Enzymatic or non-enzymatic biodiesel refining method

Abstract

The present invention provides a method for reducing the free fatty acid level in biodiesel / fatty acid alkyl esters. The method includes providing a composition comprising a fatty acid alkyl ester, a free fatty acid, and / or a fatty acid feedstock, reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester; and / or reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts to produce a fatty acid alkyl ester.

Description

[0001] This application is a divisional application of the patent application for invention with the application date of October 7, 2016, application number 201680058215.6, and invention title "Method for Refining Enzymatic or Non-enzymatic Biodiesel".

[0002] Citation of Sequence Listing

[0003] This application contains a sequence listing in computer-readable form. This computer-readable form is incorporated herein by reference. Technical Field

[0004] The present invention provides a method for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters. The method includes reducing the amount of free fatty acids in the oil phase / light phase by

[0005] i) reacting the free fatty acids and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipolytic enzymes to produce fatty acid alkyl esters; and / or

[0006] ii) reacting the free fatty acids and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts to produce fatty acid alkyl esters. Background Art

[0007] Fatty acid alkyl esters can be used as fuels in standard diesel engines, i.e., biodiesel. Biodiesel can be used alone or in mixture with petrochemical diesel. Currently, due to its environmental benefits, biodiesel has become more attractive.

[0008] Although currently biodiesel is mainly chemically produced (using, for example, NaOH and / or sodium methoxide as catalysts), there are several related problems that limit its development, such as the pre-treatment of oils due to high levels of free fatty acids, the removal of chemical catalysts from the ester and glycerol phases due to the need for a high alcohol surplus in the reaction, and the removal of inorganic salts during glycerol recovery.

[0009] By using lipolytic enzymes as catalysts, these disadvantages caused by chemical catalysts are greatly prevented, and in recent years, the use of lipases in the transesterification for producing biodiesel has attracted people's interest.

[0010] Biodiesel produced by enzymatic bioconversion is more environmentally friendly (compared with chemical conversion). However, with very few exceptions, currently enzyme technology is not used in commercial-scale biodiesel production.

[0011] The enzymatic production process of fatty acid alkyl esters using liquid enzymes is described, for example, in WO 2006 / 072256, Lv et al. (Process Biochemistry 45 (2010) 446-450) and WO 2012 / 098114.

[0012] In a method for producing fatty acid alkyl esters or biodiesel, a fatty acid feedstock is reacted with an alcohol (typically methanol) to produce fatty acid alkyl esters and glycerol. After the fatty acid feedstock has reacted with the alcohol to produce fatty acid alkyl esters, the oil phase / lighter phase contains residual free fatty acids. Generally, the presence of free fatty acids is undesirable, and the level of free fatty acids must be reduced as much as possible: for example, the European biodiesel standard requires the level of free fatty acids to be less than 0.25% (w / w). The industrial use of resins for esterifying free fatty acids in glyceride-based oils is well known as a pretreatment step in alkaline chemical biodiesel methods. However, esterifying free fatty acids in methyl esters to an FFA level below 0.25% is very challenging due to the higher water activity and the hygroscopicity of methyl esters. It is generally recognized that additional purification or "refining" of fatty acid methyl esters to remove free fatty acids results in losses of fatty acid methyl esters. Therefore, a trade-off is usually made between production yield and free fatty acid level.

[0013] Accordingly, there is a need for more efficient methods for producing fatty acid alkyl esters or biodiesel with reduced free fatty acid content. Summary of the Invention

[0014] The present invention provides a method for reducing the level of free fatty acids in biodiesel / fatty acid alkyl esters, the method comprising

[0015] i) providing a composition comprising

[0016] a. an oil phase / lighter phase containing fatty acid alkyl esters, free fatty acids and optionally a fatty acid feedstock; and

[0017] b. an aqueous phase / heavier phase containing an alcohol and water;

[0018] ii) reducing the amount of water in the composition, for example reducing the water content of the aqueous phase / heavier phase to be in the range of 0 - 15% by weight of the aqueous phase / heavier phase and / or reducing the water content of the oil phase / lighter phase to be in the range of 200 - 600 ppm;

[0019] and then reducing the amount of free fatty acids in the oil phase / lighter phase and optionally the amount of the fatty acid feedstock

[0020] iii) reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipases to produce a fatty acid alkyl ester; and / or

[0021] iv) reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts to produce a fatty acid alkyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : A schematic diagram showing a method for producing a fatty acid alkyl ester by an enzymatic transesterification reaction of a fatty acid or a fatty acid feedstock with an alcohol.

[0023] Figure 2 Showing four main embodiments of the present invention.

[0024] Figures 3 - 9 Showing the results obtained when using the method according to the present invention to produce fatty acid methyl esters with a reduced free fatty acid content.

[0025] These figures are included for illustrative purposes only and should in no way be construed as limiting the present invention. DETAILED DESCRIPTION

[0026] DEFINITIONS

[0027] Biodiesel: Fatty acid alkyl esters (FAAE) of short-chain alcohols (such as fatty acid methyl esters (FAME) and fatty acid ethyl esters (FAEE)) are also known as biodiesel because they can be used as additives or substitutes for petrochemical diesel.

[0028] Alcohol: The alcohol used in the method of the present invention is preferably a short-chain alcohol having 1 to 5 carbon atoms (C1, C2, C3, C4, or C5).

[0029] Fatty acid feedstock: The term "fatty acid feedstock" is defined herein as a substrate containing any source of fatty acids (including triglycerides, diglycerides, monoglycerides, or any combination thereof). In principle, any plant or animal oil and fat containing fatty acids can be used as a substrate in the method of the present invention to produce fatty acid alkyl esters.

[0030] Lipase

[0031] One or more lipolytic enzymes applied in the method of the present invention are selected from lipase, phospholipase, cutinase, acyltransferase, or a mixture of one or more of lipase, phospholipase, cutinase and acyltransferase. The one or more lipolytic enzymes are selected from the enzymes in EC 3.1.1, EC 3.1.4, and EC 2.3. The one or more lipolytic enzymes may also be a mixture of one or more lipases. The one or more lipolytic enzymes may include lipase and phospholipase. The one or more lipolytic enzymes include the lipase of EC 3.1.1.3. The one or more lipolytic enzymes include lipases active against tri-, di-, and mono-glycerides.

[0032] LipaseSuitable lipolytic enzymes can be polypeptides with lipase activity. For example, they can be selected from Candida antarctica lipase A (CALA) as disclosed in WO 88 / 02775; Candida antarctica lipase B (CALB) as disclosed in WO 88 / 02775 and shown in SEQ ID NO:1 in WO 2008065060; Thermomyces lanuginosus (formerly Humicola lanuginosus) lipase as disclosed in EP 258 068; Thermomyces lanuginosus variants as disclosed in WO 2000 / 60063 or WO 1995 / 22615 (specifically, the lipase at positions 1 - 269 of SEQ ID NO:2 shown in WO 95 / 22615), lipases of the genus Hyphozyma (WO 98 / 018912), and Rhizomucor miehei lipase (SEQ ID NO:5 in WO 2004 / 099400); lipases from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218 272), Pseudomonas cepacia (EP331 376), Pseudomonas stutzeri (GB 1,372,034), Pseudomonas fluorescens, Pseudomonas sp. strain SD 705 (WO95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus genus lipases, such as those from Bacillus subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253 - 360), Bacillus stearothermophilus (JP 64 / 744992), or Bacillus pumilus (WO 91 / 16422). Also preferred are lipases from any of the following organisms: Fusarium oxysporum, Absidia reflexa, Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar, Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Penicillium camemberti, Aspergillus foetidus, Aspergillus niger, Aspergillus oryzae, and Thermomyces lanuginosus, for example, lipases selected from any of SEQ ID NOs:1 - 15 in WO 2004 / 099400.

