HK1174668B - A process for the enzymatic synthesis of fatty acid alkyl esters - Google Patents

Description

Method for enzymatic synthesis of fatty acid alkyl esters

Technical Field

The present invention discloses an enzymatic process for the preparation of fatty acid alkyl esters for use in the biofuel, food and detergent industries. In the method, a fatty acid source and an alcohol or an alcohol donor are reacted in the presence of an enzyme immobilized on a hydrophobic resin in the presence of an alkaline aqueous buffer or water. The disclosed processes can be operated batch-wise or continuously using a continuously stirred tank reactor or a packed bed column reactor.

Background

Immobilization of enzymes has been described by a number of techniques aimed essentially at reducing the cost contribution of enzymes in the overall enzymatic process, facilitating recovery of enzymes from the product, and enabling continuous operation of the process.

Immobilization techniques are generally divided according to:

1. physical adsorption of enzymes to solid supports such as silica and insoluble polymers.

2. Adsorption on ion exchange resin.

3. Covalent attachment of enzymes to solid support materials, such as epoxidized inorganic or polymeric supports.

4. Entrapment of the enzyme in the growing polymer.

5. Restriction of enzymes in membrane reactors or in semi-permeable gels.

6. Cross-linked enzyme crystals (CLECS) or aggregates (CLEAS).

All the aforementioned enzyme immobilization procedures consist of the following steps:

1. with regard to pH, temperature, type of buffer salts and ionic strength, the enzyme is dissolved in a suitable buffer system.

2. The solid support is added to the enzyme solution and mixed for a certain time until the enzyme molecules are immobilized on the solid support.

3. The solid support containing the immobilized enzyme was filtered.

4. The solid support is then dried by washing the support with an appropriate buffer to remove loosely bound enzyme molecules.

Interfacial enzymes, primarily lipases, have been immobilized following the aforementioned techniques. These provide immobilized enzyme preparations with low synthetic activity and/or short operating half-lives. In order to increase the synthetic activity and stability of immobilized lipases and other interfacial enzymes, different activation methods have been applied. These methods include:

1. the surface functional groups of the enzyme are bound to hydrophobic residues, such as fatty acids or polyethylene glycol.

2. The surface of the enzyme is coated with a surfactant, such as a polyol fatty acid ester.

3. The enzyme is contacted with a hydrophobic support, typically polypropylene, which has been pre-treated with a hydrophilic agent, such as ethanol or isopropanol.

None of the above methods yields satisfactory results with respect to the stability and cost-effectiveness of immobilized interfacial enzymes for carrying out industrial quantities of enzymatic reverse conversions. Moreover, it has been reported that most enzymes either lose a significant part of their synthetic activity or they do not show their full activity performance when immobilized according to the aforementioned procedure, due to certain limitations imposed by the immobilization procedure, or due to the presence of certain enzyme inhibitors in the reaction medium.

Another major drawback of lipases and phospholipases is their low tolerance to hydrophilic substrates, in particular short chain alcohols and short chain fatty acids (below C4). It has been observed in many studies that short-chain alcohols and short-chain fatty acids (such as methanol and acetic acid, respectively) are responsible for separating the essential water molecules from the quaternary structure of those enzymes, leading to denaturation of the enzymes and thus loss of their catalytic activity. This disadvantage has prevented the use of lipases for the preparation of commercial quantities of fatty acid methyl ester "biodiesel" using oil triglycerides and methanol as substrates.

Another disadvantage of using immobilized lipases for the transesterification/esterification of fatty acid sources with free alcohols is that the glycerol and water by-products formed accumulate on the biocatalyst, thus preventing the substrate from freely reaching the active site of the immobilized enzyme. When the same batch of biocatalyst is used, these biocatalysts usually lose their catalytic properties after several cycles.

The present inventors have developed specific immobilized enzyme preparations that exhibit good stability, long-lasting activity over many production cycles. Examples of such enzyme preparations are disclosed in particular in WO/2008/084470, WO/2008/139455 and WO 2009/069116.

The conditions under which the catalytic reaction is carried out can adversely affect the stability and efficiency of the immobilized enzyme preparation. It is important to have an enzyme preparation that retains stability and activity under the reaction conditions.

These and other objects of the invention will become apparent as the description proceeds.

Disclosure of Invention

In one embodiment, the present invention relates to a process for the transesterification/esterification of a fatty acid source with an alcohol to form fatty acid alkyl esters, comprising reacting the fatty acid source with the alcohol or alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized onto a hydrophobic porous support, and the reaction medium contains an aqueous alkaline buffer solution.

The aqueous alkaline buffer solution may be an aqueous weakly alkaline buffer solution. The aqueous alkaline buffer solution may be included in the reaction mixture in an amount of up to 5 wt.% of the fatty acid source. The aqueous buffer solution can have a pH of from 7 to about 11, e.g., any of 7-8.5, 7-9, 7-9.5, 7-10, and 7-11. The pKa of the supplemental weakly basic reagent comprising the buffer solution is higher than or equal to the pKa of the acid comprising the fatty acid source.

In another embodiment, the present invention relates to a process for the transesterification/esterification of a fatty acid source with an alcohol to form fatty acid alkyl esters, comprising reacting a fatty acid source with an alcohol in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized onto a hydrophobic porous support and the reaction medium contains water. The water is in the form of an aqueous solution having a pH of 3 to 11. The reaction medium may contain water or an aqueous solution of up to 5% by weight of the fatty acid source.

In all embodiments and aspects of the invention, the alcohol may be a short chain alcohol, such as C₁-C₆Alkyl alcohol, more particularly C₁-C₄Alkyl alcohols, in particular methanol or ethanol. When the alcohol is methanol, the resulting fatty acid ester is a fatty acid methyl ester (FAME-biodiesel). The alcohol may also be a medium chain fatty alcohol (C)₆-C₁₀) Or long-chain fatty alcohols (C)₁₂-C₂₂). The alcohol donor may be a monoalkyl or dialkyl carbonate, such as dimethyl carbonate or diethyl carbonate.

In all embodiments and aspects of the present invention, the immobilized lipase is capable of catalyzing esterification of free fatty acids to produce fatty acid alkyl esters and water as byproducts, and is capable of catalyzing transesterification of triglycerides and partial glycerides to produce fatty acid alkyl esters and glycerol as byproducts.

In all embodiments and aspects of the invention involving the use of an alkaline buffer or alkaline solution, the amount of alkaline buffer or solution in the reaction medium is from 0.001 to 5% by weight of the fatty acid source.

In all embodiments and aspects of the invention, the at least one lipase may be a lipase derived from any one of the following: rhizomucor miehei, Pseudomonas, Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes, Achromobacter, Burkholderia, Thermomyces lanuginosus, Chromobacterium viscosum, Candida antarctica B, Candida rugosa, Candida antarctica A, papaya seeds, and pancreatin. The lipase preparation may comprise at least two lipases, which may each be immobilized separately or co-immobilized on the same hydrophobic carrier. The lipases may have the same or different regiospecificities. The lipase is capable of simultaneously or sequentially catalyzing esterification of free fatty acids to produce fatty acid alkyl esters and water as a byproduct, and transesterification of triglycerides and partial glycerides to produce fatty acid alkyl esters and glycerol as a byproduct.

In all embodiments and aspects of the present invention, the carrier may be any one of a hydrophobic aliphatic polymer-based carrier and a hydrophobic aromatic polymer-based carrier. The hydrophobic polymeric carrier may be comprised of linear or branched organic chains. The carrier may comprise a macroreticular organic polymer or copolymer chain. The support may be a porous or non-porous inorganic support, which may be hydrophobic, or coated with a hydrophobic organic material. The organic material may be a linear, branched or functionalized hydrophobic organic chain.

In all embodiments and aspects of the invention, when a basic buffer solution is used, the aqueous basic buffer solution may be a solution of an inorganic base salt or an organic base. The alkaline buffer solution may be any one of the following solutions and any mixture thereof: alkali metal hydroxides, carbonates, bicarbonates, phosphates, sulfates, acetates and citrates, primary, secondary and tertiary amines. In particular embodiments, the alkaline buffer solution may be a solution of a weak base selected from sodium or potassium bicarbonate and carbonate. In some particular embodiments of the process of the invention, the alkaline buffer solution may be added to the fatty acid source in a pre-mixing stage, or the alkaline buffer solution may be added directly to the reaction medium.

In all embodiments and aspects of the invention, when a basic buffer solution is used, the basic buffer solution can be present in the transesterification/esterification reaction medium in an amount in the range of from 0.001 to 5 weight percent of the oil feedstock, such as from 1 to 2 weight percent of the oil feedstock.

