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CN105462692A - Biodiesel preparation method - Google Patents

Biodiesel preparation method Download PDF

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Publication number
CN105462692A
CN105462692A CN201410412072.7A CN201410412072A CN105462692A CN 105462692 A CN105462692 A CN 105462692A CN 201410412072 A CN201410412072 A CN 201410412072A CN 105462692 A CN105462692 A CN 105462692A
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China
Prior art keywords
crude product
content
deacidite
ester crude
lipase
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李会
郑妍
杨武林
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Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
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Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
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Priority to CN201410412072.7A priority Critical patent/CN105462692A/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention relates to a biodiesel preparation method. The method comprises the following steps: preprocessing a grease raw material to reduce the content of free fatty acids in the grease, and allowing the above obtained crude alkyl fatty acid ester to be in contact with immobilized lipase and lower alcohol in order to carry out a catalysis reaction. The content of free fatty acids in the grease raw material is regulated to a certain rage in advance, so the service life of the immobilized lipase in the biodiesel preparation process is greatly prolonged.

Description

Biodiesel oil preparing process
Technical field
The present invention relates to the method preparing biofuel, particularly by the lipase-catalyzed method preparing biofuel.The invention still further relates to the method that can improve immobilized lipase work-ing life in preparation of biodiesel.
Background technology
The day by day exhausted and environmental problem of fossil resources, has become one of the most serious crisis that the mankind face.Each state is all at the environmentally friendly energy finding a kind of alternative fossil energy in the past few decades.Biofuel, due to biodegradable, nontoxic renewable, not high, the flash-point advantages of higher of sulfur-bearing and aromatic hydroxy compound, cetane value, is paid close attention to widely.
Commercial biofuel is mainly fatty acid methyl ester, can by animal-plant oil, and biodiesel etc. generate with the transesterification reaction of methyl alcohol.Chemical catalyst especially alkaline catalysts due to speed of response fast, efficiency is high, widely uses in suitability for industrialized production.But chemical catalyst to there is energy consumption large, to ingredient requirement high (free fatty acids be less than 0.5% and anhydrous condition), and the aftertreatment technology of complexity, also can produce the shortcomings such as a large amount of contaminated wastewater environment simultaneously.
Process for preparing biodiesel by enzyme is because reaction conditions is gentle, adaptability to raw material is strong, without side reaction and the feature such as product separation is simple, avoid a lot of shortcomings of chemical process, meet the trend of green chemistry, therefore cause and more and more study interest.And one of the high cost of enzyme industrialized major obstacle that is it, in general, by being fixed of enzyme significantly can be reduced the cost of enzyme the work-ing life of extending enzyme.
In the production process of biofuel, raw materials cost accounts for about about 80% of whole production cost, therefore uses cheap high acid value raw material of fatty acid as more realistic demands such as sewer oil, acidification oil, Palm fatty acid distillates (PFAD).But the stability of immobilized enzyme and the impact of reaction substrate can be subject to work-ing life, when using glyceride stock or the high-moisture glyceride stock of high acid value, the activity of immobilized enzyme and using batch can significantly decline, and causes the cost of immobilized enzyme to increase.This is very disadvantageous for immobilized enzyme in the suitability for industrialized production being substrate with these high-acid-value materials.
Summary of the invention
First aspect, this application provides a kind of method preparing biofuel, it comprises the following steps:
(1) pre-treatment is carried out to glyceride stock, obtains lipid acid alkane ester crude product, the free fatty acids in wherein said lipid acid alkane ester crude product relative to the mass percent of lipid acid alkane ester crude product not higher than 30%;
(2) the lipid acid alkane ester crude product obtained in step (1) is contacted with lower alcohol with immobilized lipase, carry out catalyzed reaction; And optionally,
(3) after the reaction of step (2) terminates, the lipid acid alkane ester products on upper strata is separated with the immobilized lipase of lower floor, and the immobilized lipase after separation is used for the described lipid acid alkane ester crude product of catalysis next batch.
In preferred embodiments, the free fatty acids mass percent in the lipid acid alkane ester crude product obtained in aforesaid method step (1) is about 8% to about 20%, such as, be about 15%.
In some embodiments, the immobilization carrier of the immobilized lipase in aforesaid method step (2) is ion exchange resin or macroporous adsorbent resin.In preferred embodiments, described ion exchange resin is deacidite, more preferably, described deacidite is selected from deacidite WR-8-5, deacidite D301, deacidite SQD-92, deacidite AmberliteIRA-900, deacidite Duolite120D and deacidite Asmit259n.
Preferably, the lipid acid alkane ester crude product obtained by above-mentioned steps (1), without the need to through except water treatment, just can be directly used in the immobilized enzyme catalysis reaction of step (2).The water content of lipid acid alkane ester crude product can be such as about 2-5%.
In some embodiments, the catalyzer that pre-treatment glyceride stock adopts can be lipase, the vitriol oil or strong acidic ion resin.In preferred embodiments, described catalyzer is liquid aliphatic enzyme, is preferably rhizomucor miehei (Rhizomucormiehei) liquid aliphatic enzyme.