[0033] Useful lipases in connection with the present invention are lipases having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity with the mature polypeptide of SEQ ID NO:2, i.e., the polypeptide at positions 1 - 269 of SEQ ID NO:2 shown in WO 95 / 22615, or the polypeptide of SEQ ID NO:1 shown in WO2008 / 065060.

[0034] Commercial lipase preparations suitable for use in the methods of the present invention include LIPOZYME CALB L, LIPOZYME (R) TL 100L, CALLERA TM TRANS and Transform (all available from Novozymes A / S).

[0035] Specifically useful lipases can be selected from the group consisting of

[0036] (a) a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

[0037] (b) a polypeptide that is a subsequence of the amino acid sequence shown in SEQ ID NO:1 or 2;

[0038] (c) a polypeptide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with any of the polypeptides defined in (a) and (b).

[0039] The lipase shown in (c) can be a variant of the amino acid sequence shown in SEQ ID NO:1, wherein the polypeptide comprises the following substitutions T231R and N233R.

[0040] The lipase shown in item (c) may have an amino acid sequence that differs from the polypeptide of SEQ ID NO: 1 or 2 by up to 40 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0041] The lipase may be a variant of a parental lipase that has lipase activity and has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with SEQ ID NO: 1, and contains substitutions at the positions corresponding to T231R+N233R of SEQ ID NO: 1 and at least one or more (e.g., several) of the positions among D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q.

[0042] In a further embodiment, the lipase is a variant that has lipase activity and has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with SEQ ID NO: 1, and contains substitutions at the positions corresponding to T231R+N233R of SEQ ID NO: 1 and at least one or more (e.g., several) of the positions selected from the following group among D96E, D111A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q:

[0043] a) D96E T231R N233R;

[0044] b) N33Q D96E T231R N233R;

[0045] c) N33Q T231R N233R;

[0046] d) N33Q D111A T231R N233R;

[0047] e) N33Q T231R N233R P256T;

[0048] f) N33Q G38A G91T G163K T231R N233R D254S;

[0049] g) N33Q G38A G91T D96E D111A G163K T231R N233R D254S P256T;

[0050] h) D27R N33Q G38A D96E D111A G163K T231R N233R D254S P256T;

[0051] i) D27R N33Q G38A G91T D96E D111A G163K T231R N233R P256T;

[0052] j) D27R N33Q G38A G91T D96E D111A G163K T231R N233R D254S;

[0053] k) D27R G38A G91T D96E D111A G163K T231R N233R D254S P256T;

[0054] l) D96E T231R N233R D254S;

[0055] m) T231R N233R D254S P256T;

[0056] n) G163K T231R N233R D254S;

[0057] o) D27R N33Q G38A G91T D96E G163K T231R N233R D254S P256T;

[0058] p) D27R G91T D96E D111A G163K T231R N233R D254S P256T;

[0059] q) D96E G163K T231R N233R D254S;

[0060] r) D27R G163K T231R N233R D254S;

[0061] s) D27R G38A G91T D96E D111A G163K T231R N233R D254S;

[0062] t) D27R G38A G91T D96E G163K T231R N233R D254S P256T;

[0063] u) D27R G38A D96E D111A G163K T231R N233R D254S P256T;

[0064] v) D27R D96E G163K T231R N233R D254S;

[0065] w) D27R D96E D111A G163K T231R N233R D254S P256T;

[0066] x) D27R G38A D96E G163K T231R N233R D254S P256T.

[0067] For example, such useful variants of the parental lipase are provided in WO 2015 / 049370.

[0068] Lipase activity:

[0069] In the context of the present invention, tributyrin can be used as a substrate to determine this lipolytic activity as lipase units (LU). The method is based on the hydrolysis of tributyrin by the enzyme, and the registration of the alkaline consumption that maintains a constant pH during hydrolysis as a function of time

[0070]

[0071] According to the present invention, one lipase unit (LU) can be defined as the amount of enzyme that releases 1 micromole of titratable butyric acid per minute under standard conditions (i.e., at 30 °C; pH 7.0; with 0.1% (w / v) gum arabic as an emulsifier and 0.16 M tributyrin as a substrate).

[0072] Alternatively, the lipolytic activity can be determined as long-chain lipase units (LCLU), Using the substrate pNP-palmitate (C:16), when incubated at pH 8.0 at 30 °C, the lipase hydrolyzes the ester bond and releases pNP, which is yellow and can be detected at 405 nm.

[0073]

[0074] Phospholipase :

[0075] The one or more lipolytic enzymes may include polypeptides having phospholipase activity, preferably phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, phospholipase D, lysophospholipase activity, and / or any combination thereof. In the method of the present invention, the one or more lipolytic enzymes may be phospholipases, for example, a single phospholipase, such as A1, A2, B, C, or D; two or more phospholipases, such as two phospholipases (including but not limited to, both type A and type B, both type A1 and type A2, both type A1 and type B, both type A2 and type B, both type A1 and type C, both type A2 and type C); or two or more different phospholipases of the same type.

[0076] The one or more lipolytic enzymes may be polypeptides having phospholipase activity and having acyltransferase activity, for example, polypeptides selected from those polypeptides disclosed in WO 2003 / 100044, WO 2004 / 064537, WO 2005 / 066347, WO 2008 / 019069, WO 2009 / 002480, and WO 2009 / 081094. The acyltransferase activity can be determined, for example, by the assay described in WO2004 / 064537.

[0077] The phospholipase may be selected from the polypeptides disclosed in WO 2008 / 036863 and WO 20003 / 2758. Suitable phospholipase preparations are PURIFINE (R) (available from Verenium) and LECITASE (R) ULTRA (available from Novozymes). Enzymes having acyltransferase activity are available as the commercial enzyme preparation LYSOMAX (R) OIL (available from Danisco A / S).

[0078] Cutinase: The one or more lipolytic enzymes may include polypeptides having cutinase activity.

[0079] For example, the cutinase may be selected from the polypeptides disclosed in WO 2001 / 92502, specifically the Humicola insolens cutinase variant disclosed in Example 2.

[0080] Preferably, the one or more lipolytic enzymes are enzymes having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with any one of the above lipases, phospholipases, cutinases, and acyltransferases.

[0081] In one embodiment, the one or more lipolytic enzymes have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with the amino acid sequence shown at positions 1 - 269 of SEQ ID NO:2 of WO 95 / 22615.

[0082] Enzyme sources and formulations: The one or more lipolytic enzymes used in the methods of the present invention can be derived from or obtainable from any of the sources mentioned herein. The term "derived from" in the context of the present invention means that the enzyme can be isolated from the organism in which it naturally occurs, i.e., the identity of the amino acid sequence of the enzyme is the same as that of the native enzyme. The term "derived from" also means that these enzymes can be recombinantly produced in a host organism, and the recombinantly produced enzyme has the same identity as the native enzyme, or has a modified amino acid sequence, e.g., having one or more deleted, inserted, and / or substituted amino acids, i.e., the recombinantly produced enzyme is a mutant and / or fragment of the native amino acid sequence. Native variants are included within the meaning of the native enzyme. In addition, the term "derived from" includes enzymes produced synthetically, e.g., by peptide synthesis. The term "derived from" also encompasses enzymes that have been modified in vivo or in vitro, e.g., by glycosylation, phosphorylation, etc. The term "obtainable" in the context of the present invention means that the enzyme has the same amino acid sequence as the native enzyme. This term encompasses enzymes that have been isolated from an organism in which the enzyme naturally occurs, or in which the enzyme has been recombinantly expressed in the same type or other type of organism, or enzymes produced synthetically, e.g., by peptide synthesis. For recombinantly produced enzymes, the terms "obtainable" and "derived from" refer to the identity of the enzyme rather than the identity of the host organism in which the enzyme is recombinantly produced.

