IL301327A

IL301327A - Processes for the production of biodiesel from fatty waste

Info

Publication number: IL301327A
Application number: IL301327A
Authority: IL
Original assignee: Univice M E Ltd
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2024-10-01
Also published as: WO2024189621A1; EP4680757A1

Description

PROCESSES FOR MANUFACTURING BIODIESEL FROM FATTY WASTE TECHNOLOGICAL FIELD The present disclosure concerns processes and systems for the manufacture of biodiesel, specifically enzymatic processes for manufacturing biodiesel from waste matter rich in free fatty acids (FFA).

BACKGROUND ARTReferences considered to be relevant as background to the presently disclosed subject matter are listed below: - PCT patent application publication no. WO2011/1079- PCT patent application publication no. WO2013/0308- PCT patent application publication no. WO2012/1309- PCT patent application publication no. WO2016/0894- PCT patent application publication no. WO2018/1916- PCT patent application publication no. WO2018/1162 Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUNDFinding renewable sources as alternatives for natural oil-based diesel has been a researching target in the past few decades. Fats and oil waste have been considered as a renewable source for various proposed manufacturing processes, recycling fat and oil waste into valuable products, one of which being biodiesel. Common processes of transforming fats and oils into biodiesel are based on transesterification chemical processes, in which triglycerides in the oil or fat are reacted with alcohols, typically methanol, to obtain fatty acid methyl esters (FAME), which constitute the biodiesel. These processes are often carried out in the presence of various catalytic agents (alkalines, acids, transition metals/alloys, enzymes, etc.), and the biodiesel production yield typically heavily depends on the reaction conditions. One of the main difficulties in such processes is increase in yield of transformation of the glycerides and free fatty acids (FFA) in the oil/fat feed stream into the biodiesel while providing strict control over the process conditions to avoid poisoning of the catalysts utilized in the process. However, oil/fat waste products typically contain relatively high amounts of water and FFA, which are often more difficult and expensive to treat. Thus, there is a need for highly controlled biodiesel manufacturing processes that can utilize FFA-rich waste streams in a high yield and cost-effective manner.

GENERAL DESCRIPTION The present disclosure concerns industrial-scale processes and systems for manufacturing biodiesel from fat sources, e.g. waste fat or waste oils, that contain relatively high amounts of free fatty acids (FFA). The processes and systems of the present disclosure involve a sequence of enzymatic treatments of the waste fats/oils, with careful control over feed streams preparation and parameters, thereby enabling utilizing the high content of the FFA in the feed stream to increase the yield of biodiesel manufacturing in a cost-effective manner. Hence, processes and systems disclosed herein are particularly suitable for treating fat/oil waste products that are relatively rich in FFA. Further, it was surprisingly found that better yield can be obtained in an industrial scale by utilizing specific feed streams preparation methods, that are different from the classic manufacturing processes that involve feeding each of the raw materials directly into the reactor. In the processes of this disclosure, specific mixing techniques and careful compositional, pH and temperature control in the various steps of the processes not only permits utilizing the high content of FFA in the waste feed and increase the yield of biodiesel manufactured therefrom, but also increase the active lifespan of the enzyme used for the transesterification/esterification step in a continuous mode of operation. Hence, in the processes of this disclosure, high contents of FFA can be used as fat sources, unlike traditional biodiesel manufacturing processes from which FFA is typically removed and not utilized. The processes of the present disclosure aim at providing a cost-effective and highly tailorable transformation process of waste fat into biodiesel on an industrial scale, with relatively short enzymatic reaction times.

Thus, by a first of its aspects, this disclosure provides a process for manufacturing biodiesel from a fat source that comprises glycerides and free fatty acids (FFA), the process comprising: (a) mixing said fat source with at least one aqueous alkaline solution and at least one C1-C6 alkyl alcohol, to obtain a first feed mixture; (b) cooling said first feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the first feed mixture into a first enzymatic reactor that holds at least one immobilized enzyme, and maintaining the first feed mixture in said first enzymatic reactor under conditions permitting concomitant enzymatic transesterification of said glycerides and esterification of said FFA by reacting with said at least one C1-Calkyl alcohol, to obtain a first crude stream of fatty acid alkyl esters; (c) transferring said first crude stream into a first separation unit while heating the first crude stream, and maintaining the first crude stream in said first separation unit to obtain a first aqueous phase and a first organic phase; (d) mixing said first organic phase with at least one aqueous alkaline solution in a first static mixer to obtain a first emulsion; (e) mixing said first emulsion with at least one C1-C6 alkyl alcohol in a second static mixer, being serially linked to said first static mixer, to obtain a second feed mixture; (f) cooling said second feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the second feed mixture into a second enzymatic reactor that holds at least one immobilized enzyme, and maintaining the second feed mixture in said second enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said second feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a second crude stream of fatty acid alkyl esters; (g) transferring said second crude stream into a second separation unit while heating the second crude stream, and maintaining the second crude stream in said second separation unit to obtain a second aqueous phase and a second organic phase, the content of unreacted glycerides and unreacted FFA in said second organic phase being smaller than in said first organic phase; and (h) treating the second organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel.

In other words, the processes of this disclosure are based on a sequence of operation cycles, in each cycle specific and different mixing conditions of the feed mixtures are applied prior to their introduction into enzymatic reactors, followed by separation treatment steps. In each cycle, the content of fatty acid alkyl esters is increased in the feed stream, until reaching a desirable purity and yield that permits further processing into biodiesel, as will be further explained below. The term biodiesel means to denote a biofuel, i.e. a fuel derived from a biological source, such as animal fat or plant-originated oil. The biodiesel typically consists of long-chain fatty acid esters, typically fatty acid alkyl esters, which are the products of chemical reactions between fatty acids and, typically, short-chain alcohols. Fatty acid alkyl esters are esters formed out of the combination of fatty acids and alkyl alcohols, typically short chain alkyl alcohols. For example combination of fatty acids with methanol will form fatty acid methyl esters (FAME), fatty acids combined with ethanol will form fatty acid ethyl esters (FAEE), etc. In the process of the present disclosure, biodiesel is manufactured from one or more fat sources, which typically include animal-fats or plant-based oils, that comprise glycerides and free fatty acids (FFA). The fat source can be a native source, e.g. pure or refined plant oil or animal fat, or a processed fat/oil or fatty waste product, such as animal fat waste, cooking oil, frying oil, etc. Glycerides are types of carbonaceous esters, which include monoglycerides, diglycerides and triglycerides, together forming the main components in natural fats and oils. Triglycerides are structured out of a glycerol backbone substituted by three fatty acid groups. Free fatty acids (or FFA) are fatty acids that are produced by hydrolysis of oils and fats. While typically found in higher amounts in processed fats and oils, FFA can also be found to some extent in unprocessed or native fats and oils. FFA encompass a carboxylic acid with an aliphatic tail (chain) of between about 2 to 30 carbon atoms, which is either saturated or unsaturated. The fatty acids can be short-chain fatty acids (1-carbon atoms in the aliphatic tail), medium-chain fatty acids (6-14 carbon atoms), or long-chain fatty acids (above 14 carbon atoms). Non-limiting examples of medium-chain fatty acids include hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid and their corresponding salts, such as sodium salts, etc. By some embodiments, the fat source can comprise monoglycerides, diglycerides, triglycerides and FFA, their mixtures at any ratio, in the absence or presence of additional fatty acid derivatives such as phospholipids, wax esters, sterol esters, etc. According to some preferred embodiments, the fat source comprises at least about wt% FFA, e.g. between about 10 and about 100 wt% FFA, between about 30 and about 100 wt% FFA, between about 40 and 100 wt% FFA, or even between about 50 and 1wt% FFA. Without wishing to be bound by theory, the inventors have found that the higher the FFA in the feed stream fed into the enzymatic stage, the less glycerin by-product is produced during the enzymatic stage, thereby requiring less separation and treatment process steps. According to some embodiments, the process further comprises, prior to step (a), one or more pre-treatments that comprise receiving a crude fat source having an FFA content of less than about 10%, and treating said crude fat source to obtain a fat source having an FFA content of at least about 10%. In other words, in cases where the crude fat source (e.g. as received from a waste management facility) contains less than about wt%, a sequence of pre-treatments may be carried out in order to obtain a fat source having an FFA content of above 10 wt%, preferably at least about 20 wt%, more preferably at least about 25 wt%, making it suitable as a raw material for processes of this disclosure. Such pre-treatments are described in Applicant’s patent application no. PCT/IL2022/050962, which is incorporated herein by reference. According to some embodiments, the fat source can be an edible fat/oil (or originating from an edible source) or a non-edible fat/oil (or originating from a non-edible source). According to other embodiments, the fat source comprises at least one oil selected from a vegetative source, animal fat, algal oil, fish oil, or any mixture or combination thereof. According to some other embodiments, the fat source comprises used cooking oil (UCO) and/or used frying oil (UFO). According to some further embodiments, the fat source can be waste cooking oil. By some embodiments, the fat source can be selected from animal-derived fat, including lard, tallow, fish oil, chicken fat, yellow grease, brown grease, and any combination thereof.

