Production Of Ethyl Esters Research Articles

Application of Spectrogel clay as a support for immobilization of lipase from Burkholderia cepacia

AbstractThe interest in maximizing the production of ethyl esters in a sustainable way and with lower energy costs has increased the use of immobilized enzymes as catalysts. This study aimed to apply the commercial clay Spectrogel® as a support for the immobilization of lipase from Burkholderia cepacia by adsorption and covalent bonding methods. The immobilizations were carried out using a 23 factorial design to study the effects of the activity offered (U g−1), pH, and the molar concentration of the enzyme solution buffer (mol L−1) on the enzyme activity obtained (U g−1). From the statistical analysis of the results, the best conditions for immobilization were pH 7.0 and 0.1 mol L−1 for both tested immobilization methods, with the best offered activity being 5709 and 7600 U g−1. The activities obtained were 1219.81 ± 7.51 and 1274.89 ± 14.99 U g−1 for adsorption and covalent bonding, respectively. The biocatalysts exhibited protein leaching of 33.55 ± 1.08% and 19.44 ± 2.43% when immobilized by adsorption and covalent bonding, respectively. The optimal activity temperature and thermal stability were obtained at 40°C. Additionally, the immobilization of lipase in Spectrogel® by both methods was efficient, showing higher thermal stability than the free enzyme. Thus, this work contributed scientifically to the development of a new and economical biocatalyst for ethyl ester production.

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Characteristics and Functions of Dominant Yeasts Together with Their Applications during Strong-Flavor Baijiu Brewing.

Yeasts are pivotal brewing microbes that are associated with the flavor and quality of Chinese baijiu, yet research on dominant yeasts in strong-flavor baijiu brewing remains limited. In this study, Saccharomyces cerevisiae, Pichia kudriavzevii, and Kazachstania bulderi were identified as predominated yeasts in strong-flavor baijiu. Each strain showed distinct characteristics in ethanol resistance, thermal tolerance, and lactic acid tolerance, severally. S. cerevisiae FJ1-2 excelled in ethanol and ethyl ester production, P. kudriavzevii FJ1-1 in ethyl acetate, and K. bulderi FJ1-3 in lactic acid generation. Subsequently, the reinforced Fuqu of each yeast were severally prepared for application in baijiu brewing to verify their functions. Results revealed that the relative abundance of fortified yeast in each group rose. Pichia, Kazachstania, and Saccharomyces emerged as the core microbe for each group, respectively, by co-occurrence network analysis, influencing the microbiota to regulate flavor substances. In short, P. kudriavzevii FJ1-1 enhanced ethyl acetate. K. bulderi FJ1-3 improved ethyl caproate production and decreased levels of ethyl acetate and higher alcohols by modulating yeast community between Pichia and Saccharomyces. This is a systematic endeavor to study the functions of yeasts of strong-flavor baijiu, providing a solid basis for improving baijiu quality.

Impact of sequential inoculation timing on the quality of wine fermented by indigenous Lachancea thermotolerans and Saccharomyces cerevisiae

Lack of acidity in wine is one of the main challenges in warm wine-producing regions. The non-Saccharomyces yeast, Lachancea thermotolerans (LT), is well known for its great potential in wine acidification through the production of lactic acid. In this study, an indigenous LT strain was sequentially inoculated with S. cerevisiae (SC). The influence of the timing of SC inoculation on wine quality was extensively studied. Inoculating SC 72 h after the inoculation of LT delayed the peak of fermentation rate, but had no significant influence on the duration of fermentation. The late inoculation (72 h) of SC effectively increased the production of lactic acid, which consequently increased the total wine acidity and decreased the pH. Higher levels of esters were also achieved by the late inoculation. Pilot-scale fermentations were carried out with both Chardonnay and Cabernet Sauvignon to verify the effect of late inoculation. Lactic acid produced in sequential fermentation was 2.73 g/L for Chardonnay wine and 5.35 g/L for Cabernet Sauvignon wine, and the production of ethyl esters was also enhanced. Overall, the results demonstrated that the late inoculation of SC in sequential fermentation with LT improved wine quality by increasing acidity and producing more balanced and aromatic wines.

