Production Of Fatty Acid Ethyl Esters Research Articles

Production of Fatty Acid Ethyl Esters from Waste Cooking Oil Using Novozym 435 in a Solvent-Free System

Fatty acid ethyl esters (FAEEs) were produced from waste cooking oil (WCO) using the immobilized Novozym 435 (Novozymes) lipase in a solvent-free system. WCO contained high molar mass degradation compounds that did not affect the lipase activity but remained as a minor component of the resulting FAEEs. The enzyme-catalyzed FAEE production was investigated using a central composite design. The best experimental conditions were obtained with an enzyme loading of 5 wt % and an ethanol/oil molar ratio of 6:1. Under these conditions and at the optimal enzyme temperature of 40 °C, FAEE mixtures with a total ester content of 77.2 and 89.5 wt % were produced after 10 and 24 h, respectively. Treatment of the spent biocatalyst with tert-butanol could restore the transesterification activity of the biocatalyst to its original value.

Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.

Continuous production of palm biofuel under supercritical ethyl acetate

The interesterification of palm oil in supercritical ethyl acetate (ETA) to produce fatty acid ethyl ester (FAEEs) or biofuel was conducted in a continuous tubular reactor. The density of the mixtures in the system was estimated using the Peng–Robinson equation of state process simulator, and the residence time was calculated. The effects of the reaction conditions, including the molar ratios of palm oil to ethyl acetate, the temperature, and the pressure, were investigated under various mass flow rates of the mixtures and optimized. The results showed that reaction temperatures above 653K and long residence times affected the content of FAEEs and triacetin, a valuable by-product. The addition of water to the mixture in a 1:30:10M ratio of palm oil to ethyl acetate to water at 653K, 16MPa, and a mixture mass flow rate of 1.5g/min increased the total production of FAEEs and triacetin from 90.9 to 101.5wt% in 42.4min. The main finding of the present study is that triglyceride associated with ETA hydrolysis used to form acetic acid protected the products from decomposition at high temperatures and long residence times. The results will aid the selection of an efficient and economical process for alternative biofuel production from palm oil in supercritical ETA.

Orthogonal Fatty Acid Biosynthetic Pathway Improves Fatty Acid Ethyl Ester Production in Saccharomyces cerevisiae.

Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms.

One-step transesterification reaction using methanol-stable lipase for omega-3 fatty acid ethyl ester production

We investigated transesterification activity of methanol-stable lipase from Staphylococcus haemolyticus L62. Lipase L62 transesterified olive oil and menhaden oil using various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. Based on the high stability of lipase L62 toward alcohols, high molar ratios (3:1 or 6:1) of alcohol to oil were employed in the reaction systems. Lipase L62 produced fatty acid esters efficiently by one-step transesterification. Reaction conditions for production of fatty acid methyl esters (FAMEs) and omega-3 fatty acid ethyl esters were further optimized. Lipase L62 produced those esters efficiently when 30–40 % water content was present in the reaction medium. The lipase L62 conversion yield was as high as 92 % for FAME and omega-3 ethyl ester production. These results indicate that lipase L62 is a suitable catalyst for producing an omega-3 ethyl ester concentrate in the pharmaceutical industry as well as for producing biodiesel in the bio-refinery industry.

Biodiesel production from microalgae oil catalyzed by a recombinant lipase

A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production.

Enhancing fatty acid ethyl ester production in Saccharomyces cerevisiae through metabolic engineering and medium optimization.

Biodiesels in the form of fatty acyl ethyl esters (FAEEs) are a promising next generation biofuel due to their chemical properties and compatibility with existing infrastructure. It has recently been shown that expression of a bacterial acyl-transferase in the established industrial workhorse Saccharomyces cerevisiae can lead to production of FAEEs by condensation of fatty acyl-CoAs and ethanol. In contrast to recent strategies to produce FAEEs in S. cerevisiae through manipulation of de novo fatty acid biosynthesis or a series of arduous genetic manipulations, we introduced a novel genetic background, which is comparable in titer to previous reports with a fraction of the genetic disruption by aiming at increasing the fatty acyl-CoA pools. In addition, we combined metabolic engineering with modification of culture conditions to produce a maximum titer of over 25 mg/L FAEEs, a 40% improvement over previous reports and a 17-fold improvement over our initial characterizations. Biotechnol. Bioeng. 2014;111: 2200-2208. © 2014 Wiley Periodicals, Inc.

