CN106755130A - It is a kind of to utilize the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation - Google Patents
It is a kind of to utilize the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation Download PDFInfo
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- CN106755130A CN106755130A CN201710004115.1A CN201710004115A CN106755130A CN 106755130 A CN106755130 A CN 106755130A CN 201710004115 A CN201710004115 A CN 201710004115A CN 106755130 A CN106755130 A CN 106755130A
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- 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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- 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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The present invention relates to a kind of using the fresh method containing sugared Ethanol Production by Biomass Fermentation of green grass or young crops, it includes the step of carrying out fermentation liquid of the fermentation acquisition rich in ethanol to fresh blue or green fresh pretreatment residue, cellulase and the producing and ethanol bacterial strain containing sugared biomass sources wood fibre Solid content containing addition in sugared biomass sources liquid containing syrup of green grass or young crops；Wherein, green grass or young crops is fresh fresh fresh containing the squeezing acquisition of sugared biomass material by green grass or young crops containing sugared biomass sources wood fibre Solid content with blue or green containing sugared biomass sources liquid containing syrup.Methods described is utilized while realizing juice free sugar and cellulose sugar in blue or green fresh sugar plant raw material, improves the utilization rate of biomass material, reduces the cost of alcohol production raw material；The fermentation method is used simultaneously, the concentration and production efficiency of ethanol in tunning is improve, and solves the problems, such as concentration of alcohol and low production efficiency in traditional fibre alcohol fermentation processes.
Description
Technical field
The invention belongs to Fermentation Engineering, biomass energy technology field, and in particular to one kind is using blue or green fresh containing sugared biomass
The method of fermentative production of ethanol.
Background technology
With scientific and technological expanding economy, human society is growing day by day to the interdependency of fossil energy.The use of fossil fuel
There are problems that consumption high,.Under energy security and the dual background of environmental protection, sustainable, renewable, height is developed
Effect, inexpensive, the convenient and safe energy seem particularly necessary.Biomass energy can effectively substitute traditional fossil fuel,
Greenization and renewableization of the energy are realized, is the generally acknowledged scheme that liquid fuel is substituted that can uniquely realize.Bio-fuel ethanol
The usage amount of gasoline can be reduced makes gasoline combustion more abundant, and then reduces the discharge of pernicious gas.
With agricultural wastes or the energy crop of marginal land plantation as raw material, production alcohol fuel has turned into many states
The important development direction of family's new energy strategy, sustainable development important in inhibiting of its industrialization to society.At present, due to hair
The limitation of ferment technology, when carrying out alcohol production with the sugar in blue or green fresh biomass material, due to blue or green fresh stalk solid biomass
The utilization rate of residue is low, causes the wasting of resources so that alcohol production is uneconomical.Additionally, the sugar in blue or green fresh stalk resource needs
Concentration is preserved, which in turns increases ethanol raw material cost.
Ethanol is directly prepared compared to using the sugar in blue or green fresh plant, the cellulose ethanol with wood fibre plant as raw material
Fermentation has more difficulty.Because the lignin in lignocellulose raw material, hemicellulose are closely wrapped in cellulose, complicated knot is formed
Structure, it is therefore desirable to pre-processed to change the natural structure of cellulose to lignocellulosic, reduce cellulose crystallinity and
The degree of polymerization, so as to be conducive to the combination and hydrolysis of cellulose enzyme-to-substrate.
The fermentation of synchronous saccharification method realizes fiber hydrolysis and the combination of alcohol fermentation, and the concentration of glucose in enzymolysis liquid is all the time
It is maintained in relatively low scope, feedback inhibition will not be produced to cellulase, improve saccharification efficiency, reduces the use of cellulase
Amount, required consersion unit is also relative to be reduced.But fibre solid residue has water imbibition, too high inventory causes bottom of fermenting
Thing viscosity increases, and fermentation system mass transfer is obstructed, it is however generally that, the inventory of wood fibre solid material is during Batch fermentation
Less than 20%, it is relatively low that this also result in tunning concentration of alcohol, therefore, it is difficult to realize high microsteping conversion ratio and high ethano simultaneously
Production concentration.Additionally, the noxious material occurred in lignocellulosic preprocessing process, to microbial fermentation and cellulase activity
Can adversely affect.