In some embodiments of the invention, the fatty acid source may be first mixed with the alkaline buffer solution or with water or an aqueous solution, then the mixture is treated with the immobilized lipase preparation, followed by addition of the alcohol, and the reaction allowed to proceed under suitable conditions until the fatty acid source is converted to a fatty acid ester.

In all embodiments and aspects of the invention, the fatty acid source may be any one of the following and any mixtures thereof: vegetable oil, animal fat, seaweed oil, fish oil and waste oil. The fatty acid source may comprise free fatty acids, mono-, di-or triglycerides, mixtures thereof in any proportion, in the absence or presence of other small amounts of fatty acid derivatives such as phospholipids and sterol esters. The fatty acid source may be unrefined, refined, bleached, deodorized, or any combination thereof.

In all embodiments and aspects of the invention, the reaction may be carried out at a temperature between 10 ℃ and 100 ℃, in particular between 25-30 ℃.

In all embodiments and aspects of the present invention, the fatty acid source may be premixed with the alcohol or alcohol donor and the water or buffer solution in a pre-reaction preparation vessel to form an emulsion, which is then fed into the transesterification/esterification reaction vessel along with the immobilized lipase preparation.

In all embodiments and aspects of the invention, the immobilized lipase may be used in a packed bed column reactor operating in batch mode or continuous mode.

According to another aspect of the present invention, there is provided a system for transesterification/esterification of fatty acids with alcohols to form fatty acid alkyl esters, comprising:

a reaction vessel configured for reacting a reaction medium in the presence of an immobilized lipase preparation, the reaction medium comprising a fatty acid and at least one of an alcohol and an alcohol donor, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support, and the reaction medium comprises at least one of an aqueous alkaline buffer solution and water.

The system may include one or more of the following features in any desired combination or permutation:

A. the reaction vessel may comprise the immobilized lipase preparation at least during operation of the system for preparing the fatty acid alkyl esters.

B. Additionally or alternatively to feature a, the reaction vessel may comprise a fatty acid and at least one of an alcohol and an alcohol donor, at least during operation of the system for preparing the fatty acid alkyl esters.

C. Additionally or alternatively to features a or B, the reaction medium comprises a mixture, the system further comprising a pre-reaction vessel in selective fluid connection with the reaction vessel, the pre-reaction vessel configured for pre-mixing at least the fatty acid and at least one of an alcohol and an alcohol donor to form the mixture and for selectively delivering the mixture to the reaction vessel at least during operation of the system for preparing the fatty acid alkyl esters. The system may optionally further comprise a fatty acid source selectively fluidly connected to the pre-reaction vessel and configured for selectively delivering fatty acids to the pre-reaction vessel at least during the operation of the system, and an alcohol source selectively fluidly connected to the pre-reaction vessel and configured for selectively delivering at least one of an alcohol and an alcohol donor to the pre-reaction vessel at least during the operation of the system. The system may optionally further comprise a buffer source selectively fluidly connected to the pre-reaction vessel and configured to selectively deliver at least one of an aqueous alkaline buffer solution and water to the pre-reaction vessel to be included in the mixture at least during the operation of the system.

D. Additionally or alternatively to features a to C, the system may be configured to selectively deliver at least one of one or more fatty acids and/or alcohols and alcohol donors and/or at least one of an aqueous alkaline buffer solution and water to the pre-reaction vessel each in a continuous manner or in discrete batches at least during the operation of the system.

E. Additionally or alternatively to features a to D, the pre-reaction vessel may be configured to selectively deliver the mixture to the reaction vessel in a continuous manner and/or in discrete batches at least during the operation of the system.

F. Additionally or alternatively to features a to E, the system may be configured to selectively deliver at least one of the following directly to the reaction vessel: at least one of a fatty acid, an alcohol, and an alcohol donor, and at least one of an aqueous alkaline buffer solution and water.

G. Additionally or alternatively to features a through F, the reaction vessel may include a thermal regulation system configured to maintain the reaction medium in the reaction vessel within a selected temperature range.

H. Additionally or alternatively to features a to G, the system may optionally further comprise a retaining device configured to retain the immobilized lipase preparation within the reaction vessel at least during operation of the system.

I. Additionally or alternatively to features a through H, the system further comprises a product separation vessel in selective fluid connection with the reaction vessel, the system configured to selectively deliver a reaction mixture comprising a reaction product from the reaction vessel to the product separation vessel, and wherein the product separation vessel is configured to selectively separate a yield of fatty acid alkyl esters from the reaction mixture delivered to the product separation vessel. For example, the product separation vessel may be one of a centrifuge and a gravity separation system.

J. Additionally or alternatively to features a to I, the reaction vessel is configured to selectively deliver the reaction mixture to the product separation vessel in a continuous manner and/or in discrete batches at least during the operation of the system.

K. Additionally or alternatively to features I through J, the system is configured to selectively deliver a yield of the fatty acid alkyl ester from the product separation vessel. For example, the system is configured to selectively deliver a production of the fatty acid alkyl esters from the product separation vessel in a continuous manner and/or in discrete batches.

Additionally or alternatively to features a to K, the system is configured to increase the yield of the fatty acid alkyl ester from the reaction mixture delivered to the product separation vessel. In one configuration of a system having this feature, the system is configured to selectively re-send the production of the fatty acid alkyl esters to the reaction vessel to further increase the production of the fatty acid alkyl esters from the reaction mixture that is subsequently delivered to the product separation vessel. In another configuration of the system with this feature, the system is configured for selectively re-sending the production of fatty acid alkyl esters to an auxiliary reactor assembly, wherein the auxiliary reactor assembly comprises an auxiliary reactor vessel and an auxiliary product separation vessel, wherein the further increased production of fatty acid alkyl esters is subsequently selectively delivered via the auxiliary product separation vessel.

Drawings

In order to understand the invention and to see 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: the transesterification activity of the lipase Thermomyces Lanuginosus (TL) immobilized on Amberlite XAD1600 (Amb. XAD 1600) as a hydrophobic resin and Duolite D568 (Duo D568) as a hydrophilic resin, and the lipase Pseudomonas (PS) immobilized on Sepabeads SP70 (SB SP 70) as a hydrophobic resin and porous silica (Sil.) as a hydrophilic resin.

Abbreviations: conv. -conversion; cyc-cycle

FIG. 2: conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction at different levels of 0.1M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from thermomyces lanuginosus immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: conv. -conversion; cyc-cycle

FIG. 3: conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction at different levels of 0.1M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from Pseudomonas immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: conv. -conversion; cyc-cycle

FIG. 4: conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction in the absence of water and at different levels of water using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from thermomyces lanuginosus immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: conv. -conversion; cyc. -cycle; DW-distilled water

FIG. 5: conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction at different levels of water using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from Pseudomonas immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: conv. -conversion; cyc. -cycle; DW-distilled water

FIG. 6: conversion of a mixture of FFA and soybean oil to biodiesel, and glycerol and water by-products after 4 hours of esterification/transesterification at different levels of 0.1M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from Pseudomonas immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: conv. -conversion; cyc. -cycle; DW-distilled water

FIG. 7: esterification of soybean oil hydrolysate to biodiesel and water after 4 hours of reaction in the presence of 2% 0.1M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst is a lipase derived from Pseudomonas immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Abbreviations: val-acid number; cyc-cycle

FIG. 8: transesterification of fish oil with ethanol after 6 hours of reaction in the presence of 1 wt% 0.1M sodium bicarbonate solution using the same batch of biocatalyst in several batch experiments. Biocatalysts are lipases derived from thermomyces lanuginosa (TL Lip.) and pseudomonas (PS Lip.) immobilized on Amberlite XAD 1600.

Abbreviations: conv. -conversion; cyc-cycle

FIG. 9: transesterification of bovine fat with ethanol after 6 hours of reaction in the presence of 2 wt% 0.1M sodium bicarbonate solution using the same batch of biocatalyst in several batch experiments. The biocatalyst is Thermomyces lanuginosus and Pseudomonas lipase (PS Lip.; TL Lip.) immobilized on Amberlite XAD 1600.

Abbreviations: conv. -conversion; cyc-cycle

FIG. 10: the transesterification/esterification medium obtained after 4 hours and containing FFA values of 7mg KOH/1g was treated with Pseudomonas or Thermomyces lanuginosus immobilized on a hydrophobic porous resin and with Candida antarctica immobilized on a hydrophobic porous resin.

