CN106755131A - A kind of method that industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol - Google Patents
A kind of method that industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol 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
- C12P7/02—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
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- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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Abstract
The invention discloses the method that a kind of industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol.The method is comprised the following steps：Ammonia-catalyzed steam blasting pretreatment is carried out to discarded sugarcane marrow first, solid content is collected by filtration, then synchronized saccharification common fermentation and obtain ethanol.The present invention can effectively remove the lignin of sugarcane marrow using the pretreatment of ammonia-catalyzed steam blasting, so as to improve the enzymolysis efficiency of raw material.Meanwhile, the pretreated raw material of ammonia-catalyzed steam blasting remains most hemicellulose, and these hemicelluloses are converted into fermentable monose in enzymolysis process, so as to improve the utilization rate of sugarcane marrow raw material.Additionally, the present invention makes yeast maintain activity higher so as to improve the yield of ethanol using gradient increased temperature, the alternate SSCF technologies of good anaerobism.
Description
Technical field
Pretreatment and fermentation arts the present invention relates to cellulosic ethanol production, more particularly, to a kind of industrial waste sugarcane marrow
The method that high efficiency synchronous saccharification common fermentation produces ethanol.
Background technology
It is used for part using lignocellulose raw material transforming fuel alcohol and substitutes increasingly exhausted fossil energy, energy can be alleviated
The problems such as source crisis, environmental pollution.For at present, cellulosic ethanol production technique includes：The pretreatment of raw material, pretreated feedstock
The step such as detoxification, enzyme hydrolysis saccharification of cellulose, alcohol fermentation, ethanol rectification and purification.Plant in nature enters in long-term
The extremely complex supermolecule being made up of cellulose, hemicellulose, lignin and a small amount of protein etc. is formd during change
Structure, to chemicals and enzyme etc. with natural biological repellence.Therefore, this biological repellence has had a strong impact on bioconversion
During the infiltration of liquid, the contact of biology enzyme and catalysis activity, ultimately cause poor biological transformation ratio.Cellulosic material it is pre-
Treatment is intended to efficiently separate and destroy combination fine and close between cellulose, hemicellulose and lignin, and removing lignin retains hemicellulose,
The crystallinity of cellulose is reduced, the porosity of raw material is improved, so as to improve the enzymolysis efficiency of raw material.
At present, the preprocess method of cellulosic material is divided into Physical, chemical method, physical-chemical method and bioanalysis.To agriculture
For industry discarded object, steam blasting pretreatment is the pretreatment mode for being best suitable for and most having industrial prospect at present.Steam blasting
Pretreatment be fibrous raw material under high temperature (160-270 DEG C), high pressure (0.69-4.83Mpa) boiling for a period of time, high steam lead to
Cross diffusion to enter inside wood fibre cell membrane, weaken the fastness of fibre structure, and by quick pressure of rushing down to reach
The effect of explosion.Additionally, research finds that steam blasting is in water, acid or alkali steeping are combined and are greatly improved preprocessing process
The utilization rate of raw material and the action effect of steam blasting.
Simultaneous saccharification and fermentation technique refers to lignocellulosic saccharification and fermentation process while carrying out, cellulosic material is in fiber
In the presence of plain enzyme it is glucogenic simultaneously, microorganism is fermented immediately and is converted into ethanol.This technique reduces fibre
Product in the plain enzymolysis process of dimension suppresses, so as to improve the efficiency of cellulase.Therefore, SSF is compared with SHF, with higher
Ethanol production.But, the shortcoming of the technique is that cellulase, zytase and saccharomycete can not simultaneously each suitable
Worked under temperature and pH, saccharomycete is mixed with the biomass that are not partly saccharified, and weakens the activity of saccharomycete.
The content of the invention
It is an object of the invention to provide the method that a kind of industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol.
To reach above-mentioned purpose, the technical solution adopted by the present invention is：Ammonia-catalyzed steam is carried out to discarded sugarcane marrow first
Explosion is pre-processed, and solid content is collected by filtration, and is then synchronized saccharification common fermentation and is obtained ethanol.The present invention is steamed using ammonia-catalyzed
It is in order to effectively remove the lignin of sugarcane marrow, so as to improve the enzymolysis efficiency of raw material that steam explosion breaks pretreatment.Meanwhile, ammonia-catalyzed is steamed
Steam explosion is broken pretreated raw material and remains most hemicellulose, and these hemicelluloses are converted into enzymolysis process can be sent out
The monose of ferment, so as to improve the utilization rate of sugarcane marrow raw material.Additionally, the present invention is using gradient increased temperature, the good alternate SSCF of anaerobism
Technology makes yeast maintain activity higher so as to improve the yield of ethanol.
