Assessing the potential of fatty acids produced by filamentous fungi as feedstock for biodiesel production

Increased costs and limited availability of traditional lipid sources for biodiesel production encourage researchers to find more sustainable feedstock at low prices. Microbial lipid stands out as feedstock replacement for vegetable oil to convert fatty acid esters. In this study, the potential of three isolates of filamentous fungi (Mucor circinelloides URM 4140, M. hiemalis URM 4144, and Penicillium citrinum URM 4126) has been assessed as single-cell oil (SCO) producers. M. circinelloides 4140 had the highest biomass concentration with lipid accumulation of up to 28?wt% at 120?hr of cultivation. The profile of fatty acids revealed a high content of saturated (SFA) and monounsaturated fatty acids (MUFA), including palmitic (C16:0, 33.2–44.1?wt%) and oleic (C18:1, 20.7–31.2?wt%) acids, with the absence of polyunsaturated fatty acids (PUFA) having more than four double bonds. Furthermore, the predicted properties of biodiesel generated from synthesized SCOs have been estimated by using empirical models which were in accordance with the limits imposed by the USA (ASTM D6715), European Union (EN 14214), and Brazilian (ANP 45/2014) standards. These results suggest that the assessed filamentous fungus strains can be considered as alternative feedstock sources for high-quality biofuel production.     

Quantitative determination of hydroxy fatty acids as an indicator of in vivo lipid peroxidation: gas chromatography-mass spectrometry methods.

A new method has been developed for the quantitation of lipid peroxidation products by gas chromatography-mass spectrometry. An important advantage over existing gas chromatography-mass spectrometry methods is the elimination of autoxidation during sample preparation. The sensitivity is sufficient to permit measurement of lipid peroxidation products under normal physiological conditions on as little as 1 mg of tissue. Lipids from whole tissue samples or cell preparations are reduced by catalytic hydrogenation during extraction. The hydrogenation stabilizes the compounds by saturating the double bonds and reducing the hydroperoxides to hydroxy derivatives. The saturated lipids are then saponified and the resulting fatty acids are converted to pentafluorobenzyl esters. Hydroxy fatty acids are further converted to trimethylsilyl ether derivatives. Quantitation is accomplished by negative ion chemical ionization gas chromatography-mass spectrometry, using deuterated internal standards. Specific products from polyunsaturated fatty acids can be quantitated, and the method differentiates between products produced by free-radical and photooxidation mechanisms. Increased levels of lipid peroxidation products, above normal physiological levels, that result from prooxidant conditions, such as exposure of animals to carbon tetrachloride, can be measured.     

Effects of free fatty acids as membrane components on permeability of drugs across bilayer lipid membranes. A mechanism for intestinal absorption of acidic drugs

Studies of the influence of fatty acids, which were the component of intestinal mucosal lipids, on the permeability of several drugs across bilayer lipid membranes generated from egg phosphatidylcholine and intestinal lipid have been pursued. The permeability coefficients of $\text{p-}\text{aminobenzoic}$ acid, salicylic acid and $\text{p-}\text{aminosalicylic}$ acid (anionic-charged drug) increased when fatty acids such as lauric, stearic, oleic, linoleic and linolenic acid were incorporated into the bilayer lipid membranes generated from phosphatidylcholine. In the presence of methyl linoleate and oleyl alcohol, no enhancing effect on $\text{p-}\text{aminobenzoic}$ acid transfer was obtained. The effect of fatty acids was more marked at pH 6.5 than at pH 4.5. In contrast, upon the addition of fatty acids to intestinal lipid membranes which originally contained fatty acids, the permeability coefficient of $\text{p-}\text{aminobenzoic}$ acid tended to decrease, though the permeability through intestinal lipid membranes was larger than that of phosphatidylcholine membranes. The permeability of $\text{p-}\text{aminobenzoic}$ acid across bilayer lipid membranes from intestinal phospholipids was significantly decreased to about equal that of phosphatidylcholine membranes, and reverted to the value of intestinal lipid membranes when fatty acids were added to intestinal phospholipids. It seemed reasonable to assume that free fatty acids in the intestinal neutral lipid fraction could contribute to the increase in the permeability of $\text{p-}\text{aminobenzoic}$ acid. On the basis of above results, possible mechanisms for good absorbability of weakly acidic drugs from the intestine are discussed.     

