Method for preparing a biofuel composition

Abstract

The present invention relates to a process for producing a FAGE-based biofuel composition, the process being characterized by an enzymatic transesterification of fatty acid alkyl esters with glycerol formal. The invention is also directed to the biofuel composition obtained by said process as well as to the uses thereof.

Description

[0001] METHOD FOR PREPARING A BIOFUEL COMPOSITION TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of biofuel production. More specifically, the present invention relates to a process for producing biofuel compositions based on fatty acid glycerol formal esters (Fatty Acids of Glycerol Formal Esters or FAGE), where the process is characterized by an enzymatic transesterification of fatty acid alkyl esters with glycerol formal. The invention is also directed to the biofuel compositions obtained by said process, as well as to the uses thereof. BACKGROUND OF THE INVENTION In the last thirty years, biodiesel has become a commercial reality, currently being integrated into diesel production supply chains worldwide.Biodiesel (usually fatty acid methyl ester (FAME)) is an optimal substitute for conventional diesel, and its use in diesel engines aims to reduce greenhouse gas emissions worldwide. However, FAME production still faces a persistent problem due to the inevitable generation of crude glycerol as a co-product. Crude glycerol is a compound of impure glycerol and other components, which, without further purification, has limited commercial applications. Despite the number of different strategies developed to find applications for crude glycerol, there are still no widespread routes for effective valorization of this impure co-product.The process described in the present patent involves the conversion of fatty acid methyl esters (FAME) to fatty acid formal glycerol esters (FAGE) through enzymatic transesterification as a means for chemically incorporating the glycerol molecule (the main component of glycerin crude) into a high value-added chemical product. The use of FAME / FAGE compositions as fuel has been previously described as a component for the production of diesel blends. The present patent advances the state of the art in the incorporation of said FAME / FAGE compositions as a component in renewable hydrocarbon blends and therefore becomes an effective solution for developing a new generation of more sustainable biofuels. Similarly, the present invention provides increased value to glycerin crude through an environmentally friendly conversion methodology.Glycerol is incorporated into FAME to become FAGE by a molecule of glycerol formal (GF). GF is the main component of an acetal mixture obtained from the conversion of oils and fats into FAME in the presence of crude glycerol, methylal, and acid catalysts. Another relevant component of the acetal mixture is glycerol formyl methoxymethane (GFoMoM), a compound with potential applications as fuel and other chemical uses. An important novel aspect of this invention is the use of lipolytic enzymes as transesterification catalysts, preferably in their immobilized forms. These have advantages in terms of process efficiency and recovery. The use of enzymes in this process is a flexible, efficient, and cleaner strategy than the previously described chemical processing. Previous developments covering the production of FAME / FAGE compositions have been described.The following literature review also includes several lipolytic enzyme / lipase-mediated transesterification reactions of different raw materials into FAGEs. However, the state of the art has never revealed reactions for the production of FAGEs based on an enzymatic reaction. EP2730567, for example, shows a process for the simultaneous production of fatty acid alkyl esters, glycerol formal, and FAGEs. The chemical reaction described in EP2730567 is mediated by a titanium catalyst and is therefore not an enzyme-catalysed reaction. EP2049623 also describes a chemical process for producing FAGE from rapeseed oil but again does not mention the use of lipolytic enzymes for this purpose. WO2014102796 describes a process and reactor for transesterification with immobilised enzymes in the production of alkyl esters of fatty acids for use as, inter alia, biofuels.According to this document, fatty acid alkyl esters are FAME or FAEE. The process described in this patent does not use formal glycerol as a starting material. There is also literature describing different hydrolytic enzymes. For example, EP287634 describes a lipase with improved thermostability in both soluble and immobilized forms. KR10-2225519 describes a modified Candida antarctica lipase B with improved hydrolytic, esterification, and amination activity. US2015 / 0031097 reports the use of selected lipase, phospolypase, and esterase catalysts for esterification and transesterification reactions. However, none of these documents disclose a transesterification process involving the specific transformation of FAME into FAGE mediated by an enzyme.The present invention is based on the surprising discovery that certain lipolytic enzymes are not only capable of formally transforming alkyl esters of fatty acids into FAGE in the presence of glycerol, but are especially specific for this type of transesterification reaction, which leads to a process that, due to its high FAGE yields, short reaction times and easy processing of the reaction crudes, is scalable at an industrial level. BRIEF DESCRIPTION OF THE FIGURES Figure 1: represents a typical gas chromatography (GC) chromatogram, which quantifies esters by area and uses octanol as an internal standard for the quantification of acetals in a mixture of FAME / FAGE + 3 v / v acetal. In the composition of the final product obtained according to the process of the invention, the acetal mixture incorporated in this composition typically contains GFoMoM: 75-90% by weight and glycerol formal 10-25% by weight.Figure 2: Extent of conversion of FAME to FAGE in the transesterification reaction mediated by Candida antarctica lipase. Figure 3: Shows the composition of the acetal mixture during the conversion mediated by Candida antarctica lipase at different times quantified by gas chromatography. Figure 4: Extent of FAME in complete conversion to FAGE in the transesterification reaction mediated by Candida antarctica. Figure 5: Extent of conversion of FAME to FAGE in the transesterification reaction mediated by Thermomyces lanuginosus lipase. DESCRIPTION OF THE INVENTION The following definitions are provided to facilitate understanding and clarify the meaning of certain terms within the context of the present invention.