Abbreviations: val-acid number; cyc-cycle

FIG. 11: a first embodiment of a system for preparing fatty acid alkyl esters according to one aspect of the present invention is schematically illustrated.

FIG. 12: a second embodiment of a system for preparing fatty acid alkyl esters according to one aspect of the present invention is schematically illustrated.

Detailed Description

In seeking improvements in enzymatic industrial processes, particularly processes for the transesterification/esterification of fatty acid sources with alcohols in the presence of immobilized lipases, the present inventors have developed specific conditions under which the stability of the immobilized lipase is maintained over many production cycles.

In one embodiment of the invention, the invention relates to a process for the preparation of alkyl esters of fatty acids, in particular short-chain alkyl esters of fatty acids, such as fatty acid methyl esters and fatty acid ethyl esters (biodiesel) in a solvent-free alkaline micro-aqueous system. In a particular embodiment, the alkaline micro-water system is a weakly alkaline micro-water system. The method comprises providing a fatty acid source and reacting the fatty acid source with a free alcohol or an alcohol donor in the presence of an immobilized lipase preparation under said alkaline or slightly alkaline conditions. Without being bound by theory, pretreatment of the fatty acid source with an alkaline buffer solution will produce a neutralizing acid that may have an inhibitory effect on the enzyme. The amount of alcohol required to complete the reaction up to 100% conversion can be added stepwise or in one portion. Furthermore, the alcohol may be a short chain alcohol, such as methanol or ethanol. Other alcohol donors may be used in the reaction with the fatty acid source in the presence of a hydrolase enzyme and allowing the reaction to proceed under suitable conditions until the fatty acid source is converted to a fatty acid alkyl ester, in particular a Fatty Acid Methyl Ester (FAME) or fatty acid ethyl ester, wherein the hydrolase enzyme preparation comprises one or more lipases separately or co-immobilized onto a suitable macroreticular porous hydrophobic polymer-based carrier.

In further embodiments, the transesterification/esterification reaction between the fatty acid source and the alcohol or alcohol donor is carried out in an aqueous microenvironment, wherein water is added to the reaction mixture. In particular embodiments, water may be added at 0.0001 to 5 wt% of the fatty acid source. As used herein, water means pure or distilled water, and "aqueous solutions" which may be, but are not limited to, tap water, seawater, or water from any other natural source or reservoir, desalinated water, chemically or enzymatically purified or treated water, and any other aqueous solution. The pH of the reaction system or aqueous solution may vary and may be, for example, from about 3 to 11, such as from 4 to 10, 5 to 9, 6 to 10, 6 to 9, or 7 to 9.

The process of the present invention can be carried out with simultaneous continuous removal of the glycerol formed and any excess water from the reaction mixture. The conversion of the fatty acid acyl groups or free fatty acids contained in the fatty acid source into fatty acid alkyl esters, in particular fatty acid methyl esters, can be monitored at various points in time during the reaction. The reaction medium can be removed by suitable means at any desired point in time during the reaction, whereby the reaction is stopped and the fatty acid methyl esters formed and optionally the glycerol formed are separated from the reaction medium. The reaction may in particular be stopped when the conversion of the fatty acid acyl groups or free fatty acids comprised in the fatty acid source to fatty acid methyl esters has reached at least 70%, such as at least 85%, or at least 90%.

The reaction system may be similar to that described in co-pending WO 2009/069116. For example, the production system may use a stirred tank reactor with a bottom sintered glass or stainless steel filter that retains the biocatalyst in the reactor but allows the reaction medium to permeate out of the reactor. This reactor configuration allows the by-products (in particular glycerol and water) self-desorbed from the immobilized enzyme to sink to the bottom of the reactor and permeate out through the filter. The result is that the desorbed glycerol formed and excess water are continuously removed from the reaction medium, resulting in a shift of the reaction to synthesis, thereby achieving a conversion of more than 98%. The biocatalyst used in this reactor consists of a single type or multiple types of lipases, as described herein, in view of their positional specificity and their origin. Alternatively, two continuous stirred tank reactors with bottom filters may be used. A settling tank or centrifuge may be used between the two reactors. The first reactor may contain an immobilized biocatalyst consisting of a single type or multiple types of lipases. The role of the settler or centrifuge between the two reactors is to remove the formed glycerol and excess water from the reaction medium, thereby increasing the conversion of the feedstocks to their corresponding fatty acid alkyl esters in the second reactor above 98% at reasonable reaction times. Some specific reaction systems and methods are described below.

The terms "reaction mixture", "reaction system" and "reaction medium" may be used synonymously herein.

As in the examples of the process of the invention, the use of lipases immobilized on hydrophobic resins in the presence of alkaline buffer solutions or water ensures a high stability of the enzymes and avoids the accumulation of hydrophilic substances (such as water and glycerol by-products formed) on the biocatalyst. In particular embodiments of the process of the invention, 0.001-5% basic or slightly alkaline buffer solutions are used, e.g. 0.01-5%, 0.05-5%, 0.1-5%, 0.5-5%, such as 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%. In particular embodiments of the methods of the present invention using water, the water is used at a level of 0.0001-5%, e.g., 0.001-5%, 0.01-5%, 0.05-5%, 0.1-5%, 0.5-5%, such as 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. As noted, when an alkaline solution is used, it can neutralize acids that are typically present in the fatty acid source or are generated as a result of side reactions. The continuous active removal of these by-products may even increase the efficiency of the process. The separated glycerin can be used industrially.

The fatty acid source used in the process of the invention may comprise at least one of the following, or any mixture of at least two of them in any desired ratio: soybean oil, canola oil, algal oil, rapeseed oil, olive oil, castor oil, palm oil, sunflower oil, peanut oil, cottonseed oil, jatropha oil, corn crude oil, fish oil, animal derived fats, waste cooking oils, brown greases, oil triglycerides derived from inedible vegetable sources, partial glycerides derived from those oils, and free fatty acids.

In all the processes of the present invention, the fatty acid short-chain alkyl esters formed by the reaction are in particular fatty acid methyl esters, fatty acid ethyl esters, fatty acid isopropyl esters or fatty tert-butyl esters (biodiesel). Other medium-chain fatty alcohols (C)₆-C₁₀) And long chain fatty alcohols (C)₁₂-C₂₂) Can also be used in the preparation method of the method. These longer alcohols may be particularly suitable for the preparation of waxes, such as cosmetic products.

The lipase may be a lipase derived from, but not limited to: thermomyces lanuginosus, Rhizomucor miehei, Mucor miehei, Pseudomonas, Rhizopus, Mucor javanicus, Penicillium rokrofulensis, Aspergillus niger, Chromobacterium viscosus, Achromobacter, Klebsiella, Candida antarctica A, Candida antarctica B, Candida rugosa, Alcaligenes, Penicillium camembertii, papaya seeds, and pancreatin.

The lipases may be co-immobilized on a suitable support, in particular a hydrophobic aliphatic polymer-based support or a hydrophobic aromatic polymer-based support. Each of the lipases may be immobilized on a suitable support, wherein the supports on which the lipases are immobilized may be identical or different. The lipases used may be regiospecific to their substrate or random. When more than one lipase is used, the lipases may be immobilized on the same or different hydrophobic supports. Lipases co-immobilized on the same support may show substrate selectivity or regiospecificity for their substrates, which may be the same or different.

The lipases can be regiospecific (or site-specific), each used alone or in combination with lipases having the same or different site-specificities. When referring to positions sn-1, sn-2 or sn-3, these are positions on the glycerol backbone of the various glycerides. Thus, the lipases used in the process of the invention may have a higher selectivity towards the sn-2 position compared to random lipases, i.e. they favour the catalytic reaction between the alcohol or alcohol donor and the fatty acyl group at the sn-2 position, whereas random lipases show the same transesterification activity towards fatty acyl groups at all three positions on the glycerol backbone. Some lipases uniquely show positional activity at the sn-2 position, particularly under specific conditions determined by the substrate, product, etc. Other lipases used in the method of the invention are sn-1,3 position specific. They can be used alone or together with random lipases, in particular lipases with affinity for part of the glycerides, and optionally a third lipase with high affinity for the sn-2 position.

The support is in particular a porous macroreticular hydrophobic support, which may be organic or inorganic. Examples of supports are porous inorganic supports (such as, but not limited to, hydrophobized silica or and alumina-based supports), and hydrophobic organic supports (such as, but not limited to, polymeric or polymer-based supports). The carrier may optionally contain reactive functional groups selected from epoxy or and aldehyde groups, or ionic groups.