In some embodiments, use liquid aliphatic enzyme to be lipid acid alkane ester crude product by glyceride stock pre-treatment in step (1), wherein the addition of liquid aliphatic enzyme is about 0.5% of described oil quality to about 3%.In one embodiment, the mol ratio of carrying out lower alcohol and the grease reacted with above-mentioned glyceride stock is 0.5:1 to 2:1, is preferably 0.8:1 to 1.2:1.In one embodiment, use liquid aliphatic enzyme to carry out pretreated step also to comprise: glyceride stock is mixed with water and glycerine in advance, and said mixture is preheated to 35-50 DEG C.Preferably, the addition of water is about 1% of oil quality to about 3%, and glycerine addition is about 1% of oil quality to about 20%.
In some embodiments, lower alcohol of the present invention is selected from methyl alcohol, ethanol, propyl alcohol, butanols and amylalcohol or its arbitrary combination, and preferably, described lower alcohol is methyl alcohol or ethanol, is more preferably methyl alcohol.In one embodiment, in aforesaid method step (2), the mol ratio of lower alcohol used and lipid acid is 1:1 to 2:1, is preferably 1.2:1.
In some embodiments, immobilized lipase in aforesaid method step (2) is from the microorganism producing lipase, preferably, described microorganism can be the thermophilic hyphomycete of thin cotton like (Thermomyceslanuginosus), rhizomucor miehei (Rhizomucormiehei), antarctic candida (Candidaantarctic), black-koji mould (Aspergillusniger), fold candiyeast (Candidarugosa) and Rhodopseudomonas (Pseudononassp.) etc.
The present invention is applicable to the high-acid value grease raw material of any kind.In one embodiment, the glyceride stock utilized can be sewer oil, acidification oil, soybean oil deodorizer distillate, Palm fatty acid distillates (PFAD), animal grease, useless food and drink oil or butter fat etc., preferably, described glyceride stock is selected from sewer oil, acidification oil, palm oil fatty acid overhead product or its mixture.
The present invention is by carrying out pre-treatment by glyceride stock, reduce the free fatty acid content in glyceride stock, thus reduce reaction raw materials and moisture content etc. to follow-up immobilized lipase enzymic activity and the impact in work-ing life, add the work-ing life of immobilized enzyme, reduce the cost utilizing immobilized enzyme production biofuel, there is good economic benefit.
On the other hand, this application provides the method improving immobilized lipase work-ing life, it comprise in advance glyceride stock is prepared as its free fatty acid mass percent not higher than 30% lipid acid alkane ester crude product.In preferred embodiments, the mass percent of the free fatty acids in above-mentioned lipid acid alkane ester crude product is about 8%-about 20%.
In preferred embodiments, the immobilized lipase in aforesaid method is fixed on macroporous adsorbent resin or ion exchange resin.In a more preferred embodiment, above-mentioned immobilized lipase take deacidite as solid carrier.
The present invention is by regulating and controlling the free fatty acids in glyceride stock to less than 30% in advance, preferred regulation and control are to about 8%-about 20%, what obviously can increase immobilized enzyme reuses number of times, such as can reuse more than 200 times, and even more than 300 times, thus the work-ing life and the catalytic activity that significantly improve immobilized enzyme.
Brief Description Of Drawings
It is the follow-up immobilized enzyme reaction batch of substrate that Fig. 1 shows with free fatty acids (FFA) the content fatty acid methyl ester crude product that is 15%, and reacted FFA content.
Fig. 2 show directly with FFA content be the PFAD of 93% for immobilized enzyme reaction batch during substrate, and reacted FFA content.
Embodiment
On the one hand, this application provides a kind of method preparing biofuel, it comprises the following steps:
(1) pre-treatment is carried out to glyceride stock, obtains lipid acid alkane ester crude product, the free fatty acids in wherein said lipid acid alkane ester crude product relative to the mass percent of lipid acid alkane ester crude product not higher than 30%;
(2) the lipid acid alkane ester crude product obtained in step (1) is contacted with lower alcohol with immobilized lipase, carry out catalyzed reaction.
In optional embodiment, the method that the present invention prepares biofuel comprises the following steps:
(1) pre-treatment is carried out to glyceride stock, obtains lipid acid alkane ester crude product, the free fatty acids in wherein said lipid acid alkane ester crude product relative to the mass percent of lipid acid alkane ester crude product not higher than 30%;
(2) the lipid acid alkane ester crude product obtained in step (1) is contacted with lower alcohol with immobilized lipase, carry out catalyzed reaction; And optionally,
(3) after the reaction of step (2) terminates, the lipid acid alkane ester products on upper strata is separated with the immobilized lipase of lower floor, and the immobilized lipase after separation being used for the described lipid acid alkane ester crude product of catalysis next batch, wherein said immobilized lipase can be reused repeatedly.
In preferred embodiments, free fatty acids mass percent in the lipid acid alkane ester crude product obtained in aforesaid method step (1) is about 8% to about 20%, be such as about 15%, this can by regulation and control lower alcohol, and the addition of such as methyl alcohol realizes.Unexpectedly, when the lipid acid alkane ester crude product being about 8% to about 20% with fatty acid content is for substrate, immobilized lipase of the present invention can reuse such as more than 300 time, significantly improves work-ing life and the catalytic activity of lipase.
