CN104404088A - Method for preparing ethanol by performing alkaline pretreatment, enzymolysis and fermentation on forestry waste acorn shells - Google Patents

Method for preparing ethanol by performing alkaline pretreatment, enzymolysis and fermentation on forestry waste acorn shells Download PDF

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Publication number
CN104404088A
CN104404088A CN201410828344.1A CN201410828344A CN104404088A CN 104404088 A CN104404088 A CN 104404088A CN 201410828344 A CN201410828344 A CN 201410828344A CN 104404088 A CN104404088 A CN 104404088A
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acorn
fermentation
forestry waste
treatment
enzymatic hydrolysis
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杨静
卫民
张宁
蒋剑春
赵剑
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Institute of Chemical Industry of Forest Products of CAF
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Institute of Chemical Industry of Forest Products of CAF
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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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • 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
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • 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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  • Engineering & Computer Science (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for preparing ethanol by performing alkaline pretreatment, enzymolysis and fermentation on forestry waste acorn shells, which comprises the following steps: treating 0.75-2% (g/ml) alkali liquor respectively in a 60-DEG C water bath and a high-pressure steam sterilization pot at 121 DEG C (0.15 MPa) for 30-90 minutes; hydrolyzing with a composite enzyme with the capacity of 5-30 FPU/g pretreated raw material for 72 hours; and after the hydrolysis finishes, carrying out solid-liquid separation on the hydrolyzate, and fermenting the obtained clear solution with Pachysolen tannophilus to obtain the ethanol. By utilizing the agriculture and forestry waste to produce the ethanol fuel, the method changes the waste into valuable substances, solves the problem of the contradiction between fuel and human, can fully utilize the biomass resources, and protects the ecological environment, thereby being beneficial to implementing sustainable development.