Insoluble supports used in the process of the invention are in particular porous, reticulated hydrophobic aliphatic or aromatic polymer-based supports, such as Amberlite^RXAD1600 and Sepabeads^RSP70 (both composed of a porous micro-network resin and made of divinylbenzene or a mixture of divinylbenzene and polystyrene), Amberlite^RXAD 7HP (which consists of a micro-network aliphatic acrylic polymer) and porous aliphatic polymers (e.g., porous polypropylene (Accurel)^R））。

The support may be a network hydrophobic polymer composed of divinylbenzene or a mixture of divinylbenzene and styrene, and a network hydrophobic aliphatic polymer composed of an aliphatic acrylic polymer or a polyolefin such as polypropylene. The specific carrier has a pore diameter of 25-1000In the range of more particularly 80 to 200Porous matrices within the scope. The support may also be a powdered or granular porous hydrophobic silica or other inorganic oxide. The support may also be a powdered or particulate porous hydrophobized silica or other inorganic oxide. In a particular embodiment, the surface area of the support resin is higher than 100m²/g。

The amount of alkaline or slightly alkaline aqueous solution to be supplemented in the lipase-catalyzed transesterification/esterification reaction between the fatty acid source and the alcohol is typically less than 5% by weight of the reaction medium. The alkaline solution is made, for example, from an inorganic base or salt, or from an organic base. Inorganic bases and salts are, for example, alkali metal hydroxides, carbonates, bicarbonates, phosphates, sulphates, acetates and citrates. The organic base can be, for example, a primary, secondary or tertiary amine. Mixtures of these alkaline agents are also contemplated. In the process according to the invention, the pH of the microenvironment of the immobilized enzyme is maintained at an alkaline or weakly alkaline value. Although the addition of distilled water to the reaction system improved the performance of the lipase immobilized on the hydrophobic carrier (resin), as shown in FIGS. 4 and 5, the addition of various alkaline buffers having different pH values depending on the type of the base used gave further stabilization of the lipase immobilized on the hydrophobic carrier (resin), as shown in FIGS. 2 and 3, for example. Carbonate and bicarbonate buffers are examples of weak bases effective to increase the stability of lipases immobilized on hydrophobic supports. Other suitable bases are described herein. Typically the complementary alkaline or weakly alkaline reagent comprising the buffer solution has a pKa equal to or higher than the pKa of the acid comprising the fatty acid source. Weakly basic solutions as used herein are typically solutions having a pH of from 7 to about 11, e.g., from 7-8.5, from 7-9, from 7-9.5, from 7-10, or from 7-11. Generally, the amount of basic or weakly basic aqueous solution used is expressed in weight percent (wt%) based on the amount of oil used in the reaction.

The use of lipase immobilized on a porous hydrophobic polymer-based support (resin) in the presence of an alkaline or slightly alkaline solution, for example in an amount of 0.01 to 5 wt.%, 0.05 to 4 wt.%, 1 to 5 wt.% or 1 to 4 wt.%, results in stabilization of the activity of the biocatalyst in the transesterification/esterification reaction between the fatty acid source and the alcohol. This is shown in the following example.

The fatty acid source is at least one of the following or a mixture of at least two of the following: triglycerides, partial glycerides, free fatty acids, phospholipids, esters and amides of fatty acids.

The preparation of the fatty acid alkyl esters is carried out simultaneously or continuously by transesterification or esterification. In such a reaction system, biocatalyst activity is maintained without significant activity loss in many applications, and also the accumulation of glycerol and water by-products or other hydrophilic compounds on the biocatalyst is avoided.

The present invention provides methods for utilizing specific immobilized interfacial enzymes that retain high activity and stability over many production cycles. In particular, lipase and phospholipase preparations are used in the transesterification/esterification reaction. These reactions are useful for the preparation of food, cosmetics and biofuels ("biodiesel"). Of particular interest, these enzymes are useful for the synthesis of short chain alkyl esters of fatty acids for use as "biodiesel".

The present invention utilizes stable immobilized interfacial enzymes with high tolerance to short chain alcohols (such as methanol, ethanol and glycerol) and short chain fatty acids (such as acetic acid). The use of these enzyme preparations also prevents the accumulation of hydrophilic substances, in particular glycerol and water, on the immobilized biocatalyst.

In one embodiment of the present invention, a process is provided for the simultaneous or sequential transesterification/esterification of a fatty acid source with an alcohol in the presence of an alkaline or slightly alkaline aqueous solution using one or more types of lipase immobilized on a hydrophobic support (resin) to obtain the desired product (i.e., fatty acid alkyl ester) at near complete conversion over a reasonable reaction time course (typically less than 5 hours). A weak base solution, such as a 0.001M, 0.1M, 0.5M or 1M sodium bicarbonate solution, may be present in the reaction system in an amount of less than about 5% by weight or less than about 4% by weight of the amount of oil used in the reaction.

As shown in the following examples, the operating life of lipases can also be extended by: the use of a hydrophobic resin support for lipase immobilization in combination with the use of an alkaline or weakly alkaline buffer solution, for example in the range of 0.001 to 5% by weight, in the transesterification/esterification medium. As further shown in the examples below, the water content of the reaction mixture can be increased regardless of pH. Thus, in another embodiment, the stability of the biocatalyst is increased with increasing water content of the reaction system by adding e.g. 0.0001-5 wt.% of said fatty acid source or any of the specific sub-intervals as defined above of water. The results show that the addition of an alkaline solution in the range of 0.0001-5 wt% of the fatty acid source (fig. 2 and 3), or the addition of water in the range of 0.001-4% of the fatty acid source (fig. 4 and 5) results in maintaining the enzyme activity and stability in many reaction cycles.

The alcohol or alcohol donor used in the process of the invention may be a short-chain alkyl alcohol, in particular C₁-C₆Alkyl alcohol, more particularly C₁-C₄An alkyl alcohol, and in particular methanol or ethanol, or the alcohol donor may be a monoalkyl ester or a dialkyl carbonate, such as dimethyl carbonate. Alcohol donors such as dialkyl carbonates can also serve as a source of alkalinity or alkalescence for the reaction system.

According to another aspect of the present invention, there is provided a system for preparing fatty acid alkyl esters. Referring to fig. 11, a first embodiment of such a system, generally designated by the reference numeral 100, includes a reactor vessel 120, a pre-reaction preparation vessel 140, and a product isolation vessel 160.

Pre-reaction preparation vessel 140 is configured for receiving feedstock and buffer (and/or water) to form a suitable emulsion therefrom, and for feeding the prepared emulsion PE (also referred to herein as emulsified feedstock) to reactor vessel 120. In particular, such feedstocks may include fatty acids FA from a fatty acid source 182 (e.g., slop oil), alcohols AL from an alcohol source 184 (e.g., methanol), and buffers (and/or water) BU from a buffer/water source 186 provided via suitable supply lines 152, 154, 156, respectively, that are fluidly connected to the pre-reaction preparation vessel 140 via vessel inlets 172, 174, 176, respectively, and suitable valves (not shown).

The pre-reaction preparation vessel 140 defines an inner volume V1 in which a reaction mixture comprising starting materials and buffer/water supplied thereto via vessel inlets 172, 174, 176 is mixed together to form an emulsion PE by a suitable stirring system 142, the stirring system 142 being driven by a power source (not shown). The pre-reaction preparation vessel 140 includes an outer jacket 149 through which a suitable working fluid may be circulated to maintain the volume V1 at the desired steady state temperature. For example, the working fluid may be oil or water that is heated or cooled in a different vessel (not shown) and pumped through the jacket 149 via suitable inlets and outlets (not shown). In an alternative variation of this embodiment, the pre-reaction preparation vessel 140 may include a system of heating and/or cooling components (e.g., electrically powered heating and/or cooling components) in place of, or in addition to, the jacket 149.

The reactor vessel 120 is configured for receiving the preparatory emulsion PE from the pre-reaction preparatory vessel 140 for reacting the feedstock therein in the presence of a suitable biocatalyst BC to produce a reaction product RP and for feeding the reaction product RP from the reaction mixture to the product separation vessel 160. Outlet line 148 provides selective fluid connection between pre-reaction preparation vessel 140 and reactor vessel 120 via suitable valves (not shown) and allows the prepared emulsion PE produced from pre-reaction preparation vessel 140 to be fed to reactor vessel 120 as desired.