“Biofuel”: Solid, gaseous, or liquid fuel derived entirely from biomass that can be used as a substitute for fossil fuels and, in the case of gases and liquids, mainly in automotive applications. “Biofuel composition”: refers to the product obtained by the process of the invention, formed by a mixture of its components comprising at least FAGE; alkyl esters of fatty acids, preferably FAME; and acetals that include GFoMoM. The composition is useful as a biofuel or as a component for the production of diesel and renewable diesel blends. “Fatty acid glycerol formal ester” or “FAGE”: Fatty acid esters resulting from the esterification of a certain fatty acid (generally C1-C28) with glycerol formal. In the context of the invention, it is produced by transesterification of alkyl esters of fatty acids, preferably FAME, with glycerol formal.“Substrate”: is the element or substance that contains the compounds or molecules on which the enzyme exerts its activity. “Fatty acid alkyl esters”: Fatty acid esters resulting, in the presence of a catalyst, from the esterification reaction of a free fatty acid (generally C1-C28) and an alcohol, or also from the transesterification reaction of triglycerides, producing glycerol as a co-product. “Fatty acid methyl ester” or “FAME”: is an alkyl ester of a fatty acid where the alkyl group is a methyl. “Source of alkyl esters of fatty acids”: Raw material, generally of natural origin, from which alkyl esters of fatty acids can be derived, such as animal fats and various vegetable oils. “Glycerol formal” or “GF”: Glycerol acetal resulting from the reaction of glycerol with methylal, formic acid or another acetalizing agent in the presence of a catalyst.“C1-C28 alkyl radicals”: ​​linear or branched saturated aliphatic radicals, preferably linear, containing 1 to 28 carbon atoms. “C1-C28 alkenyl radicals”: ​​linear or branched unsaturated aliphatic radicals, preferably linear, containing 1 to 28 carbon atoms and having 1 to 7 double bonds. “Animal fats”: Lipid fractions extracted from the processing of animal matter, the main components of which are triglycerides and free fatty acids. Animal fats are usually solid at room temperature. “Vegetable oils”: Lipid fractions extracted from the processing of seeds or plant matter, the main components of which are triglycerides and free fatty acids. Vegetable oils are typically liquid at room temperature. “Food waste”: Any food and inedible parts thereof removed from a supply chain for recovery, reuse, or disposal.In the context of the present invention, food waste refers to the fatty or oily fraction of food waste. “Used cooking oils”: UCOs are oils and fats that have been used for cooking or frying in the food processing industry, restaurants, fast food, and at the consumer level, in homes. “Non-food crops”: Crops grown for the production of goods for technical use, rather than food for consumption. In the context of the invention, it refers to the oily fraction of such crops. “Algal oils”: Lipid fractions extracted from the processing of algae and microalgae, the majority of which are triglycerides and free fatty acids. Algal oils are typically liquid at room temperature.“Oleochemical waste”: A lipid substance or by-product generated in the processing of vegetable fats and oils, which remains unused when the main components have been removed for their specific use. “Lipolytic enzyme”: Lipolytic enzymes are a genetically diverse group of industrial or non-industrial enzymes capable of catalyzing both the hydrolysis and synthesis of lipids, as well as esterification and transesterification reactions on lipid substrates. In the context of the present invention, relevant lipolytic enzymes are those capable of transforming alkyl esters of fatty acids, preferably FAMEs, into FAGEs by transesterification reaction. “Thermomyces lanuginosus lipase”: The natural or modified lipolytic enzyme from the thermophilic fungus Thermomyces lanuginosus. “Candida antarctica lipase B”: The natural or modified lipolytic enzyme from the yeast Candida antarctica. “Proteus sp. lipase K107”: The natural or modified lipolytic enzyme from the yeast Candida antarctica.”: the natural or modified lipolytic enzyme from the bacteria Proteus sp.“Immobilized enzyme”: enzyme bound to an inert material that is insoluble in the reaction medium. In this form, it provides the enzyme with greater mechanical resistance or resistance to changes in temperature or pH. Immobilized enzymes remain in place throughout the reaction, facilitating their recovery and reuse.“Lipolytic enzyme loading”: Quantity of enzyme immobilized on a given support, generally expressed as mg of protein / quantity of solid or as activity units (U), failing that. “Aqueous microenvironment”: The minimum quantity of water necessary to guarantee the activity of lipolytic enzymes in a hydrophobic environment. “Acetal mixture”: mixture of acetals where the main component is glycerol formal and secondarily GFoMoM. “GFoMoM”: acetal derived from glycerol formal where the free hydroxyl group of glycerol formal has reacted with methylal.This product is generated in the transacetalization reaction and is represented as follows: Esquema 1