The Physicochemical Basis for the Production of Rapeseed Oil Fatty Acid Esters in a Plug Flow Reactor

This article describes the results of a comprehensive comparative study of the production of fatty acid ethyl esters (FAEEs) for use as biodiesel in perfect mixing reactors (PMRs) and plug flow reactors (PFRs). The products obtained on a laboratory scale at all stages of the separation and purification of the FAEE phase were analyzed using the FTIR, XRF and GC-MS methods. We compared distillation methods for the separation of stoichiometrically excessive ethanol from the reaction mixture. Neutralization methods with H2SO4 solution and carbonation with CO2 were applied for FAEE phase purification from the catalyst. Emulsions formed during the water flushing stage were analyzed via the optical microscopy method. The optimal conditions of stirring speed and temperature were selected to maintain a high level of FAEE–water phase contact area with minimum phase separation time. The efficiency of the carbonation method for catalyst neutralization in the FAEE phase has been proven, allowing us to consider this method as an alternative to the traditional acid neutralization method. According to the results of experimental studies, we have developed a new high-performance technological scheme for the production of fatty acid esters in PFRs. The synthesis of FAEEs in a stoichiometric excess of ethanol of about 1:50 allowed us to increase the reaction rate and productivity of the synthesis unit after the transition from a PMR to a PFR. The yield of the product amounted to 86.7%. The purified FAEE fraction complied with most EN14214 specifications.

Co-culture fermentation by Saccharomycopsis fibuligera and lactic acid bacteria improves bioactivity and aroma profile of wheat bran and the bran-containing Chinese steamed bread

Co-culture fermentation with yeast and lactic acid bacteria (LAB) exhibits advantages in improving the bioactivity and flavor of wheat bran compared to single-culture fermentation, showing application potentials in bran-containing Chinese steamed bread (CSB). To explore the effects of combination of yeast and different LAB on the bioactivity and flavor of fermented wheat bran, this study analyzed the physicochemical properties, phytate degradation capacity, antioxidant activities, and aroma profile of wheat bran treated with co-culture fermentation by Saccharomycopsis fibuligera and eight different species of LAB. Further, the phenolic acid composition, antioxidant activities, texture properties, aroma profile, and sensory quality of CSB containing fermented wheat bran were evaluated. The results revealed that co-culture fermentation brought about three types of volatile characteristics for wheat bran, including ester-feature, alcohol and acid-feature, and phenol-feature, and the representative strain combinations for these characteristics were S. fibuligera with Limosilactobacillus fermentum, Pediococcus pentosaceus, and Latilactobacillus curvatus, respectively. Co-culture fermentation by S. fibuligera and L. fermentum for 36 h promoted acidification with a phytate degradation rate reaching 51.70 %, and improved the production of volatile ethyl esters with a relative content of 58.47 % in wheat bran. Wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus for 36 h had high relative content of 4-ethylguaiacol at 52.81 %, and exhibited strong antioxidant activities, with ABTS•+ and DPPH• scavenging rates at 65.87 % and 69.41 %, respectively, and ferric reducing antioxidant power (FRAP) at 37.91 μmol/g. In addition, CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. fermentum showed a large specific volume, soft texture, and pleasant aroma, and received high sensory scores. CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus, with high contents of 4-ethylguaiacol, 4-vinylguaiacol, ferulic acid, vanillin, syringaldehyde, and protocatechualdehyde, demonstrated strong antioxidant activities. This study is beneficial to the comprehensive utilization of wheat bran resources and provides novel insights into the enhancement of functions and quality for CSB.

Differences in the Volatile Profile of Apple Cider Fermented with Schizosaccharomyces pombe and Schizosaccharomyces japonicus

In this study, two strains of Schizosaccharomyces pombe (NCAIM Y01474T and SBPS) and two strains of Schizosaccharomyces japonicus (DBVPG 6274T, M23B) were investigated for their capacity to ferment apple juice and influence the volatile compounds of cider compared to Saccharomyces cerevisiae EC1118. The ethanol tolerance and deacidification capacity of Schizosaccharomyces yeasts could make them potential substitutes for the commonly used S. cerevisiae starter cultures. Despite different time courses (10–30 d), all strains could complete the fermentation process, and Schizosaccharomyces strains reduced the concentration of malic acid in the apple juice. Results indicated that each yeast exerted a distinctive impact on the volatile profile of the apple cider, giving final products separated using a principal component analysis. The volatile composition of the cider exhibited significant differences in the concentration of alcohols, esters, and fatty acids. Particularly, the flocculant strain S. japonicus M23B increased the levels of ethyl acetate (315.44 ± 73.07 mg/L), isoamyl acetate (5.99 ± 0.13 mg/L), and isoamyl alcohol (24.77 ± 15.19 mg/L), while DBVPG 6274T incremented the levels of phenyl ethyl alcohol and methionol up to 6.19 ± 0.51 mg/L and 3.72 ± 0.71 mg/L, respectively. A large production of terpenes and ethyl esters (e.g., ethyl octanoate) was detected in the cider fermented by S. cerevisiae EC1118. This study demonstrates, for the first time, the possible application of S. japonicus in cider-making to provide products with distinctive aromatic notes”.