Reaction vessel 120 defines an inner volume V2 in which a ready emulsion PE reaction in the reaction mixture therein is provided via vessel inlet 122, and the reaction mixture may be stirred by a suitable stirring system 124 to form reaction product RP, the stirring system 124 being driven by a power source (not shown). The biocatalyst BC may comprise a suitable enzyme and is provided in the form of immobilized enzyme beads which remain in the reactor vessel 120 until they become ineffective or insufficiently effective, at which time they may be removed and replaced with new biocatalyst BC. For example, the biocatalyst BC may comprise a lipase derived from thermomyces lanuginosa immobilized on a hydrophobic porous polystyrene-divinylbenzene resin.

Reactor vessel 120 includes a thermal regulation system in the form of an external jacket 129 through which a suitable working fluid may be circulated to maintain volume V2 at the desired steady state temperature. For example, the working fluid may be oil or water that is heated or cooled in a different vessel (not shown) and pumped through the jacket 129 via suitable inlets and outlets 123. In an alternative variation of this embodiment, the thermal conditioning system includes a system of heating and/or cooling components (e.g., electrically powered heating and/or cooling components) in place of the jacket 129 or in addition to the jacket 129.

The lower part of the reactor vessel 120 comprises an outlet 127 and suitable retaining means in the form of a filter 125 is provided upstream of the outlet 127 and is configured for filtering the reaction mixture, in particular the reaction product RP, before removing the reaction mixture from the reactor vessel 120 and for preventing the biocatalyst BC from being removed together with the reaction product RP.

The product separation vessel 160 is configured to separate the desired product P (fatty acid alkyl ester) from the reaction product RP, from the by-products (including excess water and glycerol G). Outlet line 147 provides selective fluid connection between product separation vessel 160 and reactor vessel 120 via suitable valves (not shown) and allows reaction product RP to be fed from reactor vessel 120 to product separation vessel 160 as desired. In this embodiment, the product separation vessel 160 comprises a centrifuge or gravity separation system for performing the aforementioned separation, and includes a first outlet 162 for outputting the product P, and a second outlet 164 for collecting excess water and glycerol G. Product P can be collected via tap 163.

The system can thus be operated in a continuous preparation mode, wherein the preparatory emulsion PE is fed to the reactor vessel 120 and the desired product P is collected in a continuous manner via tap 163. The emulsion PE can be prepared and delivered to the reactor vessel 120 in a continuous manner to fill the volume of reactants in the reactor vessel 120 at the same rate as the reaction product RP is removed from the outlet 127. Alternatively, the emulsion PE may be prepared in bulk and delivered to the reactor vessel 120 to fill the volume of reactant in the reaction mixture at discrete intervals whenever the reactant level in the reactor vessel 120 drops to a specified minimum level after the reaction product RP is continuously removed via outlet 127. Of course, it is also possible to operate the system 100 so as to provide the desired product P in batches rather than continuously.

Alternatively, system 100 can be operated in an enhanced-yield mode, wherein product P is not immediately collected via tap 163, but is instead re-sent to reactor vessel 120 via an optional re-sending system comprising line 165, vessel inlet 121, and valve 166, wherein valve 166 can be selectively operated to divert product P from tap 163. When product P is re-sent to reactor vessel 120, product P can be further reacted therein with alcohol AL provided from source 184 via a separate line (not shown), from a different alcohol source (not shown), or from source 184 via pre-reaction preparation vessel 140 to produce a higher yield of product P that can be re-separated from the by-products using product separation vessel 160. When alcohol is supplied via the preparation vessel 140, the preparation vessel 140 is first emptied of the preparation emulsion PE and appropriate valves prevent the fatty acids FA and optional buffer/water from being supplied from the respective sources 182 and 186.

Suitable pumps or gravity feed and controllable valves may be provided to selectively deliver the respective materials through the respective lines 152, 154, 156, 148, 147, 165, and a suitable controller (not shown) monitors and controls the operation of the system.

In at least some optional variations of the first embodiment, the pre-reaction preparation vessel 140 may be integral with the reaction vessel 120. For example, the respective internal volumes V1 and V2 may be separated by a wall having an opening means corresponding to line 148. Alternatively, the respective internal volumes V1 and V2 may be contiguous, but the internal volume V1 is sufficiently spaced from the biocatalyst BC to provide sufficient time for the emulsion PE to form before reaching the biocatalyst BC.

In an alternative variation of the first embodiment, one, two, or all of fatty acid FA, alcohol AL, and buffer/water BU may be provided directly to reactor vessel 120 bypassing pre-reaction preparation vessel 140. For example, one or more of fatty acid source 182, alcohol source 184, and buffer/water source 186 may bypass pre-reaction preparation vessel 140 and be selectively fluidly connected directly to reactor vessel 120 via a suitable supply line (not shown).

It will be appreciated that all of the components of the system 100 according to the first embodiment or alternative variants thereof are of a suitable form and made of suitable materials known in the art, for example to enable each component to perform its respective function under respective conditions (including temperature, pressure, pH, etc.).

Referring to fig. 12, a second embodiment of the system, designated by the reference numeral 200, includes all of the components and features of the first embodiment, including optional variations thereof, including all of the similarly numbered components of fig. 11 with some distinction mutatis mutandis. For example, system 200 also includes: reactor vessel 120, pre-reaction preparation vessel 140, product separation vessel 160, fatty acid source 182, alcohol source 184, buffer/water source 186, supply lines 152, 154, 156, vessel inlets 172, 174, 176, agitation system 142, outer jacket 149, outlet line 148, vessel inlet 122, agitation system 124, biocatalyst BC outer jacket 129, inlet and outlet 123, outlet 127, filter 125, outlet line 147, first outlet 162, second outlet 164, as disclosed in the first embodiment and mutatis mutandis.

However, in the second embodiment, the line 165, tap 163 and valve 166 of the first embodiment are omitted, and instead the auxiliary reactor assembly 300 is optionally connected to the first outlet 162 of the product separation vessel 160.

Auxiliary reactor assembly 300 includes an auxiliary reactor vessel 220 and an auxiliary product separation vessel 260, in this embodiment auxiliary reactor vessel 220 and auxiliary product separation vessel 260 are substantially similar to reactor vessel 120 and product separation vessel 160, respectively, mutatis mutandis. In operation, the desired product P from product separation vessel 160 is passed to auxiliary reactor vessel 220 via line 266, valve 267, and vessel inlet 221. Upon passing into auxiliary reactor vessel 220, product P may be further reacted therein with alcohol AL provided from source 184 or from a different alcohol source (not shown) via a separate line (not shown) to produce further reacted product FRP. Line 249 enables the further reacted product FRP to be transported to the auxiliary product separation vessel 260, which auxiliary product separation vessel 260 is then operated to separate a higher yield of product P' from the by-products.

System 200 may operate in a manner similar to system 100 (mutatis mutandis).

Although disclosed and described, it is to be understood that this invention is not limited to the particular examples, method steps, and materials disclosed herein as such, as the method steps and materials may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular examples only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples represent techniques used by the inventors in carrying out aspects of the invention. It should be understood that while these techniques illustrate preferred embodiments for practicing the invention, those skilled in the art will, in light of the present disclosure, appreciate that many changes can be made without departing from the intended scope of the invention.

Examples of the invention

Overview

All experiments were carried out either in a glass tube with a volume of 30ml with a centered glass filter at the bottom or in a mechanically stirred reactor with a volume of 500ml with a sintered glass filter with a porosity of 150 and 250 μm at the bottom. A typical reaction medium contains a fatty acid source, an alcohol (usually methanol or ethanol) on a 1:1 molar basis relative to the fatty acids (whether free or bound to the glycerol backbone) (1: 1 for free fatty acids and monoglycerides, 1:2 for diglycerides, and 1:3 for triglycerides, favouring the alcohol). The fatty acid source was premixed with varying amounts of alkaline buffer (sodium bicarbonate in a particular example). The reaction is initiated by the addition of lipase (10-15% by weight) immobilized on a hydrophobic resin, the reaction medium being either mechanically shaken or stirred at 30 ℃. Unless otherwise indicated, the alcohol amount was added equally in three steps, each of which was spaced 1 hour apart. The reaction conversion was followed by taking samples from the reaction medium at different time intervals and analyzing the fatty acid composition. The conversion to biodiesel was calculated as: 100 peak area of fatty acid alkyl ester/sum of all peak areas.

Immobilization of lipases: the lipase is immobilized according to standard procedures, wherein a lipase derived from a certain microorganism is dissolved in a 0.1M buffer solution at a certain pH value (e.g. 7.5). Introducing an organic or inorganic polymer resin into the lipase solution. The mixture was shaken at room temperature for 8 hours. Cold acetone is optionally added to the mixture to increase protease precipitation on the resin. The mixture was filtered and the enzyme beads were dried to reduce the water content to less than 5%.