[0002] “Recovered lipolytic enzyme”: Immobilized enzyme separated, treated, and regenerated from the reaction crude, which is then reused for a subsequent reaction. “Direct filtration”: Processing step consisting of the separation of the immobilized enzyme by filtration for the purpose of its subsequent treatment, regeneration, and reuse. “Sequential washings”: In the context of enzyme recovery, the use of consecutive solvent washes to clean the lipolytic enzyme for reuse or subsequent disposal. “Diesel”: Fuel types with defined specifications generally used in internal combustion engines for automotive or heating purposes. In a first aspect, the present invention is directed to a process for preparing a biofuel composition based on fatty acid glycerol formal esters (FAGE) of formula (I): comprising the reaction of a substrate of fatty acid alkyl esters of formula (II): or a source of fatty acid alkyl esters of formula (II) with glycerol formal in the presence of at least one lipolytic enzyme. wherein Het is a group selected from: R is a C1-C28 alkyl radical or a C1-C28 alkenyl radical; and R1 is a C1-C6 alkyl radical. This process will be referred to below as the process according to the invention or simply as the process of the invention. In a particular and preferred embodiment, R1 represents a methyl group, resulting in the fatty acid alkyl ester used as a substrate for the reaction being a fatty acid methyl ester (FAME) of formula (IIa): Where R is as defined above. The reaction between the compound of the particularly preferred compound of formula (IIa) and glycerol formal according to the invention is represented in Scheme 1 below: Esquema 2 The reaction substrate may be a purified mixture of fatty acid alkyl esters of formula (II), preferably of formula (IIa), or a source of fatty acid alkyl esters of formula (II) and / or of formula (IIa). Any lipid source of fatty acid alkyl esters of formula (II) and / or of formula (IIa) is useful as a substrate for the reaction in the process of the invention, although in a preferred embodiment the source of fatty acid alkyl esters of formula (II) and / or of formula (IIa) comprises at least one of animal fats, vegetable oils, food waste, used cooking oils, non-food crops, algal oils, and oleochemical residues, or mixtures thereof.The possibility of using any of these sources of fatty acid alkyl esters has the advantage of providing the process of the invention with great versatility and the possibility of reusing and recycling waste materials to give a product with significant added value. One of the most characteristic aspects of the process of the invention is that it is an enzyme-mediated process. In particular, the transesterification reaction is catalyzed by a lipolytic enzyme. Although the inventors studied several commercial and non-commercial lipolytic enzymes well known for their transesterification capacity, they surprisingly found that the specific transesterification reaction between fatty acid alkyl esters of formula (II) and / or formula (IIa) to produce a product enriched in formal glycerol esters of fatty acids (FAGE) was extremely dependent on the type of lipolytic enzyme used.Most of the enzymes tested were either not selective for this particular transesterification reaction or gave very low FAGE transformation yields. However, the inventors have found that when the lipolytic enzyme is selected from Thermomyces lanuginosus lipase, Candida antarctica lipase B, Proteus sp. lipase K107, or a mixture thereof, the reaction was not only highly selective for FAGE production but also produced a reaction product with a high proportion of FAGE. The lipolytic enzyme may be present in the reaction medium in free form; however, in a preferred embodiment, the lipolytic enzyme is present in immobilized form. The immobilized enzyme provides higher conversion rates and facilitates its handling after use and its recovery for subsequent additional uses.The inventors have found that the lipolytic enzyme, after at least one recovery process, retains an enzymatic activity greater than 80% with respect to the fresh enzyme. To optimize the reaction, the load of lipolytic enzyme or mixture of lipolytic enzymes in the reaction must be between 2-20% by weight, preferably 2-10% by weight, more preferably between 2-5% by weight with respect to the fatty acid alkyl ester substrate. Furthermore, to obtain the desired biofuel composition, the molar ratio between glycerol formal and the fatty acid alkyl ester substrate is especially relevant. In a particular and preferred