WO2010005391A1

WO2010005391A1 - Biodiesel production via enzymatic hydrolysis followed by chemical/enzymatic esterification

Info

Publication number: WO2010005391A1
Application number: PCT/SG2008/000244
Authority: WO
Inventors: Md. Mahabubur Rahman Talukder; Jinchuan Wu
Original assignee: Agency For Science, Technology And Research
Priority date: 2008-07-08
Filing date: 2008-07-08
Publication date: 2010-01-14
Also published as: CN102144035B; CN102144035A; WO2010005391A8

Abstract

A method for biodiesel production via enzymatic hydrolysis of oils or fats followed by chemical/enzymatic esterification of free fatty acids produced in a hydrolysis step. The process comprises enzymatically hydrolyzing a triglyceride to form a fatty acid and glycerol; and esterifying a lower alcohol with the fatty acid to form biodiesel.

Description

BIODIESEL PRODUCTION VIA ENZYMATIC HYDROLYSIS FOLLOWED BY CHEMICAL/ENZYMATIC ESTERIFICATION

BACKGROUND OF THE INVENTION

[0001] Biodiesel (alkyl esters of long chain fatty acids) is an environmentally friendly fuel, synthesized by transesterification of natural triglyceride (e.g. vegetables oils, animal fats) with low molecular weight alcohols, typically methanol. Biodiesel is industrially produced via chemical catalysis using strong bases as a catalyst (see, Connemann J and Krallmann A., Process for the continuous production of lower alkyl esters of higher fatty acid., U.S. Pat. No. 5,354,878; Hanna MA, Transesterification process for production of Biodiesel., U.S. Pat. Publication No. 2003/0032826 Al; Lee JH, One-stage process for feed and Biodiesel and Lubricant oil., U.S. Pat. Publication No. 2004/0022929 Al). However, a strong base process suffers from several drawbacks such as difficulties in recovery of glycerol, the need for removal of the base catalyst from the product and the treatment of alkaline waste water. Furthermore, the feedstock must comply with rigorous specifications (see, Lotero E. et al., Ind. Eng. Chem. Res. Rev, 44:5353-5363 (2005)). For example, it must be essentially anhydrous and FFA content must not exceed 0.5 wt %. The existence of impurities will cause the base process to generate soap, increasing the viscosity. High viscosity causes difficulties in down stream separation, thereby seriously hindering the production of fuel grade biodiesel. To conform to such demanding feedstock specifications, it is necessary to use highly refined vegetable oils whose price can account for 60-75% of the final cost of biodiesel (see, Lotero E. et al, Ind, Eng. Chem. Res. Rev, 44:5353-5363 (2005)). Further, the refining process (250°C and 1-3 mmHg) destroys the antioxidant (vitamin E and Carotene), reducing the oxidative stability of biodiesel.

[0002] The aforementioned drawbacks of a base process have led researchers to seek catalytic and processing alternatives that could ease these difficulties and lower production costs. Methodologies based on acid-catalyzed reactions have the potential to achieve this since acid catalysts are less sensitive to FFAs and can simultaneously catalyze esterification and transesterification. However, the liquid acid-catalyzed transesterification process does not have the same popularity in commercial applications as its counterpart, the base-catalyzed process. The fact that the liquid acid-catalyzed reaction is much slower than the liquid base- catalyzed reaction has been one of the main reasons (see, Srivastava A and Prasad R., Renewable Sustainable Energy Rev., 4:111-133 (2000)). Although reaction rates can be increased by increasing temperature, side reactions such as alcohol etherification may occur at high temperature and pressure. Furthermore, liquid catalysts lead to serious contamination problems that make essential the implementation of good separation and product purification protocols. To minimize the separation and purification problem, solid acid catalysts have been reported, but the process still requires high reaction temperature and pressure. For instance, activated montmorllonite KSF showed a 100% conversion at 22O⁰C and 52 bar (see, Lotero E. et al., Ind. Eng. Chem. Res. Rev, 44:5353-5363 (2005)). Amberlyst 15 has been studied for esterification and transesterification reactions. Although, it is very active for esterification of carboxylic acid, its activity for transesterification of oils or fats is very low and mild reaction conditions is necessary to avoid degradation of the catalyst. At a relatively low temperature (60°C), the conversion of sunflower oil was reported to be only 0.7%, when carrying out the reaction at atmospheric pressure and a 6:1 methanol to oil initial molar ratio (see, Vicente G et al.Jnd. Crops Products, 8:29-35 (1998)).

[0003] Enzymatic transesterification using lipase has become more attractive for biodiesel fuel production, since the glycerol produced as a by-product can easily be recovered and the purification of fatty methyl esters is simple to accomplish. The utilization of enzymes (e.g. immobilized Candida Antarctica lipase) for methanolysis of vegetable oils has been shown to be useful in producing biodiesels (see, U.S. Pat. No. 5,713,965; Sbimada, Y. et al. J. Am. Oil Chem. Soc. 1999, 76, 789; Nelson, L. A. et al. J. Am. Oil Chem. Soc. 1996, 73, 1191). However, the enzyme is either easily poisoned by the lower alcohols (e.g. methanol) resulting in low enzyme activity or inhibited by the by-product's adsorption on the surface of the enzyme, thereby blocking the substrate's access to the active site of the enzyme.

