CN103421716B - Lipase-producing bacterium and application thereof to preparation of biodiesel - Google Patents
Lipase-producing bacterium and application thereof to preparation of biodiesel Download PDFInfo
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention discloses a lipase-producing bacterium and application thereof to preparation of biodiesel. The preservation number of the lipase-producing bacterium is CGMCC No. 7644. The lipase-producing bacterium is applied to preparation of the biodiesel, wherein the lipase-producing bacterium is immobilized by taking a magnetic chitosan microsphere as a carrier and utilizing an adsorption method to prepare a lipase-producing bacterium magnetic whole-cell catalyst and the biodiesel is prepared by utilizing the catalyst. According to the technical scheme, the lipase-producing bacterium is simple in process and low in cost, has a long service life, high mass transfer efficiency and high conversion rate, is easy to separate and can be used for many times.
Description
Technical field
The invention belongs to biotechnology and derived energy chemical field, more particularly, relate to a kind of method preparing biofuel.
Background technology
Biofuel, belongs to the one of renewable energy source, refers to the fatty acid methyl ester obtained by transesterification by glyceride stock (main component is triglyceride level or lipid acid) and short chain alcohol (being generally methyl alcohol).At present, countries in the world prepare biofuel raw material mainly Vegetable oil lipoprotein as soya-bean oil, rapeseed oil, Viscotrol C, Cortex jatrophae wet goods.But in China, owing to being limited by the national conditions that China has a large population and a few land, be therefore difficult to carry out long-term a large amount of production.Utilizing sewer oil as being raw material, significantly can not only reduce production cost, reduce environmental pollution and the risk that it is back to dining table can be reduced.At present, the preparation method of biofuel mainly comprises chemical method and biological enzyme, wherein the technology relative maturity of chemical method biodiesel synthesis, but industrially also have obvious shortcoming at present, i.e. this method complex process, equipment requirements is high, alcohol consumption is large, and product not easily reclaims, and environmental pollution is large.Enzymatic Synthesis of Biodiesel has the advantages such as mild condition, alcohol consumption is little, non-pollutant discharge, stock oil wide adaptability, product easily reclaim, and causes increasing concern, is study hotspot in recent years.But the subject matter of process for preparing biodiesel by enzyme is the use cost of lipase compared with the catalyst acid of chemical method or alkali for high, is thus difficult to economically compete with chemical method.
Directly utilizing yielding lipase microorganism cells to be solve the new Research Thinking of of lipase production cost as biodiesel, is a study hotspot of current process for preparing biodiesel by enzyme.Adopt whole-cell biocatalyst (whole-cellbiocatalyst, WCB) not only can remove the technique of lipase separation and purification from, stopped a large amount of losses that enzyme is lived in the process, a large amount of facility investments and working cost can also be saved.Japan Kobe university research group reported by this method for the preparation of biofuel the earliest, and H.Fukuda professor was carrying out the study mechanism of lipase-catalyzed production biodiesel always from 1999.On this basis, start to select lipase to produce the cell of bacterial strain Rhizopus oryzae (Rhizopus oryzae) in calendar year 2001, prepare whole cell catalyzer (BSPs) with polyurethane cellular foam, and the transesterification of successful catalyzing glycerol three acid esters.But, current whole-cell catalytic is in the process of catalytic production diesel oil, also there is the deficiency of following aspects, first be the unicity of bacterial classification, the bacterial classification of current report is only concentrated on several specific bacterial classification, above the filamentous funguss such as Rhizopus oryzae, zhizopchin, aspergillus niger, and these bacterial classifications reported with moisture and that free fatty acid concentration is high grease for raw material time, effect is not very desirable, adopts non-filamentous bacterial classification such as bacterium to prepare the rarely found report of whole-cell catalyst catalysis for preparing biodiesel oil.And some problems of existence existed when making catalyzer with bacterial whole cell as in reaction process because the sedimentation of cell and cohesion reduce mass-transfer efficiency, the recovery of cell and recycling more difficult.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of yielding lipase bacterium and the application in preparation biofuel thereof are provided.