embodiment of the invention, the molar ratio of glycerol formal and the fatty acid alkyl ester substrate is between 1-4 equivalents, preferably between 1-3 equivalents.Glycerol formal can be obtained from crude glycerol by an acetalization process, to produce a product with a purity greater than 98%. The transformation of glycerol into glycerol formal with a purity greater than 98% can be carried out by any known process such as that described, for example, in document DE19648960 using heterogeneous catalysts that allow obtaining a water-free crude glycerol as described in document WO 2005093015. However, in a preferred embodiment of the invention, the glycerol formal used in the process is not in pure form but is generally present in a mixture of acetals. Said mixture of acetal reactants may contain from 1-99.9% by weight of glycerol formal, more preferably between 30-75% by weight and even more preferably 30-70% by weight of the total acetal mixture.The remaining amount of the acetal mixture is generally derived from glycerol formal, such as GFoMoM (Glycerol formyl methoxymethane). Regarding the process conditions, the reaction is preferably carried out in an aqueous microenvironment with water in an amount of 0.01-1% by weight with respect to the fatty alkyl ester substrate or the source of fatty acid alkyl esters. Furthermore, although the process can be carried out at room temperature, it is preferred to carry out the reaction at a temperature between 40 and 70°C. The process according to the invention can be carried out in a batch reactor, a continuous stirred tank reactor, a packed bed column reactor, or an expanded bed reactor. The process of the invention has the advantage that it optionally allows for an additional step of recovering the lipolytic enzyme for subsequent uses.The recovery of the lipolytic enzyme comprises: a) direct filtration, optionally followed by b) sequential washes with organic solvents, such as acetone and heptane, and c) vacuum drying at a temperature below 40°C. Another aspect of the invention is also the lipolytic enzyme recovered according to the process described herein. In a preferred embodiment, the recovered enzyme is selected from Thermomyces lanuginosus lipase, Candida antarctica lipase B, Proteus sp. lipase K107, Mucor miehe lipase, or a mixture thereof. The lipolytic enzyme after the recovery process retains an enzymatic activity greater than 80% with respect to the fresh enzyme. This makes the recovered lipolytic enzyme perfectly suitable for other subsequent uses, which makes the process of the invention highly sustainable. Another aspect of the present invention is the biofuel composition that can be obtained by the process of the invention.This will be referred to below as the biofuel composition of the invention or simply as the composition of the invention. The biofuel composition of the invention is characterized by a fatty acid glycerol formal ester (FAGE) comprising formula (I), fatty acid alkyl esters of formula (II), preferably of formula (IIa) and acetals. The acetals present in the composition of the invention comprise glycerol formal and derivatives thereof, mainly GFoMoM. The composition of the biofuel of the invention is defined by the ratio of its components. In a particular embodiment, the composition has a fatty acid glycerol formal esters (FAGE) / fatty acid alkyl esters / acetals ratio of 1-96.5 / 96.5-1 / 3-4; preferably 46-50 / 50-46 / 3-4; and even more preferably 19-39 / 78-58 / 3-4. Another aspect of the invention relates to the use of the biofuel composition of the invention.The biofuel composition of the present invention has technical specifications compatible with commercial automotive diesel according to the EN590 diesel standard and with the Hydrotreated Vegetable Oil (also known as renewable hydrocarbons or HVO) standard according to EN14590. Therefore, the composition can be used with adequate technical and quality requirements and is suitable for use in automotive diesel engines. The biofuel composition of the present invention meets the requirements of current regulations to be considered a sustainable biofuel: it can be obtained from feedstocks included in Annex IX of “Directive (EU) 2018 / 2001: European Renewable Energy Directive”, and also complies with the current specifications and blending conditions for the quality standards relevant for its use as automotive fuel.The composition of the invention can be used as a component for the development and production of diesel blends and also of renewable diesel for automobiles