CN108103117A - A kind of method using saccharomycetes to make fermentation corncob production biodiesel and its manufactured biodiesel - Google Patents
A kind of method using saccharomycetes to make fermentation corncob production biodiesel and its manufactured biodiesel Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention belongs to biomass energy source domains, disclose a kind of method using saccharomycetes to make fermentation corncob production biodiesel and its manufactured biodiesel.Method is as follows：By corncob cutting and grinding, after 1ml mass concentrations are 0.5% hydrochloric acid and appropriate distilled water is pre-processed, add in cellulase and xylan enzyme solution is saccharified, saccharified liquid is filtered, filtrate sterilizing is spare；Actication of culture is obtained into liquid seeds, then seed is transferred to fermentation in saccharified liquid and generates grease, microbial grease is extracted, further using preparing biodiesel by ester interchange.The present invention produces biodiesel by raw material of carbonaceous refuse, has not only reached waste utilization, the purpose of environmental protection, and can reduce production cost.
Description
Technical field
It is more particularly to a kind of to produce biodiesel using saccharomycetes to make fermentation corncob the invention belongs to biomass energy field
Method and its manufactured biodiesel.
Background technology
Currently, the whole world is faced with this short huge challenge of petroleum-based energy.Many countries all look for one kind and have actively
Effect, the reproducible energy meet the needs of mankind are growing to the energy.Biodiesel as it is reproducible, clean,
One of alternative biomass liquid fuel, its exploitation and application realize that social economy can to realizing circular economy and Green GDP
Sustainable development will generate tremendous influence.But the method for production biodiesel is mainly turned using animal and plant fat at present
Change.For animals and plants because being influenced by many factors, grease yield is unstable, and growth cycle is very long, it is necessary to substantial amounts of labour, warp
Cost of helping is higher.And the next life generation diesel oil such as oil foot, waste edible oil from catering trade are used, it can for the demand of entire society
Call an utterly inadequate amount, it is impossible to tackle the problem at its root.Biodiesel is greatly developed, just has to look for an effective new way
Footpath.
Microbial grease is also known as Unicell Oils and Fats, refers to be synthesized under certain condition by microorganism and be stored in thalline
Glyceride, aliphatic acid composition it is similar to general vegetable fat, be mainly C16、C18It is aliphatic acid, such as palmitic acid, tristearin
The R and D of this new oil resource by microbial grease such as acid, oleic acid and leukotrienes, are most promising approach
One of.And oleaginous microorganism, because it is with fat content is high, aliphatic acid composition is similar to vegetable oil, and from season, weather and
Site influence, can the advantages such as continuous production, vegetable fat is gradually substituted to become the new sources of biodiesel.Ferment in oleaginous microorganism
Female bacterium oil fatty acid composition is similar to plant, and content is high, is seen as the important sources of future biological diesel oil.
Corncob, maize straw account for the 40% of agricultural crop straw, and yield is big, source is wide, cheap, and containing big
The lignocellulosic of amount has very big developing and utilizingpotentiality.But the utilization rate of current maize straw is also very low, is mainly used for
Straw-returning and producing fodder, but the also insufficient stalk total amount of the summation as straw-returning and livestock roughage, remaining is burnt
It burns up, causes the great wasting of resources and environmental pollution.If microbial fermentation maize straw can be utilized to produce microorganism
Grease and obtain biodiesel, not only can be to avoid substantial amounts of maize straw be directly burned and causes environmental pollution, simultaneously
The energy crisis that a clean energy resource Regeneration Ways solution faces now can be opened up again.
The content of the invention
The shortcomings that in order to overcome the prior art and shortcoming, primary and foremost purpose of the invention, which is to provide, a kind of utilizes yeast
The method that bacterium fermented maize core produces biodiesel；This method is micro- by using the saccharomycetes to make fermentation corncob production of high Lipid-producing
Bio oil has not only reached waste utilization, the purpose of environmental protection to obtain biodiesel, and can reduce production cost, to be real
Now large-scale production biodiesel lays the foundation.
