METHOD FOR THE ENZYMATIC SYNTHESIS OF A BIODIESEL FROM USED LIPIDS

Description

Object of the invention

[0001] The present invention relates to the field of enzymatic synthesis and concerns a process for synthesizing a biodiesel composed of a mixture of ethyl or methyl esters, from used lipids from used non-acidic vegetable oil, used acidic oil and / or used animal fat. State of the art

[0002] Currently, only vegetable oil methyl esters (VOME) and animal fats are authorized in Europe and used as biofuel, particularly as biodiesel, by blending them with diesel up to a maximum of 30%. However, these vegetable oil methyl esters, like VOME derived from used vegetable oils, must comply with the DIN EN14214 standard, which covers all the physicochemical parameters of biodiesel, except for the amount of residual alcohol, which is defined only for methanol.

[0003] However, with legislation evolving rapidly, vegetable oil ethyl esters (VEEs) are currently undergoing customs authorization for sale and use, provided a declaration is made to that effect. Nevertheless, the DIN EN14214 standard is not suitable for measuring the quantities of used vegetable oil ethyl esters due to the standard it employs.

[0004] Patent document WO2012 / 098114 discloses a process for producing alkyl esters of fatty acids, in which a solution comprising a raw material including fatty acids, alcohol, water, and glycerol is contacted with a lipolytic enzyme. In this process, the raw material including fatty acids can be a used oil or grease. Objectives of the invention

[0005] The present invention aims to obtain an eco-friendly, simple, efficient, low-cost design process applicable from locally available lipid sources to produce biodiesel from lipids of normal used vegetable oil, acidic used oil and / or used animal fat.

[0006] Furthermore, the invention aims to provide such a process that reduces or eliminates the steps for chemically pretreating raw materials and washing the resulting biodiesel.

[0007] Another objective of the invention is to simplify the steps of the process and the equipment used in order to reduce costs. Summary of the invention

[0008] The invention relates to a process for obtaining a biodiesel consisting of a mixture of ethyl esters or methyl esters, by an enzymatic transformation of lipids, chosen from the group consisting of used vegetable oil, acidic vegetable oil and / or used animal fat, in which said enzymatic transformation comprises an enzymatic transesterification and an enzymatic hydroesterification, an addition of alcohol, an addition of water or a base in aqueous solution and an addition of glycerol, characterized in that prior to the addition of alcohol, the lipids, the water or the base and the glycerol form a homogeneous medium and in which this medium is brought into contact with one or more enzymes of the triacylglycerol acyl hydrolase type.

[0009] Preferably, the process of the invention also has one or more of the following characteristics: The enzyme is a lipase selected from the group consisting of immobilized or non-immobilized lipases of the triacylglycerol acyl hydrolase (EC3.1.1.3) type of bacterial origin or fungal extracts, such as specific 1,3 lipases or CALBs from Pseudomonas cepacia, Pseudomonas aeruginosa, Candida Antarctica, Candida Rugosa, Rhizomucor miehei, Aspergilus niger or Thermomyces lanuginosus, the enzyme being possibly immobilized on a resin, in particular a poly(methyl)methacrylate resin crosslinked with methylbenzene, and these enzymes being identified by the trade names of the enzymes Novozym 40116, Novozym 40086, Novozym 435, Lipozyme RM IM, Lipozyme TL IM, CalB Sigma, CalB Immoplus, NovoLime, TL Sigma, Callera TM, Eversa, Pallatase, Eversa Transform 2.0 free in liquid form from Thermomyces lanuginosus or a mixture of two or more of these enzymes.

[0010] Prior to enzymatic transformation into biodiesel, lipids are subjected to one or more cascade decantation steps to extract solid particles present in these lipids.

[0011] Lipids made from used palm oil or used animal fat are heated to a temperature of 40°C or more, preferably 45°C or more, before the filtration step(s).

[0012] Prior to enzymatic transformation into biodiesel, lipids are subjected to one or more water removal steps, resulting in lipids containing less than 1% water content by weight relative to the weight of lipids.

[0013] Prior to enzymatic transformation into biodiesel, the lipids from used vegetable oil and / or acidic vegetable oil are subjected to one or more filtration and collection steps of vegetable oil with a turbidity or cloudiness in the liquid close to 0.

[0014] Prior to enzymatic transformation into biodiesel, lipids are subjected to a decantation stage at ambient temperature for used oils and at 45°C or more for animal fats for a period of between 48 and 72 hours.

[0015] Water or a base is added at a concentration of less than 0.5%, and glycerol is added at a concentration of 1% to 10% relative to the lipids. This mixture is then stirred for 15 minutes to 1 hour to create a homogeneous reaction medium (or homogeneous mixture). This reaction medium (or mixture) is then incubated with the enzyme for 30 minutes, preferably 1 to 2 hours, to verify enzymatic efficiency through hydrolysis and the generation of free fatty acids. This process stimulates the esterification reaction, thereby improving enzymatic efficiency and reducing reaction time. The enzyme's efficiency is measured to identify parameters to modify in the biodiesel synthesis process in order to optimize its production. This activity measurement is performed on a sample before the addition of alcohol and before initiating the reaction.If the enzyme releases less than 10% free fatty acids after 1 hour, parameters such as increasing the enzyme concentration, reducing acidity, more efficient mixing in the reaction medium, or modifying the temperature to achieve an optimum activity for each type of enzyme must be modified.

[0016] Alcohol is added at a molar ratio (alcohol:oil) ranging from (3:1) to (16:1) sequentially from 1 to 4 times, after a period of between 15 minutes and 4 hours, preferably after a period of between 30 minutes and two hours, to trigger the enzymatic transformation of lipids into biodiesel, by the addition of ethanol or methanol.

[0017] The process includes a recycling step of the enzyme used, of the glycerol used and generated or of the water used and generated, preferably with a re-use step of the free enzyme with the water and / or base and glycerol.