[0083] Thus, the one or more lipolytic enzymes can be obtained from microorganisms by using any suitable technique. For example, enzyme preparations can be obtained by fermenting suitable microorganisms and subsequently isolating the enzyme preparation from the resulting fermentation broth or microorganisms by methods known in the art. The enzyme can also be obtained by using recombinant DNA techniques. Such methods generally include culturing host cells transformed with a recombinant DNA vector that contains a DNA sequence encoding the enzyme in question, and the DNA sequence is operably linked to appropriate expression signals such that the DNA sequence can express the enzyme in the culture medium under conditions that permit enzyme expression, and recovering the enzyme from the culture. The DNA sequence can also be incorporated into the genome of the host cell. The DNA sequence can be of genomic, cDNA, or synthetic origin, or any combination thereof, and can be isolated or synthesized by methods known in the art.

[0084] The one or more lipolytic enzymes can be applied in any suitable formulation, for example, as a lyophilized powder or in an aqueous solution.

[0085] Sequence identity

[0086] The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0087] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman - Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443 - 453) as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276 - 277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0088] (Identical residues x 100) / (Length of alignment - Total number of gaps in the alignment)

[0089] Method design

[0090] The present invention provides a method for reducing the free fatty acid level in biodiesel / fatty acid alkyl esters, the method comprising

[0091] i) providing a composition comprising

[0092] a. an oil phase / light phase containing fatty acid alkyl esters, free fatty acids, and optionally a fatty acid feedstock; and

[0093] b. an aqueous phase / heavy phase containing an alcohol and water;

[0094] ii) reducing the amount of water in the composition, for example, reducing the water content of the aqueous phase / heavy phase to be in the range of 0 - 15% by weight of the aqueous phase / heavy phase and / or reducing the water content of the oil phase / light phase to be in the range of 200 - 600 ppm;

[0095] and then reducing the amount of free fatty acids in the oil phase / light phase and optionally the amount of the fatty acid feedstock

[0096] iii) reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester; and / or

[0097] iv) reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts to produce a fatty acid alkyl ester.

[0098] Specifically, the method according to the present invention may comprise

[0099] i) providing a composition comprising

[0100] a. an oil phase / lighter phase containing a fatty acid alkyl ester, a free fatty acid and optionally a fatty acid feedstock; and

[0101] b. an aqueous phase / heavier phase containing an alcohol and water;

[0102] ii) reducing the amount of water in the composition, for example reducing the water content of the aqueous phase / heavier phase to within the range of 0 - 15% by weight of the aqueous phase / heavier phase and / or reducing the water content of the oil phase / lighter phase to within the range of 200 - 600 ppm;

[0103] iii) reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / lighter phase by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester;

[0104] And, optionally

[0105] iv) further reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / lighter phase by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts to produce a fatty acid alkyl ester.

[0106] The composition in i) may be provided by a reaction in which a free fatty acid and / or a fatty acid feedstock is reacted with an alcohol to produce a fatty acid alkyl ester until the reaction has substantially reached equilibrium. Specifically, "equilibrium" may be defined as the point at which there is no further net reduction in the free fatty acid in the reaction mixture. Thus, for the purposes of the present invention, the composition in i) may specifically be provided by a reaction which has been allowed to proceed to the point at which there is no further net reduction or substantially no further net reduction in the free fatty acid.

[0107] The composition in i) can be specifically provided by the following reaction, in which the fatty acid raw material reacts with an alcohol in the presence of glycerol in an amount corresponding to 0 - 70% by weight of the aqueous phase / heavy phase, water in an amount corresponding to 10% - 70.0% by weight of the aqueous phase / heavy phase, and an alcohol (such as methanol) in an amount in the range of 10% - 50% by weight of the aqueous phase / heavy phase.

[0108] In step ii), the water content of the aqueous phase / heavy phase can be reduced to within the range of 2% - 15% by weight of the aqueous phase / heavy phase, such as within the range of 5% - 15% by weight of the aqueous phase / heavy phase, within the range of 7% - 15% by weight of the aqueous phase / heavy phase, within the range of 10% - 15% by weight of the aqueous phase / heavy phase, within the range of 0 - 10% by weight of the aqueous phase / heavy phase, within the range of 2% - 10% by weight of the aqueous phase / heavy phase, within the range of 5% - 10% by weight of the aqueous phase / heavy phase, within the range of 0 - 9% by weight of the aqueous phase / heavy phase, within the range of 2% - 9% by weight of the aqueous phase / heavy phase, or within the range of 5% - 9% by weight of the aqueous phase / heavy phase.

[0109] The water content of the oil phase / light phase can also be reduced to within the range of 200 - 600 ppm, such as within the ranges of 300 - 600 ppm, 400 - 600 ppm, 200 - 500 ppm, 200 - 400 ppm, or within the range of 300 - 500 ppm.

[0110] The glycerol content can be equivalent to 0 to 60% by weight of the aqueous phase / heavy phase, such as 0 to 50%, 0 to 40%, 0 to 30%, 0 to 20%, 2% to 60%, 5% to 60%, 10% to 60%, 20% to 60%, 30% to 60%, 30% to 50%, 5% to 50%, 10% to 50%, 20% to 50%, 30% to 50%, 2% to 40%, 5% to 40%, 10% to 40%, 20% to 40%, 2% to 30%, 5% to 30%, or equivalent to 10% to 30% by weight of the aqueous phase / heavy phase.

[0111] The water content can be equivalent to 10% to 60% by weight of the aqueous phase / heavy phase, such as 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 12% to 60%, 15% to 60%, 20% to 60%, 30% to 60%, 30% to 50%, 10% to 50%, 20% to 50%, 30% to 50%, 10% to 40%, 20% to 40%, or equivalent to 10% to 30% by weight of the aqueous phase / heavy phase. As those skilled in the art will understand, the water content in the aqueous phase / heavy phase depends on the fatty acid raw material used: if free fatty acids are mainly used as substrates, the aqueous phase / heavy phase will mainly contain water, while using a fatty acid raw material with a relatively large amount of bound glycerol will increase the amount of glycerol in the aqueous phase / heavy phase and reduce the amount of water.

[0112] Preferably, the amount of alcohol (such as methanol) is in the range of 10% to 45% by weight of the aqueous phase / heavy phase, such as in the ranges of 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 15% to 25%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 25% to 50%, 25% to 45%, 25% to 40%, or in the range of 25% to 35% by weight of the aqueous phase / heavy phase.

[0113] In the method according to the present invention, the composition in i) can be provided by a reaction that includes reacting a free fatty acid and / or a fatty acid raw material with an alcohol until at least 90% (w / w) or for example at least 95% (w / w) of the fatty acid acyl groups or free fatty acids in the fatty acid raw material have been converted into fatty acid alkyl esters.

[0114] In some embodiments of the present invention, the fatty acid and / or the fatty acid raw material in step iii) and / or step iv) is reacted with an alcohol until at least 80% (w / w), at least 85% (w / w), at least 90% (w / w) or such as at least 95% (w / w) of the free fatty acid and / or the fatty acid acyl groups have been converted into fatty acid alkyl esters.

[0115] Preferably, the composition in step i) is provided by a reaction in which the one or more lipolytic enzymes are lipases. Preferred lipases are provided above.