By other embodiments, the fat source can be an oil from a vegetative source selected from soybean oil, canola oil (rapeseed oil), algae oil, olive oil, castor oil, palm oil, palm olein, palm stearin, grapeseed oil, hemp oil, sunflower oil, safflower oil, peanut oil, cotton seed oil, Jatropha oil, corn oil, avocado oil, sesame oil, rice bran oil, coconut oil, mustard oil, flaxseed oil, jojoba oil, tall oil, oils derived from inedible plant sources, partial glycerides and free fatty acids derived oils, and mixtures and combinations thereof. According to some other embodiments, the fat source can be a waste FFA stream from biodiesel manufacturing processes, e.g. FFA separated as waste material from the raw materials or biodiesel product produced therefrom (on in intermediate steps in such processes). In order to form consistent and repeatable feed mixtures that are fed into the enzymatic reactors, uniform mixtures of the fat source (i.e. oily components) need to be intimately mixed with an aqueous solution of one or more alkaline materials and with C1-C6 alkyl alcohols. In order to form homogenous mixtures, the inventors have found that different mixing means and mixing sequences need to be applied at different steps of the process. Hence, according to some preferred embodiments, mixing at step (a) to obtain said first feed mixture is carried out in a high shear mixing unit. High shear mixer (or high shear mixing unit) refers to mixing means in which high shear forces are applied onto the ingredients, thereby enforcing intimate dispersion of water droplets in a continuous oily phase to form water-in-oil emulsions, or dispersion of oil droplets in a continuous aqueous phase to form oil-in-water emulsions – depending on the water content of the mixture. Such intimate mixture of the fat source with the alkaline materials and the alcohols enable forming close proximity of ingredients and uniform distribution thereof within the mixture, thereby increasing the efficiency of the enzymatic process once introduced into the enzymatic reactor and coming into contact with the immobilized enzymes held therein, assisting in reducing the residence time of the ingredients in the reactor in order to obtain a target yield. The high shear mixing was found to assist in increasing yield and reducing overall enzymatic process time in an industrial process when compared to simple mixing techniques. Further, the enzymes used in the enzymatic transformation of the glycerides and FFA into fatty acid alkyl esters are typically sensitive to variations in pH. The formation of fine dispersions (or emulsions) of the alkaline materials in the fat source was found to permit better and quicker control over the pH of the feed mixtures prior to their introduction into the enzymatic reactor. In order to improve formation of a fine dispersion, mixing at step (a) is, by some embodiments, carried out at a temperature of between about 40ºC and about 80ºC, for example between 50ºC and 75ºC. Before introduction into the enzymatic reactor(s), the feed mixture is cooled to a temperature of between about 25ºC and about 35ºC, thereby introducing the feed mixture into the reactor at a temperature that does not damage the immobilized enzyme. The enzymatic reactors are configured to hold at least one immobilized enzyme, which is suitable to permit reaction of the glycerides and the FFA in the fat source with one or more alcohols within the feed mixture, to thereby obtain fatty acid alkyl esters. In the enzymatic reactor(s), the glycerides (at least the triglycerides) are transesterified while the FFA are esterified concomitantly in order to obtain the fatty acid alkyl esters. Hence, in the enzymatic reactor, the fat source and the alcohol undergo an enzyme-assisted chemical reaction. The transesterification of triglycerides with methanol is shown in eq. 1, while the esterification of FFA with methanol is shown in eq. 2: HC HC HC O C ROOC RO OC ROCHOH HC HC HC O C ROOC RO OC RO HC HC HC OH OH OH (Eq. 1) RC OHOCHOHRC OOCHHO (Eq. 2) In eqs. 1 and 2, R, R1, R2 and R3 represent C1-C24 hydrocarbon chains, saturated or unsaturated, typically aliphatic, linear or branched. The triglycerides can be short chain triglycerides, medium chain triglycerides, long chain triglycerides, or any combination thereof. The reaction is assisted by one or more immobilized enzymes, which comprise at least one substrate associated with one or more suitable enzymes, e.g. one or more lipase enzymes. Commonly, enzymatic processes in the production of biodiesel are carried out by addition of the enzyme into the reaction vessel in a free form (i.e. mobile), and the enzyme is typically washed out during the process and needs to be continuously replenished. Contrary to these common methods, the transesterification/esterification reactions in the enzymatic stages are carried out by utilizing an immobilized enzyme, that is physically and/or chemically associated with or fixated to a substrate, the substrate being in a form and/or size that prevents its unintended extraction from the reactor. Therefore, the immobilized enzyme can be utilized for continuous production of biodiesel, without requiring frequent replenishment of enzyme. The substrate can be of any suitable form, e.g. powder, agglomerated particles, flakes, discs, pellets, blocks, rods, etc. The enzyme is typically associated with or affixed to the surface of the substrate. The enzyme can be associated with one or more surface portions of the substrate or be associated substantially with the entire surface of the substate. In order to increase the surface available for enzymatic reaction, the substrate may be perforated or porous (for example having a surface area of at least 80 m/g or even at least 100 m/g). When more than one enzyme is utilized, the substrate can be associated with one or more enzymes. Alternatively, a portion of the substrate can be associated with one of the enzymes, and another portion of the substrate can be associated with another one of the enzymes. According to some embodiments, the substrate is a hydrophobic substrate, therefore being stable under the reaction conditions for a prolonged period of time. The substate may be made of or coated by one or more hydrophobic coatings. According to some embodiments, the substate can be made of or is coated by one or more hydrophobic polymers. The hydrophobic polymer(s) can be aliphatic or aromatic, linear or branched, thermoplastic or thermosetic. According to some embodiments, the one or more enzymes is a lipase enzyme. By such embodiments, the lipase enzyme can be selected from lipases derived from Thermomyces lanuginosus, Rhizomucor miehei, Mucor miehei, Pseudomonas sp., Rhizopus sp., Mucor javanicus, Penicilhum roqueforti, Aspergillus niger, Chromobacterium viscosum, Acromobacter sp., Burkholderia sp., Candida antarctica A, Candida antarctica B, Candida rugosa, Alcaligenes sp., Penicillium camembertii, papaya seeds, pancreatin or any other lipase source, and any mixture thereof.