A kind of method that industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol, comprises the following steps：
(1) get the raw materials ready：The wet sugarcane marrow that factory discards is placed in into ventilation to air-dry, until mass dryness fraction reaches more than 90%；
(2) pre-preg：By pre-preg in step (1) gained sugarcane marrow and ammoniacal liquor closed wide-mouth bottle of the addition with stirring, obtain pre-
Maceration extract；
(3) steam blasting pretreatment：Locate steam blasting is carried out in pre-preg liquid addition Moveable steam demolition set in advance
Reason；
(4) separate：The pretreated solid of steam blasting and product liquid are separated using pulp washing bag；
(5) deaerate：Separating obtained solid content is placed in the fume hood with blower fan and is purged, the ammonia in removing solid content
Water is remained；
(6) air-dry：Step (5) gained solid content is placed in into outdoor ventilation to air-dry, until solid content mass dryness fraction reaches 90%
More than；
(7) synchronous saccharification common fermentation：Solid content, cushioning liquid, fermented nutritive liquid, cellulase, fiber after air-drying
Plain disaccharidase, zytase and yeast extract mix in adding fermentation tank, obtain mixed liquor；Synchronized at 30-50 DEG C again
Saccharification common fermentation, obtains ethanol.
Preferably, step (1) the industrial waste sugarcane marrow starches paper plant from Guangxi.
Preferably, the condition of step (2) described pre-preg is：Solid-liquid ratio is 1:3-1:5, the concentration of ammoniacal liquor is 30wt%,
Temperature is normal temperature, and the time is 6-12h, and speed of agitator is 150-250r/min.
Preferably, the condition of step (3) the steam blasting pretreatment is：Solid-liquid ratio is 1:3-1:5, reaction temperature is
140-170 DEG C, carrying pressure is 0.4-0.8Mpa, and the reaction time is 5-20min.
Preferably, the specification of step (4) the pulp washing bag is 80-120 meshes.
Preferably, the time of step (5) described purging is 30-60min.
Preferably, the condition of step (7) the synchronous saccharification common fermentation is：Solid content is 6-12%, and pH is 4.5-5.5,
Rotating speed is 150-250rpm, and fermentation time is 72-108h, and the consumption of the cellulase is 20-40FPU/g, cellobiose
The consumption of enzyme is 40-80CBU/g, and (g in " FPU/g ", " CBU/g ", " U/g " refers to for 10-20U/g for the consumption of zytase
For the quality of the sugarcane marrow of synchronous saccharification common fermentation).
Preferably, step (7) described fermented nutritive liquid is (NH4)2SO4、KH2PO4、MgSO4、CaCl2With the mixture of water；
(the NH4)2SO4、KH2PO4、MgSO4、CaCl2Concentration with yeast extract in mixed liquor is 0.5-1.5g/L (" g/L "
In L refer to synchronous saccharification common fermentation liquid cumulative volume, similarly hereinafter), 2-3g/L, 0.3-0.5g/L, 0.05-0.15g/L and 2-5g/
L。
Preferably, step (7) the synchronous saccharification common fermentation uses gradient increased temperature program, and concrete operations are：45-55 first
DEG C keep 24-36h, then drop to 37-40 DEG C holding 24-36h, be finally down to 28-30 DEG C holding 48-72h.The synchronous saccharification
Common fermentation uses alternately ventilation treatment, and concrete operations are：0-24h stuffiness, leads to nitrogen 5-10min after 24h, oxygen is led to after 48h
5-10min。
Preferably, step (7) described cushioning liquid is the citrate buffer solution of 50mmol/L；The cellulase is
Celluclast1.5L；The cellobiase is Novozyme 188.
Preferably, step (7) described yeast extract is zymohexose saccharomycete and pentose fermentation saccharomycete.
It is further preferred that the zymohexose saccharomycete is saccharomyces cerevisiae (CICC1416), using anaerobic fermentation；It is described
Pentose fermentation saccharomycete is shehatae candida (GIM2.175), using aerobic fermentation.
Compared with prior art, the present invention has the following technical effect that：
1st, the present invention is in order to effectively remove the lignin of sugarcane marrow, so as to improve using the pretreatment of ammonia-catalyzed steam blasting
The enzymolysis efficiency of raw material.Meanwhile, the pretreated raw material of ammonia-catalyzed steam blasting remains most hemicellulose, these
Hemicellulose is converted into fermentable monose in enzymolysis process, so as to improve the utilization rate of sugarcane marrow raw material.