At present, the high cost of alcohol production raw material, Fermentation Substance Concentration is low and low production efficiency of ethanol, height are separated into
This, makes lignocellulosic ethanol technology be difficult to carry out business application.
The content of the invention
The technical problems to be solved by the invention are directed to the deficiencies in the prior art and provide a kind of using blue or green fresh containing sugared biological
The method of matter fermentative production of ethanol, the biomass production ethanol in the method energy high-efficiency comprehensive utilization green grass or young crops fresh plant raw material, fully
Biomass material is make use of, the cost of raw materials for production is reduced, while solving second in conventional synchronization saccharification alcohol fermentation processes
Alcohol low yield, concentration is low and separation costs problem high.
Therefore, the invention provides a kind of using the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation, it is included to green grass or young crops
It is fresh containing adding blue or green fresh degradation treatment residue, fiber containing sugared biomass sources wood fibre Solid content in sugared biomass sources liquid containing syrup
The step of plain enzyme and producing and ethanol bacterial strain carry out fermentation liquid of the fermentation acquisition rich in ethanol；Wherein, green grass or young crops is fresh contains containing sugared biomass sources
Syrup liquid is fresh fresh containing the squeezing acquisition of sugared biomass material by green grass or young crops containing sugared biomass sources wood fibre Solid content with blue or green.
According to the present invention, the green grass or young crops is fresh to contain liquid containing syrup and wood fibre Solid content containing sugared biomass material.
In some embodiments of the invention, the wood fibre Solid content includes cellulose and/or hemicellulose.
In the present invention, the green grass or young crops is fresh to include water content in the blue or green fresh corn stalk of more than 40wt% containing sugared biomass material
One or more in stalk, blue or green fresh broomcorn straw, blue or green fresh sugarcane bar and blue or green fresh and sweet dish stem tuber.
According to the present invention, the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of the green grass or young crops contains with green grass or young crops is fresh
The mode of sugared biomass sources liquid containing syrup mixing is added, or is fermented degraded in the fresh sugar containing in sugared biomass sources liquid containing syrup of green grass or young crops
Added in the way of fed-batch or continuous feeding before completely.
In some preferred embodiments of the invention, the fresh degraded containing sugared biomass sources wood fibre Solid content of the green grass or young crops
Treatment residue was added before the fresh sugar containing in sugared biomass sources liquid containing syrup of green grass or young crops is fermented degraded completely in the way of fed-batch
Enter.
In the present invention, the producing and ethanol bacterial strain is that can be metabolized produce the bacterium of ethanol, can be metabolized the true of generation ethanol
Bacterium and one or more can be metabolized in the genetic engineering bacterium of generation ethanol；It is preferred that the producing and ethanol bacterial strain is that can be metabolized product
The fungi of raw ethanol.
In the present invention, the cellulase includes Endoglucanase, exoglucanase, beta-glucosidase and wood
One or more in dextranase.
In the present invention, the fresh total sugar content ＜ 300g/L containing in sugared biomass sources liquid containing syrup of the green grass or young crops.
In some embodiments of the invention, the producing and ethanol bacterial strain is added in the form of seed liquor, or with active dry bacterium
The form of powder is added；
When the producing and ethanol bacterial strain in the form of seed liquor when to add, the addition of the seed liquor is fresh containing sugared biological with green grass or young crops
The total volume meter of matter mixture is 1v%-20v%, and the addition of preferably described seed liquor is fresh containing sugared biomass mixture with green grass or young crops
Total volume meter is 5v%-10v%；
When the producing and ethanol bacterial strain in the form of active dry bacterium powder when to add, the addition of the active dry bacterium powder is fresh with green grass or young crops
Gross weight meter containing sugared biomass mixture is 0.005w%-0.1w%.
In some embodiments of the invention, the addition of the cellulase is fresh containing sugared biomass sources wood fibre with green grass or young crops
The gross weight meter of the degradation treatment residue of Solid content is 1-60FPU/g；The addition of preferred cellulose enzyme is fresh containing sugared biological with green grass or young crops
The gross weight meter of the degradation treatment residue of matter source wood fibre Solid content be 5-40FPU/g, further preferred cellulase plus
Enter amount with the gross weight meter of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of green grass or young crops be 5-20FPU/g.