Another object of the present invention is to provide biodiesel made of a kind of above method.
The purpose of the present invention is realized by following technical proposals：
A kind of method using saccharomycetes to make fermentation corncob production biodiesel, this method include step in detail below：
S1. by corncob cutting and grinding to 30-40 mesh, 100g corncob powders are weighed, adding 1ml mass concentrations is
0.5%~1.5% dilute hydrochloric acid is pre-processed, and adds in distilled water to adjust the pH value of mixture to 5.0~5.5；
S2. MgCl is added in into mixed liquor obtained by step S12, tween, cellulase and xylan enzyme solution, obtain corncob
Mixed liquor；
S3. it is saccharified to corncob mixed liquor, after saccharification, saccharified liquid is filtered with ordinary filter paper, filtrate is taken to be obtained after sterilizing
It is spare to corncob saccharified liquid；
S4. the oil-producing saccharomyces oleaginosus strain of Cord blood is connected in solid slope culture medium, cultivates 72h in 32 DEG C, obtain
Activated spawn；
S5. above-mentioned activated spawn is accessed in liquid seed culture medium, in 25 DEG C~35 DEG C, 140r/min shaking table cultures 18
~for 24 hours, obtain saccharomycete liquid seeds；
S6. above-mentioned gained saccharomycete liquid seeds are transferred in corncob saccharified liquid and carry out production fat fermented and cultured, inoculation
It measures as the 5%~15% of corncob saccharification liquid quality；
S7. extracted using acid heat organic solvent method in the zymotic fluid obtained by the above-mentioned fermented culture of saccharomycete liquid seeds
Intracellular grease；
S8. using the microbial grease obtained by above-mentioned extraction as raw material, further using preparing biodiesel by ester interchange.
Pretreatment is to pre-process 60min under the conditions of 121 DEG C described in step S1；What the distilled water and process pre-processed
The liquid of corncob powder consolidates mass ratio as 10:1.
MgCl described in step S22Concentration in corncob mixed liquor is 2.5nmol/L, the addition of the Tween 80
It is to add 1.5ml Tween 80s according to every liter of corncob mixed liquor；The addition of the cellulase is mixed according to every 100g corncobs
It closes liquid and adds 2.5g cellulases；The addition of the xylan enzyme solution is to add 2ml xylans according to every 100g corncobs mixed liquor
Enzyme solution；The cellulose enzyme activity is 1.76 × 106u/ml；The xylan enzyme solution enzyme activity is 2.07 × 106u/ml。
Corncob mixed liquor described in step S3 is the 48h that is saccharified under conditions of temperature is 50~55 DEG C；The filtrate is
It is spare after sterilizing 20min under 121 DEG C of temperature conditionss.
Barms described in step S4 is Trichosporon pullulans, oil-producing saccharomyces oleaginosus, trichosporon cutaneum, fermentation sex pilus
Spore yeast or Rhodotorula glutinus.
Rotating speed is 130~150r/min to the condition of production fat fermented and cultured described in step S6 in order to control, and cultivation temperature is
25 DEG C~35 DEG C, incubation time is 3~5d.
It is extracted described in step S7 specifically according to following steps：Zymotic fluid is centrifuged, washes centrifugal sediment three times, every gram
Wet thallus adds in the hydrochloric acid that 10mL concentration is 4mol/L, and vibration shakes up, after being placed at room temperature for half an hour, boiling water bath 10min, and -20 DEG C
Quickly cooling 30min；Volume ratio is added as 1：The mixed liquor 20mL vibrations of 1 methanol and chloroform shake up, 4000r/min centrifugations
10min extracts chloroform layer, repeats operation twice, merges the chloroform layer of extraction gained three times；In 105 DEG C, vacuum drying removes
Chloroform, drying to constant weight up to intracellular grease.
A kind of biodiesel being prepared according to above-mentioned method；The biodiesel is joined by gas-chromatography and one matter of gas
With the analysis of technology, main component is methyl myristate, methyl palmitate, octadecenoic acid methyl ester and Methyl Stearate.