Decreasing acid value of fatty acid ethyl ester products using complex enzymes.

Recently, enzymatic method has been used to prepare biodiesel using various oils. But the high acid value of the biodiesel product using enzyme as a catalyst has been one issue. In this work, an attempt to reduce the acid value of fatty acid ethyl ester (FAEE) product to satisfy the specified requirement (AV ≤ 0.5mgKOH/g), a complex enzyme-catalyzed method was used for the ethanolysis of Semen Abutili seed oil (SASO) (AV = 5.5 ± 0.3mgKOH/g). The effects of various variables (constituents of complex enzyme, type and addition of water removal agent, time, temperature, enzyme addition load, substrate ratio) on the enzymatic reaction were investigated. The optimal reaction conditions were: 1% addition of liquid lipase Eversa® Transform 2.0% and 0.8% of enzyme dry powder CALB, reaction temperature 35°C, alcohol-oil ratio 9:1 (mol/mol), 0.8g/g of 4A-MS and reaction time 24h. Under the optimal reaction conditions, the FAEE yield was 90.8% ± 1.5% and its acid value was decreased from 12.0 ± 0.2mgKOH/g to 0.39 ± 0.10mgKOH/g. In further evaluating the feasibility of preparing FAEE from SASO, the FAEE products obtained under the optimal reaction conditions were purified and evaluated with reference to the ASTM D6751 standard for the main physicochemical indexes. The results obtained were in accordance with the requirements except for the oxidative stability.

Preparation of High-Purity Docosahexaenoic Acid Ethyl Ester from Algal Oil through Enzymatic Ethanolysis.

Docosahexaenoic acid plays a crucial role in infant brain function, and the market demand of high-purity docosahexaenoic acid is continuously increasing. The availability of docosahexaenoic acid in natural fish oil is limited, prompting the exploration of alternative sources like microalgae. For algal oil, enzymatic ethanolysis is preferred to chemical methods because the former is milder and can avoid docosahexaenoic acid oxidation. However, enzymatic methods have generally low yield due to the poor substrate-specificity of lipase to long-chain polyunsaturated fatty acids, affecting the yield and purity of docosahexaenoic acid. Therefore, we developed an efficient process to produce high-purity docosahexaenoic acid ethyl ester from algal oil, by screening lipases, optimizing enzymatic ethanolysis and applying molecular distillation. Lipase UM1 was the best lipase to produce ethyl ester from algal oil with the highest ethyl ester yield (95.41%). Meanwhile, it was a catalyst for the reaction of long-chain polyunsaturated fatty acids with ethanol. The fatty acid docosahexaenoic acid conversion rates exceeded 90%. After molecular distillation, a final product containing 96.52% ethyl ester was obtained with a docosahexaenoic acid content up to 80.11%. Our findings provide an highly effective enzymatic method for the production of high-purity docosahexaenoic acid ethyl esters, with potential commercial applications.

Biocatalysts Based on Immobilized Lipases for the Production of Fatty Acid Ethyl Esters: Enhancement of Activity through Ionic Additives and Ion Exchange Supports.

Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme-support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase®, immobilized in commercial supports like Lewatit®, Purolite® and Q-Sepharose®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods.

Higher alcohols as cosolvents of mixtures of ethanol and soybean oil: Solubility and physical properties of ternary systems

Mixtures of soybean oil (SO) and ethanol (EtOH) are of interest for several applications, such as the addition of the mixtures to diesel, the production of ethyl esters (ethyl biodiesel) from SO and the extraction of vegetable oils with a renewable solvent. However, the solubility between the components of this mixture is limited, which may hinder the possible applications. Thus, the present study aimed to determine the solubility of mixtures composed of SO and EtOH with the addition of cosolvents, n-butanol (nBut) and isoamyl alcohol (IAA), which are higher alcohols present in fusel oil, a coproduct of the production of EtOH. The physical properties of the ternary mixtures were also determined, which are necessary data for proposing the use of these renewable inputs. The regions of phase separation for the systems were determined using the cloud point method at constant temperature (25, 40 and 55 °C). The physical properties, density (ρ, kg·m−3) and dynamic viscosity (η, mPa·s) were experimentally determined for the binary (EtOH + nBut and EtOH + IAA, temperatures from 20.0 to 70.0 ± 0.1 °C) and ternary (SO + EtOH + nBut and SO + EtOH + IAA, 25.0, 40.0 and 55.0 ± 0.1 °C) mixtures. The experimental ρ data were described mathematically by the simple mixing rule (SMR) and the Jouyban-Acree (JA) model. For the description of η, the modified Kay rule (MKR), the Kendall and Monroe (KM) model, the Grunberg-Nissan (GN) model and the JA model were considered. Regarding the binary mixtures, the SMR and the GN model showed the lowest average relative deviation (ARD) values, 0 to 0.31% for ρ and 0.21 to 5% for η. For the ternary systems, the JA model best described the behavior of both physical properties (ARD from 0.02 to 4.51% for ρ and from 0.85 to 7.14% for η). Both cosolvents enabled the reduction of the immiscibility region, with similar performance in the evaluated temperature range.