According to some embodiments, the ratio between the immobilized enzyme (i.e. the weight of the enzyme together with the immobilization substrate) and the first mixture is between about 1:4 and about 1:12. The enzyme, according to some embodiments, is selected as to permit concomitant transesterification of the glycerides and esterification of the FFA, thereby permitting one-pot reaction. In the process described herein, the pre-treatment stages of the process prior to the enzymatic reaction are designed to increase the content of FFA in the crude oil (i.e. instead of other known processes which aim to entirely remove FFA at various stages of the process), thereby enabling utilizing FFA as raw material for biodiesel production without requiring its separate treatment. This, in turn, increases the yield of biodiesel production, thereby obtaining a process yield of at least 75% in an industrial process scale, as well as reducing the production of glycerin by-products. Process yield, in the context of this disclosure, means to refer to the amount of final product (i.e. biodiesel) divided by amount of fat source entering the enzymatic stages of the process. In the enzymatic reactor(s), the fat source and alcohol are brought into contact in the presence of the immobilized enzyme under conditions permitting the concomitant enzymatic transesterification and esterification reactions. According to some embodiments, the first feed mixture is maintained in said first enzymatic reactor for a period of time of between about 1 and about 8 hours, at a temperature of between about 25ºC and about 35ºC, and under stirring conditions. By some other embodiments, the temperature in the first enzymatic reactor is maintained at between about 28ºC and about 35ºC. In some embodiments, the first feed mixture has a pH of between about 5.5 and 8, preferably between about 6 and 7.5. By some embodiments, the first feed mixture is maintained in said first enzymatic reactor for a period of time of between about 1 and about 6 hours, between about 1.5 and about 5 hours, or even between about 2 and about 4.5 hours, depending on the quality (e.g. wt% of FFA and presence of various contaminants) of the fat source in the first mixture. According to some embodiments, the fat source is present in the first mixture in excess with respect to the C1-C6 alkyl alcohols. By some embodiments, the first feed mixture comprises between about 8 wt% and about 12 wt% of said at least one C1-C6 alkyl alcohol. According to such embodiments, the ratio between the fat source and the C1-C6 alkyl alcohol (e.g. methanol) in the first feed mixture is between about 5:1 and 15:1. According to some embodiment, the ratio between the fat source and the C1-C6 alkyl alcohol (e.g. methanol) in the first feed mixture is between about 5:1 and 10:1. The first crude stream of fatty acid alkyl esters is the product of the enzymatic reaction in the first enzymatic reactor. The crude stream contains fatty acid alkyl esters, unreacted components (e.g. unreacted glycerides, FFA, alcohol, etc.), water, by-products (such as polyols), and various residual contaminants. Thus, the first crude stream is transferred to a first separation unit in step (c), in which the first crude stream is allowed to separate into two phases: a first aqueous phase which contains water, alcohol, polyols, and other miscible components, and a first organic phase which contains predominantly fatty acid alkyl esters, unreacted glycerides and unreacted FFA. The polyols (e.g. glycerol) can be subsequently separated from the aqueous phase for various industrial uses. Alternatively, in some embodiments, the polyols are returned into the fat source at step (a) for further treatment. The first organic phase is then used as a raw material for the second enzymatic stage (step (f)) carried out in the second enzymatic reactor. The first organic phase is mixed, prior to introduction into the second enzymatic reactor, with an alkaline aqueous solution and C1-C6 alkyl alcohol, in a gradual manner. The inventors have found that such gradual mixing enables better control of the pH of the second mixture prior to introduction into the second enzymatic reactor, as well as obtaining better homogenization of the reactants in the second feed mixture. Therefore, the first organic phase is first mixed with the at least one aqueous alkaline solution in a first static mixer to obtain a first emulsion (step (d)), and then the first emulsion is mixed with at least one C1-C6 alkyl alcohol in a second static mixer that is serially linked to the first static mixer, to obtain a second feed mixture (step (e)). A static mixer is a mixing unit that has no moving parts. Static mixing is based on concomitantly force-feeding the components to be mixed into a mixing unit that typically comprises an array of stationary flow diverters or baffles, such that the components are forced to intermix as they flow along the static mixer. The first and second static mixers can be the same or different. Separation between the addition of the at least one aqueous alkaline solution and the C1-C6 alkyl alcohol in order to obtain the second feed mixture enables careful control over the pH of the second feed mixture. According to some embodiments, step (c) comprises measuring the pH of said first aqueous phase to obtain a first pH value, and determining the amount of said at least one aqueous alkaline solution added to the first organic phase in step (d) based on said first pH value, to obtain a pH of between about 5.5 and 8 in said first microemulsion. In other words, the pH value of the first aqueous phase is used to calculate the required amount of aqueous alkaline solution to be added to the first organic phase in the first static mixer in order to obtain a pH of between about 5.5 and about 8, preferably between about 6 and about 7.5. According to some embodiments, following formation of a first emulsion in the first static mixer, the C1-C6 alkyl alcohol is added in the second static mixture to form an intimate and homogenous second feed mixture. By some embodiments, the second feed mixture comprises between about 5 wt% and about 10 wt% of said at least one C1-Calkyl alcohol. The second feed mixture is then cooled down to a temperature of between about 25ºC and 35ºC and fed into the second enzymatic reactor (step (f)), in which the unreacted glycerides and FFA in the second feed mixture are allowed to react with the C1-C6 alkyl alcohol to product further fatty acid alkyl esters. Such a second enzymatic treatment is designed to increase the overall yield of the process, namely, to increase the formation of fatty acid alkyl esters and result in higher production capability of biodiesel from the fat source. According to some embodiments, the second feed mixture is maintained in the second enzymatic reactor for a period of time of between about 1 and about 8 hours (e.g. between 2 and 5 hours), at a temperature of between about 25ºC and about 35ºC (preferably between 28ºC and 35ºC), and under stirring conditions. According to some embodiments, second feed mixture has a pH of between about 5.5 and 8, preferably between about 6 and 7.5. By some embodiments, the second feed mixture is maintained in said second enzymatic reactor for a period of time of between about 1 and about 6 hours, between about 1.5 and about 5 hours, or even between about 2 and about 4.5 hours. According to some embodiments, the ratio between the immobilized enzyme (i.e. the weight of the enzyme together with the immobilization substrate) and the second mixture is between about 1:4 and about 1:12 The resulting second crude stream is then transferred from the second enzymatic reactor to the second separation unit at step (g) of the process, in which the second crude stream is allowed to separate into a second aqueous phase which contains water, alcohol, polyols, and other miscible components, and a second organic phase which contains predominantly fatty acid alkyl esters, unreacted glycerides and unreacted FFA. As noted, the conditions of the second enzymatic treatment are designed to permit conversion of at least some of the unreacted glycerides and FFA in the second feed mixture, thereby resulting in a content of unreacted glycerides and unreacted FFA in said second organic phase that is smaller than that in the first organic phase. In other words, the second enzymatic step is designed to increase overall glyceride and FFA conversion yield into fatty acid alkyl esters. According to some embodiments, the content of unreacted glycerides and unreacted FFA in said second organic phase is smaller by at least about 25% than in said first organic phase. According to some embodiments, steps (d)-(g) are repeated at least one time before carrying out step (h), to further increase conversion of unreacted glycerides and FFA in the organic phase until reaching a final stream that contains a desired content of fatty acid alkyl esters. Namely, several cycles of mixing the ingredients in two consecutive static mixers, treating in an enzymatic reactor and subsequently separating the organic phase from the resultant crude stream can be carried out before applying finishing treatments to obtain the final biodiesel product. According to some embodiments, the process further comprises, instead of step (h), the following steps: (i) mixing said second organic phase with at least one aqueous alkaline solution in a third static mixer to obtain a second emulsion; (j) mixing said second emulsion with at least one C1-C6 alkyl alcohol in a fourth static mixer, being serially linked to said third static mixer, to obtain a third feed mixture; (k) cooling said third feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the third feed mixture into a third enzymatic reactor that holds at least one immobilized enzyme, and maintaining the third feed mixture in said third enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said third feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a third crude stream of fatty acid alkyl esters; (l) transferring said third crude stream into a third separation unit while heating the third crude stream, and maintaining the third crude stream in said separation unit to obtain a third aqueous phase and a third organic phase, the content of unreacted glycerides and unreacted FFA in said third organic phase being smaller than in said second organic phase; and (m) treating the third organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel. According to some embodiments, step (g) comprises measuring the pH of said second aqueous phase to obtain a second pH value, and determining the amount of said at least one aqueous alkaline solution added to the second organic phase in step (i) based on said second pH value, to obtain a pH of between about 5.5 and 8 in said second microemulsion, preferably a pH of between about 6 and 7.5. According to some embodiments, the third feed mixture comprises between about wt% and about 10 wt% of said at least one C1-C6 alkyl alcohol. According to some embodiments, the third feed mixture has a pH of between about 5.5 and 8, preferably between about 6 and 7.5. By some embodiments, the third and fourth static mixers may be the same or difference. By some other embodiments, the first, second, third and fourth static mixers can be independently selected to be the same or different from one another. According to some embodiments, the third feed mixture is maintained in said third enzymatic reactor for a period of time of between about 1 and about 8 hours, at a temperature of between about 25ºC and about 35ºC (preferably between 28ºC and 35ºC), and under stirring conditions. By some embodiments, the third feed mixture is maintained in said third enzymatic reactor for a period of time of between about 1 and about 6 hours, between about 1.5 and about 5 hours, or even between about 2 and about 4.5 hours. According to some embodiments, the ratio between the immobilized enzyme (i.e. the weight of the enzyme together with the immobilization substrate) and the third mixture is between about 1:4 and about 1:12.

According to some embodiments, after separation of the third aqueous phase from the third organic phase, the content of unreacted glycerides and unreacted FFA in said third organic phase is smaller by at least about 30% than in said second organic phase. The processes described herein can be carried out in a batch mode, a semi-continuous mode, or a continuous mode. According to some embodiments, the process is carried out in a continuous mode, and step (c) comprises measuring the pH of said first aqueous phase to obtain a first pH value, and determining the amount of said at least one aqueous alkaline solution added in step (a) based on said first pH value, to obtain a pH of between about 5.5 and 8 in said first feed mixture. According to some other embodiments, the process is carried out in a continuous mode, and step (g) comprises measuring the pH of said second aqueous phase to obtain a second pH value, and determining the amount of said at least one aqueous alkaline solution added in steps (a) or (i) based on said second pH value, to obtain a pH of between about 5.5 and 8 in said first feed mixture or said second feed mixture, respectively. In any one of steps (a), (d), and (i), at least one aqueous alkaline solution is added to the organic liquid (i.e. the fat source or the organic phases) in order to bring the organic liquid to a desired and controlled pH value, suitable for the enzymatic reaction steps. The aqueous alkaline solution is a water-based solution of one or more alkaline, water-soluble materials, that render the solution with a pH value of above 7, typically between 9 and 14. The alkaline material is typically one or more alkaline salts. According to some embodiments, in each of steps (a), (d), and (i), the said at least one aqueous alkaline solution is independently selected from a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, and any mixture thereof. According to some other embodiments, in each of steps (a), (d), and (i), the said at least one aqueous alkaline solution is independently selected from a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, and any mixture thereof. According to some embodiments, the at least one aqueous alkaline solution in all of steps (a), (d), and (i) is the same aqueous alkaline solution.