in accordance with the aforementioned standards for conventional diesel, hydrotreated vegetable oil (also known as renewable hydrocarbons or HVO), respectively, or mixtures thereof. The invention will be described in more detail by means of the following examples such that a person skilled in the art can use the invention on the basis of the description. Not all steps of the embodiments are described in detail, since many of them will be apparent to a person skilled in the art. EXAMPLES EXAMPLE 1: Selection of lipolytic enzymes for the synthesis of deglycerol formal esters of fatty acids Different commercial and non-commercial lipases (see Table 1) were selected to investigate the activity for the production of FAGE.Enzymatic transesterification reactions were performed using 800 μL of FAME (2.7 mmol), 200 μL of GF (2.3 mmol), 50 μL of water, and 3% by weight of biocatalyst. The reactions were carried out at 40 °C using the Carousel 12 multipoint reactor plus reaction station (Radleys, Saffron Walden, UK). Reactions were carried out in 50 mL reaction tubes, continuously mixed using a magnetic stirrer (200 rpm) and under vacuum to remove methanol formed during the reaction, which shifted the reaction towards synthesis. To obtain the time course of the reaction, 50 μL of samples were taken at different times and then analyzed by gas chromatography (GC-FID). The FAME and FAGE content was estimated by area correlation using, in this case, methyl heptadecanoate as an internal standard following a procedure adapted from EN14103.The direct correlation between mass and response is evaluated, with an identical response factor for each methyl and each formyl ester of glycerol. The analysis of FAME present in any oily sample is carried out by dissolving 1.00 g of the sample directly in 10 ml of heptane and followed by 10 ml of the 2% (w / v) internal standard solution. 1 μL of the 50 mg / ml FAME / FAGE solution or FAME mixture was analyzed on a Shimadzu 2100 gas chromatograph, equipped with a Zebron ZB-5MS GC capillary column of 30 mx 0.25 mm x 0.25 µm. Injection temperature was 320 ° C, separation ratio was 50: 1 and FID temperature at 320 ° C, N2 was used as auxiliary gas at a flow rate of 30 ml / min, in addition to H2 at 40 ml / min. and air 400 ml / min. Total column flow rate 1 ml / min. Linear velocity 25.1 cm / s. Carrier gas pressure (helium) 80.9 kPa. The ramp used was as follows: Initial T = 40°C maintained for 1 minute, subsequently rising at 10°C / min. up to 150°C then held for 3 min., followed by two sequential ramps of 5°C / min each, first to 200°C (held for 5 min), second and finally to 320°C (held for 5 min), with a total duration of 59 minutes. The 50 μL samples were diluted in 950 μL of the methyl heptadecanoate internal standard solution prepared in heptane, and 1 μL was injected at a split ratio of 1:50. The conversion rate (%) was calculated using Eq. (1):In which, ∑fage is the sum of all FAGE peak areas; Amhd is the peak area for methyl heptadecanoate (internal standard); ∑fame is the sum of all FAME peak areas and Fr is the response factor. As shown in Table 1 below, most lipases rarely produced the FAGE compound, while Novozym 435 (Candida antarctica) showed the highest catalytic activity. Table 1. FAGE synthesis. aLipase-catalyzed Source Regioselective Conversionb (%)Pseudomonas fluorescens Non-selective NDThermomyces lanuginosus sn-1.3 11.14Mucor miehei sn-1.3 2.28Rhizopus oryzae sn-1.3 NDCandida rugosa Non-selective NDCandida antarctica Non-selective 40.85Pseudomonas cepacia Non-selective NDRHizopus niveus sn-1.3 NDRHizopus arrhizus sn-1.3 NDPenicillium camemberti Non-selective NDAspergillus oryzae sn-1.3 NDPseudomonas lipoprotein lipaseNon-selective NDsp. Burkholderia sp. lipoprotein lipase Non-selective NDPorcine pancreatic lipase sn-1.3 NDProteus sp. lipase K107 Non-selective 5.52 a Reaction conditions: FAME (2.7 mmol), GF (2.3 mmol), water (50 µl); and lipases (0.03 g) were mixed using a magnetic stirrer (200 rpm), at 40 °C for 12 h. bDetermined by GC. EXAMPLE 2: Synthesis of FAME / FAGE mixture using Eversatransform 2.0 (Lipase from Thermomyces Lanuginosus). 