[0018] The process includes the enzymatic transformation of used lipids into biodiesel, a refining step of the reaction medium including the separation of the light (organic) part containing the alcohol, glycerol and esters formed during the enzymatic transformation, and the heavy (aqueous) part containing the enzyme, water and glycerol of the so-called reaction medium (mixture) and the biodiesel obtained.

[0019] The process includes a refining step which includes evaporation of the alcohol from the so-called reaction medium (or mixture), and optionally collection by condensation of the evaporated alcohol and / or addition of the recycled alcohol in the process of obtaining biodiesel according to the invention.

[0020] The refining stage includes decantation of the glycerol and / or the aqueous part of the so-called reaction medium (or mixture).

[0021] A recycling step can be carried out of the (heavy) aqueous part of the so-called reaction medium (or mixture) incorporating the enzyme is added with alcohol to the so-called reaction medium (or mixture) containing the used lipids for obtaining biodiesel according to the invention, without the addition of base water, glycerol and enzyme.

[0022] The process includes, after the refining step, a step of collecting the refined biodiesel obtained, followed by a step of adding an antioxidant, for example in liquid form of a phenolic base, to the refined and collected biodiesel, and / or a step of blending the refined and collected biodiesel with another fuel (diesel).

[0023] Also disclosed herein (but not as part of the invention) is an installation for obtaining biodiesel by implementing the process described above, said installation comprising: a lipid pretreatment module comprising means for collecting, cascading decantation and filtering lipids from used non-acidic vegetable oil, used acidic vegetable oil and / or used animal fat, said module preferably consisting of one or more filters, preferably one or more filter screens with opening sizes between 3 mm and 1 mm, and optionally an oil separator, suitable for collecting pretreated lipids, said lipid pretreatment module being connected to a reaction module comprising a reactor, a reactor heating device, a device for stirring the reagents present in the reactor and means for adding pretreated lipids to the reactor, means for adding water and / or a base, means for adding glycerol and means for adding alcohol,said reactor being connected to a collection and refining module for a mixture of the biodiesel obtained and the reagents present in the reaction medium (or mixture) from the reactor, said collection and refining module comprising means for collecting by decantation the refined biodiesel, glycerol and the aqueous part of the reaction medium (or mixture) and means for recycling the glycerol and the enzyme; means for evaporating the excess alcohol and possibly means for recycling the alcohol present in the reaction medium, said collection and refining module being connected to a processing and collection module for the refined biodiesel obtained comprising a tank and means for adding an antioxidant to the tank, this tank being connected to a tank for mixing the refined biodiesel obtained with another fuel.

[0024] The present invention will be described in detail in the examples below with reference to the attached figures and presented as illustrations of a preferred and non-limiting embodiment of the invention. Brief description of the figures

[0025] There figure 1 represents participatory reactions to the transformation of used lipids into biodiesel. figure 2 represents a comparative analysis of the compounds obtained after two different synthesis reactions. figure 3 represents the installation Detailed description of the invention

[0026] The present invention relates to a process for synthesizing biodiesel from so-called used lipids, this biodiesel being defined as a mixture of ethyl or methyl esters from used vegetable oils (EEHVU and EMHVU), acid oils and / or used animal fats.

[0027] The method of the invention, as well as the installation disclosed herein (but not forming part of the invention), essentially comprises several of the modules or steps described below: A. A first module or first step, called lipid pretreatment (forming the incoming materials to be treated) by cascade decantation, by filtration steps and by an oil separator at ambient temperature for used vegetable oils and at 45°C for animal fats corresponds to module or step 1, followed by B. a second module or second step called reaction corresponds to module or step 2, this reaction includes, via suitable means present in the module, to obtain a transesterification (by alcoholysis) and a hydroesterification (by hydrolysis and then by esterification) of the lipids, with conditions adapted to each type of oil (used vegetable oils, acidic vegetable oils and / or animal fats), followed by C.a third module or third step called recovery of the enzyme used, the excess alcohol, the glycerol in the second step and then the biodiesel obtained, corresponds to module or step 3, followed by D. a fourth module or fourth step called collection and processing of the biodiesel (called "refined") obtained in the third step is obtained with the addition of an antioxidant and possibly a mixture with another fuel (diesel).

[0028] In the process and installation disclosed herein, three types of so-called used lipids (used as input materials) can be processed for the preparation of the biodiesel of the invention.

[0029] One type of lipid is obtained from used vegetable oil (UVO), which is oil resulting from the cooking of plant-based or meat-based foods, such as potatoes (fries), bananas, batters (such as doughnut and churro batter), or meats. Preferably, used oil is chosen from the group consisting of palm oil, sunflower oil, and / or rapeseed oil. Used vegetable oil has undergone deterioration due to cooking, resulting in the formation of polar compounds—that is, compounds not present in the original oil and therefore degraded products—and / or various polymers such as starch, as well as an increase in the level of free fatty acids, although this level does not exceed 5%. Furthermore, since the cooking temperature promotes the polymerization reaction, the viscosity of the vegetable oil increases due to the polymers formed, compared to unheated vegetable oil.

[0030] A second type of lipid is that of acidic vegetable oil, which originates from industrial sites or wastewater treatment plants. The main characteristic of acidic oil is its high content of free fatty acids, ranging from 5% to 95% in highly degraded acidic vegetable oils. Their diverse origins make them a complex mixture to process, particularly given the presence of water, solid residues, and pollutants.

[0031] A third type of lipid is obtained from used animal fat, which is typically found in restaurants and other food service establishments used for frying or cooking meats. Used animal fat has a different profile than used vegetable oil, notably due to the presence of animal sterols such as cholesterol. Furthermore, like palm oil, animal fat is solid at room temperature, but the free fatty acid content does not exceed 5%. The module and preprocessing step A

[0032] Module or step A comprises the pretreatment of the lipids forming the input materials of the process of the invention. This pretreatment allows the removal from the lipids of residues or solid particles, polymeric compounds and most of the residual water in the used vegetable oil, acidic vegetable oil and / or used grease, to achieve a water content in the lipids of between 300 ppm and 1000 ppm.