Description

A kind of method of forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production
Technical field
The present invention relates to the preparation technology of alcohol fuel, be specifically related to the method for a kind of forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production
Background technology
For many years, a large amount of uses of traditional energy result in energy dilemma and environmental degradation.In order to realize the Sustainable development of economy and environment, renewable, free of contamination biomass energy has become current study hotspot.
Lignocellulose is the renewable energy source material that on the earth, quantity is maximum, and whole world annual output is approximately 10,000,000,000 tons according to the preliminary statistics.Utilize agriculture and forestry organic waste material as raw material production ethanol, realize the higher value application of biomass resource, be conducive to improving the ecological environment, meet the requirement of recycling economy development, the income of peasant can also be increased.
Acorn nut is the fruit of Fagaceae Quercus acutissima genus and evergreen sweet oak evergreen chinquapin class wild plant Oak Tree.The whole world has 8 genus more than 900 to plant, and be distributed in temperate zone, subtropics and the torrid zone, China accounts for 6 genus 301 kinds.There is abundant acorn nut resource in China, and acorn nut woods about has (1.3 ~ 1.67) × 10 7hm 2, 60 ~ 7,000,000,000 kilograms of acorn nuts can be produced every year.Acorn kernel both can be used as animal-feed, industrial raw material, also can be used to processing health food, and consequent acorn cup output is large, source is wide, price is low, but cannot well be utilized, not only contaminate environment but also cause energy dissipation.
Natural cellulose class material contains xylogen, Mierocrystalline cellulose and hemicellulose, and cellulosic effective hydrolysis, by the spatial obstacle of xylogen and the obstruction of the high-crystallinity of Mierocrystalline cellulose own and the polymerization degree, therefore, must carry out pre-treatment to raw material for hydrocellulose.Conventional pretreated method mainly contains Physical, chemical method, biological process and combined pretreatment method etc.Alkaline process pre-treatment effectively can remove the xylogen in raw material by ester bond crosslinked between destruction xylogen and xylan, comparatively acid system is comparatively gentle, and in treating processes, the loss of sugar is less, and final pretreating effect is better.After diluted alkaline process, fibrous material expands, and interior surface area increases, and the polymerization degree reduces, and degree of crystallinity declines, thus improves hydrolysis efficiency.
Summary of the invention
The object of this invention is to provide the method that a kind of acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation produces ethanol, acorn cup is turned waste into wealth, reduces the production cost of alcohol fuel, improve the economy of the production process of alcohol fuel.
Technical scheme of the present invention is: a kind of method of forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production, and comprise cellulase hydrolysis and yeast fermentation, concrete steps are as follows:
(1) acorn cup alkalescence pre-treatment: carry out alkaline purification with alkali lye to acorn-case powder, temperature condition is respectively 30 ~ 90 DEG C of water bath processing 30min ~ 90min, and processes 30min ~ 90min under 121 DEG C/0.15Mpa;
(2) cellulase hydrolysis: the acorn-case powder good by pre-treatment, adds the cellulase of 5 ~ 30FPU and the zytase of beta-glucosidase and 1.5FXU according to every gram of pretreating raw material, and in 45 ~ 60 DEG C, 100 ~ 150r/min is hydrolyzed 48 ~ 72h;
(3) yeast fermentation: by hydrolyzate centrifugation after enzymatic hydrolysis reaction terminates, after adding nutritive salt in clear liquid, in 28 ~ 32 DEG C after inoculation Pachysolen tannophilus, rotating speed 100 ~ 150r/min, fermentation 48 ~ 72h.
Acorn-case powder used is the air-dry rear shelling of acorn nut of picking up from Anhui Chuzhou, gets acorn cup oven dry pulverizing and obtains.
The ratio adding 1g acorn-case powder according to every 10ml alkali lye during pre-treatment feeds intake.
Described cellulase and beta-glucosidase are that 1 ︰ 1 mixes according to molar ratio.
During cellulase hydrolysis, controlling enzymic hydrolysis concentration of substrate is 5%g/ml.
Described nutritive salt consists of yeast extract 2g/L, (NH 4) 2sO 410g/L, KH 2pO 42g/L, MgSO 42g/L.
During fermentation, the inoculum size of Pachysolen tannophilus is 8%v/v.
Described acorn-case powder particle diameter is 0.25 ~ 0.3mm.
Described alkali lye is the aqueous sodium hydroxide solution of 0.75% ~ 2%g/ml.
Beneficial effect: the inventive method with forestry waste acorn cup for raw material, after raw materials pretreatment and cellulase saccharification, utilize Pachysolen tannophilus producing fuel ethyl alcohol by ferment, reduce the cost of fuel ethanol production process, improve the economy of the production process of alcohol fuel.The inventive method utilizes agriculture and forestry organic waste material to produce alcohol fuel, turns waste into wealth, and solves the problem that fuel and people strive grain, while can make full use of biomass resource, preserve the ecological environment, be conducive to realizing Sustainable development.
Embodiment
Below in conjunction with embodiment the present invention done and further explain.Should be appreciated that following examples are only intended to illustrate, should not be regarded as limitation of the scope of the invention.
The method of acorn cup producing fuel ethyl alcohol by ferment of the present invention comprises following content, the pre-treatment of acorn cup alkalescence, cellulase hydrolysis and yeast fermentation steps.
In the inventive method, alkaline pre-treatment is carried out to acorn cup, with NaOH, pre-treatment is carried out to acorn-case powder (0.25 ~ 0.3mm), NaOH concentration is 0.75% ~ 2% (g/ml), temperature is 30 ~ 90 DEG C (water-baths) and 121 DEG C/0.15Mpa (high-pressure steam sterilizing pan), and the time is 30min ~ 90min;