[0116] Specifically, the composition in (i) can be provided through the following reaction, in which the total amount of the one or more lipases is in the range of 0.005 - 5 g of enzyme protein (EP) / kg of oil phase / light phase or fatty acid raw material, such as 0.005 - 2.5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 1 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.75 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.25 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.1 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.075 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.05 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.025 g EP / kg of oil phase / light phase or fatty acid raw material, 0.005 - 0.01 g EP / kg of oil phase / light phase or fatty acid raw material, 0.01 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.02 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.03 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.04 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.05 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.06 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.07 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.08 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.09 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.1 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.2 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.3 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.4 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.5 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.6 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.7 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.8 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.9 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 1 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 2 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 3 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 4 - 5 g EP / kg of oil phase / light phase or fatty acid raw material, 0.01 - 4 g EP / kg of oil phase / light phase or fatty acid raw material, 0.02 - 3 g EP / kg of oil phase / light phase or fatty acid raw material, 0.03 - 2 g EP / kg of oil phase / light phase or fatty acid raw material, 0.04 - 1 g EP / kg of oil phase / light phase or fatty acid raw material, 0.05 - 0.In the range of 9 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.06 - 0.8 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.07 - 0.7 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.08 - 0.6 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.09 - 0.5 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.1 - 0.4 g EP / kg of the oil phase / lighter phase or fatty acid raw material, 0.1 - 0.3 g EP / kg of the oil phase / lighter phase or fatty acid raw material, or in the range of 0.1 - 0.25 g EP / kg of the oil phase / lighter phase or fatty acid raw material.

[0117] In step ii) of the method, the amount of water can be reduced by applying heat (such as by convection, conduction, and / or radiation).

[0118] Specifically, an air stream can be used in step ii) to remove the water as moisture.

[0119] In an alternative embodiment, a vacuum is used in step ii) to remove the water as moisture.

[0120] In a currently preferred embodiment, the amount of water is reduced by flash drying in step ii).

[0121] In the method according to the invention, the amount of alcohol in step iii) can be equivalent to 5% - 10% by weight of the oil phase / lighter phase, such as 6% to 10%, 7% to 10%, 8% to 10%, 5% to 9%, 5% to 8%, or equivalent to 5% to 7% by weight of the oil phase / lighter phase.

[0122] The amount of alcohol in step iv) can be equivalent to 10% to 25% by weight of the oil phase / lighter phase, for example 11% to 25%, 12% to 25%, 13% to 25%, 14% to 25%, 15% to 25%, 16% to 25%, 17% to 25%, 18% to 25%, 19% to 25%, 20% to 25%, 10% to 24%, 10% to 23%, 10% to 22%, 10% to 21%, 10% to 20%, 10% to 19%, 10% to 18%, 10% to 17%, 10% to 16%, 10% to 15%, or equivalent to 12% to 15% by weight of the oil phase / lighter phase.

[0123] In the method according to the invention, step iv) can include separating the aqueous phase / heavier phase from the oil phase / lighter phase before further reducing the amount of free fatty acids and optionally the amount of fatty acid raw material.

[0124] The duration of step iv) in the method according to the invention can be 0.5 - 7 hours, such as 0.5 - 6 hours, 0.5 - 5 hours, 0.5 - 4 hours, 0.5 - 3 hours, 0.5 - 2 hours, 0.5 - 1 hour, 1 - 7 hours, 2 - 7 hours, 3 - 7 hours, or such as 4 - 7 hours.

[0125] One or more lipolytic enzymes in step iii) can specifically be lipase or one or more lipases, such as any one of the lipases disclosed previously above.

[0126] The total amount of the one or more lipolytic enzymes in step iii) can be in the range of 0.01 - 0.10 g enzyme protein (EP) / kg oil phase / light phase, or such as in the range of 0.02 - 0.10 g EP / kg oil phase / light phase, 0.03 - 0.01 g EP / kg oil phase / light phase, 0.04 - 0.10 g EP / kg oil phase / light phase, 0.05 - 0.1 g EP / kg oil phase / light phase, 0.06 - 0.1 g EP / kg oil phase / light phase or fatty acid raw material, 0.07 - 0.1 g EP / kg oil phase / light phase, 0.08 - 0.01 g EP / kg oil phase / light phase, 0.01 - 0.09 g EP / kg oil phase / light phase, 0.01 - 0.08 g EP / kg oil phase / light phase, 0.01 - 0.07 g EP / kg oil phase / light phase, 0.01 - 0.06 g EP / kg oil phase / light phase, 0.01 - 0.05 g EP / kg oil phase / light phase, 0.01 - 0.04 g EP / kg oil phase / light phase, 0.01 - 0.03 g EP / kg oil phase / light phase, 0.02 - 0.08 g EP / kg oil phase / light phase or fatty acid raw material, or such as in the range of 0.03 - 0.06 g EP / kg oil phase / light phase.

[0127] In the method according to the invention, one or more non - enzyme catalysts used in step iv) can be selected from the group consisting of: acid catalysts (such as sulfonic acid, sulfuric acid, phosphoric acid and hydrochloric acid) and base catalysts (such as metal alkoxides (e.g., sodium alkoxide or potassium alkoxide)). In a currently preferred embodiment, the catalyst is sulfonic acid.

[0128] In a specific embodiment according to the invention, one or more non - enzyme catalysts in step v) are immobilized on a solid resin such as a porous polymer - based resin. An example of such a commercially available resin is Lewatit GF 101 from Lanxess.

[0129] The reaction in step iv) can be carried out in a resin bed or column containing one or more immobilized non-enzymatic catalysts, a stirred reactor, or a continuous stirred reactor. Specifically, using a continuously stirred reactor or a resin bed or column offers the following advantages: The reaction can be carried out as a continuous reaction.

[0130] If the reaction is chosen to be run in batch mode, a conventional stirred reactor can be used. Alternatively, a more complex reactor with an integrated water removal system, such as an air bobbled batch reactor, can be employed. Such a complex reactor will allow water to be continuously removed throughout the reaction.

[0131] However, a major advantage of the present invention is the use of a main resin esterification step without using any complex reactor with an integrated water removal system. Applying one or more non-enzymatic catalysts in step iv) in a continuously stirred reactor or a resin bed or column as described in the present application allows for a simple continuous process during the esterification process without water removal, or at least without significant water removal.

[0132] The solid resin is preferably packed in a resin bed or column at a height of at least 1 meter, such as at least 1.5 meters, at least 2 meters, at least 2.5 meters, or at least 3 meters. Particularly preferred columns have a height ranging from 1 - 3 meters, such as from 1 - 2.5 meters, from 1 - 2 meters, from 1 - 1.5 meters.

[0133] According to the embodiment in which the resin is packed in a resin bed or column, the residence time on the resin is preferably in the range of 1 - 4 hours, such as 1.5 - 4 hours, 1.5 - 3 hours, 2 - 3 hours, or preferably in the range of 2.2 - 2.4 hours.

[0134] Step iv) can be carried out in a pressurized system at a temperature in the range of 75°C - 95°C, such as in the range of 80°C - 95°C, 85°C - 95°C, 90°C - 95°C, 75°C - 90°C, 75°C - 85°C, 75°C - 80°C, or such as in the range of 80°C - 90°C.

[0135] The alcohol used in each step of the method according to the present invention is preferably a C1 - C5 alcohol, more preferably ethanol or methanol.

[0136] The fatty acid raw materials used according to the present invention can be derived from one or more of the following: algal oil; canola oil; coconut oil; castor oil; coconut oil; coconut kernel oil; corn oil; corn oil from distillers' grains; cottonseed oil; linseed oil; fish oil; grape seed oil; hemp oil; jatropha oil; jojoba oil; mustard oil; canola oil; palm oil; palm stearin; palm olein; palm kernel oil; peanut oil; rapeseed oil; rice bran oil; safflower oil; soybean oil; sunflower oil; tall oil; oil from halophytes; and / or animal fats, including fats from pigs, cows, and sheep, lard, chicken fat, fish oil; palm oil free fatty acid distillate; soybean oil free fatty acid distillate; soap stock fatty acid material; yellow grease; and brown grease or any combination thereof.

[0137] In the method according to the present invention, after step iii) or iv), the following step may be carried out: in this step, in the presence of the alcohol / the light phase, soap / salt is formed from the remaining free fatty acids in the oil phase / light phase by treating with one or more basic reagents.