Biocatalyzed Transesterification of Waste Cooking Oil for Biodiesel Production Using Lipase from the Amazonian Fungus Endomelanconiopsis endophytica

The demand for biodiesel worldwide is skyrocketing as the need to replace fossil diesel with renewable energy sources becomes increasingly pressing. In this context, biocatalysis is emerging as an environmentally friendly and highly efficient alternative to chemical catalysis. When combined with the utilization of waste materials, it has the potential to make the process of biodiesel production sustainable. In the study, the potential of an extract rich in lipase produced by an Amazonian endophytic fungus as a biocatalyst in the transesterification of waste cooking oil for biodiesel production has been systematically investigated. The fungus Endomelanconiopsis endophytica exhibited an enzyme production of 11,262 U/mL after 120 h of cultivation. The lipolytic extract demonstrated its highest catalytic activity at 40 °C and a pH of 5.5. Using soybean oil and frying residue as raw materials, biodiesel was produced through biocatalytic transesterification, and yields of 91% and 89% (wt.), respectively, were achieved. By evaluating the process parameters, a maximum biodiesel yield of 90% was achieved using ethanol at a ratio of 3:1 ratio within 120 min. The experimental results demonstrate the feasibility and sustainability of applying a fungal enzymatic extract as a biocatalyst in the production of ethyl esters using waste cooking oil as a raw material.

Biocatalysts Based on Immobilized Lipases for the Production of Ethyl Esters of Fatty Acids including Bioactive Gamma-Linolenic Acid from Borage Oil

In the present work, borage oil (Borago officinalis) was used as the main source of gamma linolenic acid (GLA) to obtain ethyl esters by enzymatic ethanolysis using immobilized enzymes for its application in the food industry. Commercial Thermomyces lanuginosus lipase (TLL) was compared to chemical ethanolysis in alkaline medium. In addition, TLL was immobilized by adsorption on hydrophobic porous support (Octadecyl-Sepabeads®) to compare the results. Fatty acid ethyl ester (FAEE) yields of both reactions were compared under the same conditions (25 °C and 200 rpm) and analyzed by GC-MS. Moreover, the conversion yield for borage oil ethanolysis catalyzed by TLL immobilized on C18-Sepabeads® supports was similar to the chemical pathway (93.4% and 99.5%, respectively). When this biocatalyst was used in a solvent-free system (at 40 °C and 200 rpm), it was possible to obtain a high FAEE yield of 84.3% in the first 24 h of reaction. Furthermore, it was possible to re-use the immobilized biocatalyst for the performance of five reaction cycles maintaining 68% of its initial activity. Thus, the use of immobilized enzymes in solvent-free systems is an eco-friendly alternative to obtain GLA ethyl esters for its possible application in cosmetics and food.

Ethyl ester production with immobilized lipase in tin modified SBA‐15: Effect of the support characteristic and reaction conditions

AbstractThis study investigated the use of immobilized lipase in SBA‐15 and pore‐expanded SBA‐15 surface modified with tin, denoted as SnS23B (mean pore size of 21.6 nm) and SnS8B (with mean pore size of 9.6 nm), respectively, to produce ethyl esters. The effects of various reaction parameters, including biocatalyst type, oil (refined and degummed soybean oil), water addition, oil‐to‐ethanol molar ratio, immobilized lipase concentration, agitation speed, reaction temperature, and glycerol addition, were examined. The optimal conditions were found to be the use of SnS23B as the biocatalyst, degummed soybean oil as the substrate, 1% (w/w oil) water, 1:3 oil‐to‐ethanol molar ratio, 5% (w/w oil) immobilized lipase, 800 rpm agitation speed, and 25°C reaction temperature. The presence of glycerol negatively impacted the reaction. Under the optimal conditions, the mass percent of ethyl ester obtained was 98.2% ± 0.16. The immobilized lipase showed stability, as no lipase activity was detected upon removing the biocatalyst from the reaction. However, the biocatalyst lost 50% of its activity after 10 successive reactions, likely due to thermal deactivation and lipase inhibition caused be the adsorption of glycerol and other chemical species. Attenuated total reflectance analysis supported this hypothesis.