In any one of steps (a), (e), and (j), at least one C1-C6 alkyl alcohol, preferably C1-C4 alkyl alcohol, is added to the organic liquid (i.e. the fat source or the emulsions) as a reagent to convert, in the presence of the enzyme once introduced into the enzymatic reactor, the glycerides and FFA into fatty acid alkyl esters. By some embodiments, in each of steps (a), (e), and (j), the C1-C6 alkyl alcohol can be independently selected from methanol, ethanol, propanol, isopropanol, butanol, and mixtures thereof. According to some preferred embodiments, the C1-C6 alkyl alcohol in each of steps (a), (e), and (j) is methanol, ethanol or combinations thereof. According to some embodiments, the at least one C1-C6 alkyl alcohol in all of steps (a), (e), and (j) is the same aqueous alkaline solution. According to some embodiments, the immobilized enzyme in each of steps (b), (f) and (k) can be the same immobilized enzyme, typically one or more immobilized lipases. According to some other embodiments, the immobilized enzyme in each of steps (b), (f) and (k) can be independently selected from one or more immobilized lipase enzymes. As noted, in steps (c), (g) and (l), the crude stream is separated in a separation unit that permits separation between the undesired aqueous phase and the desired organic phase. The separation units can be independently selected from decanters, centrifuges, settling tanks, and any other suitable means for obtaining said phase separation. According to some embodiments, in each of steps (c), (g) and (l), the separation unit is one or more decanters. The decanters in each of steps (c), (g) and (l) can be the same or different, and independently selected from gravitational decanters, centrifugal decanters, and gravitational separation tanks. According to some embodiments, in each of steps (c), (g) and (l), the separation unit is one or more decanters, each being operated under different conditions. According to some other embodiments, the decanters in each of in each of steps (c), (g) and (l), are operated under the same conditions. By some embodiments, the separation unit in each of steps (c), (g) and (l), is a decanter, and the said first, second and third crude streams, respectively, are independently retained in said first, second and third separation units for a period of time of between about 2 and about 5 hours.

As noted, the crude streams are heated after their removal from the enzymatic reactors and before introduction into the separation units. Such heating was found to promote controlled phase separation of the aqueous phase from the organic phase. According to some embodiments, heating during separation steps (c), (g) and (l) is independently to a temperature of between about 40º and about 60ºC. As noted, the fat source utilized in the process of this disclosure comprises at least about 10 wt% of FFA. As also noted, as crude fat source can vary, it is sometimes required to carry out one or more pre-treatments in order to increase the content of FFA in the fat source before carrying out the enzymatic processes. Crude fat source refers to a fat source as received from a supplier. When the crude fat source contains over 10 wt% FFA, it can be used as-is as the fat source of the processes of this disclosure, or undergo pre-treatment to concentrate the FFAs. The crude fat source can be selected from an edible fat/oil (or originating from an edible source) or a non-edible fat/oil (or originating from a non-edible source), such that at least one oil selected from a vegetative source, animal fat, algal oil, fish oil, or any mixture or combination thereof; used cooking oil (UCO) and/or used frying oil (UFO); waste cooking oil; animal-derived fat, including lard, tallow, fish oil, chicken fat, yellow grease, brown grease, and any combination thereof; an oil from a vegetative source selected from soybean oil, canola oil (rapeseed oil), algae oil, olive oil, castor oil, palm oil, palm olein, palm stearin, grapeseed oil, hemp oil, sunflower oil, safflower oil, peanut oil, cotton seed oil, Jatropha oil, corn oil, avocado oil, sesame oil, rice bran oil, coconut oil, mustard oil, flaxseed oil, jojoba oil, tall oil, oils derived from inedible plant sources, partial glycerides and free fatty acids derived oils, and mixtures and combinations thereof. Such pre-treatments of the crude fat source are described in Applicant’s patent application no. PCT/IL2022/050962, which is incorporated herein by reference. According to some embodiments, the pre-treatment comprises, before step (a): (I) heating crude fat source to a temperature of at least 60 ºC to obtain heated crude fat source; (II) treating said heated crude fat source in one or more tricanters, to thereby separate the heated crude fat source into a solids stream, a water stream, and a crude oil stream; (III) contacting said crude oil stream with at least one aqueous acidic solution at a pH of at most about 3, to hydrate phosphatides in the crude oil stream, thereby obtaining gum residue and degummed crude oil; and (IV) separating between said gum residue and said degummed crude oil to obtain said fat source. According to some embodiments, pre-treatment may optionally comprise treating the heated crude fat source in one or more decanters arranged in a sequence prior to treatment in said one or more tricanters at step (II). By some embodiments, the crude oil stream, after exiting the tricanter(s), is further treated to remove residual water and/or solids therefrom, e.g. by centrifugation, before transferring the stream into the next pre-treatment step (step (III)). As noted, the organic phase at step (g) or step (l) of the process, contains predominantly fatty acid alkyl esters, e.g. above 85%, typically above 87%, preferably above 90%, and more preferably above 92% fatty acid alkyl esters. The organic phase in step (h) or step (m) of the process is then further treated (i.e. post-treated) to remove undesired contaminants therefrom, thereby obtaining said biodiesel. Such post-treatments are also described in Applicant’s patent application no. PCT/IL2022/050962, which is incorporated herein by reference. According to some embodiments, the treatment at step (h) and/or (m) comprises: (V) distilling a stream of fatty acid alkyl esters to separate a mid-fraction from light fractions and heavy fractions; (VI) contacting the mid-fraction with one or more bases to cause saponification of residual FFA, and separating saponified FFA therefrom, to obtain de-saponified mid-fraction; and (VII) neutralizing the de-saponified mid-fraction by contacting with one or more concentrated acids, thereby obtaining biodiesel. Unless otherwise specifically noted, the term light fractions refers to hydrocarbons having a boiling point of at most 200ºC (measured according to ASTM D86), the mid-fractions mean to denote hydrocarbons having a boiling point of between about 200ºC and about 340ºC (according to ASTM D86), and heavy fractions mean to denote hydrocarbons having a boiling point of at least 340ºC (according to ASTM D86).

Saponification means to denote conversion of residual FFA that remain in the final organic phase separated at from the stream of fatty acid alkyl esters in the final separation unit in the process (and, at times, after distillation) into removable soap by contacting with an alkali solution. After saponification, the saponified FFA are washed out of the biodiesel, and the biodiesel is neutralized by adding one or more acids (e.g. sulfuric acid solution or concentrated sulfuric acid). The biodiesel can then undergo further purification steps, as known per se, such as washing (e.g. with water, demineralized water, distilled water, etc.), vacuum drying, etc. The saponified FFA can be disposed as waste. Alternatively, and preferably, the saponified FFA undergoes soap splitting to obtain de-saponified FFA (e.g. by contacting the saponified FFA with one or more acids, such as hydrochloric acid), the de-saponified FFA being re-used as a fat source in the processes described herein. By another one of its aspects, the present disclosure provides a process for manufacturing biodiesel from a fat source that comprises glycerides and free fatty acids (FFA), the process comprising: (a) mixing said fat source with at least one aqueous alkaline solution and at least one C1-C6 alkyl alcohol, to obtain a first feed mixture; (b) cooling said first feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the first feed mixture into a first enzymatic reactor that holds at least one immobilized enzyme, and maintaining the first feed mixture in said first enzymatic reactor under conditions permitting concomitant enzymatic transesterification of said glycerides and esterification of said FFA by reacting with said at least one C1-Calkyl alcohol, to obtain a first crude stream of fatty acid alkyl esters; (c) transferring said first crude stream into a first separation unit while heating the first crude stream, and maintaining the first crude stream in said first separation unit to obtain a first aqueous phase and a first organic phase; (d) mixing said first organic phase with at least one aqueous alkaline solution in a first static mixer to obtain a first emulsion; (e) mixing said first emulsion with at least one C1-C6 alkyl alcohol in a second static mixer, being serially linked to said first static mixer, to obtain a second feed mixture; (f) cooling said second feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the second feed mixture into a second enzymatic reactor that holds at least one immobilized enzyme, and maintaining the second feed mixture in said second enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said second feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a second crude stream of fatty acid alkyl esters; (g) transferring said second crude stream into a second separation unit while heating the second crude stream, and maintaining the second crude stream in said second separation unit to obtain a second aqueous phase and a second organic phase, the content of unreacted glycerides and unreacted FFA in said second organic phase being smaller than in said first organic phase; (i) mixing said second organic phase with at least one aqueous alkaline solution in a third static mixer to obtain a second emulsion; (j) mixing said second emulsion with at least one C1-C6 alkyl alcohol in a fourth static mixer, being serially linked to said third static mixer, to obtain a third feed mixture; (k) cooling said third feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the third feed mixture into a third enzymatic reactor that holds at least one immobilized enzyme, and maintaining the third feed mixture in said third enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said third feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a third crude stream of fatty acid alkyl esters; (l) transferring said third crude stream into a third separation unit while heating the third crude stream, and maintaining the third crude stream in said third separation unit to obtain a third aqueous phase and a third organic phase, the content of unreacted glycerides and unreacted FFA in said third organic phase being smaller than in said second organic phase; and (m) treating the third organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel.