A comparative study of direct transesterification of camelina oil under supercritical methanol, ethanol and 1-butanol conditions

Transesterifications of camelina oil under supercritical methanol, ethanol, and 1-butanol conditions are investigated and compared to find the suitable alcohols for the esters conversion process. The factors affecting the yields of fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), and fatty acid butyl eaters (FABE), such as reaction temperature and time, are studied and discussed in detail. The increase of the reaction temperature and time is proved to be favorable to the production of FAME, FAEE, and FABE initially since the transesterification is a reversible, endothermal reaction; however, the persistent increase of reaction temperature and time will decrease the yield of the Camelina sativa biofuels due to their thermal instability. The composition and thermal stability of biodiesel produced under various supercritical alcohol conditions are characterized by gas chromatography-mass spectrometry and thermogravimetric analysis methods. The saturated degree of fatty acid esters in C. sativa oil decreases with the increase of the alkyl chain of the alcohols from methanol, ethanol, to 1-butanol. The physical properties of biodiesels including the specific gravity, viscosity, calorific value, cold properties, and cetane number are tested and compared with the American Society for Testing and Materials (ASTM) standards. According to the results, the specific gravity and the viscosity of the C. sativa biodiesels are comparable to the ASTM standards. The calorific value, cetane number, and cold properties of biodiesel are improved with the increase of the length of the carbon chain of alcohols, while they decrease with the increase of unsaturated degree of compositions of biodiesels.

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

BackgroundDue to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors.ResultsHere, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408 ± 270 μg FAEE gCDW−1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100 ± 509 and 4670 ± 379 μg FAEE gCDW−1, an up to 1.7-fold improvement.ConclusionTwo different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid ethyl esters, an advanced biofuel, by eliminating non-essential fatty acid utilization pathways

Microbial production of fatty acid derived chemicals and fuels is currently of great interest due to the limited resources and increasing prices of petroleum and petroleum-based products. The development of Saccharomyces cerevisiae as a fatty acid ethyl ester (FAEE) cell factory would represent an opportunity for biodiesel production due to its successful history in the biotechnology area. However, fatty acid (FA) biosynthesis is highly regulated and usually not high enough for developing an efficient production process. In S. cerevisiae, FAs are degraded by β-oxidation and a large fraction is utilized to synthesize steryl esters (SEs) and triacylglycerols (TAGs), which are not essential for the cell. Here, by eliminating non-essential FA utilization pathways, we developed a metabolic engineering strategy resulting in a S. cerevisiae strain that can overproduce FAs and in turn use these for producing FAEEs (biodiesel). Compared to the wild-type, there is an about 3-fold increase in free FA content in a strain devoid of both TAG and SE formation, a 4-fold increase in free FA content in a strain that is incapable of β-oxidation, and a 5-fold increase of free FAs in a strain lacking all of these non-essential FA utilization pathways. It is also demonstrated that there are similar positive effects on FAEE production in these deletion strains. The highest production of FAEEs is 17.2mg/l in the strain in which all these pathways were blocked. The results of this study serve as a basis for further strategies to improve the production of FA derivatives in S. cerevisiae.

Fatty acid ethyl esters production in aqueous phase by the oleaginous yeast Rhodosporidium toruloides

Fatty acid ethyl esters (FAEEs) are attractive biofuel molecules. Conventional FAEEs production process uses triglycerides and ethanol as feedstocks and is sensitive to water contents. In this work, we show that the oleaginous yeast Rhodosporidium toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. Up to 73% of cellular neutral glycerides could be converted into FAEEs when lipid-rich cells were incubated for 84 h at 35°C, pH 6.0 in a broth containing 10 vol% ethanol. It was found that neutral glycerides were first hydrolyzed to free fatty acids followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources.

Microbial recycling of glycerol to biodiesel

The sustainable supply of lipids is the bottleneck for current biodiesel production. Here microbial recycling of glycerol, byproduct of biodiesel production to biodiesel in engineered Escherichia coli strains was reported. The KC3 strain with capability of producing fatty acid ethyl esters (FAEEs) from glucose was used as a starting strain to optimize fermentation conditions when using glycerol as sole carbon source. The YL15 strain overexpressing double copies of atfA gene displayed 1.7-fold increase of FAEE productivity compared to the KC3 strain. The titer of FAEE in YL15 strain reached to 813 mg L(-1) in minimum medium using glycerol as sole carbon source under optimized fermentation conditions. The titer of glycerol-based FAEE production can be significantly increased by both genetic modifications and fermentation optimization. Microbial recycling of glycerol to biodiesel expands carbon sources for biodiesel production.

Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.

Lipids from heterotrophic microbes: advances in metabolism research

Heterotrophic oleaginous microorganisms are capable of producing over 20% of their weight in single cell oils (SCOs) composed of triacylglycerols (TAGs). These TAGs contain fatty acids, such as palmitic, stearic and oleic acids, that are well-suited for biodiesel applications. Although some of these microbes are able to accumulate SCOs while growing on inexpensive agro-industrial biomass, the competition with plant oil resources means that a significant increase in productivity is desired. The present review aims to summarize recent details in lipid metabolism research and engineering (e.g. direct fatty acid ethyl ester production), as well as culture condition optimization and innovations, such as solid-state or semi-solid-state fermentation, that can all contribute to higher productivity and further advancement of the field.