In some embodiments, the catalyzer that pre-treatment glyceride stock adopts can be lipase, the vitriol oil or strong acidic ion resin.Optionally, lipase can be liquid enzymes or immobilized enzyme.In one embodiment, described catalyzer is liquid aliphatic enzyme, is preferably rhizomucor miehei (Rhizomucormiehei) liquid aliphatic enzyme.Contriver finds, by the FFA mass percent regulation and control in glyceride stock to less than 30%, time preferably between 8% to 20%, adopts the significant difference that different pretreatment processs can't cause the catalytic activity of same immobilized enzyme and to reuse batch.
In some embodiments, use liquid aliphatic enzyme to be lipid acid alkane ester crude product by glyceride stock pre-treatment in step (1), wherein the addition of liquid aliphatic enzyme is about 0.5% of described oil quality to about 3%.In one embodiment, the mol ratio of carrying out lower alcohol and the grease reacted with above-mentioned glyceride stock is 0.5:1 to 2:1, is preferably 0.8:1 to 1.2:1.
In one embodiment, use liquid aliphatic enzyme to carry out pretreated step also to comprise: glyceride stock is mixed with water and glycerine in advance, and said mixture is preheated to 35-50 DEG C.Preferably, the addition of water is about 1% of oil quality to about 3%, and glycerine addition is about 1% of oil quality to about 20%.
" lower alcohol " of the present invention includes but not limited to methyl alcohol, ethanol, propyl alcohol, butanols or amylalcohol etc.In preferred embodiments, the lower alcohol of use is selected from methyl alcohol or ethanol, is preferably methyl alcohol.
In one specific embodiment, use the operation of liquid aliphatic enzyme pre-treatment glyceride stock as follows:
A. a certain amount of glyceride stock containing free fatty acids, water and glycerine are joined 2L water-bath jacketed reactor, control bath temperature at 35-50 DEG C;
B. add the liquid enzyme formulation of substrate quality 0.5%-3%, methyl alcohol adopts continous way stream to add, and within 2-4 hour, add, the reaction times is 5 hours;
C. after reaction terminates, stratification 30 minutes, lower floor is liquid enzymes, water, glycerin layer, and upper strata is fatty acid methyl ester phase, releases in lower floor's liquid enzymes to beaker for subsequent use, obtains the fatty acid methyl ester crude product of upper strata FFA content at 8-20%;
D. liquid enzymes continued to repeat above-mentioned steps, liquid enzymes can reaction repeated 5-8 time.
In some embodiments, " glyceride stock " of the present invention comprises Vegetable oil lipoprotein, animal grease, microbial oil, chemical industrial waste grease, recycled wood materials etc.Such as, glyceride stock can be sewer oil, acidification oil, soybean oil deodorizer distillate, Palm fatty acid distillates (PFAD), animal grease, useless food and drink oil or butter fat etc.In preferred embodiments, glyceride stock is sewer oil, acidification oil, PFAD or its mixture.
In preferred embodiments, the lipid acid alkane ester crude product obtained by step (1), without the need to through except water treatment, is directly used in the catalyzed reaction of immobilized lipase.The water content of lipid acid alkane ester crude product can be such as about 2-5%.
In another embodiment, in the step (2) of the method for the invention, the mol ratio of lower alcohol used and lipid acid is 1:1 to 2:1, is preferably 1.2:1.
In one specific embodiment, above-mentioned steps (2), namely the operation of follow-up immobilized enzyme catalysis reaction is as follows:
A. the fatty acid methyl ester crude product being 8-20% by a certain amount of FFA content joins in 250ml there-necked flask, and bath temperature is 35-50 DEG C;
B. add the immobilized enzyme of substrate quality 5%, methyl alcohol addition is according to 1-1.2 mole of interpolation of FFA content in raw material, and divided in 1 hour and add for three times, the reaction times is 2.5 hours;
C., after reaction terminates, stratification 30 minutes, extracts upper-layer fat acid methyl ester product out, dewaters, obtain methyl ester content more than 95%, the biofuel product that FFA content is less than 3% through revolving steaming;
D. lower floor's immobilized enzyme by dehydration, continues to reuse, and can use more than 300 batches continuously.
The present invention's lipase used comprises liquid aliphatic enzyme and immobilized lipase.Lipase is the enzyme that a class has multiple catalytic capability, can the hydrolysis of catalysis triglyceride and some other water-insoluble ester class, alcoholysis, esterification, transesterification and ester class reverse reaction reaction.Lipase is extensively present in animals and plants and microorganism.The seed of what in plant, fatty enzyme was more is oil crops, as castor seeds, Semen Brassicae campestris etc.; Animal body include lipase more be pancreas and the fatty tissue of higher animal; Lipase content in bacterium, fungi and yeast more horn of plenty.Because microbe species is many, breeding soon, easily heritable variation occurs, there is the action pH wider than animals and plants, operative temperature scope and Substratspezifitaet, and microbe-derived lipase is all generally the extracellular enzyme of secretion property, be suitable for industrialized production and obtain high purity sample, therefore microbial lipase is the important sources of industrial lipase.
Therefore, preferably, the present invention is used " liquid aliphatic enzyme " and " immobilized lipase " is from the microorganism producing lipase.Such as, described microorganism can be the thermophilic hyphomycete of thin cotton like (Thermomyceslanuginosus), rhizomucor miehei (Rhizomucormiehei), De Shi head mold (Rhizopusdelemar), Rhizopus oryzae (Rhizopusoryzae), aspergillus oryzae (Aspergillusoryzea), antarctic candida (Candidaantarctic), black-koji mould (Aspergillusniger), fold candiyeast (Candidarugosa), Candida cylindracea (Candidacylindracea) and Rhodopseudomonas (Pseudononassp.) etc., wherein dredge cotton like to bite hot hyphomycete (Thermomyceslanuginosus) lipase and be a kind of basophilic and there is the enzyme of fine thermostability, existing commercial thin cotton like bites heated filament spore bacteria liquid enzyme and immobilized enzyme.