By another one of its aspects, the present disclosure provides a system for manufacturing biodiesel from a fat source that comprises glycerides and free fatty acids (FFA), the system comprising: a high shear mixing unit configured for mixing said fat source with at least one aqueous alkaline solution and at least one C1-C6 alkyl alcohol, to obtain a first feed mixture; a first enzymatic reactor, being in liquid communication with said high shear mixing unit via a heat exchanger, the first enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the first feed mixture in said first enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of said glycerides and esterification of said FFA by reacting with said at least one C1-C6 alkyl alcohol, to obtain a first crude stream of fatty acid alkyl esters; a first separation unit, being in liquid communication with said first enzymatic reactor via a heat exchanger, and configured to maintain the first crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the first crude stream into a first aqueous phase and a first organic phase; a first static mixture, in liquid communication with said first separation unit, and configured to mix said first organic phase with at least one aqueous alkaline solution to obtain a first emulsion; a second static mixture, in liquid communication with said first static mixer, and configured to mix first emulsion with at least one C1-C6 alkyl alcohol to obtain a second feed mixture; a second enzymatic reactor, being in liquid communication with said second static mixer via a heat exchanger, the second enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the second feed mixture in said second enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said second feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a second crude stream of fatty acid alkyl esters; a second separation unit, being in liquid communication with said second enzymatic reactor via a heat exchanger, and configured to maintain the second crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the second crude stream into a second aqueous phase and a second organic phase; and one or more purification units, in liquid communication with said second separation unit, configured for treating the second organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel. According to some embodiments, the system further comprises: a third static mixer, in liquid communication with said second separation unit, and configured to mix said second organic phase with at least one aqueous alkaline solution obtain a second emulsion; a fourth static mixer, in liquid communication with said third static mixer, and configured to mix said second emulsion with at least one C1-C6 alkyl alcohol to obtain a third feed mixture; a third enzymatic reactor, being in liquid communication with said fourth static mixer via a heat exchanger, the third enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the third feed mixture in said second enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said third feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a third crude stream of fatty acid alkyl esters; and a third separation unit, being in liquid communication with said third enzymatic reactor via a heat exchanger, and configured to maintain the third crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the third crude stream into a third aqueous phase and a third organic phase; said one or more purification units being in liquid communication with said third separation unit, and configured for treating the third organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel. According to some embodiments, said first enzymatic reactor and said second enzymatic reactor are identical. According to some other embodiments, said first enzymatic reactor, said second enzymatic reactor, and said third enzymatic reactor are identical. According to some embodiments, the system optionally comprises one or more fat source holding tanks, in liquid communication with the high shear mixing unit, for receiving and holding said fat source prior to application of the processes of this disclosure.

The one or more purifications units can, by some embodiments, comprise at least one distillation module, downstream the last separation unit (e.g. said second and/or third separation units), configured to be operable under conditions permitting separation of said stream of fatty acid alkyl esters into, light fractions, mid-fractions (crude biodiesel) and heavy fractions. In such embodiments, the system can further comprise one or more saponification units, in liquid communication with said one or more distillation modules, and configured to receive said crude biodiesel and one or more aqueous basic solutions, to saponify unreacted FFA in the crude biodiesel, and separate the saponified FFA from said biodiesel. By another aspect of this disclosure, there is provided an enzymatic reactor configured for the processes and systems described herein. The enzymatic reactor comprises: a reactor body having a longitudinal axis and a total internal volume V, the internal volume being configured to hold at least one immobilized enzyme; at least one inlet fitted at a top portion of the reactor body, and at least one outlet fitted at a bottom portion of the reactor body; a plurality of filtration units arranged within the reactor body in a spaced apart arrangement and being in liquid communication with said at least one outlet, the plurality of filtration units being configured to substantially prevent transfer of said immobilized enzyme into said outlet; and at least one stirring arrangement comprising two or more pedals, vertically displaced from one another along said longitudinal axis. The term filtration unit(s) refers to a unit that permits passage of liquids therethrough, however prevents passage of solid particles above a pre-defined size. in the context of the present disclosure, the filtering units permit passage of liquids (e.g. reaction products) from the reactor volume to the outlet while preventing passage of the immobilized enzyme therethrough (thereby maintaining the immobilized enzyme within the reactor for further, subsequence reactions). Typically, the filtration units is selected according to the size and shape of the substrate carrying the enzyme. By some embodiments, each of the filtration units has a hole size of between about 100 μm and about 500 μm.

According to some embodiments, the plurality of filtration units comprises 2, 3, 4, 5, 6, 7, 8 or even more filtration units. According to some embodiments, the plurality of filtration units comprises between 4 and 24 filtering units. The filtering units are arranged within the reactor body in a spaced apart arrangement, i.e. spaced apart from one another, and each is in liquid communication with the at least one outlet to permit filtration and removal of the reaction products from the reactor volume. According to some embodiments, each of the filtration units in said plurality is linked to at least one common conduit fitted at said bottom portion of the reactor body, said conduit being in liquid communication with said outlet and defines a plane perpendicular to the longitudinal axis. The conduit is typically a common tube that collects all of the reaction products (filtrate) from the filtering units and directs the filtrate into the outlet. According to some embodiments, the plurality of filtration units are arranged on said conduit circumferentially about the longitudinal axis. According to other embodiments, the at least one conduit comprises two concentric conduits, such that some of the plurality of filtration units are arranged on a first of said conduits, while the remaining of filtration units of said plurality are arranged on a second of said conduits. According to some embodiments, the top portion of the reactor is defined above the plane defined by the conduit and has a volume Vt, and the bottom portion of the reactor is defined below said plane and has a volume Vb, such that Vb The stirring pedals can be rotated at the same rate by a mutual motor. Alternatively, each pedal can be configured to rotate in a different rate than the others, according to process requirements. According to some embodiments, the reactor has a length to diameter (L/D) ratio of between about 1:0.3 and about 1:0.8. According to some embodiments, the enzymatic reactor comprises a heating/cooling arrangement for maintaining a temperature of between about 25ºC and about 35ºC in the reactor. According to some embodiments, the enzymatic reactor further comprises one or more baffles; for example, one or more baffles that are located in one or both of the top section and the bottom section of the reactor. The structure of the enzymatic reactor permits obtaining uniform conditions throughout the reactor for effective conversion of the glycerides and FFA into fatty acid alkyl esters, by maintaining the immobilized enzyme homogeneously dispersed in the reactor, which permitting effective filtration of the reaction products out of the reactor. As used herein, the term about is meant to encompass deviation of ±10% from the specifically mentioned value of a parameter, such as temperature, pressure, concentration, etc. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. Generally it is noted that the term "…at least one…" as applied to any component of the processes of systems of this disclosure should be read to encompass one, two, three, four, five, six, seven, eight, nine or ten different occurrences of said component in the processes or systems of this disclosure. It is appreciated that certain features of this disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment described herein. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

The processes of the present disclosure involve numerous process steps which may or may not be associated with other common physical-chemical processes so as to achieve the desired purity and form of each of the isolated components (e.g. biodiesel). Unless otherwise indicated, such process steps, if present, may be set in different sequences without affecting the workability of the process and its efficacy in achieving the desired end result. As a person skilled in the art would appreciate, a sequence of steps may be employed and changed depending on various economical aspects, material availability, raw materials, environmental considerations, etc.

BRIEF DESCRIPTION OF THE DRAWINGSIn order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Fig. 1 is a block diagram of an exemplary process according to an embodiment of this disclosure. Figs. 2Aand 2B are a schematic longitudinal cross section (Fig. 2A) and top-view cross section (Fig. 2B) representation of an enzymatic reactor suitable for the processes described herein according to an embodiment of this disclosure.

