their fatty acid profiles. Six lipid extraction methodologies in three microalgae species were tested in comparison with the direct transesterification (DT) of microalgal biomass method. The choice of extraction method affected both the oily extract yield and the FAME composition of the microalgae and consequently may affect the properties of biodiesel. The efficiency of different lipid extraction methods is affected by the solvent polarity, which extracts different target compounds from lipid matrix. Dichloromethane/methanol extraction and Folch extraction produced the largest oil extract yields, but extraction with hexane/ethanol resulted in the best ester profile and levels. Performing DT reduces the volume of extractor solvent, the time and cost of FA composition analysis, as well as, presents less steps for fatty acid quantification. DT provided biomass FAME levels of 50.2, 636.4, and 258.2 mg.g^?1 in Nannochlorophisis oculata, Chaetoceros muelleri, and Chlorella sp., respectively. On the basis of an analysis of the fatty acids profiles of different species, C. muelleri is a promising microalga for biodiesel production. Depending on the extraction method, Chlorella sp. and N. oculata can be considered as an alternative in obtaining arachidonic (Aa) and eicosapentaenoic (EPA) acids.