[0004] For the forgoing reasons, there is a need to develop an efficient and economically viable method for the production of biodiesel from low-cost feedstock such as crude palm oil (CPO) containing high water and FFA. The method should provide a system where catalysts (chemical or enzyme) are recyclable and less sensitive to water, FFA and methanol. The present invention satisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides inter alia, efficient methods for biodiesel production from oils or fats via enzyme catalyzed hydrolysis followed by esterification. In certain embodiments, the methods require mild reaction conditions and utilize low cost feedstock such as for example, crude palm oil ("CPO"), containing a high water content and free fatty acids. The processes herein minimizes the problems associated with the commercially used base- catalyzed processes.

[0006] As such, in one embodiment, the present invention provides a method for producing a biodiesel, the method comprising: enzymatically hydrolyzing a triglyceride to form a fatty acid and glycerol; and esterifying a lower alcohol with the fatty acid to form the biodiesel.

[0007] In certain aspects, the esterifying step is chemically or enzymatically catalyzed. For example, in one embodiment, the chemical esterification of the fatty acid comprises: admixing the fatty acid with the alcohol and an acid catalyst; and incubating the admixture for a time and temperature sufficient to form biodiesel. [0008] In other aspects, the enzymatic esterification of the fatty acid comprises: admixing the fatty acid with the alcohol and a lipase; and incubating the admixture for a time and temperature sufficient to form the biodiesel. [0009] Compared with prior art technologies, the invented method provides higher yields, easier catalyst recovery and simpler product separation. These and other objects, aspects, and embodiments will become more apparent with the detailed description of the invention and figures that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 illustrates an embodiment of the present invention.

[0011] Figure 2 illustrates an embodiment of the present invention.

[0012] Figure 3 A-B illustrates a UV spectra of the samples before (A) and after hydrolysis (B) of degummed CPO.

[0013] Figure 4 A-B illustrates a UV spectra of the samples before (A) and after esterification (B) of palm fatty acids.

[0014] Figure 5 illustrates hydrolysis of CPO at different lipase concentrations in buffer. Reaction conditions: CPO 2 g, lipase solution 2 ml, shaking speed 250 rpm, temperature 4O⁰C.

[0015] Figure 6 illustrates a hydrolysis reaction of degummed CPO at different temperatures. Reaction conditions: CPO 2 g, lipase solution (1 mg/ml) 2 ml, shaking speed 250 rpm. [0016] Figure 7 illustrates a hydrolysis reaction of degummed CPO at different buffer to oil ratio (v/v). Reaction conditions: CPO 2 g, lipase 2 mg, shaking speed 250 rpm, temperature 40°C.

[0017] Figure 8 illustrates Amberlyst 15-catalyzed methyl and ethyl esterification of palm FFA in isooctane. Reaction conditions: 10 ml feedstock (FA in isooctane, 0.32 M), 1 g Amberlyst 15, temperature 60°C, 400% of stoichiometric amount of methanol or ethanol, shaking at 250 rpm.

[0018] Figure 9 illustrates Novozym 435-catalyzed methyl and ethyl esterification of palm FFA. Reaction conditions: 10 ml feedstock (FFA in isooctane, 0.32 M)), 0.04 g Novozym 435 [15?], temperature 4O⁰C, 120% of stoichiometric amount of methanol or ethanol, shaking at 250 rpm.

[0019] Figure 10 illustrates the comparison of Novozym 435- and Amberlyst 15-catalyzed methyl esterification of palm FFA.

[0020] Figure 11 illustrates the results of repeated use of Amberlyst 15 (A) and Novozym 435 (B) in one embodiment of the present invention.

[0021] Figure 12 illustrates the results of enzymatic hydrolysis of degummed CPO and Amberlyst 15- or Novozym 435-catalyzed esterification which provide good yields (98-99%) in embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0022] As used herein, the term "lower alkyl" includes a saturated straight, branched, or cyclic hydrocarbon Of C₁ to C₆, and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0023] As used herein, the term "alcohol" includes a hydrocarbon compound containing one or more hydroxy groups, and includes alcohols such as methanol, ethanol, propanol, butanol, isopropanol, isobutanol, t-butanol, pentanol, cyclopentanol, isopentanol, neopentanol, hexanol, isohexanol, cyclohexanol, 3-methylpentanol, 2,2-dimethylbutanol, 2,3-dimethylbutanol and isomers thereof. [0024] As used herein, the term "oil" or "oils" includes plant oils or fats, animal oils or fats, marine oils or fats, oils or fats from microbial origin, waste oils or greases, rendered product, or any mixture thereof.

[0025] As used herein, the term "rendered product" includes a fat that has been treated, usually with heat, to remove water, solids, and other impurities.

[0026] As used herein, the term "biodiesel" includes fatty acid alkyl esters used as a transportation and power generation fuel or additives.

[0027] As used herein, the term "feedstock" includes crude palm oil, or any oils or fats from any source of plant oils or fats, animal oils or fats, marine oils or fats, oils of microbial origin, and artificial or synthetic glycerides, as well as wastes, effluents and residues from the processing of such materials.

[0028] As used herein, the term "plant oil" includes fats, oils, or lipids derived from plant sources, such as agricultural crops and forest products, as well as wastes, effluents and residues from the processing of such materials, such as soap stock.

[0029] As used herein, the term "animal oil" includes fats, oils or lipids derived from animal sources, as well as wastes, effluents and residues from the processing of such materials.