Technical purpose of the present invention is achieved by following technical proposals:
Yielding lipase bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preserving number is CGMCC No.7644, preservation date is on May 27th, 2013, and suggestion Classification And Nomenclature is pseudomonas mendocina Pseudomonas mendocina.
By above-mentioned yielding lipase bacterium 37 DEG C of cultivation 48h on LB flat board.Shown in result accompanying drawing 1, bacterium colony is faint yellow, and circular, intermediate projections, in oil droplet shape, transparent, smooth surface is moistening, neat in edge, and bacterium colony is not of uniform size, mean diameter 1.5 ~ 3mm.Under the microscope, thalline is shaft-like, about 0.5 × 1.0mm, has pod membrane, Gram-negative, and wherein LB substratum is as follows:
Luria.Bertani (LB) liquid nutrient medium (g/l): peptone 10; Yeast extract paste 5; NaCl10.PH is regulated to be 7.0.Autoclaving, 121 DEG C of steam sterilizing 30min.
Luria.Bertani (LB) solid medium (wt%): add the agar powder of 2% on the basis of Luria.Bertani (LB) liquid nutrient medium.
By above-mentioned yielding lipase bacterium for the preparation of biofuel, utilize yielding lipase bacterium magnetic whole-cell catalyst to carry out catalysis to reaction, described yielding lipase bacterium magnetic whole-cell catalyst adopts the method for absorption to be prepared from being fixed of yielding lipase bacterium by chitosan magnetic micro-sphere as carrier.
In described chitosan magnetic micro-sphere, adopt lipophilicity single magnetic domain Fe
3o
4nano-magnetic colloid and chitosan are prepared, wherein lipophilicity single magnetic domain Fe
3o
4the preparation of nano-magnetic colloid is prepared (applying date is on November 10th, 1998, and application number is 981245161, and publication date is on May 17th, 2000) according to method disclosed in Chinese invention patent " superparamagnetism polymerization microballoon and preparation method thereof ".
By the lipophilicity single magnetic domain Fe prepared according to foregoing invention method
3o
4nano-magnetic colloid and chitosan carry out load preparation, carry out with reference to method disclosed in Chinese invention patent " superparamagnetism polymerization microballoon and preparation method thereof " that (applying date is on November 10th, 1998, application number is 981245161, publication date is on May 17th, 2000), specifically carry out according to following step:
Step 1, chooses each raw material, and wherein each raw material according to mass fraction is: choose the magnetic particle (Fe that particle diameter is less than 100nm
3o
4) 1-2 mass parts, chitosan 0.5-1.5 mass parts, oleic acid 2-4 mass parts, mass percent is the aqueous hydrochloric acid 20-25 mass parts of 1%, condensation water 70-75 mass parts, described condensation water to be mass percent be 20% the NaOH aqueous solution (i.e. the ratio of NaOH quality and bulk solution quality) and percent by volume be 30% formalin (i.e. the ratio of formaldehyde volume and bulk solution volume) carry out mixing forming according to equal-volume ratio; Preferably each raw materials quality part sum is 100 mass parts;
Step 2, by magnetic particle (Fe
3o
4) to dissolve each other supersound process with oleic acid, to realize mixed uniformly object, form the oleic acid solutions of magnetic particle, such as, use vibration of ultrasonic wave 10-20min, use laboratory to commonly use ultrasonic instrument;
Step 3, the oleic acid solutions of magnetic particle step 2 prepared is transferred to chitosan and is mixed with the mixing solutions of hydrochloric acid, dropwise adds condensation water, and stirs with magnetic force, forms the polymerizing microballoons of 2-5mm; In above-mentioned steps, select magnetic stirring apparatus to stir with 300-500r/min speed, after preparation, collect chitosan magnetic microsphere with Nd-Fe-B permanent magnet, with deionized water repetitive scrubbing several, finally vacuum-drying at 20 DEG C.