[0138] The one or more basic reagents can be added in an amount equivalent to 1.0 - 2.0 molar equivalents of the amount of free fatty acids, such as 1.2 - 2.0 molar equivalents, 1.3 - 2.0 molar equivalents, 1.4 - 2.0 molar equivalents, 1.5 - 2.0 molar equivalents, 1.6 - 2.0 molar equivalents, 1.7 - 2.0 molar equivalents, 1.8 - 2.0 molar equivalents, 1.0 - 0.9 molar equivalents, 1.0 - 0.8 molar equivalents, 1.0 - 0.7 molar equivalents, 1.0 - 0.6 molar equivalents, 1.0 - 0.5 molar equivalents, 1.0 - 0.4 molar equivalents, 1.0 - 0.3 molar equivalents, or such as 1.3 - 1.8 molar equivalents of the amount of free fatty acids.

[0139] Treating with one or more basic reagents can include bringing the oil phase / light phase and optionally the water phase / hydrophilic phase into contact with a basic reagent or base selected from KOH or NaOH or a mixture thereof.

[0140] Treating with one or more basic reagents can preferably be carried out at a temperature in the range of 35°C to 70°C, such as in the range of 40°C to 70°C, in the range of 45°C to 70°C, in the range of 50°C to 70°C, in the range of 55°C to 70°C, or such as in the range of 35°C to 65°C.

[0141] The basic reagent can specifically be sodium methoxide or potassium methoxide or a mixture of the two.

[0142] The method according to the present invention may comprise the following steps: reducing the amount of soap / fatty acid salt in the composition by acidifying the soap / fatty acid salt, such as by chemically titrating the soap / fatty acid salt with an acid to produce free fatty acids, such as by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4.

[0143] The step of reducing the amount of soap / fatty acid salt may specifically be carried out before step iv).

[0144] The method according to the present invention may further comprise separating an oil phase / lighter phase containing fatty acid alkyl esters from a hydrophilic phase / heavier phase.

[0145] As those skilled in the art will recognize, the oil phase / lighter phase can be separated from the hydrophilic phase / heavier phase by gravity sedimentation, decantation and / or centrifugation.

[0146] The method according to the present invention may comprise drying the glycerol to remove, for example, water and an alcohol, such as methanol or any other C1-C5 alcohol as disclosed herein, from the glycerol.

[0147] Preferably, the glycerol is purified, such as by drying and / or removing the alcohol, to produce a composition: wherein the content of glycerol is higher than 95% (w / w), such as higher than 97% (w / w), higher than 97.5% (w / w), higher than 98% (w / w), higher than 98.5% (w / w), higher than 99% (w / w), higher than 99.5% (w / w), higher than 99.75% (w / w), higher than 99.8% (w / w) or higher than 99.9% (w / w).

[0148] Specifically, the glycerol can be subjected to thermal-vacuum distillation.

[0149] The method according to the present invention may comprise distilling the fatty acid alkyl ester, such as thermal-vacuum distillation, wherein the fatty acid alkyl ester is evaporated and then concentrated.

[0150] In a specific embodiment, the fatty acid alkyl ester is subjected to thermal-vacuum distillation at 240 °C - 260 °C.

[0151] One advantage of the method provided according to the present invention is that, in addition to the distillation as described above, purification of the fatty acid alkyl ester (including any fatty alkyl ester produced in step iii) and / or iv)) is unnecessary and can be avoided. However, if still desired, the fatty acid alkyl ester can be further purified.

[0152] The purification can be carried out by subjecting the fatty acid alkyl ester to a water wash.

[0153] Specifically, purification can be carried out by allowing the fatty acid methyl ester to settle (such as by gravitational settling), and then subjecting the settled fatty acid alkyl ester to water washing.

[0154] The method according to the present invention can be a batch process, such as the following method, in which steps i), ii) and iii) in their entirety or steps i), ii) and iv) in their entirety are carried out batchwise. Alternatively, the method can be semi - continuous, such as the following method, in which one or more but not all of steps i), ii) and iii) are carried out continuously, or in which one or more but not all of steps i), ii) and iv) are carried out continuously. Preferably, the method is a continuous process, in which steps i), ii) and iii) in their entirety or steps i), ii) and iv) in their entirety are carried out continuously.

[0155] In a specific embodiment according to the present invention, they are shown as "Option 1" in Figure 2 and the method according to the present invention comprises

[0156] i) providing a composition comprising

[0157] a. an oil phase / light phase containing a fatty acid alkyl ester, free fatty acid and optionally a fatty acid feedstock; and

[0158] b. an aqueous phase / heavy phase containing an alcohol and water;

[0159] ii) separating the oil phase / light phase containing the fatty acid alkyl ester from the hydrophilic phase / heavy phase; and

[0160] iii) reducing the water content of the oil phase / light phase to within the range of 200 - 600 ppm;

[0161] iv) generating free fatty acid by acidifying the soap / fatty acid salt, such as by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4, and optionally reducing the amount of the soap / fatty acid salt in the composition;

[0162] v) reacting the free fatty acid and / or fatty acid feedstock with an alcohol in the presence of one or more non - enzymatic catalysts to produce a fatty acid alkyl ester, further reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / light phase; and

[0163] vi) purifying and / or distilling the fatty acid alkyl ester, including the fatty acid alkyl ester produced in step v).

[0164] In other specific embodiments according to the present invention, they are shown as "Option 2" in Figure 2 and the method comprises

[0165] i) Provide a composition, which composition comprises

[0166] a. an oil phase / light phase containing a fatty acid alkyl ester, a free fatty acid, and optionally a fatty acid raw material; and

[0167] b. an aqueous phase / heavy phase containing an alcohol and water;

[0168] ii) Reduce the amount of water in the composition, for example, to a range of 0 - 15% by weight of the aqueous phase / heavy phase;

[0169] iii) Reduce the amount of the free fatty acid and optionally the amount of the fatty acid raw material in the oil phase / light phase by reacting the free fatty acid and / or the fatty acid raw material with an alcohol and one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester;

[0170] iv) Contact the composition with one or more basic reagents under conditions allowing the formation of a soap / salt from the remaining free fatty acid in the oil phase / light phase;

[0171] v) Separate the oil phase / light phase containing the fatty acid alkyl ester from the hydrophilic phase / heavy phase; and

[0172] vi) Purify and / or distill the fatty acid alkyl ester, including the fatty acid alkyl ester produced in step iii).

[0173] In still other specific embodiments according to the invention, they are shown as "Option 3" in Figure 2 and the method comprises

[0174] i) Provide a composition, which composition comprises

[0175] a. an oil phase / light phase containing a fatty acid alkyl ester, a free fatty acid, and optionally a fatty acid raw material; and

[0176] b. an aqueous phase / heavy phase containing an alcohol and water;

[0177] ii) Reduce the amount of water in the composition, for example, to a range of 0 - 15% by weight of the aqueous phase / heavy phase;

[0178] iii) Reduce the amount of the free fatty acid and optionally the amount of the fatty acid raw material in the oil phase / light phase by reacting the free fatty acid and / or the fatty acid raw material with an alcohol and one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester;

[0179] iv) Produce free fatty acid by acidifying the soap / fatty acid salt, such as by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4, and optionally reduce the amount of the soap / fatty acid salt in the composition.

[0180] v) Separating the oil phase / lighter phase containing the fatty acid alkyl ester from the hydrophilic phase / heavier phase;

[0181] vi) Further reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / lighter phase by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts; and

[0182] vii) Purifying and / or distilling the fatty acid alkyl ester, including the fatty acid alkyl ester produced in steps iii) and vi).