[0030] As used herein the term "marine oils" includes fats, oils or lipids derived from marine sources in water streams, as well as wastes, effluents and residues from the processing of such materials.

[0031] Glycerides useful in the present invention include molecules of the chemical formula (R)CH(R)CH(R')(R")CH(R")₅ wherein R, R' and R" are selected from -H, -OH or a fatty acid group given by the formula -0(C=O)R'", wherein R'" is a saturated, unsaturated or polyunsaturated, straight or branched carbon chain with or without substituents, wherein the glyceride has at least one -0(C=O)R'". R, R', R" on a given glyceride can be the same or different. R, R' and R" can be obtained from any of the free fatty acids described herein. Glycerides for the present invention include triglycerides which have three fatty acid groups, diglycerides, which have two fatty acid groups; and mono glycerides, which has one fatty acid group. Glycerides useful as starting materials of the invention include natural, processed, refined and artificial or synthetic fats and oils.

[0032] The terms "fatty acid groups" or "acid groups" includes chemical groups given by the formula: -0(C=O)R"¹. Such "fatty acid groups" or "acid groups" are connected to the remainder of the glyceride via a covalent bond to the oxygen atom that is singly bound to the carbonyl carbon. In contrast, the terms "fatty acid" or "free fatty acid" both refer to HO(C=O)R"' and are not covalently bound to a glyceride. In "fatty acid groups," "acid groups," "free fatty acids," and "fatty acids," R'" is a saturated, unsaturated or polyunsaturated, straight or branched carbon chain with or without substituents, as discussed herein. One of skill in the art will recognize that R'" of the "free fatty acids" or "fatty acids" (i.e., HO(C=O)R'") described herein are useful as R'" in the "fatty acid groups" or "acid groups" attached to the glycerides or to other esters used as substrates in the present invention. That is, a substrate of the present invention may comprise fats, oils or other esters having fatty acid groups formed from the free fatty acids or fatty acids discussed herein.

II. Embodiments

[0033] The present invention provides methods for producing biofuels (e.g. , biodiesel) using crude or refined vegetable oils or animal fats. In one aspect, the present invention provides for the production of fatty acid esters from a feedstock of animal fats, vegetable oils or mixtures thereof, which comprise a triglyceride. The process comprises introducing a triglyceride feedstock to produce free fatty acids by enzymatic hydrolysis. An alcohol is thereafter introduced and esterified with the free fatty acid to form a product comprising fatty acid esters.

[0034] In accordance with the present invention, a process is provided for the economical production of fatty acid esters from the hydrolysis of triglyceride feedstock and subsequent esterification of an alcohol. The processes herein may be conducted continuously and in a manner that provides higher yields than prior art processes. The fatty acid esters produced are beneficial in producing lubricating agents and fuels such as biodiesel.

[0035] Figure 1 illustrates one embodiment of a method 100 for producing biodiesel. In one aspect, a feedstock having a triglyceride such as vegetable oil 110 is enzymatically hydrolyzed 112 to produce a free fatty acid 115, glycerol and water 120. The triglyceride can be from vegetable oil, an animal fat, tallow, a grease, or a recycled rendered oil or fat. Thereafter, the fatty acid 115 can be used to esterify an alcohol using a catalyst (e.g., acid or enzyme catalyst). Typically, the fatty acid is a C₁₂ to C₂₂ fatty acid. This esterification reaction 122 produces biodiesel 130 and water 132. The glycerol and water products from the hydrolysis of the crude vegetable oil can optionally be refined wherein the water is distilled off to produce refined glycerol 161, or glycerol can to be used in for example, a fermentation reaction of other chemicals 141. [0036] Turning now to Figure 2, a more detailed process flow 200 of an inventive method is shown wherein crude palm oil (CPO) or vegetable oil or fats comprising triglycerides 202 is hydrolyzed using for example, an enzyme catalyst 210 (e.g., lipase) in a reactor 225 with water 212 (e.g., recycled) and an organic solvent 215 (e.g., recycled). From reactor 225, the product containing free fatty acid, glycerol and water is transferred to a separation vessel 230 wherein the free fatty acid 233 can be transferred to a second reactor 248 or 250 (e.g., one in use and another in regeneration) containing catalyst, for example lipase or acid. The mixture of glycerol, water and lipase 235 can be transferred to a hollow fiber membrane 236 and the lipase 211 is recycled to a reactor 225. Glycerol and water 237 from membrane 236 can be used to ferment other chemicals 240. m reactor 248 or 250, free fatty acid 233 is used to esterify an alcohol, such as a lower alcohol (e.g., C₁- C₆ alcohol, e.g., methanol or ethanol). The esterification of the lower alcohol 242 can be catalyzed using lipase (e.g., Novozym 435) or acid catalyst (e.g., Amberlyst 15). The catalyst in reaction vessel 248 or 250 can be regenerated using polar solvents such as t-butanol. Once the reaction is finished, the ester from the reaction mixture can be separated by distillation 271, where alcohol 238, solvent 258 and water 262 will be distilled off and recycled. The final biodiesel product 260 can be used or admixed with petrol diesel. Suitable reactors include, but are not limited to, a fixed bed reactor, a fluidized reactor and a stirred-tank reactor.