[0033] In this first module and pretreatment step A, which precedes the enzymatic treatment, the lipids in tank 1 are treated by a cascade settling step in tanks 2. This removes large solid residues that could clog the filters 4 of the installation from the treated lipids, and the used lipids are then filtered at ambient temperature. Since palm oil and used edible fats are solid at ambient temperature, they must be heated to 40°C or higher to remain liquid during filtration before being subjected to the same conditions described below in the pretreatment and enzymatic treatment process. The settling phase is carried out, without additives, at a temperature between 40°C and 50°C, preferably around 45°C, for a duration of one to three days. The material transfers (by pumping) are performed at the same temperature.The installation includes suitable heating means to obtain and maintain this temperature.

[0034] The pretreatment step by filtration can be carried out by passing the used oils or melted animal fats through one or more filters, preferably one or more filter screens. The diameter or size of the openings in the first screen is largest, decreasing with each subsequent screen. The first filter screen has openings, such as holes, with a diameter between 2 mm and 4 mm, preferably around 3 mm. Preferably, a second filter screen with openings, such as holes, has a diameter or size between 0.5 mm and 2 mm, with an opening size of approximately 1 mm.

[0035] This preliminary filtration allows the removal of particles made up of the largest residues (food and non-food), such as pieces of plants, meat, fish bones or pieces of paper.

[0036] Next, the oil obtained preferably passes through an oil separator 3, which performs the reverse function, removing water and separating the fats from the treated lipids. During this operation, the oil flows through the multi-compartment oil separator at a flow rate preferably between 2 m³ / hour and 5 m³ / hour. Water is regularly removed either by manual valves or by an automatic valve that detects excess water. The reduced water content of the treated oil after this operation is preferably less than 1%.

[0037] The oil obtained reduced to water or the animal fat is conveyed, preferably via several filters 4, preferably filter screens having openings of 100 µm to 5 µm, to a storage tank 5 in order to collect an animal fat or an oil reduced to water having a turbidity close to 0. Turbidity is the measure of cloudiness in a liquid measured by a Metler Toledo InFit turbidimeter. The reaction module or step B

[0038] In the process and installation disclosed herein, the used lipids present in the storage tank 22 undergo initial settling before being added to a reactor 21 for enzymatic transformation. The reactor 21 includes means (23, 24, 25, 26 & 27) for adding various reagents (used lipids, enzymes, water, base, alcohol, glycerol, etc.) useful for the reaction and for collecting the products obtained or recycled products (alcohol, glycerol, biofuel, enzymes, etc.) to treatment tanks or modules (31, 32, 33, 34, 35, 37 & 38). A.1. Operating conditions for the treatment of a used non-acidic vegetable oil

[0039] The transformation of a non-acidic used vegetable oil into biodiesel involves hydrolyzing, esterifying, and transesterifying the various glycerides (triglycerides, diglycerides, and monoglycerides) present in the used non-acidic vegetable oil through enzymatic catalysis. In the process of the invention, the enzyme's efficiency was increased by preparing a homogeneous medium of the used oil and added water, then reacting the enzyme with the used oil to generate free fatty acids and initiate the esterification reaction, before adding the alcohol to trigger transesterification, as the rate of the esterification reaction is faster than that of the transesterification reaction. These two steps improved the enzyme's efficiency, thereby reducing the amount of enzyme required and the reaction time to complete the process.The enzyme's efficiency is also improved by adding a low-concentration base, which reduces the amount of enzyme needed for the process and therefore the reaction time. The addition of glycerol reduces the amount of water required to generate enzymatic activity, thus lowering the final acidity (by nearly 30%) in the resulting biodiesel. Furthermore, the preferred enzyme in this process is available in liquid form, is inexpensive, can be advantageously reused at least twice, and does not present any problems related to the type of mechanical stirring.

[0040] The enzymatic reaction was optimized with the addition of ethanol or methanol). A.2. Addition of the enzyme

[0041] In order to carry out the enzymatic transformation of used vegetable oil into biodiesel, the enzymes used are lipases, scientifically known as triacylglycerol acyl hydrolase (EC3.1.1.3), of bacterial origin or extracted from fungi.

[0042] Several commercial lipases were tested, and the reaction proved effective with several of them, including lipases derived from the strains Thermomyces lanuginosus, Pseudomonas cepacia, Pseudomonas aeruginosa, Candida Antarctica, Candida Rugosa, Rhizomucor miehei or Aspergilus niger.

[0043] Enzymes can be used in three different states: immobilized, non-immobilized, or lyophilized. Immobilizing the enzyme on a solid support makes it easier to recover and improves stability and thermal resistance, but generally increases the cost of the enzyme used, partially offset by potential reuse of the enzyme, as well as the possibility of damaging the support through mechanical agitation.

[0044] On the other hand, while free enzymes are inexpensive, their recovery is difficult and their stability is low; however, it is possible to reuse in the same reactor the said enzyme present in the residual aqueous portion of the reaction.

[0045] The three states were tested for different enzymes. In particular, the three states were tested for enzymes from Thermomyces lanuginosus, Candida rugosa, Candida antarctica, and Rhizmucor miehei.

[0046] Some trials were also conducted with non-immobilized enzymes, including Eversa, Callera TM Trans L, Novozym 40116, Lipozyme TL IM, and Pallatase. Some trials were also carried out with lyophilized enzymes (particularly the enzyme derived from Pseudomonas cepacian and Porcine pancreas). Among the enzymes that demonstrated high efficiency in converting normal or acidic used oil and animal fat into biodiesel, the commercial enzyme "Eversa Transform 2.0" achieved the best yield. Using this enzyme, the percentage of esters in the biodiesel reached over 96%. The Eversa Transform 2.0 used is in free liquid form, derived from Thermomyces lanuginosus.