In the inventive method, cellulase hydrolysis method is, by pretreated for NaOH acorn-case powder, adds the cellulase of 5 ~ 30FPU/ gram of pretreating raw material and the zytase of beta-glucosidase and 1.5FXU, in 45 ~ 60 DEG C, 100 ~ 150r/min is hydrolyzed 48 ~ 72h;
In the inventive method, by hydrolyzate centrifugation after enzymatic hydrolysis reaction terminates, add yeast extract 2g/L, ammonium sulfate 10g/L, potassium primary phosphate 2g/L, magnesium sulfate 2g/L is mixed with fermentation culture, and inoculation 8% (v/v) Pachysolen tannophilus (Pachysolentannophilus 1770) is in 28 ~ 32 DEG C, rotating speed 100 ~ 150r/min, fermentation 48 ~ 72h.Yeast starter is the logarithmic phase yeast culture liquid of PDA (potato glucose) culture medium culturing.
The method of acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production of the present invention, compared with steam explosion, alkaline purification utilizes xylogen can be dissolved in the feature of basic solution, and lignin structure is destroyed, improve the clearance of xylogen, thus be convenient to the carrying out of sequential hydrolysis.
The present invention's acorn cup tannin content used is higher, and Pachysolen tannophilus be a kind of can the bacterial classification of simultaneously zymohexose and wood sugar, tannin residual in hydrolyzed solution can be tolerated, traditional bacterial classifications such as yeast saccharomyces cerevisiae of comparing simultaneously, be more suitable for the fermentation containing tannin raw material.
In following examples, the content of various sugar and ethanol all adopts high performance liquid chromatography (HPLC) to measure.Chromatographic condition is as follows: chromatographic instrument: Agillent1100 high performance liquid chromatograph; Chromatographic column: Bio-Rad Aminex HPX-87H; Moving phase: 0.005mol/L sulfuric acid, flow velocity: 0.6mL/min; Column temperature: 55 DEG C; Tester: differential refraction tester; Sample size: 10 μ L.External standard method.
Pretreatment condition is on the impact of acorn cup enzymic hydrolysis effect
Embodiment 1
By acorn cup 105 DEG C oven dry, pulverize and sieve the acorn-case powder of 0.25 ~ 0.3mm is for subsequent use with pulverizer.With the NaOH process acorn cup that concentration is 0.75% (g/ml), solid-to-liquid ratio 1 ﹕ 10 (g/ml), processes 30min in 60 DEG C of water-baths.After process terminates, with distilled water, raw material is washed till neutrality, filter, gained fraction solids pretreating raw material treats enzymic hydrolysis in-20 DEG C of preservations, gets partially desiccated and measures acid insoluble lignin content and calculate solid recovery rate.Take a certain amount of sample, add 0.1M, the citrate buffer solution of pH4.8, solid-to-liquid ratio is 5% (g/ml), adds enzyme, and carrying capacity is 20FPU cellulase, 20CBU beta-glucosidase and 1.5FXU xylosidase every gram of pretreating raw material, in 50 DEG C, 150r/min is hydrolyzed 72h.After end, stop enzymic hydrolysis with ice cube immediately, filter, carry out efficient liquid phase chromatographic analysis.
Result shows, under 60 DEG C/0.75%NaOH/30min pretreatment condition, the solid recovery rate of acorn cup is 82.09%, and the degradation rate of acid insoluble lignin is 12.77%, and total reducing sugar burst size is 504.9mg/g.
Embodiment 2
By acorn cup 105 DEG C oven dry, pulverize and sieve the acorn-case powder of 0.25 ~ 0.3mm is for subsequent use with pulverizer.With the NaOH process acorn cup that concentration is 1% (g/ml), solid-to-liquid ratio 1 ﹕ 10 (g/ml), processes 30min in 121 DEG C of water-baths.After process terminates, with distilled water, raw material is washed till neutrality, filter, gained fraction solids pretreating raw material treats enzymic hydrolysis in-20 DEG C of preservations, gets partially desiccated and measures acid insoluble lignin content and calculate solid recovery rate.Take a certain amount of sample, add 0.1M, the citrate buffer solution of pH 4.8, solid-to-liquid ratio is 5% (g/ml), adds enzyme, and carrying capacity is 20FPU cellulase, 20CBU beta-glucosidase and 1.5FXU xylosidase every gram of pretreating raw material, in 50 DEG C, 150r/min is hydrolyzed 72h.After end, stop enzymic hydrolysis with ice cube immediately, filter, carry out efficient liquid phase chromatographic analysis.
Result shows, under 121 DEG C/1%NaOH/30min pretreatment condition, the solid recovery rate of acorn cup is 69.55%, and the degradation rate of acid insoluble lignin is 28.00%, and total reducing sugar burst size is 588mg/g.
Embodiment 3
By acorn cup 105 DEG C oven dry, pulverize and sieve the acorn-case powder of 0.25 ~ 0.3mm is for subsequent use with pulverizer.With the NaOH process acorn cup that concentration is 2% (g/ml), solid-to-liquid ratio 1 ﹕ 10 (g/ml), processes 60min in 121 DEG C of water-baths.After process terminates, with distilled water, raw material is washed till neutrality, filter, gained fraction solids pretreating raw material treats enzymic hydrolysis in-20 DEG C of preservations, gets partially desiccated and measures acid insoluble lignin content and calculate solid recovery rate.Take a certain amount of sample, add 0.1M, the citrate buffer solution of pH4.8, solid-to-liquid ratio is 5% (g/ml), adds enzyme, and carrying capacity is 20FPU cellulase, 20CBU beta-glucosidase and 1.5FXU xylosidase every gram of pretreating raw material, in 50 DEG C, 150r/min is hydrolyzed 72h.After end, stop enzymic hydrolysis with ice cube immediately, filter, carry out efficient liquid phase chromatographic analysis.
Result shows, under 121 DEG C/1%NaOH/30min pretreatment condition, the solid recovery rate of acorn cup is 62.94%, and the degradation rate of acid insoluble lignin is 39.34%, and total reducing sugar burst size is 606.36mg/g.
Glucose 40.25g/L is consisted of, wood sugar 0.91g/L, pectinose 0.02g/L in this hydrolyzed solution of liquid chromatogram measuring.Add yeast extract 2g/L, ammonium sulfate 10g/L, potassium primary phosphate 2g/L, magnesium sulfate 2g/L, 121 DEG C of sterilizing 30min, as fermentation culture.According to Pachysolen tannophilus kind liquid amass account for fermentation culture volume 8% inoculum size (v/v) inoculation.Culture condition is 32 DEG C, rotating speed 120r/min, and fermentation time is 60h, and after fermentation ends, filtering fermentating liquid, mensuration alcohol concn is 17.46g/L, is 85.4% of theoretical yield, reaches good effect.