[0037] In certain instances, the feedstock and enzyme (e.g., lipase) are charged into a reactor with water and optionally an organic solvent. Advantageously, the feedstock comprising triglycerides may contain water. The feedstock (e.g., degummed CPO) is charged into the reactor and optionally preheated to about 20-60⁰C, preferably about 25°C to about 50°C (e.g., 4O⁰C) and a hydrolyzing enzyme is added (e.g., lipase). Lipases can be derived from plants, bacteria, a fungus and higher eukaryotes. The lipase can be derived extracellularly or intracellularly or is in the form of a whole-cell. Suitable lipases for either the hydrolysis reaction or the esterification reaction include, but are not limited to, lipases from Candida rugusa, Candida cylindracea, Rhizopus oryzae, Chromobacterium viscosum, Pseuodomonus cepacia, Hog pancrease, Pseudomonus fluorescent, Candida antarctica B, and Novozym 435 (Immobilized Candida antarctica lipase B). The lipase can be immobilized onto acrylic resin or any solid support. Those of skill in the art will know of other lipases suitable for use in the present invention. Optionally, the lipase is regenerated by washing with polar solvent such as tert-butanol. Other suitable polar solvents include, but are not limited to, C₁- C₆ alkanols and other sterically hindered alkanols. [0038] Immobilization of the enzyme can be performed by any known method such as a carrier binding including an inorganic carrier covalent bond method and an organic carrier covalent bond method, cross-linking, entrapment and adsorption {see, U.S. Pat. Nos. 4,798,793; 5,166,064; 5,219,733; 5,292,649; and 5,773,266). The carrier binding method is preferable in view of handling. Carrier binding includes chemical adsorption or physical adsorption by which the enzyme(s) is adsorbed to any solid support (e.g. an ion-exchange resin). In the present invention, physical adsorption using a porous carrier is preferable.

[0039] Several useful carriers have been described for enzyme immobilzation {see, U.S. Pat. Nos. 4,940,845 and 5,219,733). Useful carriers are preferably microporous and have a hydrophobic porous surface. Usually, the pores have an average radius of about 10 A to about 1,000 A, and a porosity from about 20% to about 80% by volume, more preferably, from about 40% to about 60% by volume. The pores give the carrier an increased enzyme bonding area per particle of the carrier. Examples of preferred inorganic carriers include, but are not limited to, porous glass, porous ceramics, celite, porous metallic particles such as titanium oxide, stainless steel or alumina, porous silica gel, molecular sieve, active carbon, clay, kaolinite, perlite, glass fibers, diatomaceous earth, bentonite, hydroxyapatite, calcium phosphate gel, alkylamine derivatives of inorganic carriers, and combinations thereof. Examples of preferred organic carriers include, but are not limited to, microporous Teflon, aliphatic olefmic polymer {e.g., polyethylene, polypropylene, a homo- or copolymer of styrene or a blend thereof or a pretreated inorganic support) nylon, polyamides, polycarbonates, nitrocellulose, acetylcellulose, and combinations thereof. Other suitable organic carriers include, but are not limited to, hydrophillic polysaccharides such as agarose gel with an alkyl, phenyl, trityl or other similar hydrophobic group to provide a hydrophobic porous surface. Microporous adsorbing resins include, but are not limited to, those made of styrene or alkylamine polymer, chelate resin, ion exchange resin such as weakly basic anion exchange resin having a tertiary amine as the exchange group, composed basically of polystyrene chains cross linked with divinylbenzene and hydrophilic cellulose resin such as one prepared by masking the hydrophilic group of a cellulosic carrier.

[0040] The immobilization of the enzyme can be conducted by immobilizing the enzyme on a suitable carrier. A number of inorganic and organic carriers can be used to immobilize an enzyme. Examples of carrier include, but are not limited to, celite, ion exchange resins, ceramics and the like. In some embodiments, an ion exchange resin is used. The material, properties and ion-exchanging groups of the ion exchange resin can be chosen in view of the adsorbability and exhibiting rate of activity of the enzyme to be adsorbed. In other embodiments, an anion exchange resin is used. Examples of anion exchange resin include phenol-formaldehyde-based anion exchange resins, polystyrene-based anion exchange resins, acrylamide-based anion exchange resins, and divinylbenzene-based anion exchange resins. A person of skill in the art recognize that other suitable carriers can be used to immobilized the enzyme used in the present invention.

[0041] The immobilization temperature is determined depending on the properties of the enzyme. It is desired to conduct the immobilization at a temperature where the enzymatic activity is not lost. The immobilization can be conducted at about 0 ⁰C to about 60 ⁰C, preferably at about 5 ⁰C to about 40 ⁰C.

[0042] Concerning the ratio of the enzyme to the immobilization carrier, it is preferred to use 0.05 to 10 parts by weight, especially 0.1 to 5 parts by weight of the enzyme per part by weight of the immobilization carrier.

[0043] Enzymatic activity generally tends to be affected by factors such as temperature, light and moisture content. Light can be kept out by using the various light blocking or filtering means known in the art. Moisture content, which includes ambient atmospheric moisture, can be controlled by operating the process as a closed system. The closed system can be under a positive inert atmospheric pressure to expel moisture. Alternatively, a bed of nitrogen gas can be placed on top of the substrate, purification bed or column, or packed lipase column. Other inert gasses such as helium or argon can also be used. These techniques have the added benefit of keeping atmospheric oxidative species such as oxygen away from the substrate, product or enzyme.