In a preferred embodiment, the liquid aliphatic enzyme that the present invention is used is rhizomucor miehei lipase.Such as, aforesaid liquid lipase can be the rhizomucor miehei lipase of market sale, also can be recombinant expressed rhizomucor miehei lipase, such as, be expressed in the restructuring rhizomucor miehei lipase in yeast.In one embodiment, by rice black root hair enzyme lipase gene, such as aminoacid sequence is the rice black root hair enzyme lipase gene of NCBI accession number A34959 (accessionnumberA34959), be cloned into plasmid, such as pAO815 plasmid, is converted into host cell by plasmid expression vector, such as Pichia pastoris GS115 bacterial strain, abduction delivering, is then separated and purifying rhizomucor miehei lipase from host cell.
The method be fixed by lipase has a variety of, and more common method comprises absorption method, covalent coupling method, entrapping method and microencapsulation etc.Lipase immobilization can only adopt a kind of aforesaid method, and two or more complex methods also can be adopted to be fixed.The fixation support that lipase is conventional includes but not limited to natural porous material, each gellike, synthetic resins and composite modification material etc.
In a particular embodiment, the solid carrier of lipase of the present invention is macroporous adsorbent resin, ion exchange resin, chitosan microball or sodium alginate etc.In some embodiments, lipase is fixed on macroporous adsorbent resin, and described macroporous adsorbent resin includes but not limited to polar macroporous adsorption resin AmberliteXAD-7HP, nonpolar macroporous adsorption resin HPD100A, macroporous absorption acrylic resin, macroporous anion exchange resin etc.The lipase being fixed on macroporous adsorbent resin can be reused repeatedly, such as, reuse more than 200 time.In preferred embodiments, the immobilization carrier of immobilized lipase is deacidite.Described deacidite includes but not limited to deacidite WR-8-5, deacidite D301, deacidite SQD-92, deacidite AmberliteIRA-900, deacidite Duolite120D and deacidite Asmit259n etc.Such as, thin cotton like is bitten hot hyphomycete lipase and be fixed on deacidite WR-8-5, be immobilized enzyme WL-821 used in Examples below.Advantageously, compared to non-alkaline ion exchange resin, the lipase be fixed on deacidite reuses number of times significantly to be increased.
In a more preferred embodiment, in advance the fatty acid content in glyceride stock is regulated and controled to 8%-20%, then subsequent reactions is carried out with the lipase being fixed on deacidite, activity and the work-ing life of immobilized enzyme can be significantly improved, such as reuse number of times and can reach more than 300 time, final product lipid acid alkane ester content such as can more than 95%, and FFA content is then reduced to such as less than 3%.
The present invention is by carrying out pre-treatment by glyceride stock, reduce the free fatty acid content in glyceride stock, thus reduce reaction raw materials and moisture content etc. to immobilized lipase enzymic activity and the disadvantageous effect in work-ing life, add the work-ing life of immobilized enzyme, reduce the cost utilizing immobilized enzyme production biofuel.
In addition, in some embodiments, the lipid acid alkane ester crude product obtained by the inventive method pre-treatment glyceride stock does not need except water treatment, just can be directly used in the catalyzed reaction of immobilized lipase, and not affecting the catalytic activity of immobilized lipase, this effect is unexpected to those skilled in the art.
On the other hand, this application provides the method improving immobilized lipase work-ing life, is that the mass percent of its free fatty acid is not higher than the lipid acid alkane ester crude product of 30%, preferably about 8%-about 20% by glyceride stock pre-treatment before it is included in catalyzed reaction.In preferred embodiments, immobilized lipase is fixed on macroporous adsorbent resin or ion exchange resin.In a more preferred embodiment, above-mentioned immobilized lipase take deacidite as carrier.
In certain embodiments, by regulating and controlling the free fatty acids in glyceride stock to not higher than about 30% in advance, the number of times of reusing of immobilized enzyme can be made obviously to increase.Such as, by the pre-treatment of liquid aliphatic enzyme to reaction substrate, the FFA content of controllable gained lipid acid alkane ester crude product is at 8-20%, within this FFA scope, follow-up immobilized enzyme reacts repeatedly reusable further, such as, reuse more than 300 batches, and final product lipid acid alkane ester content is such as more than 95%, FFA content is reduced to such as about 3%, therefore by significantly improving the work-ing life of immobilized enzyme to the pre-treatment of glyceride stock.
In the specification and claims, word " comprises ", " comprising " and " containing " mean " including but not limited to ", and be not intended to get rid of other parts, additive, component or step.
Should be appreciated that, the feature described in particular aspects of the present invention, embodiment or embodiment, characteristic, component or step, be applicable to any other aspect, embodiment or embodiment described herein, unless contradiction with it.
Above-mentioned disclosure generally describes the present invention, by the further example the present invention of the following examples.Describe these embodiments and be only explanation the present invention, instead of limit the scope of the invention.Although employ special term and value herein, these terms and value are understood to exemplary equally, not delimit the scope of the invention.Unless specifically stated otherwise, the experimental technique in this specification sheets and technology are Method and Technology known in those skilled in the art.