DETAILED DESCRIPTION OF EMBODIMENTSAn exemplary process and system according to an embodiment of this disclosure will now be described. While the described process is typically continuous, it is contemplated that the processes, or stages thereof, can be carried out in a semi-continuous mode or batch-wise. As used herein, the term container, tank, reactor (i.e., reaction vessels), or any alternative term used interchangeably, refers to a device for carrying a unit operation. Typically, such a device may be of any size, shape and constructed of any material suitable for the process step that is to be carried out within the device, unless otherwise specifically indicated. Depending on its operational requirements, each of the reaction vessels may comprise a temperature control unit, such as a heating/cooling unit or a heat exchanger, along with means for controlling said unit in response to autothermic or the absence of autothermic conditions within the reaction chamber; internal temperature gauges for monitoring the reaction's temperature; condensation units, scrubbing units and absorption columns, to afford treatment of gaseous reaction products and gaseous contaminants; baffles of various geometries for controlling the flow profile of substance within the reactor; a top plate that is movable with respect to an outer body of the reactor; a base plate that is movable with respect to an outer body of the reactor; reactants inlets at various angles; and products outlets at various angles. Suitable pumps or gravity feeds and controllable valves may be provided for selectively transporting the respective materials between units of the system and a suitable controller monitors and controls operation of the system. Turning to Fig. 1, schematically described is a process 10 for manufacturing biodiesel from a fat source. The fat source 102 , e.g. animal-fats and/or plant-based oils, that comprise glycerides and at least 10 wt% free fatty acids (FFA), preferably at least 20 wt% FFA, are fed into a high shear mixer 100 , together with an aqueous alkaline solution 104 (e.g. sodium carbonate or bicarbonate) and C1-6 alkyl alcohol 106 (e.g. methanol or ethanol) and mixed in the high shear mixer under shearing conditions to obtain a homogenous first feed mixture 108 having a pH of between about 5.5 and 8, more typically between 6 and 7.5. In cases where the fat source as received from the supplier contains less than 10 wt% of FFA, one or more pretreatment stages (not shown) can be applied on the crude fat source in order to increase the content of FFA in the fat source to a suitable value for a process according to this disclosure. Such pre-treatments are described in Applicant’s application no. PCT/IL2022/050962, which is incorporated herein by reference. In the industrial process described herein, the utilization of fat sources that contain at least 30 wt% FFA, preferably at least about 20 wt%, more preferably at least about wt% FFA, was found to increase the yield of biodiesel production, thereby obtaining a process yield of at least 85%, more typically in the range of 90-95%, and reduces the formation of glycerin as a by-product.

First feed mixture 108 is then cooled in heat exchanger 110 , typically to a temperature of between about 25ºC and 35ºC, and fed into the first enzymatic reactor 120 . While in Fig. 1 the enzymatic reactor is generally shown, preferably the enzymatic reactors 120 , 180 and 240 have a structure that will be described with reference to Figs. 2A-2B further below. First enzymatic reactor 120 holds at least one enzyme, typically one or more lipase enzymes, that are immobilized onto particulate substrate. The enzyme promotes the concomitant transesterification and esterification chemical reactions of the glycerides and FFA in the fat source with the C1-6 alkyl alcohol, in order to obtain fatty acid alkyl esters, which are the main components in biodiesel. In the enzymatic reactor, the first feed mixture is brought into intimate contact with the immobilized enzyme by mild mixing. Typically, the first feed mixture is maintained in the first enzymatic reactor for a period of time of between about 2 and about 5 hours, at a temperature of between about 25ºC and about 35ºC (preferably between 28ºC and 35ºC), and under stirring conditions. The first crude stream of fatty acid alkyl esters 122 is transferred from the first enzymatic reactor to first separation unit 140 , in which the first crude stream 122 is allowed to phase separate into two phases: a first aqueous phase 142 which contains water, alcohol, polyols, and other miscible components, that is removed from the first separation unit 140 and separately treated, and a first organic phase 144 which contains predominantly fatty acid alkyl esters, unreacted glycerides and unreacted FFA. In order to assist in phase separation, the first crude stream 122 is typically heated in heat exchanger 130 , typically to a temperature of between about 40º and about 60ºC , before introduction into the separation unit 140 . Separation unit 140 can be, for example, a decanter (which can also be heated). The first organic phase 144 is then used as a raw material for the second enzymatic stage carried out in the second enzymatic reactor 180 . The first organic phase 144 is mixed, prior to introduction into the second enzymatic reactor 180 , with an alkaline aqueous solution and C1-C6 alkyl alcohol, in a gradual manner. The first organic phase 144 is first mixed with the at least one aqueous alkaline solution 152 in a first static mixer 150to obtain a first emulsion 154 . The first emulsion 154 is then mixed with at least one C1-C6 alkyl alcohol 162 in a second static mixer 160 that is serially linked to the first static mixer 150 , thereby obtaining second feed mixture 164 having a pH of between about 5.5 and 8, more typically between about 6 and 7.5. Separation between the addition of the at least one aqueous alkaline solution and the C1-C6 alkyl alcohol in order to obtain the second feed mixture enables careful control over the pH of the second feed mixture. Preferably, the pH of the first aqueous phase 142 is measured to obtain a first pH value, and the addition of the aqueous alkaline solution at 152 is determined by said first pH value, to obtain the desired pH in the first microemulsion 154 . The second feed mixture 164 is then cooled down to a temperature of between about 25ºC and 35ºC in heat exchanger 170 and fed into the second enzymatic reactor 180 , in which the unreacted glycerides and FFA in the second feed mixture are allowed to react with the C1-C6 alkyl alcohol to product further fatty acid alkyl esters. Such a second enzymatic treatment is designed to increase the overall yield of the process, namely to increase the formation of fatty acid alkyl esters and result in higher production capability of biodiesel from the fat source. Typically, the second feed mixture is maintained in the second enzymatic reactor for a period of time of between about 2 and about 5 hours, at a temperature of between about 25ºC and about 35ºC (preferably between 28ºC and 35ºC), and under stirring conditions. The resulting second crude stream 182 is then transferred from the second enzymatic reactor 180 to the second separation unit 200 , e.g. a decanter (which may be heated), typically via heat exchanger 190 that increases the temperature of the second crude steam. In the second separation unit 200 the second crude stream 182 is allowed to separate into a second aqueous phase 202 which contains water, alcohol, polyols, and other miscible components and is treated separately, and a second organic phase 204 which contains predominantly fatty acid alkyl esters, unreacted glycerides and unreacted FFA. The conditions of the second enzymatic treatment are designed to permit conversion of at least some of the unreacted glycerides and FFA in the second feed mixture, thereby resulting in a content of unreacted glycerides and unreacted FFA in said second organic phase that is smaller than that in the first organic phase. In other words, the second enzymatic step is designed to increase overall glyceride and FFA conversion yield into fatty acid alkyl esters. Typically, the content of unreacted glycerides and unreacted FFA in said second organic phase 204 is smaller by at least about 25% than in said first organic phase 144 . In this specific example, the second organic phase is treated in one additional enzymatic stage. It is to be understood, however, that the second organic phase 204 can be transferred to one or more finishing processes in order to obtain the final biodiesel product without a third enzymatic stage. Alternatively, the disclosure also encompasses processes in which a third, fourth, fifth, etc. enzymatic stages are carried out. In the example of Fig. 1, the second organic phase 204 is transferred into third static mixer 210 , in which the second organic phase is mixed with an aqueous alkaline solution 212 to obtain a second emulsion 214 having a pH of between 5.5 and 8, preferably a pH of between about 6 and 7.5. Preferably, the pH of the second aqueous phase 202 is measured to obtain a second pH value, and the amount of aqueous alkaline solution 212 is determined based on said second pH value to obtain the desired pH in said second microemulsion. Second microemulsion 214 is then mixed with C1-C6 alkyl alcohol 222 in fourth static mixer 220 in order to obtain a third feed mixture 224 . The third feed mixture 224 is then cooled to a temperature of between about 25ºC and about 35ºC in heat exchanger 230 , and fed into third enzymatic reactor 240 . The third feed mixture is typically maintained in said third enzymatic reactor for a period of time of between about 2 and about 5 hours, at a temperature of between about 25ºC and about 35ºC (preferably between 28ºC and 35ºC), and under stirring conditions. The third crude stream of fatty acid alkyl esters 242 is then transferred into a third separation unit 260 , typically via heat exchanger 250 in which the temperature of the third crude stream is increased to facilitate easier separation between the third aqueous phase 262 and the third organic phase 264 . Typically, the content of unreacted glycerides and unreacted FFA in said third organic phase 264 is smaller by at least about 30% than in said second organic phase 204 . The third organic phase 264 now typically contains at least 90 wt% of fatty acid alkyl esters, more typically at least 92 wt%, and is then transferred into one or more purification and finishing steps to remove undesired contaminants and obtain the final biodiesel product. An enzymatic reactor suitable for the processes and systems described herein, e.g. enzymatic reactors 120, 180 and/or 240 is demonstrated in Figs. 2A-2B. Reactor 120 comprises a reactor body 1002 , typically elongated, and having a longitudinal axis 1004 . For example, the reactor typically has a length to diameter ( L / D ) ratio of between about 1:0.3 and about 1:0.8.

