[Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography].

The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R2)>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae.

Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. KEY POINTS: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.

Metabolic engineering Yarrowia lipolytica for a dual biocatalytic system to produce fatty acid ethyl esters from renewable feedstock in situ and in one pot.

Given the grave concerns over increasing consumption of petroleum resources and dramatic environmental changes arising from carbon dioxide emissions worldwide, microbial biosynthesis of fatty acid ethyl ester (FAEE) biofuels as renewable and sustainable replacements for petroleum-based fuels has attracted much attention. As one of the most important microbial chassis, the nonconventional oleaginous yeast Yarrowia lipolytica has emerged as a paradigm organism for the production of several advanced biofuels and chemicals. Here, we report the engineering of Y. lipolytica for use as an efficient dual biocatalytic system for in situ and one-pot production of FAEEs from renewable feedstock. Compared to glucose with 5.7% (w/w) conversion rate to FAEEs, sunflower seed oil in the culture medium was efficiently used to generate FAEEs with 84% (w/w) conversion rate to FAEEs by the engineered Y. lipolytica strain GQY20 that demonstrates an optimized intercellular heterologous FAEE synthesis pathway. In particular, the titer of extracellular FAEEs from sunflower seed oil reached 9.9 g/L, 10.9-fold higher than that with glucose as a carbon source. An efficient dual biocatalytic system combining ex vivo and strengthened in vitro FAEE production routes was constructed by overexpression of a lipase (Lip2) variant in the background strain GQY20, which further increased FAEEs levels to 13.5 g/L. Notably, deleting the ethanol metabolism pathway had minimal impact on FAEE production. Finally, waste cooking oil, a low-cost oil-based substance, was used as a carbon source for FAEE production in the Y. lipolytica dual biocatalytic system, resulting in production of 12.5 g/L FAEEs. Thus, the developed system represents a promising green and sustainable process for efficient biodiesel production. KEY POINTS: • FAEEs were produced by engineered Yarrowia lipolytica. • A Lip2 variant was overexpressed in the yeast to create a dual biocatalytic system. • Waste cooking oil as a substrate resulted in a high titer of 12.5 g/L FAEEs.

Applications of an electronic nose in the prediction of oxidative stability of stored biodiesel derived from soybean and waste cooking oil.

Waste cooking oil (WCO) is a valuable feedstock for the synthesis of biodiesel but the product exhibits poor oxidative stability. Techniques available for assessing this parameter are generally expensive and time-consuming, hence the purpose of this study was to develop and validate a rapid and reliable predictive system based on signals from the sensors of a commercial hand-held e-nose instrument. Biodiesels were synthesized from soybean oil and six samples of WCO, and their physicochemical characteristics and oxidative stabilities determined before and after storage in different types of containers for 30 or 60 days at room temperature or 43 °C. Linear regression models were constructed based on principal component analysis of the signals generated by all 32 e-nose sensors and stochastic modeling of signal profiles from individual sensors. The regression model with principal components as predictors was unable to explain the oxidative stability of biodiesels, while the regression model with stochastic parameters (combining signals from 11 sensors) as predictors showed an excellent goodness of fit (R2 = 0.91) with a 45-sample training set and a good quality of prediction (R2 = 0.84) with a 18-sample validation set. The proposed e-nose system was shown to be accurate and efficient and could be used to advantage by producers/distributors of biodiesel in the assessment fuel quality.

Fatty acid ethyl esters in meconium and substance use in adolescence.