[0047] The amount of enzyme has a very significant influence on the efficiency of the reaction. If too little is used, the reaction is too slow and does not proceed within a cost-effective timeframe. If too much enzyme is used, the enzyme will self-inhibit by binding too many reactants without continuing the reaction.

[0048] Advantageously, an optimal quantity combined with all other parameters allows the reaction to take place over a period of less than 24 hours.

[0049] The test range for which experiments were feasible extends from approximately 1% (v / v) to approximately 10% (v / v) for free enzymes, and from 0.5% (w / v) to 5% (w / v) for immobilized and lyophilized enzymes. By optimizing the amount of water, the amount of alcohol, the addition of additives (sodium carbonate or sodium hydroxide), the addition of glycerol, and the method of preparing the reaction medium before adding the enzyme, the amount of enzyme added can be reduced to 1% free enzyme and 0.5% immobilized enzyme, with a yield of approximately 97% esters in the biodiesel. A.3. Addition of alcohol

[0050] Alcohol serves as a reagent for hydro-esterification / trans-esterification (acyl acceptor) and also as a solvent to improve the solubility of the oil and thus reduce its viscosity.

[0051] The reaction between oil and alcohol in the presence of the enzyme is explained by the following diagram:

[0052] The addition of several short-chain carbon alcohols (methanol or ethanol) was tested.

[0053] Ethanol is chosen for optimal conversion of oils into biodiesel because this alcohol is relatively non-toxic and, like methanol, can be obtained through a process that treats renewable biological material, such as biomass fermentation. Although biodiesel derived from ethyl esters is not yet authorized like biodiesel derived from methyl esters, the process has also been tested with ethanol, as ethanol can also be obtained through a process that treats renewable biological material.

[0054] Various alcohol-to-oil ratios, ranging from approximately 3:1 (mol / mol) to approximately 16:1 (mol / mol), were tested. Since triglycerides contain three fatty acids, the amount of alcohol must be three times greater than the amount of triglycerides. Therefore, a molar ratio of 3:1 represents the minimum ratio required to achieve this. An excess of alcohol is necessary to direct the reaction toward ester synthesis, provided that this excess does not inhibit the enzyme. In this study, a molar ratio of 16:1 represents the maximum ratio that does not cause enzyme inhibition. Consequently, the range of molar ratios from 3:1 to 16:1 resulted in ester content in the biodiesel ranging from 80% to 97%.

[0055] Like other chemical reagents, some alcohols can distort the normal three-dimensional conformation of enzymatic proteins, causing them to lose their activity. This effect depends on the nature and state of the enzyme. To avoid this effect, alcohol was preferably added sequentially during the reaction, ideally before adding the enzyme, to minimize direct contact between a high concentration of alcohol and the enzyme. Therefore, alcohol addition was tested with intervals ranging from a single addition to four or more additions after varying time intervals, from approximately 30 minutes to approximately 2 or 3 hours.Indeed, up to a molar ratio of 6:1 (ethanol:oil), ethanol is added all at once at the beginning of the reaction before the enzyme, without any enzymatic inhibition. However, from a molar ratio of 7:1 onward, ethanol is added in several stages to avoid enzymatic inhibition, because at this ratio, the enzyme loses approximately 15% of its activity when ethanol is added all at once. For methanol, the addition is carried out in several stages from a molar ratio of 3:1 onward, because methanol is more inhibiting than ethanol for the enzyme; its shorter carbon chain allows it to block the enzyme's active site. A.4. Addition of water

[0056] The presence of water ensures the stability of enzyme activity and the progress of the reaction, as the aqueous layer around the enzyme helps maintain the normal three-dimensional conformation of enzymatic proteins. Water activity is specific to each enzyme (type, nature). Here, as with other parameters, the amount of water has been optimized to achieve the best enzymatic activity and yield. Even without water, the reaction occurs, but with extremely slow kinetics. A large amount of water leads the reaction to hydrolysis, which in turn reduces ester synthesis and therefore the final biodiesel yield. Beyond a certain amount of water, the reaction ceases, as the water inhibits the synthesis reaction and leads to 100% hydrolysis.The optimal reaction occurs at water contents between 0% (v / v) with no water (but preferably with the addition of glycerol) and an ester percentage of approximately 65%, benefiting from the water generated by the esterification reaction, and 5% (v / v) with an ester percentage of approximately 55% due to hydrolysis induced by the high water content in the reaction medium. Between 0% and 5% water, a maximum ester percentage of 97% has been observed. To prevent hydrolysis caused by both added and generated water, glycerol is added at the beginning of the treatment, preferably before the enzyme is added to the reaction medium. This reduces the amount of added water and creates hydrophilic microdroplets where the enzyme can work. The percentage of glycerol ranging from 1% to 20% is tested in the absence and in the presence of a small amount of water.In general, the addition of glycerol improves the yield of the free enzyme by reducing the possibility of hydrolysis, particularly at moderate concentrations around 5%. Furthermore, the addition of a low concentration of base, ranging from 1% to 4%, instead of water, has been shown to improve enzymatic activity and thus reduce the amount of enzyme by approximately 25% to 40% and the reaction time by about 8 hours compared to the reaction without added base, because the enzymatic catalyst exhibits better activity in a basic medium (at a value above 7, preferably at a pH of 8). A.5. Temperature Change