Different resins are used to obtain a resin with hydrophobic properties, including hydrophobic polymer resins based on polystyrene/divinylbenzene, paraffin wax or any combination thereof. Typical hydrophobic resins used include Amberlite^RXAD 1600（Rohm&Haas, USA) and Sepabeeds^RSP70 (Resindex, Italy). Typical hydrophilic resins used include Duolite^RD568（Rohm&Haas) and porous silica gels. The lipases may be immobilized separately on the resin, or different lipases may be co-immobilized on the same resin.

Example 1

Immobilization to Amberlite as hydrophobic resin^RDuolite on XAD1600 and as hydrophilic resin^RLipase derived from Thermomyces lanuginosus at D568, and Sepabeads immobilized as a hydrophobic resin^RTransesterification activity of lipase derived from Pseudomonas on SP70 and on porous silica as hydrophilic resin.

Reaction conditions are as follows: refined and bleached soybean oil (20 g) contained 1 wt.% 0.1M sodium bicarbonate solution. Methanol (2.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. The reaction medium containing 10% by weight of the lipase preparation was shaken at 300rpm and 30 ℃. The results are shown in FIG. 1.

The results shown in FIG. 1 show that both Thermomyces lanuginosus and Pseudomonas lipases immobilized on different resins exhibit high transesterification activity during the first 5 cycles with the same batch of enzyme in the presence of a 1 wt.% sodium bicarbonate solution. It was observed that after batch 5, when the same batch of enzyme was used, it was due to immobilization on a hydrophilic resin (i.e., Duolite)^RD568 and porous silica) and the formation of a gelatinous precipitate around the beads of the two lipases, filtration of the reaction medium from the system became difficult. In further consecutive batches, the transesterification activity of the two lipases immobilized on the hydrophilic resin dropped sharply and they became inactive after the 10 th cycle. In contrast, immobilization on hydrophobic resin Sepabeads^RPseudomonas lipase immobilized on SP70 retained more than 80% of its initial activity after 70 cycles, whereas Amberlite, a hydrophobic resin, immobilized^RThermomyces lanuginosa lipase on XAD1600 retains more than 20% of its initial activity after more than 70 cycles.

Example 2

A. Conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.

Reaction conditions are as follows: refined and bleached soybean oil (20 g) contained varying concentrations of 0.1M sodium bicarbonate solution. Methanol (2.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. A lipase derived from Thermomyces lanuginosus (10 wt.%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 2.

B. Conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.

Reaction conditions are as follows: refined and bleached soybean oil (20 g) contained varying concentrations of 0.1M sodium bicarbonate solution. Methanol (2.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. A lipase derived from pseudomonas (10 wt%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 3.

FIGS. 2 and 3 show that the amount of sodium carbonate in the reaction medium has a major effect on the lifetime of the Thermomyces lanuginosus and Pseudomonas lipases immobilized on hydrophobic resins. As can be seen in fig. 2 and 3, both immobilized lipases significantly lost their activity after several cycles in the absence of alkaline solution, whereas the same immobilized lipases retain their transesterification activity in multiple uses in the presence of sodium bicarbonate solution as the base in the reaction system. The results for both immobilized enzymes show that increasing the amount of sodium bicarbonate solution in the reaction medium in the range of 0-4 wt.% reduces the loss of enzyme activity over multiple uses of the same batch of immobilized enzyme.

Example 3

A. Conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments. Reaction conditions are as follows: refined and bleached soybean oil (20 g) contained various concentrations of distilled water. Methanol (2.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. A lipase derived from Thermomyces lanuginosus (10 wt.%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 4.

B. Conversion of soybean oil to biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments. Reaction conditions are as follows: refined and bleached soybean oil (20 g) contained various concentrations of distilled water. Methanol (2.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. A lipase derived from pseudomonas (10 wt%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 5.

FIGS. 4 and 5 show that the transesterification activity of Thermomyces lanuginosus and Pseudomonas using the same batch of lipase immobilized on a hydrophobic resin in multiple experiments is also affected by the amount of water in the reaction system. It can be seen that increasing the amount of water from none (zero) to 4 wt% maintains the higher residual transesterification activity of the biocatalyst when the biocatalyst is used in a continuous cycle. The results shown in fig. 2 to 5 clearly show that the use of a weak base (such as sodium bicarbonate solution) in the transesterification reaction is advantageous for maintaining the activity of the lipase immobilized on the hydrophobic resin when used in continuous cycles.

Example 4

Conversion of a mixture of Free Fatty Acids (FFA) and soybean oil to biodiesel and glycerin and water by-products after 4 hours of esterification/transesterification using the same batch of biocatalyst in multiple batch experiments.

Reaction conditions are as follows: a mixture of soy hydrolysate (50 wt%) and soy oil (50 wt%) with an initial FFA value of 72mg KOH/1g of free fatty acids contained varying amounts of 0.1M sodium bicarbonate solution. Methanol (4.5 ml) was added stepwise in three equivalent batches, each 1 hour apart. A lipase derived from pseudomonas (20 wt%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 6.

Figure 6 shows that different amounts of alkali solution have a major effect on the simultaneous esterification of FFA present in the reaction mixture consisting of equal proportions of soybean oil hydrolysate and soybean oil triglycerides. It can be seen that the Pseudomonas lipase immobilized on the hydrophobic resin lost its esterification activity when no alkaline solution was added to the esterification/transesterification reaction system, while the same biocatalyst maintained its activity in continuous cycles when 1 and 2 wt% of 0.1M sodium bicarbonate solutions were added to the reaction system separately. The results shown in FIG. 6 show that the use of Pseudomonas lipase immobilized on a hydrophobic resin in the presence of 1 and 2 wt.% of 0.1M sodium bicarbonate solution reduced the FFA content from an initial value of 72mg KOH/1g to an average of 8 and 6mg KOH/1g, respectively, and maintained this activity over 22 consecutive cycles.

Example 5

Esterification of soybean oil hydrolysate to biodiesel and water after 4 hours of reaction using the same batch of biocatalyst in multiple batch experiments.

Reaction conditions are as follows: free fatty acid soy hydrolysate with FFA value of 150mg KOH/1g (20 g) contained 1 wt.% 0.1M sodium bicarbonate solution. Methanol (2 ml) was added to the reaction medium in one batch. A lipase derived from pseudomonas (10 wt%) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin was used. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 7.

FIG. 7 shows that Pseudomonas lipases immobilized on hydrophobic resins are also capable of catalyzing the esterification of free fatty acids to form fatty acid methyl esters and water by-products. The results show that, using the same batch of biocatalyst, the lipase preparation retains its esterification/transesterification activity in a medium containing 1% of a 0.1M sodium bicarbonate solution over more than 25 cycles, without any significant loss of activity being observed.

Example 6

The transesterification of fish oil with ethanol was performed after 6 hours of reaction using the same batch of biocatalyst in several batch experiments.

Reaction conditions are as follows: the refined fish oil (20 g) contained 1% 0.1M sodium bicarbonate solution. Ethanol (2.5 ml) was added stepwise to three equivalent batches, each 1 hour apart. Respectively using Amberlite as the immobilizing agent^RLipases from Thermomyces lanuginosus and Pseudomonas on XAD1600 (10 wt%). The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 8.

FIG. 8 shows that two lipases derived from Thermomyces lanuginosus and Pseudomonas immobilized on hydrophobic resins are also capable of catalyzing the transesterification of fish oil triglycerides with ethanol to form fatty acid ethyl esters and glycerol byproducts. The results also show that using the same batch of biocatalyst, both biocatalyst formulations maintained their transesterification activity in the presence of 1% sodium bicarbonate solution over more than 20 cycles without significant activity loss.

Example 7

Transesterification of bovine fat with ethanol after 6 hours of reaction using the same batch of biocatalyst in several batch experiments.

Reaction conditions are as follows: bovine fat (16 g) containing fatty acid ethyl esters of bovine fat (4 g) and 1% 1M potassium carbonate solution. Ethanol (2.5 ml) was added stepwise to three equivalent batches, each 1 hour apart. Immobilization on Amberlite^RLipases from Thermomyces lanuginosus and Pseudomonas (10% by weight) on XAD1600 were used separately or in combination in equal proportions. The reaction medium is shaken at 300rpm and 37 ℃. The results are shown in FIG. 9.

