Supercritical carbon dioxide extraction and analysis of lipids from <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> using gas chromatography

The influence of six different supercritical carbon dioxide extraction conditions on the quantity and quality of the extracted lipids from Chlorella vulgaris (C. vulgaris) was examined using experimental design. The investigated parameters included dynamic extraction times (45 and 90 min), pressures (300 and 350 bars) and a modifier (with and without ethanol). By using a grinding method prior to the extraction, cells were disrupted resulting in an enhanced extraction yield. The results showed that the total lipid yields increased by increasing the pressure, extraction time and the modifier. Applying harsher extraction conditions increased the extraction of longer-chain fatty acids (FAs), changed the FA compositions and enhanced the total lipid yield. Using transesterification, the extracted lipids were converted to biodiesel. The biodiesel physiochemical properties were estimated using empirical equations based on the fatty acid methyl ester contents. Furthermore, analysis of the protein content of remaining biomass showed a decrease in protein content with increasing the lipid extraction yield.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Determination of fatty acid methyl esters derived from algae Scenedesmus dimorphus biomass by GC–MS with one‐step esterification of free fatty acids and transesterification of glycerolipids

Algae can synthesize, accumulate and store large amounts of lipids in its cells, which holds immense potential as a renewable source of biodiesel. In this work, we have developed and validated a GC–MS method for quantitation of fatty acids and glycerolipids in forms of fatty acid methyl esters derived from algae biomass. Algae Scenedesmus dimorphus dry mass was pulverized by mortar and pestle, then extracted by the modified Folch method and fractionated into free fatty acids and glycerolipids on aminopropyl solid‐phase extraction cartridges. Fatty acid methyl esters were produced by an optimized one‐step esterification of fatty acids and transesterification of glycerolipids with boron trichloride/methanol. The matrix effect, recoveries and stability of fatty acids and glycerolipids in algal matrix were first evaluated by spiking stable isotopes of pentadecanoic‐2,2‐d₂ acid and glyceryl tri(hexadecanoate‐2,2‐d₂) as surrogate analytes and tridecanoic‐2,2‐d₂ acid as internal standard into algal matrix prior to sample extraction. Later, the method was validated in terms of lower limits of quantitation, linear calibration ranges, intra‐ and inter‐assay precision and accuracy using tridecanoic‐2,2‐d₂ acid as internal standard. The method developed has been applied to the quantitation of fatty acid methyl esters from free fatty acid and glycerolipid fractions of algae Scenedesmus dimorphus .     

Screening,Growth Medium Optimisation and Heterotrophic Cultivation of Microalgae for Biodiesel Production

This article presents a study on screening of microalgal strains from the Peking University Algae Collection and heterotrophic cultivation for biodiesel production of a selected microalgal strain. Among 89 strains, only five were capable of growing under heterotrophic conditions in liquid cultures and Chlorella sp. PKUAC 102 was found the best for the production of heterotrophic algal biodiesel. Composition of the growth medium was optimised using response surface methodology and optimised growth conditions were successfully used for cultivation of the strain in a fermentor. Conversion of algal lipids to fatty acid methyl esters (FAMEs) showed that the lipid profile of the heterotrophically cultivated Chlorella sp. PKUAC 102 contains fatty acids suitable for biodiesel production.     

Fast identification of mycobacterium species by GC analysis with trimethylsulfonium hydroxide (TMSH) for transesterification

Trimethylsulfonium hydroxide (TMSH) reproducibly converts fatty acids bound in, e.g., biomolecules such as phospholipids and/or glycerides, into the corresponding fatty acid methyl esters (FAMEs). The transesterification can be performed at room temperature in a fast single step reaction. Surprisingly, secondary alcohols and mycolic acid cleavage products (MACPs) are also released from mycobacteria under these conditions. The complex reaction mixtures containing FAMEs, MACPs, and secondary alcohols can easily be separated by high resolution temperature-programmed capillary GC. Different species of mycobacteria give rise to characteristic chromatographic patterns and the amount of lipids from a single colony of mycobacteria is sufficient for reliable identification of the bacteria. The profiles of the chromatograms match well those obtained from other sample preparation techniques. The TMSH method of identification of mycobacteria from the patterns of the gas chromatograms is faster and more sensitive than conventional methods, which also involve transesterification. The identification of mycobacterial species by microbiological culture techniques is difficult to perform and requires several weeks.     