[0044] In certain instances, the lipase is immobilized, such as on a porous support or a powder. The enzymatic hydrolysis reaction is preferably incubated at a temperature from about 2O⁰C to about 5O⁰C. In one aspect, the enzymatic hydrolysis reaction is incubated for a time of about 0.5 hours to about 24 hours to effectuate conversion of the triglycerides to free fatty acids. Preferably, the enzymatic hydrolysis reaction is carried out in the presence of water. In certain aspects, the enzymatic hydrolysis reaction is carried out in a buffered solution. In one aspect, the water to triglyceride ratio is about 1:10 (v/v) to about 20:1 (v/v). The lipase concentration can be about 0.125 to about 2 mg/ml. In certain aspects, the enzymatic hydrolysis reaction further comprises an organic solvent, such as C₅- Cj₂ alkane or a sterically hindered alkanol. [0045] In a preferred embodiment, the enzyme is added (e.g. , 1 mg/ml) to start the reaction and the reaction mixture is shaken and mixed for 2 hours to about 10 hours, preferably about 2 to 6 hours (e.g., 3 hours). In certain instances, the hydrolysis reaction rate increases proportionally with an increase in lipase concentration.

[0046] Although temperatures and pressures for the hydrolysis reaction are not critical, these parameters can be adjusted to increase the rate of reaction. In certain instances, temperature can influence the rate of hydrolysis. In one instance, hydrolysis of the triglyceride is carried out at about 20°C to about 60⁰C, preferably, at about 30°C to about 50°C, and more preferably at about 35⁰C to about 45°C. In one preferred embodiment, the reaction is carried out at 45⁰C.

[0047] In addition, in certain aspects, buffer concentration can influence the rate of hydrolysis. In certain preferred aspects, the buffer concentration to triglyceride is about 0.5 (v/v) to about 2.0 v/v (0.5:2), preferably the ratio of buffer concentration to triglyceride is about 1:1 (v/v). Suitable buffers include, but are not limited to, phosphate, citrate, succinate and the like.

[0048] After finishing the reaction, an organic solvent is added (e.g., isooctane or hexane) to separate produced fatty acids (FA) from glycerol and water. The admixture can thereafter be optionally centrifuged, and the upper phase can be directly used as a feedstock for the esterification reaction. In certain aspects, the fatty acid feedstock and a catalyst (e.g., acid, or enzyme) are then combined with an alcohol to begin the esterification reaction.

[0049] Although temperatures and pressures for the esterification reaction are not critical, these parameters can be adjusted to increase the rate of reaction. In the case for chemical catalysis, the FFA feedstock is optionally preheated to about 40⁰C to about 8O⁰C, preferably about 50⁰C to about 7O⁰C (e.g., 6O⁰C). In the case of enzyme catalysis, the FFA feedstock is optionally preheated to about 2O⁰C to about 60⁰C, preferably about 25°C to about 5O⁰C (e.g., 4O⁰C). The reaction is initiated by adding a lower alcohol. After the reaction is complete, the catalyst is removed.

[0050] Suitable alcohols for use in the esterification reaction include, but are not limited to, primary and secondary monohydric aliphatic alcohols having one to eight carbon atoms. Preferred alcohols for use in the esterification process are methanol, ethanol, propanol, butanol and amyl alcohol, with methanol and ethanol being most preferred. A particularly preferred alcohol, for example, is methanol, ethanol and combinations thereof. [0051] Suitable acid catalysts include inorganic acid catalysts (e.g., a mineral acid such as HCl, H₂SO₄, and the like) or an organic acid catalyst (e.g., benzoic acid). The acid catalyst can be a liquid or a solid. The acid can take the form of molecular sieves. In one instance, the acid catalyst is an acidic styrene-divinylbenzene sulfonated ion exchange resin. Typically, the esterifying reaction mixture comprises an organic solvent, such as a C₅-C₁₂ alkane or a sterically hindered alkanol. In one embodiment, the incubation temperature is from about 2O⁰C to about 9O⁰C. In certain instances, the incubation time is about 0.2 hours to about 24 hours. In addition, the ratio of solid acid catalyst to fatty acid is typically about 1:10 to about 10:1 (w/w). Optionally, the acid catalyst is regenerated, by washing it with for example, a polar solvent such as methanol. Other suitable polar solvents include a C₁-C₆ alkanol or a sterically hindered alkanol.

[0052] In certain instances, the esterification processes has a molar ratio of alcohol to free fatty acid of about 6:1 (mol: mol) to about 1:1, more preferably about 4:1 to about 3:1, or about 2:1, or about 1.2:1 or about 1:1. For acid catalysis, the alcohol to triglyceride molar ratio can be about 6: 1 to about 3 : 1 (e.g. , 4:1). In one example, when Amberlyst 15 is used, a 4: 1 alcohol to FFA ratio is used.

[0053] For the enzyme catalyzed esterification, suitable enzymes include Upases, as well as other enzymes that catalyze ester formation. Typically, the reaction comprises an organic solvent, such as a C₅-C₁₂ alkane or a sterically hindered alkanol. In certain instances, the reaction is carried out at a temperature from about 20⁰C to about 70⁰C, for about 0.2 hours to about 24 hours.