[0057] Increasing the temperature improves the reaction and allows for an estimation of the cost of producing the resulting biodiesel. Very high temperatures cause irreversible thermal denaturation of the enzyme protein, distorting its normal three-dimensional conformation. Even below the optimal temperature, the reaction occurs, but with extremely slow kinetics. The optimal temperature is specific to each enzyme and is independent of the operator's choices. In the case of the Novozym 40086 enzyme, the optimal reaction temperature is 30°C. Some enzymes have a greater tolerance to temperature variations, such as Novozym 435, whose optimal temperature is 40°C, but which still exhibits high activity between approximately 30°C and approximately 60°C.In general, the reaction temperature used in the process of the invention is between 30 °C and 70 °C, preferably between 35 °C and 55 °C, preferably between 40 °C and 50 °C. A.6. Agitation in the reaction medium

[0058] This operational parameter ensures the even and homogeneous distribution of enzymes and reactants within the reaction medium, forming a homogeneous emulsion that facilitates the function of the enzyme acting at the interface of an organic / aqueous medium (here, oil / alcohol). Indeed, enzymes adopt an active open form upon transitioning from an aqueous to an organic medium. Insufficient agitation leads to poor distribution of enzymes and reactants, slowing the reaction rate and thus lengthening the time required to complete the reaction, as well as inhibiting the enzyme in areas where the alcohol concentration is high. Excessive agitation can deform enzymes, particularly immobilized ones, by damaging the support or detaching the enzyme from the support, rendering it unusable and unrecoverable.For this reason, the reaction takes place at stirring speeds between 200 rpm and 1000 rpm, preferably between 300 rpm and 900 rpm. Within this range, the percentage of esters in the biodiesel was found to be between 70% and 97%, and the best yield was obtained at moderate stirring speeds.

[0059] The reactor (21) of module B of the invention is cylindrical with a conical bottom and a double-walled jacket, featuring a propeller-shaped blade or another suitable shape. The double wall of the reactor (21) ensures a stable and optimal temperature during the reaction. Furthermore, the propeller-shaped blade ensures even agitation of the reaction medium to obtain a homogeneous emulsion. At laboratory scale, the optimization of operating parameters was carried out in glass batch reactors with volumes ranging from 100 ml to 10 liters. At pilot scale, an optimal reaction was tested at a volume of 500 liters in a metal-walled reactor. At industrial scale, biodiesel production was performed in a metal-walled reactor with a volume of 5000 liters. A.7. Preparation of the reaction medium

[0060] Because the enzyme (lipase) acts at the interface of an organic / aqueous medium, a preparation to form a homogeneous emulsion is carried out before adding the enzyme. This facilitates efficient enzyme activity and ensures an optimal and homogeneous temperature throughout the reaction medium. Before adding the enzyme, a mixture of vegetable oil and water (or a base) is stirred for a period of 15 minutes to 2 hours, preferably between 30 minutes and 1 hour.

[0061] To increase the surface area for interaction between the enzyme (aqueous phase) and the triglycerides (organic phase), and to reduce the possibility of hydrolysis by water, glycerol is added. Next, lipase is added to the reaction medium for a period of time to adapt to the medium before the alcohol is added. Simultaneously, lipase hydrolyzes a certain amount of triglycerides, accelerating the reaction rate and initiating the esterification reaction, which is faster than the transesterification reaction. This step improves the enzyme's efficiency, which in turn increases the reaction rate and reduces the time required to convert the triglycerides from lipids to biodiesel. Finally, alcohol is added in stages to trigger the transesterification of the triglycerides. B. Operating conditions for the treatment of an acidic vegetable oil

[0062] Vegetable oil can have a high content of free fatty acids. The goal is to utilize all the glycerides and fatty acids present in the used acidic vegetable oil through two reactions: esterification and transesterification, both catalyzed by enzymes. Esterification is a reaction that transforms acids into esters by condensing them with an alcohol.

[0063] Three methods can be used to carry out this reaction. Either the reaction occurs in two steps. During the first step, the pretreated used acidic vegetable oil is reacted with ethanol or methanol and a first enzyme to esterify the free fatty acids into ethyl or methyl esters. This mixture is then collected and reacted with a second enzyme to transesterify the remaining glycerides. All the compounds in the used acidic vegetable oil are thus converted into ethyl or methyl esters. Esterification, unlike transesterification, will generate water.

[0064] A second method involves reacting the ethanol or methanol / acidic vegetable oil mixture with a single enzyme that performs esterification and transesterification.

[0065] A third method involves transforming the acids in used acidic vegetable oil into triglycerides by esterification with glycerol at high temperature and then transforming these triglycerides into biodiesel in the presence of ethanol or methanol by enzymatic catalysis.

[0066] Several commercial immobilized enzymes such as Lipozyme TL IM, Novozym 40086, Lipozyme RM IM, CalB Sigma, CalB Immoplus, NovoLime, Novozym 435, as well as commercial free-floating enzymes in liquid form such as TL Sigma, Callera TM, Eversa, Novozym 40116, liquid RM, Pallatase Candida rugosa were tested.

[0067] These three methods depend primarily on the nature of the enzyme. Some enzymes are 1,3-specific, 3-specific, or have no specificity. Similarly, some enzymes are more suited to esterification, transesterification, or both.

[0068] For example, Novozym 435 lacks specificity and can therefore react with both free fatty acids and glycerides, thus undergoing esterification and transesterification simultaneously. However, this alters the reaction kinetics of each reaction.

[0069] Lipozyme RM IM is a highly effective esterification enzyme. Lipozyme TL IM is highly effective in transesterification. It is possible to react acidic oil and ethanol first with Lipozyme RM IM, then with Lipozyme TL IM.

[0070] The immobilized enzyme Novozym 435 can transform acid oil with 75% free acids into biodiesel containing 95% ethyl esters, after 24 hours in the presence of ethanol as an acyl acceptor. C. Operating conditions for the processing of used animal fat (beef fat)

[0071] For the processing of used animal fat, similar to vegetable oil, an enzymatic reaction is optimized.