2nd, the present invention makes yeast maintain activity higher so as to carry using gradient increased temperature, the alternate SSCF technologies of good anaerobism
The yield of ethanol high.
Brief description of the drawings
Fig. 1 is the XRD spectra analysis of sugarcane marrow before and after pretreatment.
Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d are respectively the FE-SEM images of sugarcane marrow before and after pretreatment.
Fig. 3 is the enzyme hydrolysis curve map of sugarcane marrow before and after pretreatment.
Fig. 4 is the synchronous saccharification common fermentation curve map of sugarcane marrow before and after pretreatment.
Specific embodiment
A kind of industrial waste sugarcane marrow high efficiency synchronous sugar of the invention is further illustrated below by way of the drawings and specific embodiments
Change the method that common fermentation produces ethanol.
Embodiment 1
(1) get the raw materials ready：The wet sugarcane marrows of 10kg that factory discards are placed in into ventilation to air-dry, until mass dryness fraction reaches more than 90%, this
The discarded wet sugarcane marrow of factory used by embodiment starches paper plant from Guangxi, and it is as shown in table 1 into being grouped into.
Table 1
(2) pre-preg：By step (1) gained sugarcane marrow (300g over dry) and concentration for 30wt% ammoniacal liquor add with stir
Pre-preg in closed wide-mouth bottle, obtains pre-preg liquid；
(3) steam blasting pretreatment：Steam is carried out during pre-preg liquid is added into Moveable steam demolition set (BL-08 types)
Explosion is pre-processed；
(4) separate：Pulp washing bag using 120 meshes is divided the pretreated solid of steam blasting and product liquid
From；
(5) deaerate：Separating obtained solid content is placed in 30min is purged in the fume hood with blower fan, remove solid content
In ammoniacal liquor residual；
(6) air-dry：Step (5) gained solid content is placed in into outdoor ventilation to air-dry, until solid content mass dryness fraction reaches 90%
More than；
The present embodiment is pre-processed with reference to steam explosion technology using ammonia dipping to sugarcane marrow raw material, and its pretreatment condition is such as
Shown in table 2.
Table 2
The present embodiment is analyzed using USDOE NREL methods to the component of sugarcane marrow raw material before and after pretreatment.Using from
Sub- chromatogram (Dionex ICS-5000) is analyzed to the saccharic composition of above-mentioned gained hydrolyzate.IC testing conditions are：Detector：
Dionex IonPacTMAG11-HC, chromatographic column：Dionex CarboPac PA20, mobile phase：2mmol/L NaOH, flow velocity：
0.5mL/min, eluent：200mmol/L NaOH and 1mol/L NaAC.The chemical composition analysis result of sugarcane marrow is such as after pretreatment
Shown in table 3.
Table 3
As shown in Table 3, pretreated sugarcane marrow #1, #2 and the reduction of #3 lignin contents, hemicellulose level compared with raw material
It is basically unchanged, this explanation ammonia-catalyzed steam blasting pretreatment effectively can remove wood while most of hemicellulose is retained
Element.This is primarily due to removing of the ammoniacal liquor to lignin during steam blasting and the protective effect to hemicellulose.Lignin
Removing is exposed more celluloses and hemicellulose, the accessibility of hydrolase is increased, so as to improve the enzyme of raw material
Solution efficiency.Additionally, during the reservation of hemicellulose makes more hemicelluloses participate in pentose fermentation, so as to substantially increase
The utilization rate of raw material.
The present embodiment is using x-ray diffractometer (D8ADVANCE, German Bruker companies) to sugarcane marrow before and after pretreatment
Crystalline texture is analyzed.XRD test conditions are：Cu targets, pipe pressure 40KV, electric current 40mA, sweep speed 35.4 seconds/step, step-length
0.04 degree, sweep limits 5-60 degree.Gained XRD spectrum is as shown in Figure 1.As seen from the figure, the He of pretreated sugarcane marrow 101Face
Diffraction maximum blending together, and the diffraction maximum in 040 face becomes strong.Additionally, the crystallinity of sugarcane marrow #1, #2 and #3 is all omited after pretreatment
Micro- to improve, the crystallinity of fibrous raw material is down to 48.4% by 43%.This explanation by ammonia-catalyzed steam blasting pretreatment after,
The noncrystalline domain of the cellulose of sugarcane marrow is destroyed so that the crystallinity of fibrous raw material slightly has raising.