Analysis of the Triglycerides and the Free and Derivatized Fatty Acids in Fish Oil by Capillary Electrochromatography

Capillary ElectroChromatography (CEC) on a fused silica capillary column (40 cm L × 100 μm i.d.) packed with 3 μm octadecylsilica (ODS) was evaluated for the analysis of the triglycerides and their fatty acids in fish oils, and more especially in the oil of Moroccan Sardinia pilchardus. The very high complexity of the lipids in fish oil is well illustrated by CEC with a nonaqueous mobile phase consisting of acetonitrile/isopropanol/n-hexane in the ratio 57/38/5 to which 50 mM ammonium acetate has been added. In order to unravel the complexity, the oil was hydrolyzed and the free fatty acids (FFAs), the methyl esters (FAMEs), and the phenacyl esters (FAPEs) were analyzed by CEC on the same column used for the analysis of the triglycerides. Isocratic elution was achieved with the mobile phase acetonitrile/50 mM MES pH 6 in ratio 9/1. The migration characteristics of FFAs, FAMEs, and FAPEs are compared. The analysis of FAPE derivatives has the advantage that quantification applying UV detection is possible and moreover that the number of double bonds in the fatty acid chains can be elucidated by measuring the UV abundance ratio 240/210 nm.     

Trans-esterification of fatty acids from microorganisms and human blood serum by trimethylsulfonium hydroxide (TMSH) for GC analysis

Summary Trimethylsulfonium hydroxide (TMSH) can convert fatty acids into the corresponding fatty acid methyl esters (FAMEs) in a single step. These fatty acids may also be bound in biomolecules such as phospholipids and/or glycerides. Complex mixtures of saturated and unsaturated FAMEs which may contain hydroxy and cylopropyl groups are obtained by trans-esterification; they can easily be separated in most cases by capillary GC. When FAMEs are generated from different microorganisms e.g. bacteria the patterns of the chromatograms are characteristic. Examples of characteristic patterns of bacteria with different cell wall structures are shown. The described method of transesterification can also be applied directly to blood serum without sophisticated sample pretreatment. The profiles of the chromatograms match well those described in the literature obtained by other methods of trans-esterification or sample preparation.     

Environmental control of lipids and fatty acid production in the diatomNavicula saprophila

The unique ability of microalgae to accumulate large amounts of lipids has led to an increase in the research on the cultivation of these organisms for the production of fuels. This paper describes the accumulation of cellular lipids by the diatom,Navicula saprophila, as a result of culture management strategies. The cells were grown in two different nutrient concentrations (nitrogen and silica) maintained at 20° and 30°C. The biomass, lipids, and fatty acids were measured. Biomass yield was higher in a nutrient (nitrogen/silica) nonstress treatment; however, the production of lipids was enhanced in a nutrient stress medium at 30 °C. The influence of the environmental conditions on the distribution of fatty acids was also observed.

     

Aalysis of the fatty acid composition of the lipid classes in human blood serum under normal diet and when supplemented with fish oil

Total lipids have been extracted from human serum with chloroform–methanol 2:1 (v/v) and separated into individual classes by TLC. After transesterification the fatty acid methyl esters were analyzed by capillary gas chromatography on an FFAP column. The quantitation of ω-3 fatty acids has been performed using internal and external standards. Internal lipid standards for each lipid class were carried throughout the entire analytical procedure. Under normal diet eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are incorporated into the lipid classes to different extents: cholesterol esters; EPA, 6.5 ± 1.9 γ/ml serum; DHA, 4.3 ± 1.9 μg/ml: phospholipids; EPA, 5.9 ± 2.7 μg/ml; DHA, 31.8 ± 8.1 μg/ml. Fish oil supplementation leads to a 4 to 6-fold rise in EPA and to an approximately 2-fold rise in DHA.     

Direct fatty acid profiling of complex lipids in intact algae by fast-atom-bombardment mass spectrometry

Fast-atom-bombardment mass spectrometry (FABMS) is used for the semiquantitative determination of the fatty acids of complex lipids directly from intact algal cells, crude algal lipid extracts, and vegetable oils. Carboxylate ions, RCOO^?, corresponding to the fatty acid moieties of the complex lipids are detected. The relative abundances of the carboxylate fatty acid ions in the FAB mass spectra agreed with the relative percentages found by gas chromatography of the fatty acid methyl esters derived from the extracted lipids. Chemical ionization/fast-atom-bombardment mass spectrometry (CI/FABMS) is discussed with respect to enhancing the molecular ions of the fatty acids and triacylglycerols from these materials. The use of FABMS requires little sample preparation, and FABMS enables rapid fatty acid screening, directly from crude biological materials.     