Embodiment
In following embodiment, the mensuration of free fatty acid content is carried out according to the method in the standard GB/T/T14489.3-1993 of the free fatty acid content mensuration of oil in oil plant.It is carry out according to the method in international standard EN14103:2011 that fatty acid methyl ester measures.Unless specifically stated otherwise, liquid aliphatic enzyme used in embodiment and immobilized lipase are all purchased from Novozymes Company, and in following embodiment of the present invention, the rhizomucor miehei liquid aliphatic enzyme of use is purchased from Novozymes Company 20000L.
The synthetic method of the resin WR-8-5 used in embodiment is as follows: at 45 DEG C, by methyl acrylate, divinylbenzene, gasoline (200# gasoline, purchased from traditional Chinese medicines chemical reagent company limited) mix by the mass ratio of 14:1:20, the benzoyl peroxide adding above-mentioned quality 0.1%, as initiator, mixes.Join in the deionized water being dissolved with gelatin (concentration is 0.1% mass ratio), sodium-chlor (3% mass ratio), start after stirring is warming up to 75 DEG C, reaction 6h, 6h is incubated at 75 DEG C, then mother liquor is pumped, washing, after drying, obtains methyl acrylate-divinylbenzene copolymer.Then, by 10g multipolymer at 100mLN, swelling 2h in dinethylformamide, adds a certain amount of 1,8-octamethylenediamine (the molar weight and 1 of methyl esters key in methyl acrylate divinylbenzene copolymer, the mol ratio of 8-octamethylenediamine is 1:2) carry out amination reaction, be warming up to 130 DEG C, isothermal reaction 8h, then mother liquor is pumped, with deionized water wash, filter, obtain weakly alkaline acrylate ionomer exchange resin WR-8-5.
In the following embodiments, the immobilized enzyme WL-821 of use adopts following absorption method to prepare:
Take 15g deacidite WR-8-5 (functional group content 2.5mmol/g, mean pore size 30nm, average specific surface area 250m 2/ g, median size 0.22mm), be placed in 100ml round-bottomed flask, add 10mlTL enzyme liquid (LipozymeTL100L, Novozymes Company), with the speed oscillation 4h of 150 revs/min in 25 DEG C of shaking tables, then enzyme is taken out and put into clean culture dish, put into stink cupboard and carry out drying, being fixed enzyme WL-821 after drying.
Except commercial immobilized enzyme, the process for fixation of other immobilized enzyme used in following examples is the same with the process for fixation of WL-821.
In the following embodiments, the lower floor's immobilized enzyme obtained after reaction dewaters through following methods: lower floor's immobilized enzyme is used Büchner funnel suction filtration 2h, removes the biofuel and moisture that remain in above immobilized enzyme.
In the following embodiment of the application, the mole number of Palm fatty acid distillates (PFAD) can be obtained by the molecular-weight average of the quality of PFAD divided by PFAD, and the molecular-weight average of PFAD then depends on the lipid acid composition of PFAD.The lipid acid of the PFAD used in following embodiment of the present invention consists of: the lauric acid (C12:0) of 1%, the myristic acid (C14:0) of 2%, the palmitinic acid (C16:0) of 46%, the stearic acid (C18:0) of 5%, the oleic acid (C18:1) of 36%, the linolic acid (C18:2) of 10%.Therefore, the molecular-weight average of the PFAD used in the embodiment of the present application is 270.
First, we are in embodiment 1-6, have detected FFA content in lipid acid alkane ester crude product not higher than 30%, be fixed on the work-ing life of the lipase of deacidite.
Embodiment 1
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 125ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1.2:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 8%, and water content is the fatty acid methyl ester crude product of 2.5%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 4 times of lower floor's liquid enzymes.
Be 8% by above-mentioned for 80g FFA content, water content be 2.5% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 1.2ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that fatty acid methyl ester is 96%, FFA content is the biofuel of 2.5%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 350 batches.
Embodiment 2
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 95%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 335 batches, see accompanying drawing 1.
Embodiment 3
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 85ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 0.8:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 20%, and water content is the fatty acid methyl ester crude product of 2.2%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 20% by above-mentioned for 80g FFA content, water content be 2.2% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.8ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 95%, FFA content is the biofuel of 3.6%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 310 batches.
Embodiment 4
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 35g (5% substrate quality) immobilized enzyme WL-821 is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 12%, and water content is the fatty acid methyl ester crude product of 2.0%.
Be 12% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 1.7ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 95%, FFA content is the biofuel of 3.1%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 340 batches.
Embodiment 5
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 21g (3% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 206ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 2:1), at the uniform velocity added by constant pressure funnel within 3h.React after 15 hours, stratification 30min, upper strata is FFA content is 5%, and water content is the fatty acid methyl ester crude product of 3%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 1 time of lower floor's liquid enzymes.
Be 5% by above-mentioned for 80g FFA content, water content be 3% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 0.8ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 96%, FFA content is the biofuel of 2.5%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 345 batches.