Synthesis of acidic modified UiO‐66 by concentrated sulphation form and its application in the production of fatty acids ethyl esters

AbstractIn this article, UiO‐66 metal–organic frameworks containing structural modifications were synthesized and characterized to increase acidic surface behaviour for application in heterogeneous acid catalysis to produce fatty acid ethyl esters (FAEE) from degummed soybean oil. The solvothermal route synthesized UiO‐66, and the post‐synthesis acid modifications were done by sulphation reaction in strong acidic medium. The synthesized materials were characterized by physicochemical techniques such as X‐ray diffraction (XRD), C‐ray fluorescence (XRF), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectrometer (FTIR), and N2 physisorption, observing a strong structural modification of the original UiO‐66, but no signal of sulphated ZrO2 was presented. However, the acidic characteristics on the material's surface were increased. A central compound centred on face 23 experiments design was carried out to evaluate three essential reaction variables: temperature, time, and alcohol/oil ratio, obtaining an optimized fit for the UiO‐66‐S35 of temperature at 140°C, time of 3 h, and molar ratio of 16. The optimized statistical adjustment was applied in the reuse cycles assays in three catalyst cycles, reaching the maximum yield of 95% in FAEE, keeping the catalyst active at the end of 3 cycles.

Investigating Catalyzed Transesterification for Castor Oil Ethyl Ester Production: A Promising Biodiesel Substitute

Abstract: This study investigates the catalysed transesterification method for producing castor oil ethyl ester (COEE), a biodiesel substitute. The research looks at a number of variables that impact the reaction, including temperature, the ratio of alcohol to oil, the concentration of the catalyst, the amount of mixing, and the purity of the reactant. In the experimental setting, biodiesel is created in a lab and tested on a diesel engine. The outcomes show that the transesterification procedure successfully lowers castor oil's viscosity and raises the fuel's cetane number. According to engine testing, a 20% blend of COEE demonstrates promising performance with conventional diesel fuel-like energy consumption and thermal efficiency. The study emphasises COEE's potential as a superior biodiesel alternative with viability.

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.

Impact of Assimilable Nitrogen Supplementation on Saccharomyces cerevisiae Metabolic Response and Aromatic Profile of Moschofilero Wine.

The concentration of nitrogen in must is critical to yeast fermentation efficiency and wine aroma profile. The present work determined the effect of the amount of yeast assimilable nitrogen (YAN) on fermentation kinetics, aroma production, and gene expression patterns of the wine yeast Saccharomyces cerevisiae. Fermentations were performed under two different YAN concentrations of must. Acetate esters, linalool, and nerol appeared to be clearly affected by the different YAN levels. Real-time-PCR results revealed that the genes involved in ethyl and acetate esters production recorded, in general, higher transcript levels under high nitrogen supplementation. In addition, an up-regulation of the BGL2 and EXG1 genes, which are related to terpenes production, was observed in the case of high nitrogen content and it is well corresponded to the terpenol concentration found. Our study revealed the impact of nitrogen supplementation on yeast metabolism and its importance to adjust wine's aromatic composition and sensory profile.

Ethyl hexanoate rich stream from grape pomace: A viable route to obtain fine chemicals from agro by-products

A novel viable route for obtaining ethyl hexanoate-rich streams from grape pomace, an agro by-product generated from the winery industry, was investigated. Highly concentrated hexanoic acid (87 wt%) was efficiently produced through the chain elongation fermentation of red and white grape pomace, easily separated, recovered and reacted with ethanol by using AlCl3·6H2O as a catalyst. Under the optimised mild reactive conditions (molar ratio acid:ethanol:catalyst of 1:2:0.1, 5 h, 348 K), a high conversion equal to 93.5 % was achieved in a single step. AlCl3·6H2O was very effective in catalysis, playing an additional key role in the separation of products, shifting the equilibrium of the reaction and making products' recovery easier. After the reaction cycle, the catalyst was easily recoverable and reusable without losing effectiveness. According to this simple layout of processes, including the anaerobic digestion of the residual part of grape pomace, a specific production cost of 0.935 €·kg−1 was calculated related to the final products. This proposed approach could represent a promising green and sustainable route to produce ethyl esters of volatile fatty acids from agro by-products.

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO

BackgroundEthyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates.ResultsWe engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium.ConclusionsThis work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future.