[0054] For enzyme catalysis, the alcohol to fatty acid ratio is about 1 : 1 to about 2:1 (mol/mol). In other instances, the ratio is preferably about 1.2:1, or 1.3:1, or 1.4:1 or about 1.5:1. In one example, when Novozym 435 is used, an equivalent alcohol amount of about 120% stoichiometric amount is used. In one example, the lipase to fatty acid ratio is about 0.005:1 to about 1:1 (w/w).

[0055] After esterification is complete, the fatty acid esters are separated from any reactants. Separation may be achieved by any means generally known in the art, preferably by gravity separation and decantation or centrifugation. The separated fatty acid ester product can be washed with recycled water and then dried. It has been found that the process of the present invention achieves an overall conversion of triglyceride feedstocks to fatty acid esters of about 95% to about 97%. [0056] Biodiesels prepared according to the methods of the present invention can be analyzed by various instrumentation well known to those of skill in the art. For example, analytical analysis can be performed using HPLC with a UV detector. In one aspect, the detector is set at 210 nm and a prevail Cl 8 5u column (250 4.6 mm, Alltech Associates, Inc., USA) can be employed. The mobile phase comprises for example, three different components: hexane, isopropanol and methanol. Reservoir A contains methanol and reservoir B contains a mixture of isopropanol and hexane (5:4, v/v). In one aspect, a gradient from 100% A to 50% A + 50% B linearly over 30 min is used. The flow rate of the mobile phase can be 1 ml/min and the sample injection volume is 10 1. This non-aqueous RP-HPLC method is a modification of the prior art method {see, Holcapek, M. et al. J ChromatogrA 1999, 858, 13).

III. Examples

1 Materials and Methods

1.1 Materials

[0057] Degummed crude palm oil was purchased from Wawasan Tehran Sdn Bhd, Johor, Malaysia. Lipase from Candia rugosa was purchased from Meito Sangyo Co., Japan. Lipases from Pseuodomonus cepacia, Chromobacterium viscosum, Candida cylindracea and Novozym 435 {Candida antarctica lipase B immobilized onto acrylic resin) are bought from Sigma- Aldrich (USA). Lipases from Hog pancrease, Pseuodomonus cepacia and Candida antarctica B (powder) are from Fluka (USA). Amberlyst 15 (Strong acidic styrene-divinylbenzene sulfonated ion exchange resin, 4.7 mequiv/g) was purchased from Sigma (USA). Isooctane was purchased from Fisher Chemical, New Jersey, USA.

1.2 Hydrolysis of degummed crude palm oil

[0058] The 80ml screw-capped glass battle containing degummed CPO (2g) was preheated at reaction temperature of 40°C for about 10 min. 2 ml lipase solution (1 mg/ml) was added to the preheated CPO and the mixture was shaken at 250 rpm for 3 h. After finishing reaction, 20 ml isooctane or hexane was mixed with reaction mixture to separate produced fatty acids (FA) from glycerol and water. The mixture was then centrifuged at 5000 rpm for 5 min. The upper phase was collected and directly used as a feedstock for esterification.

1.3 Esterification of palm free fatty acids

[0059] The reaction mixture consisting of 10 ml feedstock (FA acid in isooctane, 0.32 M) and Ig Amberlyst 15 or 0.04g Novozym 435 was preheated at reaction temperature of 6O⁰C (for Amberlyst 15) and 4O⁰C (for Novozym 435) with shaking speed of 250 rpm. The reaction was initiated by adding methanol or ethanol equivalent to 400 and 120% stoichiometric amount for Amberlyst 15 and Novozym 435, respectively. After finishing reaction, Novozym 435 and Amberlyst 15 were filtered out and washed with tert-butanol (HPLC grade) and methanol, respectively. They were freeze dried for over night and further used.

1.4 Analysis

[0060] The fatty acids content in reaction mixture was measured by titration against 0.2 M NaOH. The conversion of triglyceride was confirmed by HPLC with a UV detector at 210 nm (see, Fig. 3 A-B). A prevail C 18 5u column (250 x 4.6 mm, Alltech Associates, Inc., USA) was employed. The mobile phase consisted of three different components: hexane, isopropanol and methanol. Reservoir A contained methanol and reservoir B contained a mixture of isopropanol and hexane (5:4, v/v). The gradient went from 100% A to 50% A + 50% B linearly over 30 min. The flow rate of the mobile phase was 1 ml/min and the sample injection volume was 10 μl. This non-aqueous RP-HPLC was modified from the method reported by Holcapek et al (Holcapek M et al., J ChromatogrA, 858:13-31 (1999)). The consumption of fatty acids in esterification reaction was monitored by titration against 0.2m NaOH. The methyl or ethyl esters (biodiesel) yield was confirmed by HPLC system as mentioned above (see, Fig. 4A-B).