[0183] In still further embodiments of the present invention, they are shown as "Option 4" in Figure 2 and the method comprises

[0184] i) Providing a composition comprising

[0185] a. An oil phase / lighter phase containing a fatty acid alkyl ester, a free fatty acid and optionally a fatty acid feedstock; and

[0186] b. An aqueous phase / heavier phase containing an alcohol and water;

[0187] ii) Reducing the amount of water in the composition, for example, to a range of 0 - 15% by weight of the aqueous phase / heavier phase;

[0188] iii) Reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / lighter phase by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol and one or more liquid lipolytic enzymes to produce a fatty acid alkyl ester;

[0189] iv) Producing a free fatty acid by acidifying the soap / fatty acid salt, such as by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4, and optionally reducing the amount of the soap / fatty acid salt in the composition;

[0190] v) Separating the oil phase / lighter phase containing the fatty acid alkyl ester from the hydrophilic phase / heavier phase;

[0191] vi) Further reducing the amount of the free fatty acid and optionally the amount of the fatty acid feedstock in the oil phase / lighter phase by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non-enzymatic catalysts;

[0192] vii) Contacting the composition with one or more basic reagents under conditions allowing the formation of soap / salt from the remaining free fatty acid in the oil phase / lighter phase; and

[0193] viii) Purifying and / or distilling the fatty acid alkyl esters, including the fatty acid alkyl esters produced in steps iii) and vi).

[0194] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0195] Examples

[0196] Example 1: Mixed refining method, 1L scale

[0197] Objective:

[0198] The objective is to test and develop a simple method with a yield > 97% that can be implemented on a large scale. The method developed herein is based on improving the conversion of FFA through an additional liquid enzyme step prior to the caustic washing step.

[0199] Procedure:

[0200] 1. Inorganic acid neutralization step

[0201] a. Heat 770 g of dry RBD soybean oil to 35°C / 95°F and add 20 ppm NaOH as a 1N solution ≈ 0.385 g of caustic solution. Mix at 530 rpm for 15 minutes.

[0202] b. Add water: Add a total of 2% water (including the caustic solution) ≈ 15.0 g of water and mix for 15 minutes.

[0203] 2. Enzymatic reaction.

[0204] a. Add the lipase (lipase having the amino acid sequence shown in SEQ ID NO: 2) in two doses: 0.2% w / w at 0 h and 0.1% addition at 23 h, approximately equal to 1.54 ml and 0.77 ml respectively.

[0205] b. The total methanol dose is 1.5 equivalents, starting with the addition of 21 ml at 0 h, continuously adding 100 ml within the first 20 h, and adding 14 ml at 26.5 h. The reaction proceeds for 32 hours with the target of < 0.5% BG. Final QTA reading: BG = 0.38%, FFA (titration) = 1.5%. Changes in triglycerides, free fatty acids, etc. during the transesterification process are as Figure 3 shown.

[0206] 3. Heating and drying

[0207] a. Heat the reaction mixture to 105°C / 220°F for 1 hour, then cool and dry at vacuum (end point 22 mbar) and 45°C / 113°F for 1 hour. Heavy phase measurement (QTA): water 15%, methanol 3.3%, MONG 4.4%.

[0208] b. Heat the mixture to 80°C / 180°F and dry (end point 20 mbar) for 1 hour. Heavy phase measurement (QTA): water 9.9%, methanol 0.1%, MONG 0.0%.

[0209] 4. Enzymatic refining reaction with lipase (lipase having the amino acid sequence shown in SEQ ID NO: 2)

[0210] a. Cool the dried mixture to 35°C / 95°F and add 0.075% lipase ≈ 0.50 ml and mix at 530 rpm

[0211] b. Total methanol is 3.5% w / w. Divide the dosage into 6 doses: 10 ml each at t = 0 and t = 1.5 h, and 5 ml each at 3 h, 5.5 h, 6.5 h, and 23.5 h. The reaction proceeds for 24.5 hours. End point FFA (titration) = 0.8%

[0212] The QTA analysis data showing the decrease of FFA during the enzymatic refining process are presented in Figure 4 in.

[0213] 5. Caustic washing

[0214] Heat the mixture to 60°C / 140°F and add 1.5 equivalents of NaOH (relative to the FFA content). The added caustic is a 3.2% w / w solution in methanol (= 62% concentration) and water. A total of 40 g of caustic solution is added to 760 g of the mixture. Mix at 530 rpm for 2 hours. Final FFA (titration) < 0.1% and BG = 0.2%. The QTA analysis of the oil phase and heavy phase is shown in Figure 5 in.

[0215] 6. Sedimentation, washing and drying

[0216] a. Gravity sedimentation for 30 min.

[0217] b. Perform static water washing by spraying with 4% v / v water, then stir for 30 min

[0218] c. Sediment and decant.

[0219] d. Dry the FAME phase at 100°C / 212°F for 30 min. QTA measurement (B100):

[0220] i. Acid value: 0.05

[0221] ii. MAG: 0.4%

[0222] iii. DAG: 0.27%

[0223] iv. TAG: 0.01%

[0224] v. BG: 0.19%

[0225] vi. Total glycerol: 0.21%

[0226] 7. Estimation of yield loss by acidification

[0227] a. Add 10 g of clear glycerol phase to 0.73 ml of 4N HCl and heat to 100 °C with shaking for 30 min.

[0228] b. Centrifuge at 1500 rpm at 22 °C for 30 min.

[0229] c. Aspirate the glycerol phase with a pipette.

[0230] d. The weight of the oil phase is equal to 0.7 g

[0231] e. Estimation of yield loss:

[0232] i. Total heavy phase 170 g

[0233] ii. Amount of oil in the heavy phase: 0.7 / 10 × 170 = 11.9 g

[0234] iii. Total yield loss: 11.9 g of oil relative to 770 g of oil is equal to = 1.5％

[0235] 8. Estimation of yield loss by QTA measurement

[0236] a. Estimation of yield loss:

[0237] i. Total heavy phase 170 g

[0238] ii. Total MONG content in the heavy phase = 11% MONG equals 11 / 100 × 170 = 18.7 g of oil

[0239] iii. Total yield loss: 18.7 g of oil relative to 770 g of oil is equal to = 2.4%

[0240] Conclusion:

[0241] The above tests successfully provided a total yield of 97%-99%. Using a crude FAME of a dry model substrate from RBD soybean oil, 0.075% lipase (having the amino acid sequence shown in SEQ ID NO:2), and an enzymatic refining reaction with the addition of 3.5% methanol, the FFA was reduced from 1.5% to 0.8% within 24 hours before the final one-pot caustic treatment.

[0242] Example 2: Resin refining on crude palm oil

[0243] Purpose:

[0244] To study different resin refining parameters such as column height, methanol dosage, reaction temperature, and FFA in CPO FAME to achieve a final FFA of <0.25%.

[0245] Part 1: Refining of 1.3% feedstock FFA with 0.4 m, 0.8 m, and 1.2 m height resins

[0246] a) At 80 °C, with 15% anhydrous methanol and a column height of 0.4 m, the FFA can be reduced from 1.3% to an average 0.54％ .

[0247] b) At 80 °C, with 20% anhydrous methanol and a column height of 0.8 m, the FFA can be reduced from 1.3% to an average 0.45％ , while a column height of 1.2 m can reduce the FFA to an average 0.37％ .

[0248] c) At 90 °C, with 20% anhydrous methanol and a column height of 1.2 m, at a flow rate of 4.3 bed volumes / hr, the FFA was reduced from 1.3% to 0.24％ (average 3 kg FAME passed through the resin bed).

[0249] Part 2: Refining with a column height of 1.2 m for 2% raw material FFA

[0250] a) At 90 °C, with 20% anhydrous methanol and a column height of 1.2 m, the FFA was able to be reduced from 2% to an average 0.45％ , and further reduced to 0.28％-0.31％ after the second pass.

[0251] b) The target FFA can be achieved with a higher column.