Fatty acid ethyl esters production using a non-commercial lipase in pressurized propane medium

The objective of this work is to investigate the production of fatty acid ethyl esters from soybean oil in compressed propane using a non-commercial lipase from Yarrowia lipolytica and two commercial ones as catalysts, Amano PS and Amano AY30. The experiments were performed in the temperature range of 35-65 °C. at 50 bar, enzyme concentration of 5 wt%, oil to ethanol molar ratio of 1:6 and 1:9, and solvent to substrates mass ratio of 2:1 and 4:1. The results indicated that low reaction conversions were generally obtained with the use of commercial and non-commercial lipases in pressurized propane medium. On the other hand, the aspects of low solvent to substrates mass ratio and mild temperature and pressure operating conditions used to produce ethyl esters justify further investigations to improve reaction yields.

Ethanol Contamination Leads to Fatty Acid Ethyl Esters in Hair Samples

The diagnosis of alcoholism is a topical subject of discussion; in fact, many studies have been published on the determination of biochemical markers useful to this target. Fatty acid ethyl esters (FAEE) are minor metabolites of ethanol, and their usefulness has been demonstrated by their detection in hair using a headspace solid-phase microextraction-gas chromatographic-mass spectrometric technique. Environmental contamination in the analysis of drugs of abuse is a well-known focus of discussion between scientists. In the same way, interference from the surroundings could be hypothesized in FAEE detection. To assess the influence of ethanol contamination, an in vitro experiment was performed, leaving hair in an atmosphere saturated with ethanol vapors for 15 days. The spontaneous production of FAEE was demonstrated by analyzing hair day by day. In fact, we observed a constant increase of ethyl myristate, palmitate, and stearate that reached very high concentrations at the end of the investigation. Although the experiment was managed in a stressed way and could not represent real life, its purpose was to focus the attention of researchers on the problem of hair contamination that can occur, for example, with ethanol-containing cosmetics. Therefore, care in interpretation must be taken into account, especially with such a volatile molecule.

Transesterification of Fish Oil to Produce Fatty Acid Ethyl Esters Using Ultrasonic Energy

AbstractThis study evaluated the production of fatty acid ethyl esters from fish oil using ultrasonic energy and alkaline catalysts dissolved in ethanol. The feasibility of fatty acid ethyl ester production was determined using an ultrasonic bath and probe, and between 0.5 and 1% KOH (added to the fish oil). Furthermore, factors such as ultrasonic device (bath and probe), catalyst (KOH and C2H5ONa), temperature (20 and 60 °C), and duration of exposure (10–90 min) were assessed. Sodium ethoxide was found to be a more efficient catalyst than KOH when transesterifying fish oil. Ultrasonic energy applied for greater than 30 min at 60 °C using 0.8% of C2H5ONa as a catalyst transesterified over 98% fish oil triglycerides to fatty acid ethyl esters. It is reasonable to conclude that the yield of fatty acid ethyl esters produced by applying ultrasonic energy to fish oil is related to the sonication time. Due to increases in the surface area contact between the reactants and the catalyst, ultrasonic energy has the potential to reduce the production time required by a conventional large‐scale commercial transesterification method that uses agitation as a way of mixing.

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.

FATTY ACID ETHYL ESTERS (FAEE); COMPARATIVE ACCUMULATION IN HUMAN AND GUINEA PIG HAIR AS A BIOMARKER FOR PRENATAL ALCOHOL EXPOSURE

To compare the incorporation rate (ICR) of fatty acid ethyl esters (FAEE) in hair between guinea pigs and humans, and to assess the relationship between ethanol exposure and FAEE concentrations in hair. Published data from pregnant guinea pigs, including maximum blood ethanol concentration (BEC), dosage regimen, and total hair FAEE concentration, were compared with published data from alcoholic patients, where dose of ethanol consumed and total hair FAEE concentration were reported. Mean values of ethanol Vmax for pregnant guinea pigs and humans were obtained from published data (26.2 and 24 mg/dl/h, respectively). Total and individual FAEE ICRs, defined as the ratio of hair FAEE to the area under the BEC-time curve (total systemic ethanol exposure), were found to be on average an order of magnitude lower in the guinea pig than in the human. The profiles of ester incorporation also differed slightly between species, with ethyl stearate being highly incorporated in guinea pig hair and less so in human hair. The results may reflect in the human greater FAEE production, greater FAEE deposition in hair, slower FAEE catabolism, differential sebum production and composition, or a combination thereof. Also, ethyl oleate was found to correlate with total systemic ethanol exposure for both guinea pigs and humans, correlation coefficients equalling 0.67 (P < 0.05) and 0.49 (P < 0.05), respectively. No other ethyl esters, nor total FAEE, were found to correlate with systemic ethanol exposure. When extrapolating FAEE concentrations in hair from guinea pigs to humans, an order of magnitude difference should be considered, with humans incorporating more FAEE per unit of ethanol exposure. Also, the results suggest caution should be taken when interpreting values of single esters because of their differential incorporation among species. Lastly, our findings suggest ethyl oleate may be of keen interest in FAEE hair analysis, particularly across species.