According to the present invention, the green grass or young crops is fresh to be carried out at degraded containing sugared biomass sources wood fibre Solid content in the way of powder
Reason.
In some embodiments of the invention, the particle diameter of the powder is in the mesh of ＞ 30；It is preferred that the particle diameter of the powder>60
Mesh.
In the present invention, the method for degradation treatment is alkaline process, acid system, hydro-thermal method, organic solvent method, ionic liquid, steam
One or more in blasting procedure, ultrasonic method, ball-milling method, high-pressure homogenization and bioanalysis, the preferably method of degradation treatment is acid
Method and/or alkaline process.
In some embodiments of the invention, the temperature of the fermentation is 20-60 DEG C, and the temperature preferably fermented is 30-50
DEG C, further preferably the temperature of fermentation is 35-45 DEG C.
Brief description of the drawings
The present invention is illustrated below in conjunction with accompanying drawing.
Fig. 1 is the concrete operations flow chart of the method for the invention
Specific embodiment
To be readily appreciated that the present invention, the present invention is described more detail below.
As it was previously stated, in the high cost of the raw materials for production of ethanol and conventional synchronization diastatic fermentation ethanol process ethanol low life
Efficiency is produced, fermentation liquid concentration of alcohol is low, makes lignocellulosic alcohol production technology be difficult to carry out business application.The present inventor grinds
Discovery is studied carefully, because the limitation of fermentation technique is without simultaneously using the blue or green fresh liquid containing syrup containing in sugared biomass in existing process
And fibrous raw material, the utilization rate for thereby resulting in raw material is low so that the cost of material of ethanol is higher, low production efficiency, fermentation liquid
Concentration of alcohol is low.
The present inventor further study show that, half simultaneous saccharification and fermentation method, i.e., first by lignocellulosic substrates a part be saccharified
Into liquid phase, then access fermentation strain and proceed simultaneous saccharification and fermentation, free and both simultaneous saccharification and fermentations can be had concurrently
Advantage, solves during conventional synchronization saccharification producing and ethanol, and the fermentation initial stage there may be the not enough situation of sugar fermentation, obtain higher
Alcohol yied.It is corresponding to this, by being previously added certain free sugar part in the initial-stage culture base that ferments, in conjunction with conventional fibre
Dimension matter simultaneous saccharification and fermentation mode, can also obtain the ferment effect similar with half synchronous saccharification method.The present inventor is first with green grass or young crops
Fresh is raw material containing sugared biomass, carries out the fermenting and producing of ethanol.The present invention is based on what the above method was made.
Therefore, the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation of utilization involved in the present invention can be understood as blue or green fresh
Containing application of the sugared biomass in fermentative production of ethanol, the concrete operations flow chart of methods described is as shown in figure 1, it is included to green grass or young crops
It is fresh containing adding blue or green fresh degradation treatment residue, fiber containing sugared biomass sources wood fibre Solid content in sugared biomass sources liquid containing syrup
The step of plain enzyme and producing and ethanol bacterial strain carry out fermentation liquid of the fermentation acquisition rich in ethanol；Wherein, green grass or young crops is fresh contains containing sugared biomass sources
Syrup liquid is fresh fresh containing the squeezing acquisition of sugared biomass material by green grass or young crops containing sugared biomass sources wood fibre Solid content with blue or green.
In certain embodiments, the concentration of ethanol is 68.83-98.1g/L in the fermentation liquid rich in ethanol.
In certain embodiments, the temperature of the fermentation is 20-60 DEG C, and the temperature preferably fermented is 30-50 DEG C, further
It is preferred that the temperature fermented is 35-45 DEG C.
In some preferred embodiments, it is green grass or young crops is fresh fresh containing the wooden fibre of sugared biomass sources with blue or green containing sugared biomass sources liquid containing syrup
After dimension Solid content mixing, the fermentation of ethanol is used further to after addition urea, potassium dihydrogen phosphate and anhydrous magnesium sulfate.
