The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters

Fatty acid alkyl esters have broad applications in biofuels, lubricant formulas, paints, coatings, and cosmetics. Traditionally, these esters are mostly produced through unsustainable and energy-intensive processes. In contrast, microbial production of esters from renewable and sustainable feedstocks may provide a promising alternative and has attracted widespread attention in recent years. At present, yeasts are used as ideal hosts for producing such esters, due to their availability for high-density fermentation, resistance to phage infection, and tolerance against toxic inhibitors. Here, we summarize recent development on the biosynthesis of alkyl esters, including fatty acid ethyl esters (FAEEs), fatty acid short-branched chain alkyl esters (FASBEs), and wax esters (WEs) by various yeast cell factories. We focus mainly on the enzyme engineering strategies of critical wax ester synthases, and the pathway engineering strategies employed for the biosynthesis of various ester products. The bottlenecks that limit productivity and their potential solutions are also discussed in this review.

Oligocat: Oligoesters as Pseudo-Homogenous Catalysts for Biodiesel Synthesis

Biodiesel production from first-generation feedstock has shown a strong correlation with the increase in deforestation and the necessity of larger areas for land farming. Recent estimation from the European Federation for Transport and Environment evidenced that since the 2000s decade, an area equal to the Netherlands was deforested to supply global biodiesel demand, mainly originating from first-generation feedstock. Nevertheless, biodiesel is renewable, and it can be a greener source of energy than petroleum. A promising approach to make biodiesel independent from large areas of farming is to shift as much as possible the biodiesel production chain to second and third generations of feedstock. The second generation presents three main advantages, where it does not compete with the food industry, its commercial value is negligible, or none, and its usage as feedstock for biodiesel production reduces the overall waste disposal. In this manuscript, we present an oligomeric catalyst designed to be multi-functional for second-generation feedstock transesterification reactions, mainly focusing our efforts to optimize the conversion of tallow fat and sauteing oil to FAME and FAEE, applying our innovative catalyst. Named as Oligocat, our catalyst acts as a Brønsted-Lowry acid catalyst, providing protons to the reaction medium, and at the same time, with the course of the reaction, it sequesters glycerol molecules from the medium and changes its physical phase during the transesterification reaction. With this set of properties, Oligocat presents a pseudo-homogenous behavior, reducing the purification and separation steps of the biodiesel process production. Reaction conditions were optimized applying a 42 factorial planning. The output parameter evaluated was the conversion rate of triacylglycerol to mono alkyl esters, measured through gel permeation chromatography (GPC). After the optimization studies, a conversion yield of 96.7 (±1.9) wt% was achieved, which allows classifying the obtained mono alkyl esters as biodiesel by ASTM D6751 or EN 14214:2003. After applying the catalyst in three reaction cycles, Oligocat still presented a conversion rate above 96.5 wt% and as well an excellent recovery rate.

Advanced Methods for Hydroxylation of Vegetable Oils, Unsaturated Fatty Acids and Their Alkyl Esters

Vegetable oils and their derivatives have great potential as renewable and sustainable raw materials for the production of polyurethanes and bio-based polyols. For industry an important process is their modification. Chemical reactions that are carried out on vegetable oils and their derivatives are: transesterification, auto-oxidation, hydrogenation, epoxidation, hydroxylation, acrylation, isocyanation and others. One of the modifications are reactions performed on double bonds and/or carbonyl moieties of plants oils and their derivatives. These reactions result in products that are actively used as binders in coating materials due to their unique structural properties. In this manuscript, we describe important technological methods for the introduction of hydroxyl groups: opening of oxirane rings by nucleophilic reagents such as: water, alcohols, glycols, amino alcohols, carboxylic acids; direct hydroxylation of unsaturated bonds with carboxylic peracids in combination with hydrolysis of carboxyl groups and hydration; hydroformylation of unsaturated bonds with subsequent hydrogenation and alkoxylation; and ozonolysis of unsaturated bonds in combination with subsequent hydrogenation and alkoxylation.