Prenatal alcohol exposure (PAE) continues to be a serious public health problem, yet no reliable clinical tools are available for assessing levels of drinking during pregnancy. Fatty acid ethyl esters (FAEEs), the nonoxidative metabolites of ethanol measured in meconium, are potential biomarkers to quantify the level of PAE. The association between the concentrations of FAEEs from meconium and adolescent substance use and related problems was examined in a prospective birth-cohort of adolescents exposed to alcohol and drugs in utero. FAEEs were quantified with gas chromatography via a flame ionization detector. Meconium was analyzed for FAEEs in 216 newborns; 183 of them (81 boys, 102 girls) were assessed at age 15 for alcohol, tobacco, and marijuana use using biologic assays and self-report. Substance use problems were assessed using the Problem Oriented Screening Instrument for Teenagers. Findings from multivariable logistic regression analyses indicated that, after controlling for other prenatal drug exposure and covariates, higher concentrations of FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) were related to a greater likelihood of marijuana use and experiencing substance use problems, but not tobacco or alcohol use, at age 15. Elevated levels of FAEEs in meconium may be promising markers for PAE, identifying newborns at risk for early substance use and developing substance use problems.

Valorization of sewage sludge via non-catalytic transesterification.

To seek a way to valorize sewage sludge (SS), it was chosen as a raw material for biodiesel production. As such, non-catalytic transesterification of dried SS was carried out, to enhance its value. Note that picking a waste material such as SS as an inexpensive lipid feedstock for biodiesel production, without lipid extraction, greatly increases the economic viability of biodiesel. Also, to enhance biodiesel sustainability, ethanol (EtOH) was employed as the acyl acceptor, in this study, and this was experimentally justified by results showing that employing EtOH as an acyl acceptor provided an effective means for compensating for the lower heating value arising from the large amount of palmitic (C16) acid in SS. This study experimentally proved that the fatty acid ethyl ester (FAEE) yield at 380 °C reached up to 13.33 wt% of dried SS. Given that the lipid content of dried SS is 14.01 ±â€¯0.64 wt%, the FAEE yield of 13.33 wt% implied that 95.14 wt% of lipid in dried SS had been converted into FAEEs. The introduced SS valorization in this study offered an excellent opportunity to address diverse environmental hazards arising from SS and associated emerging contaminants. Given that the optimal temperature for the non-catalytic conversion for biodiesel production from SS was found to be 380 °C, emerging contaminants, such as microplastics and antimicrobials, were simultaneously degraded, due to their inferior thermal stabilities. Lastly, considering that the introduced biodiesel conversion process is thermally induced, the SS reside after the biodiesel conversion process can be further used in thermo-chemical processes as raw materials for gasification and pyrolysis (future work).

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated ß-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

Ultrasound-assisted in-situ transesterification of wet Aurantiochytrium sp. KRS 101 using potassium carbonate.

A new in-situ transesterification method was developed for wet biomass: K2CO3 was used as an alkaline catalyst and, Aurantiochytrium sp. KRS 101 as oleaginous DHA-producing microalgae. It was found that the presence of water greatly impaired the overall efficiency even with the powerful catalyst that had worked surpassingly well with dry biomass, and thus a mechanical aid like ultrasonication was needed to make advantage of full potential of the alkaline catalyst. The total fatty acid ethyl ester (FAEE) recovery yield of 94.6% was achieved with sonication at 100 g/L of biomass (40% moisture), 3% of K2CO3, 70 °C and 30 min. All these suggest that the ultrasound assisted in-situ transesterification can offer a feasible means for FAEE recovery and it was so by way of overcoming the physical limitation of mass transfer caused the presence of water and providing effective contacts between reactants.

Trastornos del espectro alcohólico fetal: biomarcadores del consumo de etanol durante la gestación / Fetal alcohol spectrum disorders: biomarkers of ethanol consumption during pregnancy / Perturbações do espectro do alcoolismo fetal: biomarcadores de consumo de etanol durante a gravidez

El consumo de alcohol durante la gestación está asociado con un amplio espectro de efectos adversos conocidos como trastornos del espectro alcohólico fetal (TEAF). Así es como a través del dosaje de los esteres etílicos de ácidos grasos (FAEEs) en meconio, como biomarcadores de exposición prenatal al consumo de etanol, nos brinda una herramienta importantísima en el screening de los recién nacidos, facilitando la confi rmación del diagnóstico y la intervención clínica adecuada en los individuos afectados por esta problemática con una mejora en las expectativas y calidad de vida. El síndrome alcohólico fetal (SAF) representa el clásico síndrome y la manifestación más grave inducido por el alcohol de forma repetida durante el embarazo. Dado el carácter heterogéneo de las alteraciones clínicas presentes en los recién nacidos expuestos a etanol, la mayoría de los cuales no son específi cos, conlleva a un diagnóstico del TEAF extremadamente difícil.