Comparison of solvent mixtures for pressurized solvent extraction of soil fatty acid biomarkers

The extraction and transesterification of soil lipids into fatty acid methyl esters (FAMEs) is a useful technique for studying soil microbial communities. The objective of this study was to find the best solvent mixture to extract soil lipids with a pressurized solvent extractor system. Four solvent mixtures were selected for testing: chloroform:methanol:phosphate buffer (1:2:0.8, v/v/v), chloroform:methanol (1:2, v/v), hexane:2-propanol (3:2, v/v) and acetone. Soils were from agricultural fields and had a wide range of clay, organic matter and microbial biomass contents. Total lipid fatty acid methyl esters (TL-FAMEs) were the extractable soil lipids identified and quantified with gas chromatography and flame ionization detection. Concentrations of TL-FAMEs ranged from 57.3 to 542.2 n mole g⁻¹ soil (dry weight basis). The highest concentrations of TL-FAMEs were extracted with chloroform:methanol:buffer or chloroform:methanol mixtures than with the hexane:2-propanol or acetone solvents. The concentrations of TL-FAMEs in chemical groups, including saturated, branched, mono- and poly-unsaturated and hydroxy fatty acids were assessed, and biological groups (soil bacteria, mycorrhizal fungi, saprophytic fungi and higher plants) was distinguished. The extraction efficiency for the chemical and biological groups followed the general trend of: chloroform:methanol:buffer ≥ chloroform:methanol > hexane:2-propanol = acetone. Discriminant analysis revealed differences in TL-FAME profiles based on the solvent mixture and the soil type. Although solvent mixtures containing chloroform and methanol were the most efficient for extracting lipids from the agricultural soils in this study, soil properties and the lipid groups to be studied should be considered when selecting a solvent mixture. According to our knowledge, this is the first report of soil lipid extraction with hexane:2-propanol or acetone in a pressurized solvent extraction system.     

Lipids from the Marine Alga <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

The composition of lipids and fatty acids from the red alga Gracilaria verrucosa, for which a high content of 20:4n-6 acid is typical, was studied. The principal lipids were digalactosyldiacylglycerides, phosphatidylcholines (PC), monogalactosyldiacylglyderides (MGDG), and sulfoquinovosyldiacylglycerides, the fraction of each was approximately the same. Sphingophospholipids, inositephosphoceramides, were identified among the polar lipids. Each lipid class differed in the ratio of fatty acids (FA). The FA of all glycerolipids contained 20:4n-6 acid but its concentration was greatest in MGDG and PC, 67.2% and 56.5% of the acid mass.__________Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 230–232, May–June, 2005.     

Production of methyl ester fuel from microalgae

Microalgae are unique photosynthetic organisms that are known to accumulate storage lipids in large quantitites and thrive in saline waters. Before these storage lipids can be used, they must be extracted from the microalgae and converted into usable fuel. Transesterification of lipids produces fatty acid methyl esters that can be used as a diesel fuel substitute. Three solvents, 1-butanol, ethanol, and hexane/2-propanol, were evaluated for extraction efficiency of microalgal lipids. Type of catalyst, concentration of catalyst, time of reaction, temperature of reaction, and quality of lipid were examined as variables for transesterification. The most efficient solvent of the three for extraction was 1-butanol (90% efficiency), followed by hexane/2-propanol and ethanol. Optimal yield of fatty acid methyl esters was obtained using 0.6N hydrochloric acid in methanol for 0.1 h at 70°C.     

Utilization of Agricultural Residues of Pineapple Peels and Sugarcane Bagasse as Cost-Saving Raw Materials in Scenedesmus acutus for Lipid Accumulation and Biodiesel Production

The aim of this study is to optimize the lipid accumulation in microalgae by using two agricultural residues of pineapple peels and sugarcane bagasse as low-cost organic carbon sources. Green microalgae Scenedesmus acutus was isolated and selected for cultivation. Effects of three initial sugar concentrations and the stage for adding sugar during cultivation on biomass and lipid production were investigated. The results clearly showed that two-stage cultivation is more suitable than one-stage. The maximum biomass concentration and productivity were obtained at 3.85 g/L and 160.42 mg/L/day when sugarcane bagasse was used. The highest lipid content and lipid yield was reached at 28.05 % and 0.93 g/L when pineapple peels were used, while in the case of sugarcane bagasse, 40.89 % and 1.24 g/L lipid content and yield were obtained. Lipid content was found in normal condition (autotrophic) at 17.71 % which was approximately 2.13-fold lower than when sugarcane bagasse was used (40.89 %). Biodiesel production via in situ transesterification was also investigated; the main fatty acids of palmitic acid and oleic acid were found. This work indicates that using agricultural residues as organic carbon sources could be able to increase lipid content and reduce the cost of biofuel production.     