0.45 g of FAME (1.5 mmol) derived from used cooking oil, (Humidity <500 ppm) was reacted with 0.6 g of glycerol formal (5.8 mmol) in the presence of Eversa transform 2.0 (5% by weight of FAME) for a period of 24 h and at 300 rpm under orbital shaking at 37 ° C. The conversion of FAME to FAGE was verified by TLC and calculated by GC. The results of the conversion of FAME to FAGE using Eversa transform 2.0, a Lipase from Thermomyces Lanuginosus, are shown in Table 1 above.EXAMPLE 3: Synthesis of FAME / FAGE and GFoMoM in the same reaction withNovozym 435 (Lipase B from Candida antarctica).2000 grams of FAME (6.76 mol) of water content below 500 ppm and derived from a mixture of animal fat and used cooking oil were charged into a 4 L reactor equipped with a vacuum distillation system., The system was closed and inerted with N2 (0.8 bar) for up to 1 hour. Heating and stirring were turned on (T=40-50ºC for the jacket reactor and 200 rpm, respectively) and the following addition curve was defined by software for the acetal phase (which had a weight content of 53% glycerol formal and 47% GFoMoM):Table 2: Addition curve for the acetal phase (reactive acetal mixture) Time Addition Volume Added Dosage. Dosado GF Dosed Dosed Dosed Dosed d e GFadded(g) GF Phase GF Phase GF Phase GF Phase (ml / h) added (g) (g / min) added (ml / min.) (ml / h) (g) (g / min.) (ml) 0Inst 100 Inst inst 120 226.20-20 375 125 150 7.5 270 (9.5 508.9 14.13 12.29 737.47ml / min, @ 5min and 12.0ml / mn @ 20 min) 20-40 275 91.67 110 5.5 380 716.2 10.36 9.01 540.8140-60 250 83.33 100 5 480 904.6 9.42 8.19 491.65 60-80 225 75 90 4.5 570 1074.3 8.48 7.37 442.48 5 80-110 200 100 120 4 690 1300.4 7.53 5.56 393.32 110-140 175 87.5 105 3.5 795 1498.3 6.59 5.74 344.15 140-170 150 75 90 3 885 1667.9 5.65 4.92 294.99 230 Inst 100 73 inst 958 (9.21 mol) 1805.5 The reaction It was conditioned for an additional 15 min, and 60 g of Novozym 435 was added. Vacuum was started at <30 mbar, as well as the addition of Phase GF as in Table 2, with simultaneous collection of methanol by vacuum distillation system. Samples were collected by suction with N2200 rpm and 30 mbar at t=0.3=, 60, 120, 150, 180, 240, 300 minutes.The crude oil composition was analyzed at these times by GC for FAME, FAGE, GF, GFoMoM, and FAGE. Figure 2 shows the kinetics of the conversion of FAME to FAGE. Figure 3 shows the compositions of the acetal mixture during the conversion at different time points. Once the desired purity of FAGE was obtained in the mixture, the reaction was cooled to 25°C with slow stirring (50 rpm). The vacuum and N2 supply were stopped, as well as the stirring. The reactor was discharged, and the enzyme was separated from the crude oil using a vacuum frit No. 3 filter. The filtered enzyme was washed sequentially with up to three acetone washes and the same number of times with heptane, in order to be reused. The enzyme could be reused directly without washing.The reaction crude was again charged to a 2 L spherical reactor (in two different batches) fitted with a vacuum distillation system. The system was inerted and fed with a continuous flow of nitrogen to prevent fatty acid oxidation as much as possible. The vacuum was then started and the GFoMoM-enriched GF phase (Total 112 g, 75 wt% GFoMoM, determined by GC) was distilled at 70–110 °C at 3–10 mbar. After the distillation was completed, the temperature was dropped by approximately 10 °C. The acid number was tested and corrected below 0.5 mg KOH / g for the distillation bottoms by neutralization with 10% KOH solution followed by sequential washing with water to remove excess alkalinity. The ester mixture was then dried to <500 ppm, under vacuum at 45 °C for up to 24 h, and the final purity of the FAME / FAGE composition was confirmed by GC as 70 / 30 wt %, respectively.EXAMPLE 4: Synthesis of pure FAGE with Novozym 435 (Candida antarctica lipase B). 2500 grams of FAME (8.45 mol), derived from a mixture of vegetable oil and used cooking oil, with a water content below 500 ppm, were loaded into a 4 L reactor equipped with a vacuum distillation system. The system was closed and inerted with N2 (0.8 bar) for up to 1 hour. Heating and stirring were turned on (T=50°C, for the jacketed reactor (T=5°C) and 200 rpm, respectively). The ester was conditioned for a further 15 minutes, 75 g of Novozym 435 were added, and then the addition curve (Table 3) for glycerol formal (ultrapure grade of glycerol formal) was followed:Table 3: Addition curve for Glycerol FormalTime Addition Vol.of added GF Added Dosed Dosed (ml / h) (ml) (g) (g / min) Added GF (g) 0Inst 125 inst inst 1250-20 468.75 156.25 187.5 9.38 312.520-40 343.75 114.58 137.5 6.88 450.040-60 312.5 104.17 125.00 6.25 575.060-80 281.25 9.75 112.50 5.63 687.580-110 250 125 150.00 5.00 837.5110-140 218.75 109.38 131.25 4.38 968.8140-170 178.5 93.75 112.5 3.75 1081.3170-200 156.25 78.13 93.75 3.13 1175.0200--240 156.25 104.17 125.00 3.13 1300.0240 inst 125.00 150.00 Ints 1450 ,(13.92 