Alcohol consumption during pregnancy is associated with a wide range of adverse effects known as fetal alcohol spectrum disorders (FASDs). Through the measurement of Fatty Acid Ethyl Esters (FAEEs) in meconium, as biomarkers of prenatal alcohol exposure, we have a very important tool in the screening of newborns, facilitating the confi rmation of diagnosis and adequate clinical intervention in individuals affected by this problem with improvements in life quality and expectancy. Fetal Alcohol Syndrome (FAS) represents the classic syndrome and the most serious condition is caused by repetitive alcohol consumption during pregnancy. Due to the heterogeneous characteristics of clinical alterations of newborns exposed to ethanol, most of which are not specifi c, diagnosis of FASDs is extremely diffi cult.

O consumo de álcool durante a gravidez é associado a um amplo espectro de efeitos adversos conhecidos, tais como transtornos do espectro do alcoolismo fetal (TEAF). É assim como através da dosagem dos ésteres etílicos de ácidos graxos (FAEEs) em mecô- nio, como biomarcadores de exposição pré-natal ao consumo de etanol, fornece uma ferramenta importantíssima na triagem de recém-nascidos, aprimorando a confi rmação do diagnóstico e a intervenção clínica adequada em indivíduos afectados por este problema com uma melhoria das expectativas e qualidade de vida. Síndrome alcoólica fetal (FAS) representa a síndrome clássica e a mais grave manifestação induzida frequentemente pelo álcool durante a gravidez. Dada a heterogeneidade das alterações clínicas em recémnascidos expostos ao etanol, a maioria das quais são não-específi cas, esta situação gera um diagnóstico de TEAF extremamente difícil.

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.

Prenatal alcohol exposure and its repercussion on newborns.

BACKGROUND: Alcohol consumption during pregnancy, even when moderate, implies a risk of impaired neurodevelopment, physical impairments and malformations. Its early identification is essential for establishing preventive measures to diminish disabilities among newborns. METHODS: To determine the frequency of consumption of substance use in pregnant women, we have used the techniques of gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry to detect drugs and markers of chronic consumption of alcohol in meconium. We performed a prospective study during a period of 10 months among 110 infants in our hospital, assessing anthropometry, neuromuscular development and determination of toxic substances in urine and meconium. Furthermore, meconium analysis identified fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg). We also conducted a survey regarding the obstetric history, toxic habits, and employment status of the mothers. RESULTS: According to early detection markers analyzed in meconium (FAEE >1000 ng/g and/or Etg >50 ng/g meconium), 34.65% of pregnant women consumed alcohol during pregnancy, and 17% were positive for both markers. Within the positive cases, 50% of those exceeding a FAEE's value of 5000 ng/g in meconium had low birth-weight children. Only 5/110 mothers (4.5%) admitted to occasional alcohol consumption during pregnancy. Nobody admitted to frequent intake. The cocaine test was positive in three cases; two of them were positive for alcohol as well. CONCLUSION: As expected, many screening devices do not accurately capture use during pregnancy and supplemental methods such as meconium analysis of biomarkers of chronic alcohol consumption may be warranted.

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.

A novel and robust recombinant Pichia pastoris yeast whole cell biocatalyst with intracellular overexpression of a Thermomyces lanuginosus lipase: preparation, characterization and application in biodiesel production.

A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84 h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73 U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production.