Gained biodiesel of the invention is that saccharomycete utilizes the biological bavin that corncob saccharified liquid is carbon source, nitrogen source fermentation produces
Oil.
The present invention is had the following advantages compared with the prior art and effect：
(1) high Lipid-producing Yeast bacterium growth adaptability is strong used by, and growth and breeding is rapid, and growth cycle is short, and metabolism is lived
Power is strong, is easy to cultivate, and genetic engineering can be facilitated to improve；It is limited from place, not climate, season limit, it can be continuously extensive
Production, it is lower than labour needed for agricultural production grease；Abundant raw materials needed for growth, it is cheap, such as starch, carbohydrate, and
Microorganism can be using discarded object in agricultural and sideline product, food service industry and paper industry, in whey, molasses, waste saccharide liquid, Starch Production
The discarded object of generation, as generated in whey, molasses, waste saccharide liquid, Starch Production waste material waste liquid, sulfurous acid, paper pulp, wood saccharification
Liquid etc.；On the one hand waste utilization can be strengthened, be on the other hand conducive to environmental protection again.
(2) used in the discarded object in agricultural using corncob as the carbonaceous refuse of representative containing substantial amounts of lignocellulosic
Microbial grease is produced using saccharomycetes to make fermentation carbonaceous refuse and obtains biodiesel, it not only can be to avoid substantial amounts of corn stalk
Stalk is directly burned and causes environmental pollution, while can open up what a clean energy resource Regeneration Ways solution faced now again
Energy crisis.
(3) it is expected to substantially reduce its production cost using micro-organisms biodiesel, accelerates the production of biodiesel, promotees
It is widely applied into it and large-scale industrial production.Research on microorganism Lipid-producing can for solving Chinese society economy
, there is important meaning in the problems such as energy shortage of sustainable development, environmental degradation.
Specific embodiment
Further the present invention is illustrated below by the mode of specific embodiment and combination, but it is not intended that this
The limitation of invention protection domain, those skilled in the art make some nonessential changes and tune according to the content of foregoing invention
It is whole, it all belongs to the scope of protection of the present invention.
Embodiment 1
(1) by corncob cutting and grinding to 30 mesh, 100g corncob powders are weighed, are 0.5% adding in 1ml mass concentrations
Dilute hydrochloric acid, after pre-processing 60min under the conditions of 121 DEG C, add in distilled water and be adjusted to pH value as 5.0, distilled water and by pre-
The liquid of the corncob powder of processing consolidates mass ratio as 10:1.It is mixed to corncob and MgCl is added in thing liquid2, tween, fiber
Plain enzyme and xylan enzyme solution obtain corncob mixed liquor (MgCl2Concentration is 2.5nmol/L, and Tween 80 concentration is 1.5ml/L, is added
Enter cellulase solution 2.5g/100g and xylan enzyme solution 2ml/100g), be saccharified 48h under conditions of 50 DEG C, through ordinary filter paper mistake
Filter takes filtrate to obtain corncob saccharified liquid after sterilizing, spare.
(2) the oil-producing saccharomyces oleaginosus strain of Cord blood is connected in solid slope culture medium, cultivates 72h in 32 DEG C, obtain
Activated spawn；Activated spawn is accessed in liquid seed culture medium, in 25 DEG C, 140r/min shaking table culture 18h, obtains yeast liquid
Body seed.
(3) and then by above-mentioned gained saccharomycete liquid seeds it is transferred in corncob saccharified liquid and carries out production fat fermented and cultured,
Inoculum concentration is the 5% of corncob saccharification liquid quality.Rotating speed is controlled as 130r/min, cultivation temperature is 25 DEG C, incubation time 3d
Afterwards, zymotic fluid is obtained.