mol)When the reaction starts, the vacuum is set to <30 mbar to collect methanol continuously through the distillation system. The conversion was monitored by gas chromatography, and samples were collected at t = 1, 4, 6, 8, 10, 14, and 23 hours. After four hours, the addition of GF was stopped, and the conversion of FAME to FAGE progressed, reaching 87% at 23 hours.After that time, an additional 100 g of glycerol formal (0.96 mol) was added to the FAME / FAGE reaction crude and the conversion progressed over an additional 8 hours until the FAME to FAGE conversion value reached 96% (by GC). The total reaction time was 31 hours. The reaction was cooled and the crude was worked up as in Example 3. After the reaction, the unreacted glycerol formal was flash distilled from the reaction crude under vacuum and N2 flow. Figure 4 shows the kinetics of the conversion of FAME to FAGE. After the completion of the distillation, the purity of the FAME / FAGE distillation bottom was confirmed by GC. The acid number (mg KOH / g) (<0.5) and the water content (<500 ppm) were verified. Where necessary, the acid number and water content were corrected as in Example 3.Example 5: Synthesis of FAME / FAGE mixture and GFoMoM in the same reaction with Thermomyces lanuginosus lipase (Lipozyme TL IM)1200 grams of FAME (4.05 mol) with a water content below 500 ppm and derived from used cooking oil were loaded into a 4L reactor equipped with a vacuum distillation system. The system was closed and inerted with N2 for up to 1 hour. Heating and stirring were turned on (T=50ºC, for the jacketed reactor and 200 rpm, respectively) and the following glycerol formal addition curve was defined in the software (<1000 ppm moisture and 99.9% purity): Table 4: Glycerol formal addition curveTime Addition Vol. of GF Added Dosage Total Total Dosage Dosage Dosage added GF(g) (ml / h) (g) (g / min) GF added GF GF (ml / min.) GF (ml / h) (ml) (g) (g / min.) 0Inst 59.76 inst inst 71.7 71.70-20 224.1 74.7 89.64 4.48 161.4 161.4 4.48 3.9 233.8420-40 164.34 54.78 65.74 3.29 227.1 227.1 3.29 2.86 171.9040-60 149.4 49.8 59.76 2.99 286.8 286.8 2.99 2.60 155.960-80 134.46 44.82 53.78 2.69 340.6 340.6 2.69 2.34 140.3180-110 119.52 59.76 71.71 2.39 412.3 412.3 2.39 2.08 124.72110-140 104.58 52.29 62.75 2.09 475.1 475.1 2.09 1.82 109.13140-170 89.64 44.82 53.78 1.79 528.9 528.9 1.79 1.56 93.54230 Inst 59.76 73 inst 601.9 601.9(5.78 mol) The reaction was conditioned for 15 min. more and 234.9 g of Lipozyme TL IM from Thermomyces lanuginosus were added over a period of 30-60 min. Vacuum was started at <30 mbar as well as the addition of glycerol formal as in table 3, with simultaneous collection of methanol by vacuum distillation system. Samples were collected by suction with N2200 rpm and 30 mbar, t=0, 30, 60, 120, 150, 180, 240, 300 min., similarly to examples 2 and 3.The crude oil composition was monitored by GC for FAME, FAGE, and the remaining GF. Figure 5 shows the kinetic rate of conversion of FAME to FAGE up to approximately 23%. After 5 hours the conversion did not seem to progress, the reaction was cooled to 25 °C, with slow stirring (50 rpm). The vacuum and N2 supply were stopped, as well as the stirring. Example 6: Synthesis of FAME / FAGE with a mixture of Candidaantarctica lipase (Novozym 453) and Thermomyces lanuginosus lipase (Lipozyme TL IM) The following proportions of Lipozyme TL IM and Novozym 435 were added in a round-bottom flask to make 10% by weight to 2.5 g (8.44 mmol) of FAME (<500 ppm moisture): T. abla 5Catalyst Assay1 0% Novozym 435; 10% Lipozyme TL IM2 0.1% Novozym 435; 9.9% Lipozyme TL IM3 1% Novozym 435; 9% Lipozyme TL IM4 5% Novozym 435; 5% Lipozyme TL IM5 10% Novozym 435; 0% Lipozyme TL IMThe mixture was started to relax with stirring (200 rpm) at 50 ° C with continuous N2 flow for an initial time of 30 min. Glycerol formal (<1000 ppm moisture) was added sequentially dropwise at a rate of 100 μl after every 5 minutes for a total period of 45 minutes. After the addition time, reflux was maintained under nitrogen for an additional 30 minutes.The apparent conversion of FAME to FAGE was qualitatively assessed by TLC with the following results: Conversion 1< Conversion 2< Conversion 3< Conversion 5< Conversion 4. These results are indicative that Candida antarctica lipase provides higher conversion rates than Thermomyces lanuginosus lipase, when used alone or with a small proportion of Thermomyces lanuginosus lipase, but at the same time reveals a synergistic effect on the conversion when used in combination in a balanced proportion.