[0072] Two transesterification methods were used: Transesterification in an organic solvent: the used animal fat is reacted with ethanol or methanol and a commercial enzyme in the presence of hexane, allowing for better fat dissolution. Transesterification without an organic solvent: the used animal fat is reacted directly with ethanol or methanol and a commercial enzyme at a temperature that keeps the fat in liquid form. This method requires much stronger stirring, especially at the beginning of the reaction, and has slower kinetics. A second way to optimize the kinetics of this reaction is to carry it out at a higher temperature (between approximately 40°C and approximately 50°C) with enzymes adapted or resistant to these temperatures. For example, the activity of Lipozyme TL IM is significantly reduced above 35°C.Conversely, some enzymes like Novozym 40086 have much wider activity ranges relative to reaction temperature.

[0073] Three types of enzymes were tested: immobilized (TL-IM, Novozyme 40086, Lipozyme RM IM, CalB Sigma, CalB Immoplus, NovoLime, Novozym 435), free in solution (TL Sigma, Callera TM, Eversa, Novozym 40116, liquid RM, Lipozyme TL IM, Pallatase, Candida rugosa) and lyophilized ( Candida rugosa ) .

[0074] The free enzyme Novozym 40116 was able to transform beef fat into biodiesel with 80% ethyl esters at 40°C after 24 hours. The module or step C of enzyme recycling and biodiesel refining obtained

[0075] Advantageously, and to improve the economic and environmental aspects of biodiesel production, immobilized enzymes can be collected from the reaction medium and cleaned for recycling and reuse, preferably in the process of the invention. For this purpose, the enzyme is washed in a closed loop in module C with absolute hexane and then dried overnight at room temperature. Using this washing method, the enzyme has been reused several times, retaining almost 95% of its activity. This module and step 3 are only useful for immobilized enzymes. For example, by applying this recycling method, Novozym 435 was reused 10 times over a period of one month, retaining almost 95% of its initial activity.

[0076] For free enzymes present in liquid form, enzyme recycling is complex because it is difficult to recover from the reaction medium. However, the Eversa enzyme was successfully reused at least twice by leaving it in the reaction medium and adding more reagents (oils and alcohol) without adding water.

[0077] To avoid generating waste and losing potential reagents or valuable by-products, the refining process of the crude biodiesel obtained is done in two sub-steps: a step of evaporating the residual alcohol in the mixture and a step of natural decantation of the glycerol and water generated from the biodiesel obtained during the reaction.

[0078] The evaporation step has been optimized to eliminate the majority, preferably all, of the excess alcohol and to recover this excess alcohol to put it back into reaction in the process of the invention.

[0079] Evaporation takes place in module C, i.e., a tank (34) maintained under a residual pressure of approximately 150 mbar and a heating temperature of the mixture of approximately 120 °C. These conditions are only slightly adjustable, except by operating under lower residual pressures down to approximately 30 mbar, thus reducing the evaporation time using a falling-float evaporator.

[0080] The liquid enters the upper part of the evaporation column and flows down the heated tubes. The apparatus is depressurized (to approximately 150 mbar) and the alcohol evaporates, to be condensed in a condenser (35). The collected alcohol (99% pure) is then reused in the transesterification reaction (see figure 3 ).

[0081] After all the alcohol (35) has evaporated, the mixture is transferred to a settling tank (36) in the module where the glycerol (37) and biodiesel separate naturally. Three phases are recovered: glycerol, a mixture of glycerol and refined biodiesel consisting of pure biodiesel, and pure biodiesel (B100). The first two products (glycerol and glycerol / biodiesel) are stored in separate 1000-liter tanks.

[0082] The glycerol obtained can be used in the chemical market (industrial soap production) and even in the food and cosmetics industries, as the glycerol-biodiesel mixture is reintroduced into the refining module. The B100, meanwhile, is transferred via a pump to the biodiesel processing module D described below. Module or stage D of refined biodiesel processing

[0083] The refined biodiesel processing module D obtained from the module or stage includes a tank (42), where an antioxidant (41) is added to the refined biodiesel obtained at a rate of 0.8% and this refined biodiesel obtained after treatment by water washing (43) and purification on resin (44), can then be transferred to another tank (36) and supplemented with diesel at a rate of 70% in order to obtain a B30 biodiesel.

[0084] The most significant advantage of the process and installation described here is the ability to use the same enzymatic catalyst to trigger the transformation of so-called normal or acidic used oils and animal fats into biodiesel. This is considered an "eco-friendly" process because, unlike chemical processes using acidic or basic catalysts, this process yields only biodiesel (as the primary product) and glycerol (as a co-product), without any secondary reactions (saponification), thanks to the specificity and selectivity of the enzyme.

[0085] In the process of the invention, the efficiency of the enzyme used is also increased by preparing a homogeneous medium of used oil and added water, and then reacting the enzyme with the used oil before adding the alcohol and initiating the transesterification. These two steps have therefore made it possible to advantageously reduce the amount of enzyme required to carry out the reaction.

[0086] The enzyme's efficiency could also be improved by adding a low-concentration base (a solution of NaOH or Na₂CO₃) and / or glycerol. This can control the acidity in the biodiesel and reduce the amount of enzyme needed for the process, thus shortening the reaction time. Furthermore, the most effective enzyme in this process is in liquid form, inexpensive, and has been reused at least twice without losing any activity or causing problems related to the type of mechanical agitation, unlike immobilized enzymes.

[0087] This process can be advantageously combined with local collection of lipids from used oils and used animal fats and with local use of this biodiesel for vehicle traffic. Examples Example 1: Transformation of a sample of vegetable oil into biodiesel

[0088] 100 ml of the obtained vegetable oil is poured into a 400 ml reactor 20. Then, 3% (v / v) water or base and 5% glycerol are mixed with the oil for 30 minutes. Next, 2% (v / v) of a triacylglycerol acyl hydrolase lipase enzyme (Eversa Transform 2.0 in liquid form) is added to the reaction mixture, which is left for 1 hour without alcohol, thus preventing the transesterification reaction from occurring. This step causes the enzyme to react with the oil to generate fatty acids and accelerates the reaction. Then, ethanol at a molar ratio of 4:1 to vegetable oil is added in three stages at time intervals of 0, 2, and 4 hours. After approximately 22 hours, the organic and aqueous phases are separated by centrifugation at 4700 rpm for 15 minutes.