In technique scheme, because chitosan is selected as carrier, as long as can microballoon be formed, further strict demand is not done to chitosan nature (such as molecular weight and distribution, deacetylation etc.), select the chitosan of deacetylation 85%-95%, can realize forming chitosan microball as molecular weight and distribution thereof, carry out selecting in the product of three kinds of different viscosity: high-viscosity products is 0.7 ~ 1Pas, medium viscosity product is 0.25 ~ 0.65Pas, low viscosity product <0.25Pas.
Yielding lipase bacterium (preserving number CGMCCNo.7644) is carried out cultivate (adopting LB substratum to cultivate), then by the cultured bacterium liquid of fixed volume under 8000rpm condition centrifugal 3 minutes, outwell supernatant liquor, again with sterilized water centrifuge washing 3 times repeatedly, finally collection thalline is dry under putting into 105 DEG C of conditions, until weight does not change, then weigh, bacterium cell dry weight can be obtained.
Described yielding lipase bacterium magnetic whole-cell catalyst is prepared according to following step:
Step 1, choose each raw material, wherein each raw material according to mass fraction is: chitosan magnetic micro-sphere 5-10 mass parts, pH be 6.8 phosphate buffer soln 88.5-93.5 mass parts, yielding lipase bacterium (preserving number CGMCCNo.7644) somatocyte (dry weight) be 0.5-1.5 mass parts, preferably each raw material sum is 100 mass parts;
Step 2, yielding lipase bacterium (preserving number CGMCCNo.7644) being joined pH is that in 6.8 phosphate buffer solns, concussion evenly, makes cell suspending liquid;
Step 3, chitosan magnetic micro-sphere is added in cell suspending liquid prepared by step 2, and 30-40 DEG C, react 18-24h, after being then separated with permanent magnet under the condition of 120-150r/min, use phosphate buffer soln repetitive scrubbing, namely obtain magnetic immobilized whole-cell catalyst.
When the present invention utilizes magnetic immobilized whole-cell catalyst to prepare biofuel, alcohol oil is added according to mol ratio 1:1, add account for oily alcohol and water cumulative volume 5-40% water and account for oil, alcohol, water and catalyzer total mass 2-8% magnetic immobilized whole-cell catalyst, be placed in shaking table to react, at least 60h is reacted, preferably 70-100h at temperature of reaction is 25-30 DEG C.In reaction process, select to add when 12h, 24h, 48h are carried out in reaction the alcohol becoming equimolar ratio with stock oil more respectively; Described grease separation soya-bean oil, rapeseed oil or sewer oil; Described alcohol is methyl alcohol.
Compared with prior art, the present invention has following beneficial effect: technique is simple, with low cost: compared with traditional fixed lipase catalyzed transesterification preparation biofuel, magnetic whole-cell catalyst provided by the invention is without the need to carrying out the extraction purification of lipase, and the equipment of immobilization process operational path and simple use is simple, which greatly simplifies technique, and reduce cost, be easy to be separated, can repeatedly use: after transesterification completes, permanent magnet can be utilized to be separated from reaction product easily by catalyzer, then the reaction carrying out next batch in new reaction substrate is directly put into.There is high work-ing life, mass-transfer efficiency and transformation efficiency: because catalyzer has magnetics effect, react therefore, it is possible to catalyzer to be put into the reactor with magnetic field, catalyst distribution can be made so more even, reduce sedimentation collision, reach the effect improving mass-transfer efficiency and transformation efficiency like this.
Accompanying drawing explanation
Fig. 1 is the form photo of yielding lipase bacterium (CGMCCNo.7644) of the present invention on LB substratum.
Fig. 2 is the environmental scanning electronic microscope photo (1) of magnetic whole-cell catalyst of the present invention.