[0252] Materials

[0253] Substrate: CPO FAME (FFA ~1.3%) accumulated from ELN-14-PSSH-0008

[0254] Resin: Lewatit GF 101 from Lanxess (CHT00077)

[0255] Chemical: anhydrous methanol (max 0.005% H2O) (Merck code: 1.06012.2500)

[0256] Methodology

[0257] 1. Wash the resin with deionized water at 80 °C to remove impurities, then fill it into a glass column with a diameter of 25 mm and a height of 250 mm (total resin height of 0.4 m).

[0258] 2. Pump anhydrous methanol through the resin column at 90 °C to reduce the water content in the resin to 0.1% water, then conduct the actual refining test.

[0259] 3. Incubate a mixture of 85% CPO and 15% or 20% MeOH in a 60 °C water bath, then pump it through the resin.

[0260] 4. Measure the water % of the collected sample, then evaporate the methanol for FFA analysis.

[0261] 5. Conduct further tests on several parameters, such as methanol dosage (15% and 20%), temperature (80 °C and 90 °C), column height (0.4 m, 0.8 m, and 1.2 m), and different FFAs (1.3% and 2%) in the feedstock FAME.

[0262] Results and Discussion

[0263] Part 1: Refining of 1.3% feedstock FFA with column heights of 0.4 m, 0.8 m, and 1.2 m

[0264] Table 1: Results after resin refining with a column height of 0.4 m

[0265]

[0266]

[0267] Initially, the resin was packed in two glass columns with dimensions of 25 mm in diameter and 250 mm in column height. This could be filled with up to 20 g of resin / column and the bed height was 20 cm / column. In this case, 1.4 liters (1.1 kg) of anhydrous methanol was passed through the resin column, and the water content was reduced from 35.3% to 0.065% in 1 hour (target: <0.1% water)

[0268] CPO FAME with 1.3% FFA and 0.011% moisture was mixed with 15% anhydrous methanol as 85% as the raw material for the refining test. Different settings such as resin temperature, methanol %, and bed volume flow rate were tried to reduce the FFA from 1.3% to 0.25%. However, with the parameters as described in Table 1, the FFA could only be reduced to 0.44%. This indicates that the resin bed height must be at least 1 meter for better conversion height.

[0269] Therefore, the failure of FFA reduction may be due to the insufficient height of the resin setting in the aquarium. Thus, for better conversion, the pretreated resin from 2 large columns (25 mm diameter x 250 mm height) with a resin height of 0.4 meters was transferred to 4 and 6 small columns (10 mm diameter x 250 mm height) with heights of 0.8 meters and 1.2 meters respectively.

[0270] Table 2: Results after refining with 0.8 m height resin (aquarium at 80 ℃ )

[0271]

[0272] Table 3: Results after refining with 1.2 - meter - high resin (aquarium at 80℃)

[0273]

[0274] All tests with 0.8 - meter and 1.2 - meter column heights were conducted at 80℃. Referring to Table 2, with a 0.8 - meter column height, the FFA could only be reduced to 0.36%. While Table 3 shows that using 80% CPO FAME and 20% methanol as the raw material, a 1.2 - meter column height can reduce the FFA from 1.3% to 0.22%. The ratio of FAME + methanol to resin is approximately 4 bed volumes.

[0275] Using a 1.2 - meter column height, 20% methanol in the raw material, and a flow rate of 4.3 bed volumes / hr, the aquarium temperature was raised to a maximum of 90℃. As Figure 6 shown, the FFA was reduced from 1.3% to 0.24% (average 3 kg FAME passed through the resin bed). The FFA range after resin treatment was (0.17% - 0.30)%. The raw material of the dried FAME sample was then added with anhydrous methanol. The moisture content was approximately 0.02%.

[0276] Refer to Figure 7 , the average moisture of FAME after resin treatment was 0.24%. The moisture was tested after resin treatment, and then methanol / water was evaporated for FFA analysis. By stoichiometric calculation, the conversion of 1% palmitic acid FFA to FAME will produce 0.07% moisture. The measured higher moisture content may be due to the absorption of moisture by biodiesel in the surrounding environment.

[0277] Part 2: Refining of 2% feedstock FFA with 1.2 m resin height

[0278] Using the same 1.2 m resin height and 90 °C aquarium temperature, higher feedstock FFA (2%) and methanol (80%:20%) in the feedstock were tested. As Figure 8 shown, the system was able to reduce FFA from 2% to 0.45% (average) after the first round of resin refining. The FAME collected from the first round of refining containing 18%-19% methanol was passed through the resin a second time. Then, the system was able to reduce FFA from 0.45% (average) to 0.28%-0.31%.

[0279] Figure 9 It is shown that when FFA was reduced from 2% to 0.45%, the water content in the FAME was 0.27% (average). Further reduction of FFA from 0.45% to 0.3% led to an increase in water content to 0.36% (average). The increase in water content in the collected samples may be due to water absorption from moisture.

[0280] Conclusion

[0281] Table 4: A brief summary overview of the FFA refining

[0282]

[0283] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. Indeed, various modifications of the invention will become apparent to those skilled in the art from the foregoing specification, in addition to those shown and described herein. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, shall prevail.

Claims

1. A method for reducing the free fatty acid level in biodiesel / fatty acid alkyl esters, the method comprising i) providing a composition comprising a. an oil phase / light phase containing fatty acid alkyl esters, free fatty acids and optionally fatty acid raw materials; and b. an aqueous / heavier phase containing an alcohol and water; ii) reducing the amount of water in the composition to within the range of 0 - 15% by weight of the aqueous / heavier phase; iii) reducing the amount of free fatty acid in the oil phase / lighter phase and optionally the amount of the fatty acid feedstock by reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more liquid lipolytic enzymes to produce fatty acid alkyl esters; iv) optionally, reducing the amount of soap / fatty acid salt in the composition by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4; v) separating the oil phase / lighter phase containing the fatty acid alkyl esters from the aqueous / heavier phase; vi) reacting the free fatty acid and / or the fatty acid feedstock with an alcohol in the presence of one or more non - enzyme catalysts to produce fatty acid alkyl esters, further reducing the amount of free fatty acid in the oil phase / lighter phase and optionally the amount of the fatty acid feedstock, wherein the one or more non - enzyme catalysts are immobilized on a solid resin; wherein the solid resin is packed in a resin bed having a height of at least one meter; vii) contacting the composition with one or more basic reagents under conditions that allow the formation of soap / salt from the free fatty acid remaining in the oil phase / lighter phase; and viii) purifying and / or distilling the fatty acid alkyl esters, including the fatty acid alkyl esters produced in steps iii) and vi).

2. The method according to claim 1, wherein the composition in i) is provided by a reaction in which the free fatty acid and / or the fatty acid feedstock is reacted with an alcohol to produce fatty acid alkyl esters until the reaction has substantially reached equilibrium.

3. The method according to claim 1, wherein the composition in i) is provided by a reaction in which the fatty acid feedstock is reacted with an alcohol in the presence of glycerol in an amount corresponding to 0 - 70% by weight of the aqueous / heavier phase, water in an amount corresponding to 10% - 70.0% by weight of the aqueous / heavier phase, and an alcohol in an amount within the range of 10% - 50% by weight of the aqueous / heavier phase.

4. The method according to claim 1, wherein the composition in i) is provided by a reaction comprising reacting the free fatty acid and / or the fatty acid feedstock with an alcohol until at least 90% w / w or at least 95% w / w of the fatty acid acyl groups or free fatty acids in the fatty acid feedstock have been converted to fatty acid alkyl esters.

5. The method according to any one of the preceding claims 1-4, wherein in step iii) and / or step vi), the fatty acid and / or the fatty acid raw material is reacted with an alcohol until at least 80% w / w, at least 85% w / w, at least 90% w / w or at least 95% w / w of the free fatty acid and / or the fatty acid acyl group in the fatty acid raw material has been converted into fatty acid alkyl esters.