The reactor body 1002 has total internal volume V , that holds the at least one immobilized enzyme. The feed mixture is introduced through inlet 1006 fitted at a top portion of the reactor body. The crude fatty acid alkyl esters stream is drained out of the reactor via outlet 1008 fitted at a bottom portion of the reactor. In the reactor, a plurality of filtration units, collectively designated 1010 are arranged in a spaced apart manner. In this example, each of the filtration units 1010 is in liquid communication with the outlet 1008 through circumferential conduit 1012 . Conduit 1012 is configured to collect the liquid filtrate from the filtration units 1010 and drain the filtrate to the outlet 1008 . As seen in Fig. 2B, in this specific example, 16 filtration units are utilized. However it will be understood that the number of filters is provided as a non-limiting example, and in other arrangements the plurality of filtration units can include 2, 3, 4, 5, 6, 7, 8 or even more filtration units, typically between 4 and 24 filtering units. The conduit defines a plane P , that is perpendicular to the longitudinal axis 1004 . The reactor volume V is divided into a top portion defined above the plane defined by the conduit and has a volume Vt , and the bottom portion of the reactor is defined below said plane and has a volume Vb , such that Vb therefrom to obtain the fat source with a content of at least 10 wt% FFA, preferably at least about 20 wt%, more preferably at least about 30 wt% FFA. This pretreatment is described in Applicant’s patent application no. PCT/IL2022/050962, which is incorporated herein by reference. The process described in this example is carried out for a process in a continuous mode at a capacity of 2,000-6,000 liter/hour. It is to be understood that the process can also be carried out in batch or semi-continuous modes. First reactor cycle: The fat source is mixed with sodium bicarbonate buffer solution and with methanol in a high shear mixing pump to obtain the first feed mixture. The first feed mixture contains 10% wt% methanol, and sufficient 1M sodium bicarbonate buffer solution to obtain a pH of 6-7.5. The first feed mixture is then cooled to 30ºC before introduction to the first enzymatic reactor. The enzymatic reactor contained lipase enzyme(s), immobilized on a hydrophobic substrate, at a ratio of immobilized enzyme to first mixture ratio of between about 1:6 and 1:10. The first feed mixture is maintained in the first enzymatic reactor for 2-4.5 hours, at a temperature of 30-32ºC in atmospheric pressure, using a multi-stage stirrer in low stirring intensity (e.g. 100-150 rpm). As the esterification / transesterification reaction is slightly exothermic, the reactor is cooled to the target temperature. The resulting products are filtered through a plurality of 180 μm filters, having an operative filtration area of 2-3m. The product stream is heated to 50ºC and transferred into a first decanter, and maintained therein for 3-5 hours to separate the undesired first aqueous phase from the desired first organic phase. The composition of the first organic phase is shown in Table 1 . Second reaction cycle: The first organic phase is mixed with 1M sodium carbonate buffer solution in a first static mixer to obtain a pH of 6-7. The amount of buffer to be added to obtain the desired pH is determined according to the pH value measured in the first aqueous phase. The resulting microemulsion is then mixed, in a second static mixer with 7 wt% methanol to obtain the second feed mixture, which is then cooled to 30ºC before introduction to the second enzymatic reactor. The second enzymatic reactor contained lipase enzyme(s), immobilized on a hydrophobic substrate. The second feed mixture is maintained in the second enzymatic reactor for 2-5 hours, at a temperature of 30-32ºC in atmospheric pressure, using a multi-stage stirrer in low stirring intensity (e.g. 100-150 rpm). The resulting products are filtered through a plurality of 180 μm filters, having an operative filtration area of 2-3m. The product stream is heated to 50ºC and transferred into a second decanter, and maintained therein for 3-5 hours to separate the undesired second aqueous phase from the desired second organic phase. The composition of the second organic phase is shown in Table 1. Third reaction cycle: The second organic phase is mixed with 1M sodium carbonate buffer solution in a third static mixer to obtain a pH of 6-7. The amount of buffer to be added to obtain the desired pH is determined according to the pH value measured in the second aqueous phase. The resulting microemulsion is then mixed, in a fourth static mixer with 7 wt% methanol to obtain the third feed mixture, which is then cooled to 30ºC before introduction to the third enzymatic reactor. The third enzymatic reactor contained lipase enzyme(s), immobilized on a hydrophobic substrate. The third feed mixture is maintained in the third enzymatic reactor for 2-5 hours, at a temperature of 30-32ºC in atmospheric pressure, using a multi-stage stirrer in low stirring intensity (e.g. 100-150 rpm). The resulting products are filtered through a plurality of 180 μm filters, having an operative filtration area of 2-3m. The product stream is heated to 50ºC and transferred into a third decanter, and maintained therein for 3-5 hours to separate the undesired third aqueous phase from the desired third organic phase. The composition of the third organic phase is shown in Table 1.

Table 1 : Composition of organic phases (wt%) Composition of organic phase Reduction in unreacted fats (%) Unreacted fats FAME Methanol Unsaponi- fiables Water Glycerides FFA st organic phase 11.6 9.8 67.2 1 9.7 0.5 - nd organic phase 4.8 7.7 74.5 3 9.5 0.5 41.6% rd organic phase 2.8 4.7 78.1 4 9.3 1 40%

Claims

- 34 -

1. CLAIMS: 1. A process for manufacturing biodiesel from a fat source that comprises glycerides and free fatty acids (FFA), the process comprising: (a) mixing said fat source with at least one aqueous alkaline solution and at least one C1-C6 alkyl alcohol, to obtain a first feed mixture; (b) cooling said first feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the first feed mixture into a first enzymatic reactor that holds at least one immobilized enzyme, and maintaining the first feed mixture in said first enzymatic reactor under conditions permitting concomitant enzymatic transesterification of said glycerides and esterification of said FFA by reacting with said at least one C1-Calkyl alcohol, to obtain a first crude stream of fatty acid alkyl esters; (c) transferring said first crude stream into a first separation unit while heating the first crude stream, and maintaining the first crude stream in said first separation unit to obtain a first aqueous phase and a first organic phase; (d) mixing said first organic phase with at least one aqueous alkaline solution in a first static mixer to obtain a first emulsion; (e) mixing said first emulsion with at least one C1-C6 alkyl alcohol in a second static mixer, being serially linked to said first static mixer, to obtain a second feed mixture; (f) cooling said second feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the second feed mixture into a second enzymatic reactor that holds at least one immobilized enzyme, and maintaining the second feed mixture in said second enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said second feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a second crude stream of fatty acid alkyl esters; (g) transferring said second crude stream into a second separation unit while heating the second crude stream, and maintaining the second crude stream in said second separation unit to obtain a second aqueous phase and a second organic phase, the content of unreacted glycerides and unreacted FFA in said second organic phase being smaller than in said first organic phase; and (h) treating the second organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel. - 35 -

2. The process of claim 1, wherein steps (d)-(g) are repeated at least one time before step (h).

3. The process of claim 1 or 2, wherein said mixing at step (a) to obtain said first feed mixture is carried out in a high shear mixing unit.

4. The process of any one of claims 1 to 3, wherein mixing at step (a) is carried out at a temperature of between about 40ºC and about 80ºC.

5. The process of any one of claims 1 to 4, wherein the first feed mixture is maintained in said first enzymatic reactor for a period of time of between about 1 and about 8 hours, at a temperature of between about 25ºC and about 35ºC, and under stirring conditions.

6. The process of any one of claims 1 to 5, wherein said first feed mixture has a pH of between about 5.5 and 8.

7. The process of any one of claims 1 to 6, wherein the second feed mixture is maintained in said second enzymatic reactor for a period of time of between about 1 and about 8 hours, at a temperature of between about 25ºC and about 35ºC, and under stirring conditions. 8. The process of any one of claims 1 to 7, wherein said second feed mixture has a pH of between about 5.5 and

8.

9. The process of any one of claims 1 to 8, wherein the content of unreacted glycerides and unreacted FFA in said second organic phase is smaller by at least about 25% than in said first organic phase.

10. The process of any one of claims 1 to 9, wherein step (c) comprises measuring the pH of said first aqueous phase to obtain a first pH value, and determining the amount of said at least one aqueous alkaline solution added to the first organic phase in step (d) based on said first pH value, to obtain a pH of between about 5.5 and 8 in said first microemulsion.

11. The process of any one of claims 1 to 10, operating in a continuous mode, and wherein step (c) comprises measuring the pH of said first aqueous phase to obtain a first pH value, and determining the amount of said at least one aqueous alkaline solution added in step (a) based on said first pH value, to obtain a pH of between about 5.5 and in said first feed mixture. - 36 -

12. The process of any one of claims 1 or 11, wherein said heating during separation steps (c) and (g) is to a temperature of between about 40º and about 60ºC.

13. The process of any one of claims 1 to 12, wherein said first separation unit is a decanter, and said first crude stream is retained in said first separation unit for a period of time of between about 2 and about 5 hours.