Claims

CLAIMS 1. Un proceso para preparar una composición de biocombustible a base de Glycerol formal esters of fatty acids (FAGE) of formula (I): comprising the reaction of a substrate of alkyl esters of fatty acids of formula (II): or a source of alkyl esters of fatty acids of formula (II) with glycerol formal in the presence of at least one lipolytic enzyme, wherein Het is a group selected from: R es un radical alquilo C1-C28 o radical alquenilo C1-C28 ; y R1 es un radical alquilo C1-C6.

2. Un proceso de acuerdo con la reivindicación 1, en el que el éster alquílico de fatty acid is a fatty acid methyl ester (FAME) of formula (IIa): en donde R es como se define en la reivindicación 1.

3. Un proceso de acuerdo con la reivindicación 1 en el que la fuente de ésteres alkyl fatty acids of formula (II) comprises at least one of animal fats, vegetable oils, food waste, used cooking oils, non-food crops, algal oils and oleochemical residues.

4. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores en el que la enzima lipolítica se selecciona de lipasa de Thermomyces sp.; lipasa de Candida sp., lipasa de Proteus sp., lipasa de Mucor sp. o una mezcla de las same.

5. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores en el que la enzima lipolítica se selecciona de lipasa de Thermomyces lanuginosus; lipasa B de Candida antarctica, lipasa K107 de Proteus sp., lipasa de Mucor miehe or a mixture thereof.

6. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores enin which the lipolytic enzyme is in immobilized form.

7. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores en el que la carga de enzima lipolítica en la reacción está entre el 2 - 20 % en peso, preferentemente entre el 2 - 10 % en peso, más preferentemente entre el 2 - 5 % en weight relative to the fatty acid alkyl ester substrate.

8. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores en el que la relación molar de glicerol formal y el sustrato de éster alquílico de ácido fat is between 1 and 4 equivalents, preferably between 1 and 3 equivalents.

9. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores wherein the reaction is carried out in an aqueous microenvironment with water in an amount of 0.01-1% by weight with respect to the substrate of alkyl esters of fatty acids.

10. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores in which the reaction is carried out at a temperature between 40 and 70ºC.

11. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores in which glycerol formal is present in a mixture of acetals.

12. Un proceso de acuerdo con la reivindicación 10, en el que la mezcla de Acetals may contain 1-99.9% by weight of glycerol formal, more preferably between 30-75% by weight and even more preferably 30-70% by weight of the total acetal mixture.

13. Un proceso de acuerdo con cualquiera de las reivindicaciones anteriores en el que el proceso se lleva a cabo en un reactor discontinuo, continuo de tanque stirred, packed bed column or expanded bed reactor.

14. Un proceso de acuerdo con cualquiera de las reivindicaciones anterioreswhich comprises an additional step of recovering the lipolytic enzyme for subsequent uses.

15. Un proceso de acuerdo con la reivindicación 14 en el que la recuperación de la lipolytic enzyme comprises: a ) filtración directa, opcionalmente seguida de b) lavados secuenciales con disolventes orgánicos c) secado al vacío a temperatura inferior a 40ºC.

16. Una composición de biocombustible que puede obtenerse mediante el process of any of claims 1-15, characterized in that it comprises fatty acid glycerol formal esters (FAGE) of formula (I), fatty acid alkyl esters of formula (II) as defined in claim 1 and acetals.

17. Una composición de biocombustible de acuerdo con la reivindicación 16 where the acetals comprise glycerol formal and GFoMoM.

18. Una composición de biocombustible de acuerdo con la reivindicación 17 en donde la relación de ésteres de glicerol formal de ácidos grasos (FAGE) / ésteres alkyl fatty acids / acetals is 1-96.5 / 96.5-1 / 3-4; preferably 46-50 / 50-46 / 3-4; and even more preferably 19-39 / 78-58 / 3-4.

19. Uso de una composición de biocombustible de acuerdo con cualquiera de claims 15 to 17 as a biofuel.

20. Uso de una composición de biocombustible de acuerdo con cualquiera de las reivindicaciones 15 a 17 como componente para la producción de diésel y mezclas de diésel renovable.

21. Una enzima lipolítica recuperada de acuerdo con el proceso de cualquiera of claims 14 or 15.

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