Kinetic and Transesterification Properties of Lipase from Sprouted Melon (Cucumeropsis manni) Seeds

Crude lipase (acetone powder) was extracted from freshly sprouted melon seeds (Cucumeropsis manni). The activity, kinetic properties (effect of time, pH, and enzyme and substrate concentration, respectively) as well as the ability of the crude lipase to catalyze the production of methyl esters (biodiesel) were examined. The enzyme activity was determined using n-hexane as the solvent (1:2 v/w solvent: substrate ratio) and the transesterification product was analyzed by HPLC. A linear relationship was observed between reaction time and rate of lipolysis with the optimal activity at 2hr of incubation. Furthermore, the lipase was optimally active at acid pH 5 and lipolysis was achieved optimally when the amount of enzyme was 2.0g. Rate of lipolysis was observed to increase linearly at concentrations up to 5.0g of substrate above which a drop in the rate, with no apparent decrease in activity, was observed. The Km (6.25g) and Vmax (13.33%FFA/hr) were also determined. Analysis of the transesterification product yielded 0.61% alkyl ester, 0.81 %FFA, 93.17% TAG, 4.15% 1, 3-DAG and 1.26% 1, 2-DAG while transesterification efficiency was determined to be at 0.588%. Biodiesel (alkyl esters) prepared with the crude lipase was had a density of 0.872 g/mL while its cloud and pour points were 22°C and 12°C, respectively. The results from this research showed that an active lipase was isolated from sprouted melon seeds. However, the fuel properties of the biodiesel produced did not meet international transportation fuel standards. In order to be used industrially, better reaction conditions need to be established for the lipase.

Understanding of the Key Factors Determining the Activity and Selectivity of CuZn Catalysts in Hydrogenolysis of Alkyl Esters to Alcohols

CuZn catalysts are perspective catalysts for esters hydrogenolysis, but more knowledge is needed to optimize their catalytic performance. In this work, we consider the impact of CuZn catalysts composition on their structure, activity, selectivity, and stability in esters hydrogenolysis. Four catalysts with various Cu/Zn ratio were synthesized by a co-precipitation and characterized in as-prepared, calcined, reduced, and spent state by XRF, XRD, N2 physisorption, CO2-TPD, NH3-TPD, and N2O chemisorption. XRD data revealed the effect of the composition on the size of Cu and ZnO particles. The catalytic performance was investigated using an autoclave. All catalysts exhibited high methyl hexanoate conversion about 48–60% after 3 h but their activity and selectivity were found to be dependent on Cu/Zn ratio. The conversion of methyl hexanoate and hexyl hexanoate was compared to explain the observed product selectivity. Moreover, the catalysts stability was investigated in three consecutive reaction cycles and correlated with changes in the size of constituent particles. Moreover, when different esters were tested, a slight decrease in conversion and increase in alcohol selectivity with a growth in molecule size was observed. Obtained results allow making a conclusion about the optimal composition that provides the good performance of CuZn catalysts in ester hydrogenolysis.

Lactiplantibacillus plantarum Used as Single, Multiple, and Mixed Starter Combined with Candida boidinii for Table Olive Fermentations: Chemical, Textural, and Sensorial Characterization of Final Products

In this study, four different kinds of table olive fermentations belonging to Olea europaea L. Itrana cultivar were evaluated: A, spontaneous fermentation; B, fermentation with a single inoculum (Lactiplantibacillus plantarum B1); C, fermentation with multiple inoculum (L. plantarum B1 + L. plantarum B51 + L. plantarum B124, 1:1:1); and D, fermentation with mixed (bacterium + yeast) inoculum (L. plantarum B1 + Candida boidinii). This research focuses on the correlation between the different mixes of inoculations and their effect under the chemical, sensorial, and textural profiles in the final products (olives) for potential applications on table olive fermentation. During the fermentation, some specific parameters were monitored: chemical characterization of oil fraction (pigments, tocopherols, fatty acids, alkyl esters, and sterol composition), Texture Profile Analysis (TPA), determination of olive color, and sensory evaluation of the final products. The use of LAB starters (single and multiple inocula) compared to spontaneous process revealed a greater performance in preventing the spoilage process and in developing favorable physico-chemical conditions during the fermentation. In fact, the highest values of fatty acid alkyl esters were reached in spontaneous fermentation (~480 mg/kg in jar A). The presence of C. boidinii as inoculum in jar D was involved in table olive softening: the fermented olives showed the lowest values of the parameters related to consistence of fruit as hardness (~2300 g) and gumminess (~990 g) and high value of fatty acid methyl esters (~110 mg/kg).