[0089] For the production of biodiesel from methyl esters, the same process for ethyl esters as described above was applied, except that the molar ratio (Methanol: oil) is 6:1.

[0090] The content of the refined biodiesel obtained was studied by chromatographic methods (HPLC and GC). Example 2: Transformation of a sample of acidic vegetable oil into biodiesel

[0091] In the present invention, the transformation of acidic vegetable oils into biodiesel was catalyzed by the same enzyme used in Example 1 for the transformation of ordinary used vegetable oils into biodiesel. During this reaction, the step of thoroughly mixing the oil with water or a base was eliminated, as the reaction proceeded without added water. The high quantity of free fatty acids will induce the esterification reaction in the presence of alcohol. Since this type of reaction yields both ester and water, the amount of water generated in the reaction medium will be sufficient to trigger the transesterification reaction between the triglycerides and the alcohol. Thus, the step of generating fatty acids between the enzyme and the oil without alcohol was eliminated, because the oil used in this process is already acidic and contains a high quantity of free fatty acids.Glycerol was added to the reaction medium, which can create a site on which the enzyme works and decrease hydrolysis, thus reducing the amount of free fatty acids.

[0092] 100 mL of an acidic vegetable oil was poured into a 400 mL reactor 20, and 0.5% NaOH and 10% glycerol were added. Then, 1% (v / v) of the Eversa® Transform 2.0 enzyme in liquid form was added. Finally, ethanol at a molar ratio of 4:1 to the acidic vegetable oil was added in three stages at time intervals of 0, 2, and 4 hours to avoid enzymatic inhibition by the alcohol. After 22 hours, the organic and aqueous phases were separated by centrifugation at 4700 rpm for 15 minutes. To study the composition of the refined biodiesel obtained, samples were analyzed by chromatographic methods (HPLC and GC). For the production of biodiesel from methyl esters, the same process for ethyl esters as described above was applied, except that the molar ratio (Methanol: oil) of 6:1 was used. Example 3: Transformation of an animal fat sample into biodiesel

[0093] In the present invention, three triacylglycerol acyl hydrolase lipase enzymes have shown satisfactory results: Lipozyme TL IM, Novozym 40086, and Eversa Transform 2.0. Therefore, the transformation of animal fats can be carried out using the same enzymatic catalyst (enzyme) used to convert normal or acidic used oils into biodiesel. Since the objective of this invention is to find an environmentally friendly process, the conversion of animal fat into biodiesel has been optimized without the addition of any organic solvent.

[0094] 100 g of animal fat was weighed into a 400 ml reactor 20. Then, 2% (w / v) of water or base was mixed with the fat for 30 minutes at 45°C to ensure the fat was thoroughly melted and mixed with the water or base. Next, 4% (v / v) of the enzyme (Eversa Transform 2.0 in liquid form) was added to the reaction mixture, which was left to stand for 1 hour without the addition of alcohol. Finally, ethanol at a molar ratio of 5:1 to the animal fat was added in three stages at 0, 2, and 4 hours to avoid enzyme inhibition by the alcohol.

[0095] After 22 hours, the organic and aqueous phases were separated by centrifugation at 4700 rpm for 15 minutes. To study the composition of the refined biodiesel obtained, samples were analyzed by chromatographic methods (HPLC and GC). For the production of biodiesel from methyl esters, the same process as for ethyl esters as described above was applied, except that the molar ratio (methanol:fat) was 6:1. Example 4:

[0096] The used oil is mixed with water (or a base) and glycerol for 30 minutes to obtain a homogeneous medium and avoid thermal and chemical shock to the enzyme. Then, the required amount of enzyme is added to initiate hydrolysis for one hour. Finally, alcohol is added gradually to prevent enzyme inhibition.

[0097] The inventors observed that the hydrolysis step improves enzymatic activity (the initial velocity of the enzyme with hydrolysis (29.5 mM / h) is almost twice that of the enzyme without hydrolysis (16.5 mM / h)).

[0098] This hydrolysis step improves the quality of biodiesel (the volume percentage of triglycerides and diglycerides at the end of the reaction following hydrolysis is close to zero, whereas without hydrolysis, these products represent 2-3% of the volume at the end of the reaction), and this hydrolysis step controls the functioning of the enzyme (it is useful to test the enzymatic activity and know if the activity of this enzyme is working correctly or not, before adding the alcohol).

[0099] The inventors also observed that the addition of a base improves enzymatic activity (by about 20%), which reduced the amount of enzyme needed (by almost 25%) and reduced the reaction time by about 8 hours compared to the reaction with water (for example, with the addition of a base, triglycerides are completely transformed after 13 hours, whereas with water, they are transformed after 22 hours).

[0100] Adding a base also improves the yield (i.e., the amount of biodiesel produced) by at least 5%: in the presence of a base, the emulsion (the heavy phase) does not form, so no biodiesel is retained in the gel network, thus increasing the amount of biodiesel obtained. The absence of the emulsion and the presence of two liquid phases also facilitate filtration and the recovery of glycerol (a by-product). For example, with the addition of a base, the biodiesel phase obtained represents almost 88% of the volume, whereas with water and the formation of the emulsion, the biodiesel phase represents around 82% of the volume.

[0101] The addition of the base also improves the quality of biodiesel (with the addition of a base, triglycerides and diglycerides are almost totally transformed (100% of the volume) and monoglycerides are present in a minimal amount (less than 1.5% by volume)).