FIG. 9 shows that two lipases derived from Thermomyces lanuginosus and Pseudomonas, respectively or in combination, immobilized on a hydrophobic resin are also capable of catalyzing the transesterification of bovine fat triglycerides with ethanol to form fatty acid ethyl esters and glycerol byproducts. The starting material for the reaction medium consists of bovine fat (80%) and fatty acid ethyl esters derived from bovine fat to lower the melting point of the reaction medium. The results shown in figure 9 show that when the same batch of biocatalyst was used in 100 consecutive cycles, all biocatalysts retained more than 80% of their initial activity in the presence of a weak alkaline solution (e.g. 1M potassium carbonate).

Example 8

In a number of batch experiments, the transesterification/esterification reaction medium obtained after 4 hours, which had an FFA value of 7mg KOH/1g, was treated with the same batch of biocatalyst (10% by weight) with Pseudomonas lipase or Thermomyces lanuginosus lipase immobilized on a hydrophobic porous resin and with Candida antarctica B lipase and methanol (respectively, in a ratio of 1:10 by moles between FFA and methanol) immobilized on a hydrophobic porous resin. The reaction medium was shaken at 300rpm and 30 ℃. The results are shown in FIG. 10.

FIG. 10 shows that the transesterification medium obtained after treatment with Thermomyces lanuginosus lipase or Pseudomonas lipase as described above, which typically has an FFA value of 3-7mg KOH/1g, can be treated with Candida antarctica B lipase immobilized on a hydrophilic or hydrophobic support to reduce the FFA value to less than 2mg KOH/1 g. The immobilized lipase can retain its activity for more than 100 cycles.

Example 9

Example 8

Using a first example of the system shown in fig. 11, waste cooking oil containing 10% FFA was transesterified/esterified with methanol to form biodiesel, water and glycerol.

Reaction conditions are as follows: slop oil (1100 g) containing 2% 0.1M sodium bicarbonate solution and methanol (140 g) were first premixed in pre-reaction preparation vessel 140 to form an emulsion, which was then introduced into reactor vessel 120 having an internal volume V2 of about 2 liters. The reaction mixture was mixed in reactor vessel 120 with a lipase derived from thermomyces lanuginosa (30 wt.% of oil) immobilized on a hydrophobic porous polystyrene-divinylbenzene resin at 30 ℃ for 6 hours. The reaction mixture is filtered through filter 125 and fed to product separation vessel 160. Glycerol and excess water are removed from the reaction mixture in the product separation vessel 160. The upper phase containing fatty acid methyl esters and unreacted glycerides is reintroduced to reactor vessel 120 via re-send line 165 and agitation in reactor vessel 120 is resumed after methanol (110 g) is added to the reaction medium in reactor vessel 120. The conversion to methyl ester after 2 hours was 98%. An emulsion reaction medium (ready emulsion) containing slop oil (83 wt%), methanol (15%) and 0.1M sodium bicarbonate solution (2%) was fed continuously into reactor vessel 120 at a flow rate of about 30 ml/min. When the same batch of biocatalyst derived from thermomyces lanuginosus lipase immobilized on a macroporous hydrophobic resin was used, the conversion to fatty acid methyl esters remained for more than 3 months without significant loss of activity.

Claims (69)

1. A process for the transesterification and/or esterification of a fatty acid source with an alcohol to form fatty acid alkyl esters, comprising reacting the fatty acid source with an alcohol or an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises a lipase immobilized on a hydrophobic porous support and the reaction medium contains an aqueous alkaline buffer solution having a pH of 7-11 in an amount of at most 5 wt% of the fatty acid source,

wherein the fatty acid source comprises a mono-, di-or triglyceride, mixture thereof in any proportion, in the absence or presence of free fatty acids or derivatives thereof selected from amides, esters, phospholipids and sterol esters,

wherein the transesterification and/or esterification is carried out simultaneously or continuously,

wherein glycerol and/or water, respectively, are formed as by-products.

2. The method of claim 1, wherein the aqueous alkaline buffer solution is an aqueous weakly alkaline buffer solution.

3. The method of claim 1 or claim 2, wherein the aqueous alkaline buffer solution is included in an amount of 0.5 wt.%, 0.75 wt.%, 1 wt.%, 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, 4.5 wt.%, or 5 wt.% of the fatty acid source.

4. The method of claim 1 or claim 2, wherein the pH is any one of 7-8.5, 7-9, 7-9.5, and 7-10.

5. A process for the transesterification and/or esterification of a fatty acid source selected from the group consisting of triglycerides, diglycerides, monoglycerides and any mixture thereof with an alcohol or an alcohol donor to form fatty acid alkyl esters, the transesterification/esterification being carried out in the absence or presence of free fatty acids or derivatives thereof selected from the group consisting of amides, esters, phospholipids and sterol esters, the process comprising reacting the fatty acid source with an alcohol in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises a lipase immobilized on a hydrophobic porous support and water or an aqueous solution is added to the fatty acid source or to the reaction medium in an amount of at most 5 wt% of the fatty acid source, wherein the pH of the aqueous solution or reaction system is between 3 and 11, and

wherein the transesterification and/or esterification is carried out simultaneously or continuously,

wherein glycerol and/or water, respectively, are formed as by-products.

6. The process of claim 5, wherein the reaction medium contains 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the fatty acid source of water or aqueous solution.

7. The method of claim 1 or claim 5, wherein the alcohol is a short chain alcohol.

8. The process according to claim 1 or claim 5, wherein the alcohol donor is a monoalkyl ester, or a dialkyl carbonate, which also acts as a source of weakly basic reagent in the reaction medium.

9. The method of claim 8, wherein the monoalkyl ester is methyl acetate and the dialkyl carbonate is dimethyl carbonate.

10. The method according to claim 1 or claim 5, wherein the at least one lipase is a lipase derived from any one of: rhizomucor miehei, Pseudomonas, Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes, Achromobacter, Burkholderia, Thermomyces lanuginosus, Chromobacterium viscosum, Candida antarctica B, Candida rugosa, Candida antarctica A, and papaya seeds.

11. The process according to claim 1 or claim 5, wherein the immobilized lipase is capable of catalyzing esterification of free fatty acids to produce fatty acid alkyl esters and water as a by-product, and is capable of catalyzing transesterification of triglycerides and partial glycerides to produce fatty acid alkyl esters and glycerol as a by-product.

12. The process according to claim 1 or claim 2, wherein the amount of the alkaline solution in the reaction medium is from 0.5 to 5% by weight of the fatty acid source.

13. The method according to claim 1 or claim 2, wherein the lipase preparation comprises at least two lipases which can each be immobilized separately or co-immobilized on the same hydrophobic support.

14. The method of claim 13, wherein the lipases have identical or different regiospecificities.

15. The process of claim 13, wherein the lipase is capable of simultaneously or sequentially catalyzing esterification of free fatty acids to produce fatty acid alkyl esters and water as a byproduct, and transesterification of triglycerides and partial glycerides to produce fatty acid alkyl esters and glycerol as a byproduct.

16. The method of claim 1 or claim 5, wherein the support is any one of a hydrophobic aliphatic polymer-based support and a hydrophobic aromatic polymer-based support.

17. The method of claim 16, wherein the hydrophobic polymeric support consists of linear or branched organic chains.

18. The method of claim 17, wherein the carrier comprises macroreticular organic polymer or copolymer chains.

19. The method of claim 1 or claim 5, wherein the support is a porous inorganic support, which may be hydrophobic or coated with a hydrophobic organic material.

20. The method of claim 19, wherein the organic material is a linear, branched, or functionalized hydrophobic organic chain.

21. The process of claim 1 or claim 2, wherein the aqueous alkaline buffer solution is a solution of an inorganic alkaline salt or an organic base.

22. The method of claim 21, wherein the alkaline buffer solution is a solution of any one of the following and any mixture thereof: alkali metal hydroxides, carbonates, bicarbonates, phosphates, sulfates, acetates and citrates, primary, secondary and tertiary amines.

23. The method of claim 22, wherein the alkaline buffer solution is a solution of a weak base selected from sodium or potassium bicarbonate and carbonate.

24. A process according to claim 1 or claim 2, wherein the alkaline buffer solution is added to the fatty acid source in a pre-mixing stage or is added directly to the reaction medium.

25. The process of claim 1 or claim 2, wherein the amount of the basic buffer solution in the transesterification/esterification reaction medium is in the range of 0.5 to 5 wt.% of the oil feedstock.

26. The method of claim 25, wherein the alkaline buffer solution is present in an amount of 1-2 wt% of the oil feedstock.