6. The method according to any one of the preceding claims 1-4, wherein the amount of water in the composition provided in step i) is in the range of 10% - 70% by weight of the aqueous phase / heavy phase.

7. The method according to any one of the preceding claims 1-4, wherein the composition in i) is provided by a reaction in which the one or more lipolytic enzymes are lipases.

8. The method according to any one of the preceding claims 1-4, wherein the composition in i) is provided by a reaction in which the total amount of the one or more lipolytic enzymes is in the range of 0.005 - 5 g of enzyme protein (EP) / kg of oil or fatty acid raw material.

9. The method according to any one of the preceding claims 1-4, wherein in step ii), the amount of water is reduced by applying heat.

10. The method according to any one of the preceding claims 1-4, wherein in step ii), an air stream is used to remove the water as moisture.

11. The method according to any one of the preceding claims 1-4, wherein in step ii), a vacuum is used to remove the water as moisture.

12. The method according to any one of the preceding claims 1-4, wherein in step ii), the amount of water is reduced by flash drying.

13. The method according to any one of the preceding claims 1-4, wherein the amount of alcohol in step iii) is equivalent to 5% - 10% by weight of the light phase.

14. The method according to any one of the preceding claims 1-4, wherein the amount of alcohol in step vi) is equivalent to 10% - 25% by weight of the light phase.

15. The method according to any one of the preceding claims 1-4, wherein the duration of step vi) is 0.5 - 7 hours.

16. The method according to any one of the preceding claims 1-4, wherein the one or more lipolytic enzymes in step iii) are lipases.

17. The method according to any one of the preceding claims 1-4, wherein the total amount of the one or more lipolytic enzymes in step iii) is in the range of 0.01 - 0.10 g of enzyme protein (EP) / kg of oil.

18. The method according to any one of the preceding claims 1-4, wherein the one or more non-enzymatic catalysts in step vi) are selected from the group consisting of: An acid catalyst, and a base catalyst.

19. The method according to any one of the preceding claims 1-4, wherein the one or more non-enzyme catalysts in step vi) are immobilized on a solid resin, wherein the solid resin is a porous polymer-based resin.

20. The method according to any one of the preceding claims 1-4, wherein step vi) is carried out at a temperature in the range of 75°C - 95°C in a pressurized system.

21. The method according to any one of the preceding claims 1-4, wherein the alcohol is a C1-C5 alcohol.

22. The method according to any one of the preceding claims 1-4, wherein the fatty acid raw material is derived from one or more of the following: algal oil; low erucic acid rapeseed oil; coconut oil; castor oil; coconut oil; coconut kernel oil; corn oil; corn oil from distillers' grains; cottonseed oil; linseed oil; fish oil; grapeseed oil; hemp oil; jatropha oil; jojoba oil; mustard oil; low erucic acid rapeseed oil; palm oil; palm stearin; palm olein; palm kernel oil; peanut oil; rapeseed oil; rice bran oil; safflower oil; soybean oil; sunflower oil; tall oil; oil from halophytes; and / or animal fats, including fats from pigs, cows, and sheep, lard, chicken fat, fish oil; palm oil free fatty acid distillate; soybean oil free fatty acid distillate; fatty acid materials for soapstock; yellow grease; and brown grease or any combination thereof.

23. The method according to any one of the preceding claims 1-4, wherein after step iii) or vi), the following step is carried out, in which in the presence of the alcohol / the light phase, soap / salt is formed from the free fatty acids remaining in the oil phase / light phase by treatment with one or more basic reagents.

24. The method according to claim 23, wherein the one or more basic reagents are added in an amount of 1.0-2.0 molar equivalents corresponding to the amount of free fatty acids.

25. The method according to claim 23, wherein the treatment with one or more basic reagents comprises contacting the oil phase / light phase and the water phase / hydrophilic phase with a basic reagent or base selected from KOH or NaOH or a mixture thereof.

26. The method according to any one of claims 24 or 25, wherein the treatment with one or more basic reagents is carried out at a temperature in the range of 35°C to 70°C.

27. The method according to any one of claims 24 or 25, wherein the basic reagent is sodium methoxide or potassium methoxide or a mixture of the two.

28. The method according to any one of the preceding claims 1-4, 24 and 25, the method comprising the step of reducing the amount of soap / fatty acid salt in the composition by acidifying the soap / fatty acid salt to produce free fatty acids.

29. The method according to claim 28, wherein the step of reducing the amount of soap / fatty acid salt is carried out before step vi).

30. The method according to any one of the preceding claims 1-4, the method comprising separating the oil phase / light phase containing the fatty acid alkyl ester from the water phase / heavy phase.

31. The method according to claim 30, wherein the oil phase / light phase is separated from the water phase / heavy phase by gravity settling, decantation and / or centrifugation.

32. The method according to any one of the preceding claims 1-4, the method comprising drying glycerol in order to remove water and alcohol from the glycerol.

33. The method according to any one of the preceding claims 32, wherein the glycerol is purified by drying and / or removing the alcohol to produce a composition wherein the content of glycerol is higher than 95% w / w, higher than 97% w / w, higher than 97.5% w / w, higher than 98% w / w, higher than 98.5% w / w, higher than 99% w / w, higher than 99.5% w / w, higher than 99.75% w / w, higher than 99.8% w / w or higher than 99.9% w / w.

34. The method according to any one of the preceding claims 1-4, wherein the glycerol is subjected to thermal-vacuum distillation.

35. The method according to any one of the preceding claims 1-4, the method comprising distilling the fatty acid alkyl ester, wherein the fatty acid alkyl ester is evaporated and then concentrated.

36. The method according to any one of the preceding claims 1-4, the method comprising subjecting the fatty acid alkyl ester to thermal-vacuum distillation at 240 °C - 260 °C.

37. The method according to claim 1, wherein purification is carried out by subjecting the fatty acid alkyl ester to a water wash.

38. The method according to claim 37, wherein purification is carried out by allowing the fatty acid methyl ester to settle, and then subjecting the settled fatty acid alkyl ester to a water wash.

39. The method according to claim 1, wherein the composition in i) is provided by a reaction in which free fatty acids and / or fatty acid raw materials are reacted with an alcohol to produce a fatty acid alkyl ester until the reaction proceeds to a substantially further net reduction in free fatty acids.

40. The method according to claim 1, wherein in step ii) the amount of water is reduced by convection, conduction and / or radiation.

41. The method according to claim 1, wherein the duration of step vi) is 0.5 - 6 hours.

42. The method according to claim 18, wherein the acid catalyst is sulfonic acid, sulfuric acid, phosphoric acid or hydrochloric acid.

43. The method according to claim 18, wherein the base catalyst is a metal alkoxide.

44. The method according to claim 43, wherein the metal alkoxide is sodium alkoxide or potassium alkoxide.

45. The method according to claim 1, the method comprising the step of reducing the amount of soap / fatty acid salt in the composition by chemically stoichiometrically titrating the soap / fatty acid salt with an acid to produce free fatty acids.

46. The method according to claim 1, the method comprising the step of reducing the amount of soap / fatty acid salt in the composition by contacting the soap / fatty acid salt with H3PO4 and / or H2SO4 to produce free fatty acids.

47. The method according to claim 1, wherein the distillation is thermal-vacuum distillation.

48. The method according to claim 1, wherein purification is carried out by allowing the fatty acid methyl ester to settle by gravity, and then subjecting the settled fatty acid alkyl ester to a water wash.

49. The method according to any one of the preceding claims 1-4, wherein the alcohol is ethanol or methanol.

50. The method according to claim 1, wherein the treatment with one or more basic reagents is carried out at a temperature of 60 °C.

Images