Fig. 3 is the environmental scanning electronic microscope photo (2) of magnetic whole-cell catalyst of the present invention.
Embodiment
Technical scheme of the present invention is further illustrated below in conjunction with specific embodiment.Select commercially available chitosan, deacetylation 85%, number-average molecular weight 20,000.
First, lipophilicity single magnetic domain Fe is prepared according to method disclosed in Chinese invention patent " superparamagnetism polymerization microballoon and preparation method thereof "
3o
4nano-magnetic colloid (applying date is on November 10th, 1998, and application number is 981245161, and publication date is on May 17th, 2000).
Then the preparation of chitosan magnetic microsphere is carried out, each each raw material feeding situation is as shown in the table, wherein said condensation water to be mass percent be 20% the NaOH aqueous solution (i.e. the ratio of NaOH quality and bulk solution quality) and percent by volume be 30% formalin (i.e. the ratio of formaldehyde volume and bulk solution volume) carry out mixing forming according to equal-volume ratio
By magnetic particle (Fe
3o
4) to dissolve each other supersound process with oleic acid, use laboratory to commonly use ultrasonic instrument vibration of ultrasonic wave 10-20min; The oleic acid solutions of magnetic particle is transferred to chitosan to mix with the mixing solutions of hydrochloric acid, dropwise add condensation water, and stir with magnetic force, form the polymerizing microballoons of 2-5mm; In above-mentioned steps, select magnetic stirring apparatus to stir with 300-500r/min speed, after preparation, collect chitosan magnetic microsphere with Nd-Fe-B permanent magnet, with deionized water repetitive scrubbing several, finally vacuum-drying at 20 DEG C.
Yielding lipase bacterium (preserving number CGMCCNo.7644) is carried out cultivate (adopting LB substratum to cultivate), then by the cultured bacterium liquid of fixed volume under 8000rpm condition centrifugal 3 minutes, outwell supernatant liquor, again with sterilized water centrifuge washing 3 times repeatedly, finally collection thalline is dry under putting into 105 DEG C of conditions, until weight does not change, then weighs, bacterium cell dry weight can be obtained, choose each raw material according to following table
Yielding lipase bacterium (preserving number CGMCCNo.7644) being joined pH is that in 6.8 phosphate buffer solns, concussion evenly, makes cell suspending liquid; Chitosan magnetic micro-sphere is added in the cell suspending liquid of preparation, and 30-40 DEG C, react 18-24h under the condition of 120-150r/min, after being then separated with permanent magnet, using phosphate buffer soln repetitive scrubbing, namely obtain magnetic immobilized whole-cell catalyst.Chitosan magnetic microsphere 1-4 is utilized to be prepared according to the described ratio of upper table respectively, different magnetic immobilized whole-cell catalysts can be obtained, different magnetic immobilized whole-cell catalysts is respectively used in the embodiment of following preparation biofuel, and carry out arithmetical mean according to the final transformation efficiency that different catalysts measures, the feed ratio of two kinds is calculated to make both equimolar ratios according to the molecular-weight average of oil and the molecular weight of methyl alcohol, respectively according to document 1 " microwave-assisted solid base catalyst prepares biofuel ", Zhang Honghao, Zhang Like, high beautiful, " Zhejiang chemical industry " the 40th volume the 3rd phase in 2009 and " rapeseed oil base catalysis legal system is for the processing parameter of biofuel ", yellow rosy clouds, Liu Ronghou, the molecular-weight average that method disclosed in " Transactions of the Chinese Society of Agricultural Engineering " the 25th volume the 12nd phase in 2009 calculates soya-bean oil and rapeseed oil is 882 and 880, because the oils molecular-weight average selected is about 880, therefore select equal in quality to test, the alcohol oil rate of calculating is 1:1.