2 Results

2.1 Effect of different Upases on hydrolysis ofdegummed crude palm oil

[0061] Table 1 shows the effect of different lipases on conversion of CPO to FFAs at fixed protein concentration. It is evident that lipases from Candida rugusa and Chromobacterium viscosum are most effective. Although, Novozym 435 (Candida antarctica lipase B immobilized onto acrylic resin) is very active for esterification and transesterification, but it's hydrolytic activity is very low. Since Candida rugosa lipase is 50 times cheaper than Chromobacterium viscosum lipase, it is selected for further studies. Table 1 Effect of different lipases on hydrolysis of crude palm oil*

Lipases FFA content (mol %)

Candida rugusa 52.0

Candida cylindracea 44.2

Rhizopus oryzae 26.9

Chromobacterium viscosum 52.2

Pseuodomonus cepacia 22.0

Hog pancrease 22.8

Pseudomonus fluorescent 24.9

Candida antarctica B 6.65

Novozym 435 5.1 (Immobilized Candida antarctica lipase B)

* experimental conditions: protein concentration in buffer for all lipases except Novozym 435 (160 mg) is kept constant at 0.5 mg/ml, buffer 2 ml; CPO 2 g, Temp 40°C, shaking speed 250 rpm, time 1 h.

2.2 Hydrolysis ofdegummed CPO at different C. rugosa lipase concentration

[0062] Fig. 5 shows that reaction rate increases with the increase in lipase concentration in buffer. The reaction reaches equilibrium at FFA yield of 99.0 % for any lipase concentrations except 0.125 mg/ml. The reaction rate at low lipase concentration is slow and more than 20 h is required for completion of reaction at 0.125 mg/ml.

2.3 Hydrolysis ofdegummed CPO at different temperature

[0063] Hydrolysis of CPO at different reaction temperatures are shown in Fig. 6. The reaction rates at 40 and 55°C are almost the same, while it is lower at 30⁰C. Since lipase is more stable at lower temperature and CPO melting point is around 35°C, reaction temperature of 40°C is chosen for the subsequent experiment.

2.4 Hydrolysis of CPO at different buffer to CPO ratio (v/v)

[0064] It can be seen that optimal buffer to CPO ratio is 1 : 1 (v/v), below or above which both reaction rate and FFA yield are low (Fig. 7). 2.5 Amberlyst 15-catalyzed methyl and ethyl esterification of palm FFA

[0065] Fig. 8 shows the comparison of Amberlyst 15-catalyzed methyl and ethyl esterification of FFA. Although in both cases, biodiesel yield reaches 99%, the methyl esterification progresses faster than ethyl esterification. The low cost of methanol makes it a good choice for the esterification reaction. Ethanol, however, could be the ideal candidate for the synthesis of a fully biogenerated fuel since it is derived from agricultural product and less toxic than methanol.

2.6 Novozym 435-catalyzed methyl and ethyl esterification of palm FFA

[0066] It is evident that although Novozym 435-catalyzed methyl esterification progresses faster than ethyl esterification, both reactions reach equilibrium at BD yield 99% after 2h (Fig. 9). Since methanol at higher concentration could be a poison to Novozym 435, ethanol is right selection for Novozym 435-catalyzed esterification of palm FFA.2

2.7 Repeated use of Amberlyst 15 and Novozym 433

[0067] Amberlyst 15 and Novozym 435 catalyzed methyl and ethyl esterification, respectively, are reused after washing with solvents and freeze-drying. The solvents used for washing Amberlyst 15 and Novozym 435 are methanol and tert-butanol, respectively. The BD yields after 1.5 h (for Amberlyst 15) and 3 h (for Novozym 435) reaction in each cycle are shown in Fig. 10. It can be seen that Amberlyst 15 and Novozym 435 can be reused more than 100 and 50 cycles, respectively (see, Fig. 11).

Comparison of invented method with publish technologies

[0068] It is evident from Table 2 that the method, C. rugosa lipase-catalyzed hydrolysis followed by Amberlyst 15- or Novozym 435-catalyzed esterification provides higher yield (98- 99%) with reasonable processing time (4-5 h). As shown in the table, in KOH- or NaOH- catalyzed transesterification the yield is relatively lower (see, Fig. 12).

Table 2 Comparison of invented method with publish technologies

Transesterification ofdegummed crude palm oil (CPO)

[0069] Transesterification of CPO with methanol was carried out in 8OmL screw-capped glass battle with shaking at 250 rpm and 40°C (for Novozym 435) or 60⁰C (for Amberlyst 15). A standard reaction mixture consisted of CPO, methanol and Novozym 435 (in presence and absence of 0.75 ml LiCl saturated solution or 10 ml tert-butanol) or Amberlyst 15. The methanol used was equivalent to 400 and 120% of stoichiometric amount for Amberlyst 15 and Novozym 435, respectively. Amberlyst 15 and Novozym 435 used are 0.2 and 0.04g per gram of CPO. For Novozym 435-catalyzed methanolysis in solvent free system, 40% of stoichiometric amount of methanol was added three times at 0, 5 and 1O h reaction.

[0070] AU publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for producing a biodiesel, said method comprising: enzymatically hydrolyzing a triglyceride to form a fatty acid and glycerol; and esterifying a lower alcohol with said fatty acid to form said biodiesel.

2. The method of claim 1, wherein said triglyceride is admixed with a lipase and incubated for sufficient time and temperature to form said fatty acid.

3. The method of any claim 1-2, wherein said triglyceride is a member selected from the group consisting of a vegetable oil, an animal fat, tallow, a grease, and a recycled rendered oil or fat.

4. The method of any claim 1-3, wherein said fatty acid is a C₁₂ to C₂₂ fatty acid.

5. The method of any claim 2-4, wherein said lipase is derived from a member selected from the group consisting of a plant, a bacteria, a fungi and a higher eukaryotes.

6. The method of claim 5, wherein said lipase is derived extracellularly, intracellularly or in the form of a whole-cell.