The present embodiment is entered using MERLIN field emission scanning electron microscopes (FE-SEM) to the exterior appearance of sugarcane marrow before and after pretreatment
Row analysis.Its scanning electron microscopic picture is as shown in Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d.As seen from the figure, sugarcane marrow surface has been covered with big size
Lignin is filled with small stomata, and most stomatas.And sugarcane marrow surface goes out under the synergy of ammoniacal liquor dipping and steam blasting
Substantial amounts of slight crack and hole are showed.Further, since the delignification of ammoniacal liquor causes that the most stomata in sugarcane marrow surface is opened.
The present embodiment is poly- using cellulase (Celluclast 1.5L), cellobiase (Novozyme 188) and wood
Carbohydrase is digested to the sugarcane marrow before and after pretreatment.Enzyme hydrolysis condition is：Gu contain：2%, pH：4.8, cellulase consumption：
30FPU/g (g in " FPU/g ", " CBU/g ", " U/g " refers to the quality for the sugarcane marrow for digesting), cellobiose enzyme dosage：
60CBU/g, zytase consumption：15U/g, rotating speed：250rpm, reaction temperature：50 DEG C, reaction time：48h.
Fig. 3 is the enzymolysis curve of sugarcane marrow before and after pretreatment.In Fig. 3, solid line is the enzymolysis curve of glucan, and dotted line is poly- for wood
The enzymolysis curve of sugar.As seen from the figure, compared with raw material pretreated sugarcane marrow #1, #2 and #3 glucans and xylan conversion ratio
Significantly improve.Specifically, the conversion ratio of glucan and xylan brings up to 85.8% by original 25.2% and 9.8% respectively
With 83.3%.Also, the most violent sample #3 of steam blasting pretreatment condition has highest cellulose and hemicellulose
Conversion ratio.It follows that the preconditioning technique used by the embodiment of the present invention has extraordinary effect to sugarcane marrow.
(7) synchronous saccharification common fermentation：Solid content, cushioning liquid, nutrient solution, cellulase, cellulose two after air-drying
Carbohydrase, zytase and yeast extract synchronize saccharification common fermentation in adding fermentation tank, obtain ethanol；The yeast extract
For zymohexose saccharomycete is saccharomyces cerevisiae CICC1416 and shehatae candida GIM2.175 (mass ratioes 1:1).
The present embodiment is fermented using synchronous saccharification co-fermentation technology to the sugarcane marrow before and after pretreatment.Synchronous saccharification is sent out altogether
The condition of ferment is：Solid content：8%, pH：4.8, nutrient solution：1g/L(NH4)2SO4(L in " g/L " refers to synchronous saccharification common fermentation
The cumulative volume of liquid, similarly hereinafter), 2.5g/L KH2PO4, 0.4g/L MgSO4, and 0.1g/L CaCl2, cellulase consumption：30FPU/
G (g in " FPU/g ", " CBU/g ", " U/g " refers to the quality of the sugarcane marrow for synchronous saccharification common fermentation), cellobiose enzyme
Consumption：60CBU/g, zytase consumption：15U/g, yeast extract：2g/L, rotating speed：150rpm, fermentation temperature：30-50 DEG C,
Fermentation time：108h.Zymohexose saccharomycete used in the embodiment of the present invention is saccharomyces cerevisiae (CICC1416), pentose fermentation
Saccharomycete is shehatae candida (GIM2.175).Synchronous saccharification common fermentation uses gradient increased temperature program, and concrete operations are：It is first
First 50 DEG C of holding 24h, then drop to 40 DEG C of holding 24h, are finally down to 30 DEG C of holding 60h；The synchronous saccharification common fermentation is used
Replace ventilation treatment, concrete operations are：0-24h stuffiness, leads to nitrogen 5min after 24h, oxygen 5min is led to after 48h.
Fig. 4 is the leaven line chart of sugarcane marrow before and after pretreatment.In Fig. 4, the fermentation diagram of solid line representative sample #3, dotted line generation
The fermentation diagram of the untreated sugarcane marrow of table.As seen from the figure, add after saccharomyces cerevisiae (24h), the glucose of two groups of samples drastically declines,
The content of ethanol is gradually stepped up.And the content of xylose tends towards stability because the saccharomyces cerevisiae used by the present embodiment can not convert wood
Sugar.These xyloses are gradually consumed after shehatae candida (48h) is added.Can be obtained by MaterialBalance Computation, ammonia-catalyzed
The ethanol production of sugarcane marrow brings up to 223kg/t by original 64.6kg/t after steam blasting pretreatment.