Swine Manure-Based Pilot-Scale Algal Biomass Production System for Fuel Production and Wastewater Treatment—a Case Study

Integration of wastewater treatment with algae cultivation is one of the promising ways to achieve an economically viable and environmentally sustainable algal biofuel production on a commercial scale. This study focused on pilot-scale algal biomass production system development, cultivation process optimization, and integration with swine manure wastewater treatment. The areal algal biomass productivity for the cultivation system that we developed ranged from 8.08 to 14.59 and 19.15–23.19 g/m²?×?day, based on ash-free dry weight and total suspended solid (TSS), respectively, which were higher than or comparable with those in literature. The harvested algal biomass had lipid content about 1.77–3.55 %, which was relatively low, but could be converted to bio-oil via fast microwave-assisted pyrolysis system developed in our lab. The lipids in the harvested algal biomass had a significantly higher percentage of total unsaturated fatty acids than those grown in lab conditions, which may be attributed to the observed temperature and light fluctuations. The nutrient removal rate was highly correlated to the biomass productivity. The NH₃-N, TN, COD, and PO₄-P reduction rates for the north-located photo-bioreactor (PBR-N) in July were 2.65, 3.19, 7.21, and 0.067 g/m²?×?day, respectively, which were higher than those in other studies. The cultivation system had advantages of high mixotrophic growth rate, low operating cost, as well as reduced land footprint due to the stacked-tray bioreactor design used in the study.     

Lipid composition of <Emphasis Type="Italic">Potamogeton pectinatus</Emphasis> as a function of water contamination

The lipid composition of Potamogeton pectinatus L. collected in different parts of the Sok River in the Lower Volga basin was studied. Differences were found in the quantitative lipid composition of the plant as a function of habitat. Plants inhabiting contaminated waters typically had a lower content of total lipids and glycolipids with an increased content of neutral lipids in addition to a lower content of polyunsaturated acids.     

Isolation,Identification and High-Throughput Screening of Neutral Lipid Producing Indigenous Microalgae from South African Aquatic Habitats

Exploring indigenous microalgae capable of producing significant amounts of neutral lipids through high-throughput screening is crucial for sustainable biodiesel production. In this study, 31 indigenous microalgal strains were isolated from diverse aquatic habitats in KwaZulu-Natal, South Africa. Eight superior lipid-producing strains were selected for further analysis, based on Nile red fluorescence microscopy screening. The microalgal isolates were identified to belong to the genera Chlorella, Neochloris and Chlamydomonas via morpho-taxonomic and molecular approach by 18S rRNA gene sequencing. Chlorella vulgaris PH2 had the highest specific growth rate (μ) and lowest doubling time of 0.24 day⁻¹ and 2.89 ± 0.05 day⁻¹, respectively. Chlorella vulgaris T4 had the highest biomass productivity of 35.71 ± 0.03 mg L⁻¹day⁻¹. Chlorella vulgaris PH2 had the highest lipid content of 34.28 ± 0.47 and 38 ± 9.2% (dcw) as determined by gravimetric analysis and the sulfo-phospho-vanillin (SPV) method, respectively. Chlorella vulgaris PH2 exhibited a high content of saturated fatty acids, while Chlorella sp. T4 exhibited a high total content of saturated and monounsaturated fatty acids with a low content of polyunsaturated fatty acids. The preponderance of neutral lipids suggests that Chlorella sp. T4 is a suitable candidate for biomass feedstock for biodiesel production.

     

Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography-high-resolution mass spectrometry

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography–mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid.     

Lipids and lipophilic components of Viburnum opulus fruits during maturation

The variation of the composition of neutral and polar lipids and lipophilic components during maturation of guelder rose (V. opulus L., fam. Caprifoliaceae) fruits was investigated. During fruit ripening, all lipid groups are accumulated, the highest accumulation rate being observed in the first period of maturation. In nonpolar lipids, this is due to a substantial increase in the content of triacylglycerides, whereas the contents of free fatty acids, methyl esters of fatty acids, monoacylglycerides, and especially esters of triterpenoid compounds decrease. During early maturation, all acyl-containing classes of lipids contain large amounts of saturated fatty acids (mainly, 16:0), whose content decreases during ripening, while the contents of unsaturated 18:1 and 18:2 acids increase.