Comparison of cis and trans fatty acid containing phosphatidylcholines on membrane properties

The ever-increasing amount of trans fatty acids in the human diet has been linked to a variety of afflictions, most notably coronary heart disease and arteriosclerosis. The mechanism of why the replacement of cis fatty acids with their trans counterparts can be detrimental to the health of an individual remains a mystery. Here, we compare the differences in membrane physical properties including molecular dynamics, lateral lipid packing, thermotropic phase behavior, "fluidity", lateral mobility, and permeability between model membranes (lipid monolayers and bilayers) composed of cis- and trans-containing phosphatidylcholines (PCs). The PCs tested have a total of zero, one, two, or four cis (oleic or linoleic) or trans (elaidic or linoelaidic) double bonds. These experiments all confirm the basic hypothesis that trans fatty acids produce membrane properties more similar to those of saturated chains than to those of acyl chains containing cis double bonds; i.e., cis double bonds induce much larger membrane perturbations than trans double bonds.     

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography.

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.     

Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.     

Analysis of the lipid moiety of lipopolysaccharide from Rhizobium tropici CIAT899: identification of 29-hydroxytriacontanoic acid.

The lipid moieties of two lipid A's isolated from the phenolic and aqueous fractions of lipopolysaccharide from Rhizobium tropici CIAT899 have been studied. Several 3-hydroxy fatty acids and two long-chain hydroxy fatty acids, 27-hydroxyoctacosanoic acid, and 29-hydroxytriacontanoic acid were identified; the ratios of these acids are the same in both lipid A's. These results can be used for chemotaxonomic purposes.     

Occurrence of trans fatty acids in rats fed a trans-free diet: A free radical-mediated formation?

Trans isomers of unsaturated fatty acids are absorbed from the diet, due to their presence in diary fat and hydrogenated vegetable oils, and health concern has risen due to their effects on lipid risk factors in cardiovascular diseases. On the basis of the efficiency of the thiyl-radical-catalyzed cis/trans isomerization in vitro and the presence of many sulfur-containing compounds in the cell, the aim of this study was to demonstrate that trans geometry of lipid double bonds can be endogenously generated within membrane phospholipids. The study reports trans fatty acids occurrence in tissue and erythrocyte phospholipids of young adult rats fed a diet completely free of trans isomers. Results show that tissues are differently prone to the endogenous isomerization and that, following a free radical attack, trans fatty acids can reach very high amounts. The effectiveness of this process is considerably inhibited in the presence of all-trans retinol, confirming previous data in model membranes. Our results suggest that geometrical isomerization of unsaturated fatty acids, which causes a structural modification of membrane lipids and may influence basic membrane properties and vital biochemical functions, can occur under radical stress conditions and could be efficiently prevented by vitamin A.     

Evidence of evolutionary optimization of fatty acid length and unsaturation

The lipid composition of cell membranes exerts a crucial influence on cell physiology. Indeed, one double bond triggers membrane fluidity, essential for cell functionality, but additional double bonds increase the susceptibility to peroxidation, which produces reactive compounds that impair the viability of cells. It has therefore been suggested, but never tested in an extensive comparative context, that the composition of membrane fatty acids has been optimized during evolution. A similar prediction has been made for fatty acid chain length, on which susceptibility to peroxidation also depends. Here I tested for stabilizing selection on fatty acid composition by evaluating the fitting of the single stationary peak (SSP) model of evolution to a large data set from 107 species of birds, against alternative evolutionary models. I found that across‐species variation in average chain length and in the proportion of monounsaturated fatty acids (MUFAs), but not in the proportion of polyunsaturated (PUFAs) nor saturated (SFAs) fatty acids, was better explained by SSP models than by other models. Results show optimum values of fatty acid chain length and proportion of MUFAs of 18 C atoms and 25.5% mol, respectively, the strength of stabilizing selection being particularly high in chain length. This is the first evidence of evolutionary optimization in fatty acid composition, suggesting that certain values may have been selected because of their adaptive capacity to minimize susceptibility to lipid peroxidation.     

Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes

About one-fourth the phosphatidylcholines (PCs) from bovine disk photoreceptor membranes contain very long chain (24-36 carbons) polyunsaturated (4, 5, and 6 double bonds) fatty acids of the n-3 and n-6 series (VLCPUFA). Such fatty acids, exclusively occurring in dipolyunsaturated species, are esterified to the sn-1 position of their glycerol backbone, docosahexaenoate being the major fatty acid at sn-2. Chromatographically, such PCs display a weakly polar character relative to other species, ascribable to their exceedingly large number of carbons. After hexane extraction of lyophilized disks, PC is the major component of the fraction of lipids that remains associated with rhodopsin, followed by phosphatidylserine, while a large proportion of the phosphatidylethanolamine is removed. The fatty acid composition of the hexane-removable and protein-bound lipid fractions markedly differs, the latter being enriched in lipid species containing long-chain and very long chain polyenes. This is observed for all lipid classes except free fatty acids. VLCPUFA-containing PCs are the most highly concentrated species in the rhodopsin-associated lipid fraction. The very long chain polyenes these PCs have at sn-1 may account for their resistance to being separated from the protein. It is hypothesized that their unusually long polyenoic fatty acids could be well suited to partially surround alpha-helical segments of rhodopsin.     

The lipid composition of flight muscle mitochondria isolated from the blowfly, Phormia regina.

The role of lipids in membrane structure and function was studied by measuring the major lipid classes in mitochondria isolated from flight muscle of the blowfly, Phormia regina. Approximately 98% of the total lipid is phospholipid. Neutral lipid constitutes the remaining 2% of the total. Phosphatidylethanolamine accounts for 55–60% of the phospholipid. A molecular ratio of 4:1:1 is found for phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (diphosphatidylglycerol). The neutral lipids include cholesterol, about 20%, and quinone, 40–45% of the total. The free fatty acid content of the neutral lipid fraction is variable, apparently being generated by endogenous phospholipase activity. The fatty acids of the neutral and phospholipid classes are predominantly 14–18 carbon acids; long-chain fatty acids of 20 and 22 carbons are essentially absent. The neutral lipid fraction contains 43% saturated and 51% monoenoic fatty acids. More than 65% of the phospholipid fatty acids are unsaturated. The principal fatty acids are palmitic, palmitoleic, oleic, linoleic, and linolenic. No trace of α- or β-tocopherol is detected. As vitamin E is considered an important naturally occuring antioxidant that prevents lipid peroxidation, the apparent absence of α- and β-tocopherol in these mitochondria coupled with intense oxidative activity of the mitochondria leads to the suggestion that blowfly flight muscle mitochondria may be particularly susceptible to peroxidative damage.     

Mechanisms of inhibition of triacylglycerol hydrolysis by human gastric lipase

In the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis. In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis.     

Diazirine based photoaffinity labeling

Diazirines are among the smallest photoreactive groups that form a reactive carbene upon light irradiation. This feature has been widely utilized in photoaffinity labeling to study ligand-receptor, ligand-enzyme and protein-protein interactions, and in the isolation and identification of unknown proteins. This review summarizes recent advances in the use of diazirines in photoaffinity labeling.     

Quantitative fatty acid analyses in cultured porcine pulmonary artery endothelial cells: the combined effects of fatty acid supplementation and oxidant exposure.

Supplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oleic acid (18:1 omega 9) or with an equivalent volume of ethanol vehicle alone (ETOH-0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 microM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemental PAEC were exposed to HBSS or H2O2 either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2-induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2 reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury.     

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.     

Interaction with membrane lipids and heme ligand binding properties of Escherichia coli flavohemoglobin

Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket.     

Lipid peroxidation and the thiobarbituric acid assay: standardization of the assay when using saturated and unsaturated fatty acids

Saturated fatty acids are less vulnerable to lipid peroxidation than their unsaturated counterparts. In this investigation, individual fatty acids of the C(16), C(18) and (20) families were subjected to the thiobarbituric (TBA) assay. These fatty acids were chosen based on their degree of saturation and configuration of double bonds. Interestingly, an assay threshold was reached where increasing the fatty acid concentration resulted in no additional decrease in the TBARS concentrations. Therefore, the linear range of TBARS inhibition was determined for fatty acids in the C(16) and C(20) families. The rate of TBARS inhibition was greater for the saturated than for unsaturated fatty acids, as measured from the slope of the linear range. These findings demonstrate the need to standardize the TBARS assay using multiple fatty acid concentrations when using this assay for measuring in vitro lipid peroxidation.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.