According to the present invention, the producing and ethanol bacterial strain is added in the form of seed liquor, or is added in the form of active dry bacterium powder；
In certain embodiments of the present invention, the producing and ethanol bacterial strain is added in the form of seed liquor, the seed liquor
Addition with the fresh total volume meter containing sugared biomass mixture of green grass or young crops be 1v%-20v%；Preferably implement more of the invention
In mode, the addition of the seed liquor with the fresh total volume meter containing sugared biomass mixture of green grass or young crops be 5v%-10v%.
In other implementation methods of the invention, the producing and ethanol bacterial strain with the form of active dry bacterium powder add when, institute
State the addition of active dry bacterium powder with the fresh gross weight meter containing sugared biomass mixture of green grass or young crops be 0.005w%-0.1w%.
In the present invention, the producing and ethanol bacterial strain is that can be metabolized produce the bacterium of ethanol, can be metabolized the true of generation ethanol
Bacterium and one or more can be metabolized in the genetic engineering bacterium of generation ethanol.
In certain embodiments, the producing and ethanol bacterial strain is H5 plants of Saccharomyces Cerevisiae in S accharomyces cerevisiae.
In certain embodiments, the addition of the cellulase is fresh containing sugared biomass sources wood fibre Solid content with green grass or young crops
The gross weight meter of degradation treatment residue is 1-60FPU/g；The addition of preferred cellulose enzyme is fresh wooden containing sugared biomass sources with green grass or young crops
The gross weight meter of the degradation treatment residue of fiber Solid content is 5-40FPU/g, and the addition of further preferred cellulase is with green grass or young crops
The gross weight meter of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content is 5-20FPU/g.
It should be understood that the addition time of the cellulase is to be less than 5g/L for the remaining sugar concentration in zymotic fluid
When.
In the present invention, the cellulase includes Endoglucanase, exoglucanase, beta-glucosidase and wood
One or more in dextranase.
Source in the present invention to cellulase has no particular limits, for example, cellulase can be commercially available or be tool
There is the microorganism of cellulase-producing ability；It is preferred that the microorganism includes trichoderma reesei, penicillium decumbens and through protein surface display
One or more in saccharomyces cerevisiae after technological transformation.
In the present invention, the green grass or young crops is fresh to contain liquid containing syrup and wood fibre Solid content containing sugared biomass material.In some realities
In applying example, for example, the green grass or young crops is fresh to include that blue or green stalk of fresh corn of the water content in more than 40wt%, green grass or young crops are fresh containing sugared biomass material
One or more in broomcorn straw, blue or green fresh sugarcane bar and blue or green fresh and sweet dish stem tuber.
In the present invention, the green grass or young crops it is fresh containing sugared biomass sources liquid containing syrup is used directly for fermentation, it is also possible to through dilution or
After concentration, then fermented, but the blue or green fresh total sugar content ＜ 300g/L containing in sugared biomass sources liquid containing syrup for fermenting.
In the present invention, the wood fibre Solid content includes cellulose and/or hemicellulose.In certain embodiments, institute
The blue or green fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content is stated with fresh mixed containing sugared biomass sources liquid containing syrup with green grass or young crops
The mode of conjunction is added, or is added in the way of batch or continuous feeding before termination of fermenting.It is preferred that the green grass or young crops is fresh containing sugared biomass
The degradation treatment residue of source wood fibre Solid content is added before termination of fermenting in the way of fed-batch.
In the present invention, the green grass or young crops is fresh to carry out degradation treatment containing sugared biomass sources wood fibre Solid content in the way of powder.
In certain embodiments, for example, the particle diameter of the powder is in the mesh of ＞ 30；It is preferred that the particle diameter of the powder>60 mesh.
In the present invention, the method for degradation treatment is alkaline process, acid system, hydro-thermal method, organic solvent method, ionic liquid, steam
One or more in blasting procedure, ultrasonic method, ball-milling method, high-pressure homogenization and bioanalysis, the preferably method of degradation treatment is acid
Method and/or alkaline process.
According to certain embodiments of the present invention, methods described also include will be enriched in ethanol fermentation liquid carry out separate it is pure
Change the step of alcohol product is obtained.