(4) intracellular in the zymotic fluid obtained by the above-mentioned fermented culture of saccharomycete seed is extracted using acid heat organic solvent method
Grease：Zymotic fluid is centrifuged, three times, every gram of wet thallus adds in the hydrochloric acid of 10mL 4mol/L to washing centrifugal sediment, and vibration is shaken
It is even, after being placed at room temperature for half an hour, boiling water bath 10min, -20 DEG C of quickly cooling 30min.Add in the mixed liquor 20mL (bodies of methanol and chloroform
Product is than being 1：1) vibration shakes up, and 4000r/min centrifugation 10min extract chloroform layer.The mixed liquor of methanol and chloroform is added, weight
Multiple above-mentioned steps twice, merge the chloroform layer of extraction gained three times；In 105 DEG C, vacuum drying removes chloroform, and drying to constant weight i.e.
Obtain microbial grease.
(5) using the microbial grease obtained by above-mentioned extraction as raw material, further using preparing biodiesel by ester interchange.
Embodiment 2
(1) by corncob cutting and grinding to 35 mesh, 100g corncob powders are weighed, are adding in 1ml mass concentrations
0.75% dilute hydrochloric acid after pre-processing 60min under the conditions of 121 DEG C, adds in distilled water and is simultaneously adjusted to pH5.4, distilled water and process
The liquid of the corncob powder of pretreatment consolidates mass ratio as 10:1.It is mixed to corncob and MgCl is added in thing liquid2, tween, fibre
The plain enzyme of dimension and xylan enzyme solution, obtain corncob mixed liquor (MgCl2Concentration is 2.5nmol/L, and Tween 80 concentration is 1.5ml/L,
Add in cellulase solution 2.5g/100g and xylan enzyme solution 2ml/100g), be saccharified 48h under conditions of 53 DEG C, through ordinary filter paper
Filtering takes filtrate to obtain corncob saccharified liquid after sterilizing, spare.
(2) the oil-producing saccharomyces oleaginosus strain of Cord blood is connected in solid slope culture medium, cultivates 72h in 32 DEG C, obtain
Activated spawn；Activated spawn is accessed in liquid seed culture medium, in 32 DEG C, 140r/min shaking table culture 22h, obtains yeast liquid
Body seed.
(3) and then by above-mentioned gained saccharomycete liquid seeds it is transferred in corncob saccharified liquid and carries out production fat fermented and cultured,
Inoculum concentration is the 10% of corncob saccharification liquid quality.Rotating speed is controlled as 140r/min, cultivation temperature is 25 DEG C~35 DEG C, during culture
Between after 4d, to obtain zymotic fluid.
(4) intracellular in the zymotic fluid obtained by the above-mentioned fermented culture of saccharomycete seed is extracted using acid heat organic solvent method
Grease：Zymotic fluid is centrifuged, three times, every gram of wet thallus adds in the hydrochloric acid of 10mL 4mol/L to washing centrifugal sediment, and vibration is shaken
It is even, after being placed at room temperature for half an hour, boiling water bath 10min, -20 DEG C of quickly cooling 30min.Add in the mixed liquor 20mL (bodies of methanol and chloroform
Product is than being 1：1) vibration shakes up, and 4000r/min centrifugation 10min extract chloroform layer.The mixed liquor of methanol and chloroform is added, weight
Multiple above-mentioned steps twice, merge the chloroform layer of extraction gained three times.In 105 DEG C, vacuum drying removes chloroform, and drying to constant weight i.e.
Obtain microbial grease.
(5) using the microbial grease obtained by above-mentioned extraction as raw material, further using preparing biodiesel by ester interchange.
Embodiment 3
(1) by corncob cutting and grinding to 40 mesh, 100g corncob powders are weighed, are 1% adding in 1ml mass concentrations
Dilute hydrochloric acid after pre-processing 60min under the conditions of 121 DEG C, adds in distilled water and is simultaneously adjusted to pH5.5, distilled water and by pretreatment
The liquid of corncob powder consolidates mass ratio as 10:1.It is mixed to corncob and MgCl is added in thing liquid2, tween, cellulase and
Xylan enzyme solution obtains corncob mixed liquor (MgCl2Concentration is 2.5nmol/L, and Tween 80 concentration is 1.5ml/L, adds in fiber
Plain enzyme solution 2.5g/100g and xylan enzyme solution 2ml/100g), be saccharified 48h under conditions of 55 DEG C, filters, takes through ordinary filter paper
Corncob saccharified liquid is obtained after filtrate sterilizing, it is spare.