The transesterification preparation biofuel of embodiment 1 catalysis soya-bean oil
Take soya-bean oil as raw material, with the magnetic immobilized whole-cell catalyst prepared for catalyzer carries out transesterification test.28.95g soya-bean oil and 1.05g methyl alcohol (molar ratio of methanol to oil 1:1) mixture are loaded in 100ml screw socket bottle with cover, add the water (accounting for 10% of oily alcohol and water cumulative volume) of 10%, add respectively again 5% above-mentioned catalyzer (account for oil, alcohol, water, catalyzer total mass 5%), be placed in shaking table to react, temperature 30 DEG C, rotating speed 150rpm.In order to make to react completely, in reaction process, add 1.05g methyl alcohol respectively again when 12h, 24h, 48h are carried out in reaction respectively, adding methyl alcohol number of times is altogether 4 times.Every sampling in 4 hours once, the sample of taking-up is got supernatant liquid and is measured methyl ester content after leaving standstill, and after reaction 100h, recording final average conversion is 92.7%.
The transesterification preparation biofuel of embodiment 2 catalysis rapeseed oil
With rapeseed oil (molecular-weight average) for raw material, carry out transesterification test to prepare magnetic immobilized whole-cell catalyst for catalyzer.28.95g rapeseed oil and 1.05g methyl alcohol (molar ratio of methanol to oil 1:1) mixture are loaded in 100ml screw socket bottle with cover, add the water of 50%, then add the above-mentioned catalyzer of 2% respectively, be placed in shaking table and react, temperature 30 DEG C, rotating speed 150rpm.In order to make to react completely, in reaction process, add 1.05g methyl alcohol respectively again when 12h, 24h, 48h are carried out in reaction respectively, adding methyl alcohol number of times is altogether 4 times.Every sampling in 4 hours once, the sample of taking-up is got supernatant liquid and is measured methyl ester content after leaving standstill, and after reaction 60h, recording final average conversion is 88.3%.
The transesterification preparation biofuel of embodiment 3 catalysis sewer oil
Take sewer oil as raw material, with the magnetic immobilized whole-cell catalyst prepared for catalyzer carries out transesterification test.Sewer oil raw material can Bio-energy Technology Ltd. (GB) Suite 1, Bristol House, 80A Southampton Row Loud provide by Tianjin is prebiotic, directly uses after the centrifugal removing moisture of mechanical purifying, bentone adsorbent adsorption filtration, washing and high speed rotating.Sewer oil main characteristic is as follows: acid number 33.14mgKOH/g; Saponification value 192mgKOH/goil; Non-saponified value 4.1wt%; Free fatty acids 21.37%; Moisture 1960ppm, molecular-weight average is 882.28.
28.95g sewer oil and 1.05g methyl alcohol (molar ratio of methanol to oil 1:1) mixture are loaded in 100ml screw socket bottle with cover, add the water of 40%, then add the above-mentioned catalyzer of 8% respectively, be placed in shaking table and react, temperature 25 DEG C, rotating speed 150rpm.In order to make to react completely, in reaction process, add 1.05g methyl alcohol respectively again when 12h, 24h, 48h are carried out in reaction respectively, adding methyl alcohol number of times is altogether 4 times.Every sampling in 4 hours once, the sample of taking-up is got supernatant liquid and is measured methyl ester content after leaving standstill, and after reaction 70h, recording final average conversion is 81.6%.
Above to invention has been exemplary description; should be noted that; when not departing from core of the present invention, any simple distortion, amendment or other those skilled in the art can not spend the equivalent replacement of creative work all to fall into protection scope of the present invention.
Claims (10)
1. a strain yielding lipase bacterium, is characterized in that, this bacterial strain be pseudomonas mendocina (
pseudomonas mendocina), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCCNo.7644.
2. the application of a strain yielding lipase bacterium as claimed in claim 1 in preparation biofuel, it is characterized in that, utilize yielding lipase bacterium magnetic whole-cell catalyst to carry out catalysis to reaction, described yielding lipase bacterium magnetic whole-cell catalyst adopts the method for absorption to be prepared from being fixed of yielding lipase bacterium by chitosan magnetic micro-sphere as carrier.