14. The process of any one of claims 1 to 13, wherein said second separation unit is a decanter, and said second crude stream is retained in said second separation unit for a period of time of between about 2 and about 5 hours.

15. The process of any one of claims 1 to 14, wherein said first feed mixture comprises between about 8 wt% and about 12 wt% of said at least one C1-C6 alkyl alcohol.

16. The process of any one of claims 1 to 15, wherein said second feed mixture comprises between about 5 wt% and about 10 wt% of said at least one C1-C6 alkyl alcohol.

17. The process of any one of claims 1 to 16, wherein said fat source comprises at least about 10 wt% FFA.

18. The process of any one of claims 1 to 17, wherein said at least one aqueous alkaline solution in steps (a) and (d) is independently selected from a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, and any mixture thereof.

19. The process of any one of claims 1 to 18, wherein said at least one C1-C6 alcohol in steps (a) and (e) is independently selected from methanol, ethanol, and mixtures thereof.

20. The process of any one of claims 1 to 19, wherein said immobilized enzyme in steps (b) and (f) comprises at least one substrate associated with one or more lipase enzymes.

21. The process of claim 20, wherein said substrate is a hydrophobic substrate.

22. The process of claim 21, wherein said hydrophobic substrate comprises one or more hydrophobic polymers.

23. The process of any one of claims 1 to 22, further comprising, instead of step (h), the following steps: (i) mixing said second organic phase with at least one aqueous alkaline solution in a third static mixer to obtain a second emulsion; - 37 - (j) mixing said second emulsion with at least one C1-C6 alkyl alcohol in a fourth static mixer, being serially linked to said third static mixer, to obtain a third feed mixture; (k) cooling said third feed mixture to a temperature between about 25ºC and about 35ºC, followed by transferring the third feed mixture into a third enzymatic reactor that holds at least one immobilized enzyme, and maintaining the third feed mixture in said third enzymatic reactor under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said third feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a third crude stream of fatty acid alkyl esters; (l) transferring said third crude stream into a third separation unit while heating the third crude stream, and maintaining the third crude stream in said third separation unit to obtain a third aqueous phase and a third organic phase, the content of unreacted glycerides and unreacted FFA in said third organic phase being smaller than in said second organic phase; and (m) treating the third organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel.

24. The process of claim 23, wherein the third feed mixture is maintained in said third enzymatic reactor for a period of time of between about 1 and about 8 hours, at a temperature of between about 25ºC and about 35ºC, and under stirring conditions.

25. The process of claim 23 or 24, wherein said third feed mixture has a pH of between about 5.5 and 8.

26. The process of any one of claims 23 to 25, wherein the content of unreacted glycerides and unreacted FFA in said third organic phase is smaller by at least about 30% than in said second organic phase.

27. The process of any one of claims 23 to 26, wherein step (g) comprises measuring the pH of said second aqueous phase to obtain a second pH value, and determining the amount of said at least one aqueous alkaline solution added to the second organic phase in step (i) based on said second pH value, to obtain a pH of between about 5.5 and 8 in said second microemulsion.

28. The process of any one of claims 23 to 27, operating in a continuous mode, and wherein step (g) comprises measuring the pH of said second aqueous phase to obtain a second pH value, and determining the amount of said at least one aqueous - 38 - alkaline solution added in steps (a) or (i) based on said second pH value, to obtain a pH of between about 5.5 and 8 in said first feed mixture or said second feed mixture, respectively.

29. The process of any one of claims 23 to 28, wherein said heating during separation step (l) is to a temperature of between about 40º and about 60ºC.

30. The process of any one of claims 23 to 29, wherein said third separation unit is a decanter, and said third crude stream is retained in said third separation unit for a period of time of between about 2 and about 5 hours.

31. The process of any one of claims 23 to 30, wherein said third feed mixture comprises between about 5 wt% and about 10 wt% of said at least one C1-C6 alkyl alcohol.

32. The process of any one of claims 23 to 31, wherein said at least one aqueous alkaline solution in step (i) is selected from a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution, a potassium carbonate aqueous solution, a potassium bicarbonate aqueous solution, and any mixture thereof.

33. The process of any one of claims 23 to 32, wherein said at least one C1-C6 alcohol in step (j) is selected from methanol, ethanol, and mixtures thereof.

34. The process of any one of claims 23 to 33, wherein said immobilized enzyme in steps (k) comprises at least one substrate associated with one or more lipase enzymes.

35. The process of any one of claims 1 to 34, further comprising, prior to step (a), one or more pre-treatments that comprise receiving a crude fat source having an FFA content of less than about 10 wt%, and treating said crude fat source to obtain a fat source having an FFA content of at least about 10 wt%.

36. A system for manufacturing biodiesel from a fat source that comprises glycerides and free fatty acids (FFA), the system comprising: a high shear mixing unit configured for mixing said fat source with at least one aqueous alkaline solution and at least one C1-C6 alkyl alcohol, to obtain a first feed mixture; a first enzymatic reactor, being in liquid communication with said high shear mixing unit via a heat exchanger, the first enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the first feed mixture in said first enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of said glycerides and esterification - 39 - of said FFA by reacting with said at least one C1-C6 alkyl alcohol, to obtain a first crude stream of fatty acid alkyl esters; a first separation unit, being in liquid communication with said first enzymatic reactor via a heat exchanger, and configured to maintain the first crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the first crude stream into a first aqueous phase and a first organic phase; a first static mixture, in liquid communication with said first separation unit, and configured to mix said first organic phase with at least one aqueous alkaline solution to obtain a first emulsion; a second static mixture, in liquid communication with said first static mixer, and configured to mix first emulsion with at least one C1-C6 alkyl alcohol to obtain a second feed mixture; a second enzymatic reactor, being in liquid communication with said second static mixer via a heat exchanger, the second enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the second feed mixture in said second enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said second feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a second crude stream of fatty acid alkyl esters; a second separation unit, being in liquid communication with said second enzymatic reactor via a heat exchanger, and configured to maintain the second crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the second crude stream into a second aqueous phase and a second organic phase; and one or more purification units, in liquid communication with said second separation unit, configured for treating the second organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel.

37. The system of claim 35, further comprises a third static mixer, in liquid communication with said second separation unit, and configured to mix said second organic phase with at least one aqueous alkaline solution obtain a second emulsion; a fourth static mixer, in liquid communication with said third static mixer, and configured to mix said second emulsion with at least one C1-C6 alkyl alcohol to obtain a third feed mixture; - 40 - a third enzymatic reactor, being in liquid communication with said fourth static mixer via a heat exchanger, the third enzymatic reactor being configured to hold at least one immobilized enzyme, and maintain the third feed mixture in said second enzymatic reactor at a temperature of between about 25ºC and about 35ºC under conditions permitting concomitant enzymatic transesterification of unreacted glycerides and esterification of unreacted FFA in said third feed mixture by reacting with said at least one C1-C6 alkyl alcohol, to obtain a third crude stream of fatty acid alkyl esters; and a third separation unit, being in liquid communication with said third enzymatic reactor via a heat exchanger, and configured to maintain the third crude stream at a temperature of between about 40ºC and about 60ºC and permit separation of the third crude stream into a third aqueous phase and a third organic phase; said one or more purification units being in liquid communication with said third separation unit, and configured for treating the third organic phase to remove undesired contaminants therefrom, thereby obtaining said biodiesel.

38. The system of claim 36, wherein said first enzymatic reactor and said second enzymatic reactor are identical.

39. The system of claim 37, wherein said first enzymatic reactor, said second enzymatic reactor, and said third enzymatic reactor are identical.

40. An enzymatic reactor configured for the process of any one of claims 1 to 35 or the system of any one of claims 36 to 39, comprising: a reactor body having a longitudinal axis and a total internal volume V, the internal volume being configured to hold at least one immobilized enzyme; at least one inlet fitted at a top portion of the reactor body, and at least one outlet fitted at a bottom portion of the reactor body; a plurality of filtration units arranged within the reactor body in a spaced apart arrangement and being in liquid communication with said at least one outlet, the plurality of filtration units being configured to substantially prevent transfer of said immobilized enzyme into said outlet; and at least one stirring arrangement comprising two or more pedals, vertically displaced from one another along said longitudinal axis.

41. The enzymatic reactor of claim 40, wherein each of the filtration units in said plurality being linked to a common conduit fitted at said bottom portion of the reactor - 41 - body, said conduit being in liquid communication with said outlet and defines a plane perpendicular to the longitudinal axis.

42. The enzymatic reactor of claim 41, wherein the plurality of filtration units are arranged on said conduit circumferentially about the longitudinal axis.

43. The enzymatic reactor of claim 41 or 42, wherein the top portion of the reactor is defined above said plane and has a volume Vt, and the bottom portion of the reactor is defined below said plane and has a volume Vb, such that Vb