(2) the oil-producing saccharomyces oleaginosus strain of Cord blood is connected in solid slope culture medium, cultivates 72h in 32 DEG C, obtain
Activated spawn；By activated spawn access liquid seed culture medium in, in 35 DEG C, 140r/min shaking table cultures for 24 hours, obtain yeast liquid
Body seed.
(3) and then by above-mentioned gained saccharomycete liquid seeds it is transferred in corncob saccharified liquid and carries out production fat fermented and cultured,
Inoculum concentration is the 15% of corncob saccharification liquid quality.Rotating speed is controlled as 150r/min, cultivation temperature is 35 DEG C, incubation time 5d
Afterwards, zymotic fluid is obtained.
(4) intracellular in the zymotic fluid obtained by the above-mentioned fermented culture of saccharomycete seed is extracted using acid heat organic solvent method
Grease：Zymotic fluid is centrifuged, three times, every gram of wet thallus adds in the hydrochloric acid of 10mL 4mol/L to washing centrifugal sediment, and vibration is shaken
It is even, after being placed at room temperature for half an hour, boiling water bath 10min, -20 DEG C of quickly cooling 30min.Add in the mixed liquor 20mL (bodies of methanol and chloroform
Product is than being 1：1) vibration shakes up, and 4000r/min centrifugation 10min extract chloroform layer.The mixed liquor of methanol and chloroform is added, weight
Multiple above-mentioned steps twice, merge the chloroform layer of extraction gained three times.In 105 DEG C, vacuum drying removes chloroform, and drying to constant weight i.e.
Obtain microbial grease.
(5) using the microbial grease obtained by above-mentioned extraction as raw material, further using preparing biodiesel by ester interchange.
The small molecular sugar conversion ratio of fermented maize core powder production is up to 85% in embodiment 1-3；Saccharomycete utilizes
Corncob saccharified liquid carries out submerged fermentation, and the fat content of generation may be up to 45%, and grease yield is up to 13g/L, oil-producing conversion
Rate is up to 10.9%.Analysis by the one matter joint technology of gas-chromatography and gas of bio-oil prepared in the present invention, it was demonstrated that
Its main component is tetradecylic acid (myristic acid) methyl esters, hexadecylic acid (palm fibre puts acid) methyl esters, octadecenoic acid (oleic acid) methyl esters and ten
Eight sour (stearic acid) methyl esters, and the main component of these all biodiesel.
The main problem of biodiesel is of high cost, and cost is feedstock oil cost in biodiesel preparation experiment.Using
Cheap raw material and raising conversion ratio are the key that can biodiesel practical.It is beautiful with the agriculture waste of abundance in the present invention
Rice core stalk produces biodiesel for raw material, compared to the mode for the production biodiesel that is converted using animal and plant fat, the present invention
The cost of raw material can be greatly reduced；Raw material sources are extensive, can save substantial amounts of labour, need not occupy cultivated land, warp
It helps at low cost；Substantial amounts of corncob stalk is avoided directly to be burned and cause environmental pollution, while one can be opened up again clearly
The energy crisis that clean energy regeneration approach solution faces now.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (9)
- A kind of 1. method using saccharomycetes to make fermentation corncob production biodiesel, it is characterised in that：This method includes following tool Body step：S1. by corncob cutting and grinding to 30-40 mesh, 100g corncob powders are weighed, add 1ml mass concentrations as 0.5% ~1.5% dilute hydrochloric acid is pre-processed, and adds in distilled water to adjust the pH value of mixture to 5.0~5.5；S2. MgCl is added in into mixed liquor obtained by step S12, tween, cellulase and xylan enzyme solution, obtain corncob mixing Liquid；S3. it is saccharified to corncob mixed liquor, after saccharification, saccharified liquid is filtered with ordinary filter paper, filtrate is taken to obtain jade after sterilizing Rice core saccharified liquid, it is spare；S4. the oil-producing saccharomyces oleaginosus strain of Cord blood is connected in solid slope culture medium, cultivates 72h in 32 DEG C, must activate Strain；S5. by above-mentioned activated spawn access liquid seed culture medium in, in 25 DEG C~35 DEG C, 140r/min shaking table cultures 18~ For 24 hours, saccharomycete liquid seeds are obtained；S6. above-mentioned gained saccharomycete liquid seeds are transferred in corncob saccharified liquid and carry out production fat fermented and cultured, inoculum concentration is The 5%~15% of corncob saccharification liquid quality；S7. the intracellular in the zymotic fluid obtained by the above-mentioned fermented culture of saccharomycete liquid seeds is extracted using acid heat organic solvent method Grease；S8. using the microbial grease obtained by above-mentioned extraction as raw material, further using preparing biodiesel by ester interchange.