Embodiment 6
Be that the rhizomucor miehei liquid aliphatic enzyme of PFAD, 10.5g of 93%, 7g glycerine and 7g water are placed in 2L water-bath jacketed reactor and mix by 700gFFA content, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 0.8:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, to be FFA content be on upper strata 30% fatty acid methyl ester crude product, isolate lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
The fatty acid methyl ester crude product being 30% by above-mentioned for 80g FFA content joins in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, add the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 4.2ml at 0h, 0.5h, 1h in three batches.React after 2.5 hours, stratification 30min, inclines and upper strata product, dewaters through revolving steaming, obtains that fatty acid methyl ester is 90%, FFA content is the biofuel of 6%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 215 batches.
Comparing embodiment 1
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 54ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 0.5:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 45%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 45% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 6.4ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 92%, FFA content is the biofuel of 4.5%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 150 batches.
Comparing embodiment 2
By 80gFFA content be 93% Palm fatty acid distillates (PFAD) join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, add the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 14.2ml at 0h, 0.5h, 1h in three batches.React after 2.5 hours, stratification 30min, inclines and upper strata product, dewaters through revolving steaming, obtains the biofuel of fatty acid methyl ester 85%, FFA content 12%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 22 batches.Concrete outcome refers to accompanying drawing 2.
Above embodiment and comparing embodiment show, free fatty acid content wherein, by carrying out pre-treatment to glyceride stock, is reduced to less than 30% by the present invention, and what obviously can increase immobilized enzyme reuses number of times.Such as, in comparing embodiment 2, glyceride stock is not through pre-treatment, and the number of times of reusing of immobilized enzyme is only 22 times; And after carrying out pre-treatment with liquid aliphatic enzyme, the number of times of reusing of immobilized enzyme increases (see embodiment 1-6).And, only when in advance by glyceride stock free fatty acids regulation and control to not higher than 30%, the work-ing life of immobilized enzyme just can be increased to more than 200 times, and when such as, in comparing embodiment 1, FFA content is 45%, immobilized enzyme only can be reusable 150 batches; When FFA content is down to 30%, the work-ing life of immobilized enzyme increases to 215 batches; When FFA content is down to 8% to 20% further, the work-ing life of immobilized enzyme is increased to more than 300 batches further, such as 350 times (see embodiment 1), meanwhile, the end product lipid acid alkane ester content obtained is all more than 95%, and FFA is then reduced to about 3%.In addition, can be found out by the result of embodiment 5, when the FFA content of lipid acid alkane ester crude product is reduced to about 5% further from 8%, the work-ing life of immobilized enzyme does not correspondingly improve, and the content of end product methyl esters and FFA content are also suitable with the result of embodiment 1 (the FFA content of lipid acid alkane ester crude product is 8%).Therefore consider the factor of production cost, it is preferred for regulating and controlling the FFA content of lipid acid alkane ester crude product at 8-20%.
To sum up, significantly improving of this immobilized enzyme work-ing life, reduces the cost utilizing immobilized enzyme production biofuel, creates good economic benefit.
In above-described embodiment 1-6, employ liquid aliphatic enzyme (such as embodiment 1-3 and 5-6) and immobilized lipase (see embodiment 4) carries out pre-treatment to glyceride stock, in ensuing embodiment 7-8, we used other catalyst pretreatment glyceride stocks, then subsequent disposal is carried out with identical immobilized enzyme, to observe the impact of different pretreatment processs on immobilized enzyme catalysis activity.
Embodiment 7
By 700gFFA content be 93% Palm fatty acid distillates (PFAD) according to bibliographical information method (see, Liu Boxuan etc. the esterification of palm-kernel oil deodorization distillate is studied. Chinese oil, 2014,39 (3): 58-60) vitriol oil is adopted to be catalyzer, preparing FFA content is 10%, and water content is the fatty acid methyl ester crude product of 3.0%.
Be 10% by above-mentioned for 80g FFA content, water content be 3.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 1.5ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 96%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 330 batches.
Embodiment 8
By 700gFFA content be 93% Palm fatty acid distillates (PFAD) according to bibliographical information method (see, Xi Limin etc. Preparation of fatty acid methyl ester catalyzed by solid acid from by-products of plam oil. daily chemical industry, 2006,36 (6): 405-408.) D001 strong acidic ion resin is adopted to be catalyzer, preparing FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.5%.
Be 15% by above-mentioned for 80g FFA content, water content be 2.5% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) immobilized enzyme WL-821, the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, incline and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 94%, FFA content is the biofuel of 3.8%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 322 batches.
Can be found out by the result of embodiment 7 and 8, no matter be use the vitriol oil as catalyzer or use strong acidic ion resin as catalyzer, when FFA content in the fatty acid methyl ester crude product prepared falls into scope of the present invention, the work-ing life of follow-up immobilized lipase all significantly improves, reusable 330 batches of such as, immobilized enzyme in embodiment 7, in embodiment 8 reusable 322 batches.This shows, the catalytic activity of immobilized lipase and work-ing life do not affect by concrete oil preprocessing method.
The deacidite used in above-mentioned experiment is deacidite WR-8-5, in ensuing embodiment 9-13, we used the deacidite fixed fat enzyme of other types, to observe the impact of different deacidites on immobilized enzyme catalysis activity.
Embodiment 9
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on deacidite D301 (Jiangsu Su Qing company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining fatty acid methyl ester is 94%, FFA content is the biofuel of 3.5%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 320 batches.
Embodiment 10
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on deacidite SQD-92 (Jiangsu Su Qing company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 95%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 325 batches.