[0102] The addition of the base also reduces the acidity and the amount of free fatty acids remaining in the biodiesel): The presence of a base has reduced the % of FFA (for example, the acidity in the presence of the base is almost 2.6%, whereas it is almost 3% with water).

[0103] The inventors observed that the addition of glycerol is useful to avoid hydrolysis caused by added water and generated water. Glycerol (hydrophilic) creates hydrophilic microdroplets that promote enzyme activity.

[0104] The addition of glycerol to the reaction medium improves acidity: (the % of non-esterified free fatty acids (FFA) is almost 2.2% by volume in the presence of glycerol, compared to almost 3% of FFA by volume with water).

[0105] The addition of glycerol also improves the yield and the reduction of AGL increases the yield of the biodiesel obtained by about 2% by volume (for example, without the addition of glycerol, the % of esters obtained is almost 90% by volume, whereas with the addition of glycerol the % of esters obtained is almost 92%). Example 5:

[0106] We carry out a normal reaction 1 on the mixture (used oil + 2% water + 25% alcohol + 2.5% enzyme) and a reaction 2 (with hydrolysis + glycerol + addition of a base) on used oil + 2% base + 4% glycerol + 30 min of mixing + 2.5% enzyme + hydrolysis 1 hour + 25% alcohol.

[0107] To compare the efficiency of the two reactions, the inventors monitored the transformation of triglycerides (TGs) and the appearance of the products (diglycerides (DGs), monoglycerides (MGs), esters, free fatty acids (FAL)) by high-performance liquid chromatography (HPLC).

[0108] The data is also shown in Table 1 below. Table 1 Reaction 1 Reaction 2 TGs % 3, 4 0 DGs 5, 6 0 Esters % 85, 2 95, 6 AGL % 3, 1 2, 6 MGs % 2, 7 1, 8

[0109] There figure 3 is an HPLC chromatogram of reaction 1 (Oil + water + enzyme + alcohol) and reaction 2 (Oil + base + glycerol + mixture 30 minutes + enzyme + hydrolysis 1 hour + alcohol).

[0110] The chromatograms show that reaction 1 is not complete, as the triglycerides (TGs) are not fully transformed after 24 hours and the intermediate products (diglycerides, monoglycerides, and free fatty acids) are not completely converted into esters. However, reaction 2 is complete after 24 hours, as the triglycerides and intermediate products are converted into esters.

Claims

1. Method for obtaining a biodiesel consisting of a mixture of ethyl esters or methyl esters by enzymatic transformation of lipids selected from the group consisting of used vegetable oil, acid vegetable oil and / or used animal fat, wherein said enzymatic transformation comprises an enzymatic transesterification and an enzymatic hydro-esterification, an addition of alcohol, an addition of water or of a base in aqueous solution, and an enzymatic addition of glycerol, characterized in that prior to the addition of alcohol, the lipids, the water or the base and glycerol form a homogeneous medium and wherein this medium is brought into contact with one or more enzymes of the triacylglycerol acyl hydrolase type.

2. Method of claim 1, wherein the medium is brought into contact with the enzyme for a duration between 30 minutes and 2 hours.

3. Method of claim 1 or 2, wherein alcohol is added to the molar ratio (alcohol: oil) from (3:1) to (16:1).

4. Method according to any one of the preceding claims, wherein the lipids are subjected, prior to enzymatic transformation into biodiesel, to a cascade decantation step and one or more steps of filtration of the solid particles present in the lipids on filters and to a water removal step, thereby obtaining lipids containing a water content of less than 1% by weight in relation to the weight of the lipids.

5. Method of claim 4, wherein the filters have apertures between 100 µm and 5 µm.

6. Method according to any one of the preceding claims, wherein the lipids consist of used palm oil or of used animal fat that is heated to a temperature of 40 °C or more, preferably 45 °C or more, prior to the filtration step(s).

7. Method according to any one of preceding claims 1 to 5, wherein the lipids are subjected, prior to the enzymatic transformation into biodiesel, to a decantation step at room temperature for used oils and at 45°C or more for animal fats for a duration of between 48 hours and 72 hours.

8. Method according to any one of the preceding claims, comprising a step of recycling the enzyme used.

9. Method according to claim 8 wherein the recycling step of the enzyme used comprises a step for reusing the free enzyme in liquid form, a step of immobilising the enzyme used, a step of collecting the immobilised enzyme used, a step of washing the enzyme with hexane, and a step of drying the collected enzyme.

10. Method according to any one of the preceding claims, comprising after the enzymatic transformation of the used lipids into biodiesel, a refining step of the medium comprising a separation by decantation of a heavy phase which contains glycerol formed during the enzymatic transformation, the water or the base into aqueous solution, and the enzyme, and of an organic phase which contains the biodiesel obtained and the alcohol, a separation of alcohol and biodiesel from the organic phase by evaporation of the alcohol, and a collection by condensation of the evaporated alcohol and / or addition of the recycled alcohol in the method of obtaining biodiesel, a decantation of glycerol and / or of the aqueous portion of the heavy phase, a refined biodiesel collection step, followed by a step of adding an antioxidant, to the refined and collected biodiesel, and / or a step of blending the collected refined biodiesel with another fuel to obtain a final 30% biodiesel (B30).

11. Method of claim 10, wherein an aqueous portion of the medium incorporating the enzyme, water or base in aqueous solution and the glycerol formed during enzymatic transformation, to which the alcohol is added and the used lipids for the obtaining of biodiesel, without separately adding the enzyme, water and / or base and the glycerol.

12. Method according to any one of the preceding claims, wherein the alcohol is selected from the group consisting of ethanol or methanol.

13. Method according to any one of the preceding claims, wherein alcohol is added to said medium sequentially 1 to 4 times, after a duration of between 15 minutes and 4 hours.