Claims (10)

1. a method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production, it is characterized in that, comprise cellulase hydrolysis and yeast fermentation, concrete steps are as follows:
(1) acorn cup alkalescence pre-treatment: carry out alkaline purification with alkali lye to acorn-case powder, temperature condition is respectively 30 ~ 90 DEG C of water bath processing 30min ~ 90min, and processes 30 min ~ 90min under 121 DEG C/0.15 Mpa;
(2) cellulase hydrolysis: the acorn-case powder good by pre-treatment, adds the cellulase of 5 ~ 30FPU and the zytase of beta-glucosidase and 1.5FXU according to every gram of pretreating raw material, and in 45 ~ 60 DEG C, 100 ~ 150 r/min are hydrolyzed 48 ~ 72 h;
(3) yeast fermentation: by hydrolyzate centrifugation after enzymatic hydrolysis reaction terminates, after adding nutritive salt in clear liquid, in 28 ~ 32 DEG C after inoculation Pachysolen tannophilus, rotating speed 100 ~ 150r/min, fermentation 48 ~ 72 h.
2., according to the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that acorn-case powder used is the air-dry rear shelling of acorn nut of picking up from Anhui Chuzhou, get acorn cup oven dry pulverizing and obtain.
3. the method for forestry waste acorn cup according to claim 1 alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production, the ratio adding 1g acorn-case powder according to every 10ml alkali lye when it is characterized in that pre-treatment feeds intake.
4. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, described cellulase and beta-glucosidase are that 1 ︰ 1 mixes according to molar ratio.
5. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, during cellulase hydrolysis, controlling enzymic hydrolysis concentration of substrate is 5%g/ml.
6. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, described nutritive salt consists of yeast extract 2g/L, (NH 4) 2sO 410g/L, KH 2pO 42g/L, MgSO 42g/L.
7. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, during fermentation, the inoculum size of Pachysolen tannophilus is 8%v/v.
8. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, Pachysolen tannophilus kind liquid is the logarithmic phase yeast culture liquid of PDA culture medium culturing.
9. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, described acorn-case powder particle diameter is 0.25 ~ 0.3mm.
10. the method for forestry waste acorn cup alkalescence pre-treatment enzymatic hydrolysis and fermentation ethanol production according to claim 1, it is characterized in that, described alkali lye is the aqueous sodium hydroxide solution of 0.75% ~ 2% g/ml.
CN201410828344.1A 2014-12-26 2014-12-26 Method for preparing ethanol by performing alkaline pretreatment, enzymolysis and fermentation on forestry waste acorn shells Pending CN104404088A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009015481A1 (en) * 2007-08-02 2009-02-05 Iogen Energy Corporation Cellulase enzyme based method for the production of alcohol and glucose from pretreated lignocellulosic feedstock
CN103305556A (en) * 2013-07-03 2013-09-18 中国林业科学研究院林产化学工业研究所 Method for producing ethanol by fermenting steam explosion quercus acutissima carr

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009015481A1 (en) * 2007-08-02 2009-02-05 Iogen Energy Corporation Cellulase enzyme based method for the production of alcohol and glucose from pretreated lignocellulosic feedstock
CN103305556A (en) * 2013-07-03 2013-09-18 中国林业科学研究院林产化学工业研究所 Method for producing ethanol by fermenting steam explosion quercus acutissima carr

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杨静等: "稀碱法预处理橡子壳制备生物乙醇", 《食品与发酵工业》 *

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