It is above desirable embodiment of the invention, those skilled in the art completely can be without departing from this invention technological thought
In the range of, carry out various modifications and optimization.And these all of modifications and optimization are included in such as the claims in the present invention institute
Within the scope of restriction, without departing from the Spirit Essence for being included of the invention and required practical scope.
Claims (10)
1. a kind of method that industrial waste sugarcane marrow high efficiency synchronous saccharification common fermentation produces ethanol, it is characterised in that including following step
Suddenly：
（1）Get the raw materials ready：The wet sugarcane marrow that factory discards is placed in into ventilation to air-dry, until mass dryness fraction reaches more than 90%；
（2）Pre-preg：By step（1）Gained sugarcane marrow and ammoniacal liquor add pre-preg in the closed wide-mouth bottle with stirring, obtain pre-preg
Liquid；
（3）Steam blasting is pre-processed：Steam blasting pretreatment is carried out during pre-preg liquid is added into Moveable steam demolition set；
（4）Separate：The pretreated solid of steam blasting and product liquid are separated using pulp washing bag；
（5）Degassing：Separating obtained solid content is placed in the fume hood with blower fan and is purged, the ammoniacal liquor in removing solid content is residual
Stay；
（6）Air-dry：By step（5）Gained solid content is placed in outdoor ventilation and air-dries, until solid content mass dryness fraction reaches more than 90%；
（7）Synchronous saccharification common fermentation：Solid content, cushioning liquid, fermented nutritive liquid, cellulase, cellulose two after air-drying
Carbohydrase, zytase and yeast extract mix in adding fermentation tank, obtain mixed liquor；Saccharification is synchronized at 30-50 DEG C again
Common fermentation, obtains ethanol.
2. method according to claim 1, it is characterised in that：Step（2）The condition of the pre-preg is：Solid-liquid ratio is 1:
3-1:5, the concentration of ammoniacal liquor is 30wt%, and temperature is normal temperature, and the time is 6-12 h, and speed of agitator is 100-250 r/min.
3. method according to claim 1, it is characterised in that：Step（3）The condition of steam blasting pretreatment is：Material
Liquor ratio is 1:3-1:5, reaction temperature is 140-170 DEG C, and carrying pressure is 0.4-0.8 Mpa, and the reaction time is 5-20 min.
4. method according to claim 1, it is characterised in that：Step（7）The condition of the synchronous saccharification common fermentation is：Gu
Content is 6-12%, and pH is 4.5-5.5, and rotating speed is 150-250 rpm.
5. method according to claim 1, it is characterised in that：Step（7）The consumption of the cellulase is 20-40
FPU/g, the consumption of cellobiose enzyme is 40-80 CBU/g, and the consumption of zytase is 10-20 U/g.
6. method according to claim 1, it is characterised in that：Step（7）The synchronous saccharification common fermentation uses gradient liter
Warm program, concrete operations are：45-55 DEG C first holding 24-36 h, then drops to 37-40 DEG C of holding 24-36 h, is finally down to
28-30 DEG C keeps 48-72 h.
7. method according to claim 1, it is characterised in that：Step（7）The synchronous saccharification common fermentation is using alternately logical
Gas disposal, concrete operations are：0-24 h stuffiness, leads to nitrogen 5-10 min after 24 h, oxygen 5-10 min are led to after 48 h.
8. method according to claim 1, it is characterised in that：Step（7）The cushioning liquid is the lemon of 50 mmol/L
Acid buffer；The cellulase is the L of Celluclast 1.5；The cellobiase is Novozyme 188.
9. method according to claim 1, it is characterised in that：Step（7）The fermented nutritive liquid is (NH4)2SO4、
KH2PO4、 MgSO4、 CaCl2With the mixture of water；(the NH4)2SO4、 KH2PO4、 MgSO4、 CaCl2And yeast extract
Concentration in mixed liquor is 0.5-1.5g/L, 2-3g/L, 0.3-0.5g/L, 0.05-0.15g/L and 2-5 g/L.
10. method according to claim 1, it is characterised in that：Step（7）The yeast extract is zymohexose yeast
Bacterium and pentose fermentation saccharomycete；The zymohexose saccharomycete is saccharomyces cerevisiae CICC1416, using anaerobic fermentation；The pentose
Fermented yeast bacterium is shehatae candida GIM2.175, using aerobic fermentation.
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