3. the application of a strain yielding lipase bacterium as claimed in claim 2 in preparation biofuel, it is characterized in that, described chitosan magnetic micro-sphere is prepared according to following step:
Step 1, chooses each raw material, and wherein each raw material according to mass fraction is: choose the magnetic particle Fe that particle diameter is less than 100nm
3o
41-2 mass parts, chitosan 0.5-1.5 mass parts, oleic acid 2-4 mass parts, mass percent is the aqueous hydrochloric acid 20-25 mass parts of 1%, condensation water 70-75 mass parts, described condensation water to be mass percent be 20% the NaOH aqueous solution and percent by volume be 30% formalin carry out mixing forming according to equal-volume ratio;
Step 2, by magnetic particle Fe
3o
4to dissolve each other supersound process with oleic acid, to realize mixed uniformly object, form magnetic particle Fe
3o
4oleic acid solutions;
Step 3, magnetic particle Fe prepared by step 2
3o
4oleic acid solutions transfer to chitosan and mix with the mixing solutions of hydrochloric acid, dropwise add condensation water, and stir with magnetic force, form the polymerizing microballoons of 2-5mm.
4. the application of yielding lipase bacterium as claimed in claim 3 in preparation biofuel, it is characterized in that, preferably each raw materials quality part sum is 100 mass parts in step 1; In step 2, use vibration of ultrasonic wave 10-20min, use laboratory to commonly use ultrasonic instrument; In step 3, select magnetic stirring apparatus to stir with 300-500r/min speed, after preparation, collect chitosan magnetic micro-sphere with Nd-Fe-B permanent magnet, with deionized water repetitive scrubbing several, finally vacuum-drying at 20 DEG C.
5. the application of yielding lipase bacterium as claimed in claim 2 in preparation biofuel, it is characterized in that, yielding lipase bacterium magnetic whole-cell catalyst is prepared according to following step:
Step 1, chooses each raw material, and wherein each raw material according to mass fraction is: chitosan magnetic micro-sphere 5-10 mass parts, pH be 6.8 phosphate buffer soln 88.5-93.5 mass parts, yielding lipase somatic cells be 0.5-1.5 mass parts;
Step 2, yielding lipase bacterium being joined pH is that in 6.8 phosphate buffer solns, concussion evenly, makes cell suspending liquid;
Step 3, chitosan magnetic micro-sphere is added in cell suspending liquid prepared by step 2, and 30-40 DEG C, react 18-24h, after being then separated with permanent magnet under the condition of 120-150r/min, use phosphate buffer soln repetitive scrubbing, namely obtain yielding lipase bacterium magnetic whole-cell catalyst.
6. the application of yielding lipase bacterium as claimed in claim 5 in preparation biofuel, it is characterized in that, in described step 1, preferably each raw material sum is 100 mass parts.
7. the application of yielding lipase bacterium as claimed in claim 2 in preparation biofuel, it is characterized in that, alcohol oil is added according to mol ratio 1:1, add account for oily alcohol and water cumulative volume 5-40% water and account for oil, alcohol, water and catalyzer total mass 2-8% yielding lipase bacterium magnetic whole-cell catalyst, be placed in shaking table to react, at temperature of reaction is 25-30 DEG C, reacts at least 60h.
8. the application of yielding lipase bacterium as claimed in claim 7 in preparation biofuel, is characterized in that, in reaction process, selects to add when 12h, 24h, 48h are carried out in reaction the alcohol becoming equimolar ratio with stock oil more respectively.
9. the application of the yielding lipase bacterium as described in claim 7 or 8 in preparation biofuel, it is characterized in that, described alcohol is methyl alcohol.
10. the application of the yielding lipase bacterium as described in claim 7 or 8 in preparation biofuel, is characterized in that, described grease separation soya-bean oil, rapeseed oil or sewer oil.
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