7. The method of any claim 2-6, wherein said lipase is immobilized.

8. The method of claim 7, wherein said immobilization is on a porous support or a powder.

9. The method of claim 8, wherein said porous support is an acrylic resin.

10. The method of claim 9, wherein said lipase is Candida antarctica lipase B.

11. The method of any claim 2-9, wherein said lipase is from Candida rugosa.

12. The method of any claim 1-11, wherein said enzymatic hydrolysis reaction is incubated at a temperature from about 20 ⁰C to about 5O⁰C.

13. The method of any claim 1-12, wherein said enzymatic hydrolysis reaction is incubated for a time of about 0.5 hours to about 24 hours.

14. The method of any claim 1-13, wherein said enzymatic hydrolysis reaction is carried out in the presence of water.

15. The method of any claim 1-14, wherein said enzymatic hydrolysis reaction is carried out in a buffered solution.

16. The method of claim 14, wherein the water to triglyceride ratio is about 1:10 to about 20:1 (v/v).

17. The method of any claim 2-16, wherein said lipase concentration is about 0.125 to about 2 mg/ml.

18. The method of any claim 2-17, wherein said lipase can be recycled using a hollow fiber membrane or other filters.

19. The method of claim 14, wherein said enzymatic hydrolysis reaction further comprises an organic solvent.

20. The method of claim 19, wherein said organic solvent is a member selected from the group consisting of a C₅- C₁₂ alkane and a sterically hindered alkanol.

21. The method of any claim 1-20, wherein said esterifying step is chemically or enzymatically catalyzed.

22. The method of claim 21, wherein said chemical esterification of said fatty acid comprises: admixing said fatty acid with said alcohol and an acid catalyst; and incubating the admixture for a time and temperature sufficient to form biodiesel.

23. The method of claim 22, wherein said alcohol is a C₁- C₅ alcohol.

24. The method of claim 22, wherein said acid catalyst is selected from an inorganic acid catalyst or an organic acid catalyst.

25. The method of claim 24, wherein said acid catalyst.is a liquid or a solid.

26. The method of any claim 22-25, wherein said acid catalyst is an acidic styrene-divinylbenzene sulfonated ion exchange resin.

27. The method of claim 22-26, wherein said acid catalyst is regenerated by washing with a polar solvent.

28. The method of claim 27, wherein said polar solvent is a member selected from the group consisting of a C₁-C₆ alkanol and a sterically hindered alkanol.

29. The method of any claim 21-28, wherein said esterifying step comprises an organic solvent.

30. The method of claim 29, wherein said organic solvent is a member selected from the group consisting a C₅- C₁₂ alkane and a sterically hindered alkanol.

31. The method of any claim 21-30, wherein the esterifying step comprises molecular sieves.

32. The method of claim 31, wherein said incubation temperature is from about 2O⁰C to about 9O⁰C.

33. The method of claim 32, wherein said incubation time is about 0.2 hours to about 24 hours.

34. The method of claim 33, wherein said alcohol to fatty acid ratio is about 1:1 to about 6:1 (mol/mol).

35. The method of claim 21-34, wherein solid acid catalyst to fatty acid ratio is about 1:10 to about 10:1 (w/w).

36. The method of claim 21, wherein said enzymatic esteriflcation of said fatty acid comprises: admixing said fatty acid with said alcohol and a lipase; and incubating the admixture for a time and temperature sufficient to form said biodiesel.

37. The method of claim 36, wherein said lipase is derived from a member selected from the group consisting of a plant, a bacteria, a fungus and a higher eukaryotes.

38. The method of claim 36-37, wherein said lipase is derived extracellularly, intracellularly or in the form of a whole-cell.

39. The method of claim 36-38, wherein said lipase is immobilized.

40. The method of claim 39, wherein said immobilization is on a porous support or a powder.

41. The method of claim 40, wherein said porous support is an acrylic resin.

42. The method of claim 36-41, wherein said lipase is Candida antarctica lipase B.

43. The method of any claim 36-42, wherein said lipase is regenerated by washing with a polar solvent.

44. The method of claim 43, wherein said polar solvent is a member selected from the group consisting of a C₁- C₆ alkanol and a sterically hindered alkanol.

45. The method of claim 36, wherein said esterification reaction comprises an organic solvent.

46. The method of claim 45, wherein said organic solvent is a member selected form the group consisting of a C₅- C₁₂ alkane and a sterically hindered alkanol.

47. The method of any claim 36-46, wherein said esterification reaction comprises molecular sieves.

48. The method of claim 47, wherein said incubation temperature is from about 2O⁰C to about 7O⁰C.

49. The method of any claim 36-48, wherein said incubation time is from about 0.2 hours to about 24 hours.

50. The method of any claim 36-49, wherein said alcohol to said fatty acid ratio is about 1:1 to about 2:1 (mol/mol).

51. The method of any claim 36-50, wherein said lipase to said fatty acid ratio is about 0.005:1 to about 1:1 (w/w).

52. The method of any claim 1-51, wherein said hydrolysis or esterification is conducted in a reactor.

53. The method of claim 52, wherein said reactor is a member selected from the group consisting of a fixed bed reactor, a fluidized reactor and a stirred-tank reactor.

54. The method of any claim 1-53, wherein said biodiesel is admixed with petrodiesel.