In some embodiments of the invention, the method for isolating and purifying includes common distillation, rectification under vacuum, liquid liquid extraction
Take, one or more in infiltration evaporation, Steam soak, stripping, vacuum extraction and absorption method.
According to the present invention, described isolating and purifying is carried out after anaerobic fermentation termination, or is coupled with anaerobic fermentation process and carried out
Original position separates.
It is described using the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation in certain specific embodiments of the invention
Comprise the following steps：
(1) raw material squeezing：It is fresh to green grass or young crops to be squeezed containing sugared biomass material, realize the blue or green fresh solid-liquid containing sugared biomass material
Separate, obtain blue or green fresh containing sugared biomass sources liquid containing syrup and blue or green fresh containing sugared biomass sources wood fibre Solid content；
(2) treatment of fiber residue：By green grass or young crops it is fresh containing the drying of sugared biomass sources wood fibre Solid content, crushing after, then use
Preprocess method destroys the structure of fiber residue, obtains the blue or green fresh degradation treatment containing sugared biomass sources wood fibre Solid content residual
Slag, to reach the purpose of effective enzymolysis；
(3) preparation of seed liquor：By producing and ethanol bacterial strain in synthetic media and/or natural medium amplification cultivation, obtain
The seed liquor of fermentation must be used for；
(4) fermentative production of ethanol：Green grass or young crops is fresh fresh containing sugared biomass sources wood fibre with blue or green containing sugared biomass sources liquid containing syrup
The degradation treatment residue mixing of Solid content, adds cellulase, and the seed liquor for accessing producing and ethanol bacterial strain is fermented, and is rich in
The fermentation liquid of ethanol；
In some preferred embodiments, it is green grass or young crops is fresh fresh containing the wooden fibre of sugared biomass sources with blue or green containing sugared biomass sources liquid containing syrup
After dimension Solid content mixing, the fermentation of ethanol is used further to after addition urea, potassium dihydrogen phosphate and anhydrous magnesium sulfate.
(5) separation of ethanol purifying：Will be enriched in the fermentation liquid of ethanol carries out isolating and purifying prepared alcohol product.The separation
The method of purifying includes common distillation, rectification under vacuum, liquid-liquid extraction, infiltration evaporation, Steam soak, stripping, vacuum extraction and suction
One or more in attached method.Described isolating and purifying can be carried out after anaerobic fermentation termination, or be coupled with anaerobic fermentation process
Carry out separation in situ.
Term " wood fibre Solid content " of the present invention is also known as " fiber residue ".
Beneficial effects of the present invention are：
(1) profit while methods described realizes juice free sugar and cellulose sugar in blue or green fresh sugar plant raw material
With, the utilization rate of biomass material is improve, reduce the cost of alcohol production raw material.
(2) use the fermentation method, due to green grass or young crops it is fresh containing sugared biomass sources liquid containing syrup ferment the initial stage addition, help
In cell proliferation rate and alcohol yied higher is obtained at the alcohol fermentation initial stage, additionally, green grass or young crops is fresh to contain syrup containing sugared biomass sources
The addition of liquid can effectively improve the substrate carbon source concentration in fermentation process, so, can further improve second in tunning
The concentration of alcohol, solves conventional synchronization saccharification alcohol fermentation low production efficiency, the low problem of mash concentration of alcohol.
Embodiment
To make the present invention easier to understand, the present invention is further described below in conjunction with embodiment, these realities
Apply example only serve it is illustrative, it is not limited to range of application of the invention.If raw material or component nothing used in the present invention
Specified otherwise can be obtained by commercial sources or conventional method.
In following embodiments, main process parameters and refer to that object detection method is as follows：
(1) HPLC methods survey sugar：It to be separated then realize using glycan analysis post using high performance liquid chromatography (HPLC) and is divided
Analysis.Sample to be analysed is centrifuged 10 minutes by after pH adjustment, taking 1.00g in 8000rpm, and supernatant dilutes 100 times, carries out liquid
Facies analysis.The glycan analysis post for being used is Bio Rad companies Aminex87P analytical columns, and liquid phase analysis condition is：Flow velocity 0.6mL/
Min, 65 DEG C of column temperature, the μ L of sample size 20；Qualitative analysis is carried out using standard items retention time；Carried out using standard curve external standard method
Quantitative analysis.
