糠醛对粘红酵母生长与油脂积累的影响

为了探究纤维素水解液中常见的发酵抑制物糠醛对粘红酵母Rhodotorula glutinis生长与油脂积累的影响,对比了不同的糠醛浓度(0.1、0.4、0.6、1.5 g/L)下粘红酵母的生物量和油脂积累情况,并探究了1.0 g/L的糠醛对粘红酵母不同碳源(葡萄糖和木糖)利用的影响。研究表明,当糠醛浓度达1.5 g/L时,粘红酵母的延迟期延长至96 h,残糖高达17.7 g/L,生物量最高6.6 g/L,仅为正常积累量的47%,油脂含量也减少了约50%;以木糖为碳源时,糠醛对粘红酵母的抑制程度小于葡萄糖为碳源时的情况;在糠醛存在的逆境中,粘红酵母倾向于生成更多的18碳脂肪酸或18碳不饱和脂肪酸。     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

Monitoring Rhodotorula glutinis CCMI 145 physiological response and oil production growing on xylose and glucose using multi-parameter flow cytometry

Flow cytometry was used to monitor the lipid content, viability and intrinsic light scatter properties of Rhodotorula glutinis CCMI 145 cells growing on batch cultures using xylose and glucose as carbon sources. The highest lipid content was observed for cells grown on glucose, at the end of the exponential phase (17.8% w/w). The proportion of cells stained with PI attaining 77% at the end of the glucose growth. Cells growing on xylose produced a maximum lipid content of 10.6% (w/w), at the stationary phase. An increase in the proportion of cells stained with PI was observed, reaching 29% at the end of xylose growth. Changes in the side and forward light scatter detected during the yeast batch cultures supported that R. glutinis cells grown on glucose experienced harsher conditions, resulting in a high level of cytoplasmic membrane damage, which did not occur when R. glutinis cells grew on xylose.     

Overproduction of free fatty acids in E. coli: implications for biodiesel production

Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.     

Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling

Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with ¹³C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the ¹³C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.

     

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.     

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L⁻¹) and lipid production (1.04 g·L⁻¹), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C_16:1, 9.4%), oleic acid (C_18:1, 47.2%), linoleic acid (C_{18:2 n−6}, 9.6%), and linolenic acid (C_{18:3 n−3}, 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L⁻¹ of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.     

Dimethyl carbonate as potential reactant in non-catalytic biodiesel production by supercritical method

In this study, the non-catalytic supercritical method has been studied in utilizing dimethyl carbonate. It was demonstrated that, the supercritical dimethyl carbonate process without any catalysts applied, converted triglycerides to fatty acid methyl esters with glycerol carbonate and citramalic acid as by-products, while free fatty acids were converted to fatty acid methyl esters with glyoxal. After 12 min of reaction at 350 degrees C/20 MPa, rapeseed oil treated with supercritical dimethyl carbonate reached 94% (w/w) yield of fatty acid methyl ester. The by-products from this process which are glycerol carbonate and citramalic acid are much higher in value than glycerol produced by the conventional process. In addition, the yield of the fatty acid methyl esters as biodiesel was almost at par with supercritical methanol method. Therefore, supercritical dimethyl carbonate process can be a good candidate as an alternative biodiesel production process.     

Triacylglycerols in prokaryotic microorganisms

Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.     

Simplifying Hansen Solubility Parameters for Complex Edible Fats and Oils

Hansen solubility parameters (HSPs), often used to predict the miscibility between two compounds, are an alternative tool in evaluating the ability of the solvent to interact via dispersion, dipole-dipole, and hydrogen bonding interactions. The aim of this paper is to find a simple way to calculate HSPs for complex mixtures of triglycerides (TAGs). HSPs were calculated using two approaches: the first assumes that the contributions to the dispersion, dipole-dipole, and hydrogen bonding interactions may be subdivided into larger functional moieties (i.e., fatty acids and fatty acid methyl esters) that are additive, while the second approach assumes that vegetable oils are comprised of mixtures of simple TAGs in the same mass fractions as the fatty acids. The HSPs obtained using the two approaches are compared to reference values determined using the “Hansen Solubility Parameters in Practice” software (HSPiP) considering the complex TAG profile for each vegetable oil.HSPs for vegetable oils, obtained with the HSPiP software, did not correspond well to the HSPs obtained from the group contribution approach, when using fatty acids, fatty acids + glycerol or fatty acid methyl esters. In contrast, the HSPs calculated for vegetable oils, assuming that all TAGs are simple and in the same mass fractions as the fatty acids, provide similar values to the HSPs obtained from the HSPiP software. Therefore, it is possible to calculate the HSPs for complex oils by simply knowing the fatty acid composition. Knowledge of HSPs may be used to rationalize the ability of certain low molecular weight molecules to develop organogels in vegetable oils as well as the crystallization of triglycerides.     

Cultural characteristics and fatty acid composition of Corynebacterium acnes

A detailed study of the cultural characteristics and cellular fatty acid composition of 27 isolates of Corynebacterium acnes was performed to establish the properties by which this organism may be identified and characterized. The fatty acids were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Each strain possessed a similar fatty acid profile which was characterized by a large percentage of C15 branched-chain acid. Uniformity in certain biochemical reactions and cultural characteristics was also observed. All strains were catalase-positive, nonmotile, and urease-negative, reduced nitrate, liquefied gelatin, failed to hydrolyze esculin and starch, and gave a positive methyl red test. Glucose, fructose, and glycerol were fermented, but not lactose, salicin, sucrose, maltose, xylose, or arabinose. Production of hydrogen sulfide and indole, fermentation of mannitol, and hemolytic activity were variable characteristics. Two species of the genus Propionibacterium were also tested and found to be similar to C. acnes both in cultural characteristics and fatty acid composition. The results strengthen previous suggestions that C. acnes should be classified in the genus Propionibacterium.     

Performance of bacterial strains used for distillery waste treatment on different substrates

Six bacterial strains were isolated and acclimatized on distillery waste. The performance of these bacterial strains in respect to growth, reduction in chemical oxygen demand (COD) values, carbon dioxide production and volatile acid production were studied on five different substrates. Glucose and xylose exhibited growth patterns similar to that on spentwash. Glucose, xylose, casein hydrolysate and amino acids led to very good reduction in COD values compared with glycerol. Rate of substrate consumption was maximum in the case of glucose followed by amino acids, casein hydrolysate, xylose and glycerol. Production of volatile acids and carbon dioxide from glucose amounted to ≈ 50% of the theoretical yield based on glycolysis and the tricarboxylic acid cycle. Production of carbon dioxide followed the usual microbiological growth pattern while volatile acids did not show any such pattern. Carbon dioxide and volatile acids appear to be the major degradation products in distillery waste treatment by these bacteria.     

Engineering of a xylose metabolic pathway in Rhodococcus strains

The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and ¹³C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly ¹³C₃ labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. ¹³C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.     

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.     

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.     

Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil

SUMMARY: A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.     

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.