- 2. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：Pretreatment is to pre-process 60min under the conditions of 121 DEG C described in step S1；The distilled water and the corn by pretreatment The liquid of core powder consolidates mass ratio as 10:1.
- 3. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：MgCl described in step S22Concentration in corncob mixed liquor is 2.5nmol/L, the addition of the Tween 80 be by Add 1.5ml Tween 80s according to every liter of corncob mixed liquor；The addition of the cellulase is according to every 100g corncobs mixed liquor Add 2.5g cellulases；The addition of the xylan enzyme solution is to add 2ml xylan enzyme solutions according to every 100g corncobs mixed liquor； The cellulose enzyme activity is 1.76 × 106u/ml；The xylan enzyme solution enzyme activity is 2.07 × 106u/ml。
- 4. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：Corncob mixed liquor described in step S3 is the 48h that is saccharified under conditions of temperature is 50~55 DEG C；The filtrate is 121 It sterilizes under DEG C temperature conditionss spare after 20min.
- 5. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：Barms described in step S4 is Trichosporon pullulans, oil-producing saccharomyces oleaginosus, trichosporon cutaneum, fermentation sex pilus spore ferment Female or Rhodotorula glutinus.
- 6. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：Described in step S6 production fat fermented and cultured condition in order to control rotating speed be 130~150r/min, cultivation temperature for 25 DEG C~ 35 DEG C, incubation time is 3~5d.
- 7. a kind of method using saccharomycetes to make fermentation corncob production biodiesel according to claim 1, feature exist In：It is extracted described in step S7 specifically according to following steps：Zymotic fluid is centrifuged, washes centrifugal sediment three times, every gram of wet bacterium Body adds in the hydrochloric acid that 10mL concentration is 4mol/L, and vibration shakes up, after being placed at room temperature for half an hour, boiling water bath 10min, and -20 DEG C of quickly coolings 30min；Volume ratio is added as 1：The mixed liquor 20mL vibrations of 1 methanol and chloroform shake up, 4000r/min centrifugation 10min, extraction Chloroform layer is taken, repeats operation twice, merges the chloroform layer of extraction gained three times；In 105 DEG C, vacuum drying removes chloroform, dries It does to constant weight up to intracellular grease.
- 8. a kind of biodiesel being prepared according to claim 1-7 any one of them methods.
- 9. biodiesel according to claim 8, it is characterised in that：The biodiesel is joined by gas-chromatography and one matter of gas With the analysis of technology, main component is methyl myristate, methyl palmitate, octadecenoic acid methyl ester and Methyl Stearate.
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Cited By (2)
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|---|---|---|---|---|
| CN108795997A (en) * | 2018-06-11 | 2018-11-13 | 南京理工大学 | The method for producing microbial grease with maize straw acid processing hydrolyzate |
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| CN108865448A (en) * | 2018-07-28 | 2018-11-23 | 钱万琦 | A kind of preparation method of biodiesel |
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