27. The process according to claim 1 or claim 2, wherein the fatty acid source is first mixed with the alkaline buffer solution, then the mixture is treated with the immobilized lipase preparation, followed by addition of the alcohol, and the reaction is allowed to proceed under suitable conditions until the fatty acid source is converted to a fatty acid ester.

28. A process according to claim 1 or claim 5, wherein the fatty acid source is any one of the following and any mixture thereof: vegetable oil, animal fat, seaweed oil, fish oil, waste oil and brown lubricating grease.

29. A process according to claim 1 or claim 2, wherein the fatty acid source comprises free fatty acids, monoglycerides, diglycerides or triglycerides, mixtures thereof in any proportion, fatty acid esters and amides, in the absence or presence of other minor fatty acid derivatives selected from phospholipids and sterol esters, which are unrefined, refined, bleached, deodorised or any combination thereof.

30. The method of claim 5, wherein the fatty acid source further comprises fatty acid esters and amides, and other minor fatty acid derivatives selected from phospholipids and sterol esters, wherein the fatty acid source is unrefined, refined, bleached, deodorized, or any combination thereof.

31. The method of claim 1 or claim 5, wherein the alcohol is a short chain alkyl alcohol.

32. The method of claim 31, wherein the short chain alkyl alcohol is C₁-C₆An alkyl alcohol.

33. The method of claim 31, wherein the short chain alkyl alcohol is C₁-C₄An alkyl alcohol.

34. The method of claim 31, wherein the short chain alkyl alcohol is methanol or ethanol.

35. The method of claim 1 or claim 5, wherein the alcohol is methanol and the resulting fatty acid ester is a fatty acid methyl ester.

36. A process according to claim 1 or claim 5, wherein the alcohol is a medium chain fatty alcohol of 6 to 10 carbon atoms or a long chain fatty alcohol of 12 to 22 carbon atoms.

37. The process according to claim 1 or claim 5, wherein the reaction is carried out at a temperature between 10 ℃ and 100 ℃.

38. The method of claim 37, wherein the reaction is carried out at a temperature between 25-30 ℃.

39. The process according to claim 1 or claim 5, wherein the fatty acid source is premixed with the alcohol or alcohol donor and the water or buffer solution in a pre-reaction preparation vessel to form an emulsion, which is subsequently fed into a transesterification/esterification reaction vessel together with the immobilized lipase preparation.

40. The process according to claim 1 or claim 5, wherein the immobilized lipase is used in a continuously stirred tank reactor or in a packed bed column reactor operating in batch mode or continuous mode.

41. The method of claim 29 wherein the other minor fatty acid derivative is a phospholipid or a sterol ester.

42. The method of claim 30 wherein the other minor fatty acid derivative is a phospholipid or a sterol ester.

43. A production system for producing fatty acid alkyl esters formed by transesterification/esterification of a fatty acid source with an alcohol, the production system comprising:

a reaction vessel configured for reacting a reaction medium in the presence of an immobilized lipase preparation, the reaction medium comprising the fatty acid source and at least one of an alcohol and an alcohol donor, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support, and the reaction medium contains an aqueous alkaline buffer solution.

44. A production system for producing fatty acid alkyl esters formed by transesterification/esterification of a fatty acid source selected from the group consisting of triglycerides, diglycerides, monoglycerides and mixtures of any thereof with an alcohol, the production system comprising:

a reaction vessel configured to react a reaction medium comprising the fatty acid source and at least one of an alcohol and an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises a lipase immobilized on a hydrophobic porous support, and water is added to the fatty acid source or the reaction medium.

45. The production system according to claim 43 or claim 44, wherein the reaction vessel contains the immobilized lipase preparation at least during operation of the production system for producing the fatty acid alkyl esters.

46. The production system according to claim 43 or claim 44, wherein the reaction vessel contains a fatty acid and at least one of an alcohol and an alcohol donor at least during operation of the production system for producing the fatty acid alkyl ester.

47. The production system as set forth in claim 43 or claim 44 wherein the reaction medium comprises a mixture, the production system further comprising a pre-reaction vessel selectively fluidly connected to the reaction vessel, the pre-reaction vessel configured for pre-mixing at least the fatty acid and at least one of an alcohol and an alcohol donor to form the mixture and for selectively delivering the mixture to the reaction vessel at least during operation of the production system for producing the fatty acid alkyl ester.

48. The production system of claim 47, further comprising a fatty acid source selectively fluidly connected to the pre-reaction vessel and configured for selectively delivering fatty acids to the pre-reaction vessel at least during the operation of the production system, and an alcohol source selectively fluidly connected to the pre-reaction vessel and configured for selectively delivering at least one of an alcohol and an alcohol donor to the pre-reaction vessel at least during the operation of the production system.

49. The production system of claim 48, further comprising a buffer source selectively fluidly connected to the pre-reaction vessel and configured for selectively delivering at least one of an aqueous alkaline buffer solution and water to the pre-reaction vessel to be included in the mixture at least during the operation of the production system.

50. The production system of claim 48 or claim 49, configured for selectively delivering one or more fatty acids and at least one of an alcohol and an alcohol donor to the pre-reaction vessel in a continuous manner at least during the operation of the production system.

51. The production system according to claim 49, configured for selectively delivering at least one of an aqueous alkaline buffer solution and water to the pre-reaction vessel in a continuous manner at least during the operation of the production system.

52. The production system of claim 48 or claim 49, configured for discontinuous batch selective delivery of one or more fatty acids and at least one of an alcohol and an alcohol donor to the pre-reaction vessel at least during the operation of the production system.

53. The production system according to claim 49, configured for selectively delivering at least one of an aqueous alkaline buffer solution and water to the pre-reaction vessel in discrete batches at least during the operation of the production system.

54. The production system of claim 47, wherein the pre-reaction vessel is configured to selectively deliver the mixture to the reaction vessel in a continuous manner at least during the operation of the production system.

55. The production system according to claim 47, wherein the pre-reaction vessel is configured for discontinuous batch selective delivery of the mixture to the reaction vessel at least during the operation of the production system.

56. A production system according to claim 43 or claim 44, configured for selective direct delivery to the reaction vessel of at least one of: at least one of a fatty acid, an alcohol, and an alcohol donor, and at least one of an aqueous alkaline buffer solution and water.

57. The production system of claim 43 or claim 44, wherein the reaction vessel comprises a thermal conditioning system configured to maintain a reaction medium in the reaction vessel within a selected temperature range.

58. The manufacturing system of claim 43 or claim 44, further comprising a retention device configured to retain the immobilized lipase preparation within the reaction vessel at least during operation of the manufacturing system.

59. The production system according to claim 43 or claim 44, further comprising a product separation vessel in selective fluid connection with the reaction vessel, the production system configured for selectively delivering a reaction mixture comprising reaction products from the reaction vessel to the product separation vessel, and wherein the product separation vessel is configured for selectively separating an output of fatty acid alkyl esters from the reaction mixture delivered to the product separation vessel.

60. The production system of claim 59, wherein the product separation vessel comprises one of a centrifuge and a gravity separation system.

61. The production system according to claim 59, wherein the reaction vessel is configured to selectively deliver the reaction mixture to the product separation vessel in a continuous manner at least during the operation of the production system.

62. The production system according to claim 59, wherein the reaction vessel is configured for discontinuous batch selective delivery of the reaction mixture to the product separation vessel at least during the operation of the production system.

63. The production system according to claim 59, configured for selectively delivering the output of fatty acid alkyl esters from the product separation vessel.

64. The production system according to claim 63, configured for selectively delivering the output of fatty acid alkyl esters from the product separation vessel in a continuous manner.

65. The production system according to claim 63, configured for selectively delivering the output of fatty acid alkyl esters from the product separation vessel in discrete batches.

66. The production system according to claim 59, wherein the production system is configured to increase the output of the fatty acid alkyl ester from the reaction mixture delivered to the product separation vessel.

67. The production system according to claim 66, wherein the production system is configured for selectively re-sending the output of fatty acid alkyl esters to the reaction vessel to further increase the output of fatty acid alkyl esters from a reaction mixture that is subsequently delivered to the product separation vessel.

68. The production system according to claim 66, wherein the production system is configured for selectively re-sending the output of fatty acid alkyl esters to an auxiliary reactor component, wherein the auxiliary reactor component comprises an auxiliary reactor vessel and an auxiliary product separation vessel, wherein the further increased output of fatty acid alkyl esters is subsequently selectively delivered via the auxiliary product separation vessel.

69. The method of claim 1 or claim 5, carried out in the manufacturing system of any one of claims 43 to 68.