Embodiment 11
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on deacidite AmberliteIRA-900 (ROHM AND HAAS chemical company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 93%, FFA content is the biofuel of 4%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 315 batches.
Embodiment 12
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on deacidite Duolite120D (ROHM AND HAAS chemical company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 95%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 340 batches.
Embodiment 13
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on deacidite Asmit259n (ROHM AND HAAS chemical company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 94%, FFA content is the biofuel of 3.5%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 330 batches.
The result of embodiment 9-13 shows, under the same reaction conditions, use different deacidite fixed fat enzyme, catalytic activity and the work-ing life of immobilized enzyme can not be affected, such as respectively with deacidite WR-8-5 (embodiment 2), deacidite D301 (embodiment 9), deacidite SQD-92 (embodiment 10), deacidite AmberliteIRA-900 (embodiment 11), the work-ing life that deacidite Duolite120D (embodiment 12) and deacidite Asmit259n (embodiment 13) is the immobilized lipase of carrier all reaches more than 300 times, catalytic activity is also without obviously difference.
The immobilization carrier used in above-mentioned experiment is deacidite, and when we have detected and use other types resin for carrier in ensuing embodiment, on the impact of immobilized lipase enzymic activity, wherein silica-gel carrier is as comparative example.
Embodiment 14
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on polar macroporous adsorption resin AmberliteXAD-7HP (ROHM AND HAAS chemical company), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 93%, FFA content is the biofuel of 4%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 210 batches.
Embodiment 15
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme that the thermophilic hyphomycete of cotton like is fixed on nonpolar macroporous adsorption resin HPD100A (Cangzhou, Hebei Bao En Chemical Co., Ltd.), at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 94%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 230 batches.
Embodiment 16
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized lipase (Lanxess Corporation of LewatitVPOC1600 Germany) that the thermophilic hyphomycete of cotton like is fixed on macroporous absorption acrylic resin, at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 95%, FFA content is the biofuel of 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 240 batches.
Embodiment 17
Be that PFAD, 10.5g rhizomucor miehei liquid aliphatic enzyme of 93%, 7g glycerine and 7g water are placed in 2L water-bath jacketed reactor and mix by 700gFFA content, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, to be FFA content be on upper strata 15% fatty acid methyl ester crude product, isolate lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
The fatty acid methyl ester crude product being 15% by above-mentioned for 80g FFA content joins in 250ml there-necked flask, bath temperature controls at 50 DEG C, add the lipase RMIM (Novozymes Company) that 4g derives from rhizomucor miehei bacterium, it is fixed on macroporous anion exchange resin (Novozymes Company).The methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, inclines and upper strata product, dewatering through revolving steaming, obtaining that methyl ester content is 93%, FFA content is the biofuel of 4%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 245 batches.
Embodiment 18
Be that PFAD, 10.5g rhizomucor miehei liquid aliphatic enzyme of 93%, 7g glycerine and 7g water are placed in 2L water-bath jacketed reactor and mix by 700gFFA content, temperature controls at 50 DEG C, add 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1), at the uniform velocity added by constant pressure funnel within 3h.React after 5 hours, stratification 30min, to be FFA content be on upper strata 15% fatty acid methyl ester crude product, isolate lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
The fatty acid methyl ester crude product being 15% by above-mentioned for 80g FFA content joins in 250ml there-necked flask, bath temperature controls at 50 DEG C, add the immobilized lipase Novozyme435 (Novozymes Company) that 4g derives from antarctic candida, it is fixed on macroporous acrylic resin.The methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of common 2.1ml is added in three batches at 0h, 0.5h, 1h, react after 2.5 hours, stratification 30min, inclines and upper strata product, dewatering through revolving steaming, obtaining the biofuel that methyl ester content is 95%, FFA is content 3%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 250 batches.
Comparing embodiment 3
Be the Palm fatty acid distillates (PFAD) of 93% by 700gFFA content, 10.5g (1.5% substrate quality) rhizomucor miehei liquid aliphatic enzyme, 7g (1% substrate quality) glycerine and 7g (1% substrate quality) water is placed in 2L water-bath jacketed reactor and mixes, temperature controls at 50 DEG C, adds within 105ml methyl alcohol (mol ratio of methyl alcohol and PFAD is 1:1) 3h and is at the uniform velocity added by constant pressure funnel.React after 5 hours, stratification 30min, upper strata is FFA content is 15%, and water content is the fatty acid methyl ester crude product of 2.0%, isolates lower floor's liquid enzymes mutually for subsequent use, reusable 5 times of lower floor's liquid enzymes.
Be 15% by above-mentioned for 80g FFA content, water content be 2.0% fatty acid methyl ester crude product join in 250ml there-necked flask, bath temperature controls at 50 DEG C, add 4g (5% substrate quality) and dredge the immobilized enzyme TL-IM (Novozymes Company) that the thermophilic hyphomycete of cotton like is fixed on silica-gel carrier, at 0h, 0.5h, 1h adds the methyl alcohol (mol ratio of methyl alcohol and substrate FFA is 1.2:1) of 2.1ml altogether in three batches, react after 2.5 hours, stratification 30min, incline and upper strata product, dewater through revolving steaming, obtaining methyl ester content is 93%, FFA content is the biofuel of 4%.Lower floor's immobilized enzyme, through dewatering, continues on for next batch reaction, reusable 15 batches.
Known by the result of above embodiment and comparing embodiment, when the FFA content of lipid acid alkane ester crude product is identical, compared with the lipase being fixed on silica-gel carrier, the number of times of reusing being fixed on the lipase of macroporous adsorbent resin obviously increases, but significantly lower than the work-ing life of lipid acid taking deacidite as carrier, the immobilized enzyme WL-821 being such as fixed on deacidite has reused 335 times (embodiment 2), and the identical lipase (embodiment 14-16) being fixed on non-alkaline ion exchange resin only can reuse more than 200 time.This shows that the immobilized enzyme be fixed on deacidite has and reuses the more advantage of number of times.In addition, result in above-described embodiment also shows, be fixed on the catalytic activity of the different lipase of same vehicle and there is no significant difference work-ing life, the thermophilic hyphomycete lipase of thin cotton like such as, being fixed on macroporous absorption acrylic resin in embodiment 16 has reused 240 times, the immobilized lipase deriving from antarctic candida being fixed on macroporous acrylic resin in embodiment 18 has reused 250 times, and both end product methyl ester content of obtaining and FFA content identical.
In addition, in all above-described embodiments, lipid acid alkane ester crude product through except water treatment, is not directly used in the catalyzed reaction of immobilized lipase, and there is no the catalytic activity of materially affect immobilized lipase, this effect is also unexpected to those skilled in the art.
Although be appreciated that the present invention is illustrated with some form, the present invention is not limited to content that is shown in this specification sheets and that describe.It should be apparent to those skilled in the art that and also can to make a variety of changes under the prerequisite not departing from scope of the present invention.These changes are all in the scope of protection of present invention.

Claims (10)

1. prepare the method for biofuel, it comprises the following steps:
(1) pre-treatment is carried out to glyceride stock, obtains lipid acid alkane ester crude product, the free fatty acids in wherein said lipid acid alkane ester crude product relative to the mass percent of lipid acid alkane ester crude product not higher than 30%;
(2) the lipid acid alkane ester crude product obtained in step (1) is contacted with lower alcohol with immobilized lipase, carry out catalyzed reaction; And optionally,
(3) after the reaction of step (2) terminates, the lipid acid alkane ester products on upper strata is separated with the immobilized lipase of lower floor, and the immobilized lipase after separation is used for the described lipid acid alkane ester crude product of catalysis next batch.
2. the method for claim 1, the mass percent of the free fatty acids in the lipid acid alkane ester crude product wherein obtained by step (1) is about 8% to about 20%.
3. method as claimed in claim 1 or 2, the immobilization carrier of wherein said immobilized lipase is ion exchange resin or macroporous adsorbent resin; Preferably, described ion exchange resin is deacidite; More preferably, described deacidite is selected from deacidite WR-8-5, deacidite D301, deacidite SQD-92, deacidite AmberliteIRA-900, deacidite Duolite120D and deacidite Asmit259n.
4. the method as described in aforementioned any one of claim, the lipid acid alkane ester crude product wherein obtained by step (1) is without the need to removing water treatment, be directly used in the catalyzed reaction of step (2), such as the water content of described lipid acid alkane ester crude product is 2%-5%.
5. the method as described in aforementioned any one of claim, the catalyzer that wherein in step (1), pre-treatment glyceride stock adopts is lipase, the vitriol oil or strong acidic ion resin, preferably, described catalyzer is liquid aliphatic enzyme, is preferably rhizomucor miehei (Rhizomucormiehei) liquid aliphatic enzyme.
6. method as claimed in claim 5, when wherein using liquid aliphatic enzyme as catalyzer in step (1), the addition of liquid aliphatic enzyme is about 0.5% of described oil quality to about 3%, and be 0.5:1 to 2:1 with the mol ratio that described grease carries out lower alcohol and the grease reacted, be preferably 0.8:1 to 1.2:1.
7. the method as claimed in any one of the preceding claims, wherein described lower alcohol is selected from methyl alcohol, ethanol, propyl alcohol, butanols and amylalcohol or its arbitrary combination, and preferably, described lower alcohol is methyl alcohol or ethanol; And preferably, the mol ratio of lower alcohol used and free fatty acids is 1:1-2:1 in step (2), be preferably 1.2:1.
8. the method as described in aforementioned any one of claim, wherein said immobilized lipase is microbial lipase, preferably, described microorganism is selected from the thermophilic hyphomycete of thin cotton like (Thermomyceslanuginosus), antarctic candida (Candidaantarctic), rhizomucor miehei (Rhizomucormiehei), black-koji mould (Aspergillusniger), fold candiyeast (Candidarugosa) and Rhodopseudomonas (Pseudononassp.).
9. the method as described in aforementioned any one of claim, wherein said glyceride stock is sewer oil, acidification oil, soybean oil deodorizer distillate, Palm fatty acid distillates (PFAD), animal grease, useless food and drink oil or butter fat, preferably, described glyceride stock is selected from sewer oil, acidification oil, palm oil fatty acid overhead product or its mixture.
10. improve the method in immobilized lipase work-ing life, it comprise in advance glyceride stock is treated to its free fatty acid mass percent not higher than 30%, be preferably the lipid acid alkane ester crude product of about 8%-about 20%, and preferably, the immobilization carrier of described immobilized lipase is ion exchange resin or macroporous adsorbent resin, is more preferably deacidite.
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