(2) composition analysis of degradation treatment fiber residue：Ethanol soxhlet type sample to be analysed is used first, to remove tree
Fat, pigment etc., the then concentrated sulfuric acid and 4% dilute sulfuric acid two-step hydrolysis sample to be analysed with 72%, in making lignocellulosic
Component is hydrolyzed into easily quantitative material.Wherein, cellulose, hydrolysis of hemicellulose generation monose, are entered using high performance liquid chromatography
Row quantitative analysis, and lignin carries out quantitative analysis using calcination method.
(3) gas chromatography determines concentration of alcohol：The analytical column for being used is cbp-20 chromatographic columns.Analysis condition is：Post
180 DEG C of temperature, 90 DEG C of injector temperature, N2It is carrier, H2It is combustion gas, detector is FID, the μ L of sample size 1.0；Protected using standard items
Staying the time carries out qualitative analysis；Quantitative analysis is carried out using standard curve external standard method.
(4) cellulase compounding liquid described in embodiment is provided by Shandong Kang Dien Bioisystech Co., Ltd.Test paper enzyme
Assay method living is as follows：Appropriate dilution enzyme liquid is taken, 50 DEG C of constant temperature hydrolyze whatman1 filter paper 60 minutes, define degraded per minute
The enzyme amount of 1ug reduced sugars produced by filter paper, is a FPU enzyme activity；Wherein, the assay method of reduced sugar using DNS colorimetric methods or
HPLC methods.
Embodiment 1：Ethanol is produced by fermenting raw materials of blue or green fresh and sweet broomcorn straw.
(1) squeezing of blue or green fresh and sweet broomcorn straw：The blue or green fresh and sweet broomcorn straw of 1kg grown in field is taken, is passed through three times after peeling
About 654ml liquid containing syrup and fiber residue are obtained after squeezing repeatedly；Determined through HPLC methods, this contains about 144g/L in containing syrup liquid
Free sugar.Additionally, measured by ICP-MS and Kjeldahl's method, this contains the inorganic ions that contains in syrup liquid and nitrogen phosphorus contains
Amount is as shown in table 1：
Inorganic ions and nitrogen and phosphorus content in the blue or green fresh and sweet broomcorn straw source liquid containing syrup of table 1
| Element | Content (mg/L) |
| K | 262.76 |
| Na | 17.6 |
| Ca | 143.86 |
| Mg | 263.45 |
| Fe | 2.07 |
| Mn | 0.34 |
| P | 128.69 |
| N | 1480 |
(2) degradation treatment of fiber residue：By remaining about 294g fiber residues in over dry is overnight dried at 105 DEG C, obtain
To 216g bone dry fiber residues；Fiber residue is crushed to 50 mesh using pulverizer, afterwards by bone dry fiber residue with solid-to-liquid ratio
It is 1:10 ratio is soaked in being added to 2% NaOH solution, in being incubated 80 minutes at 121 DEG C, afterwards in filtration under diminished pressure mode
Realize separation of solid and liquid；Take filter residue neutral through being originally washed to pH, over dry is overnight dried at 105 DEG C, obtain at through alkaline process degraded
Fiber residue after reason, its main component is shown in Table 2
The composition of the blue or green fresh and sweet broomcorn straw fiber residue after the alkaline process degradation treatment of table 2
| Composition | Percentage composition (%) |
| Glucoside | 54.03 |
| Xyloside | 21.54 |
| Arabinoside | 0.43 |
| Galactoside | 0.13 |
| Mannoside | 0.05 |
| Klason lignin | 4.33 |
| Ash content | 0.08 |
(3) preparation of zymotic fluid：118.5g is crushed to below 60 mesh through the fiber residue after alkaline process degradation treatment, with step
Suddenly obtained liquid containing syrup is sufficiently mixed in (1), inserts in fermentation tank, under conditions of without other exogenous nutrition compositions,
In being sterilized 25 minutes at 121 DEG C, zymotic fluid is obtained standby.
(4) preparation of seed liquor：Producing and ethanol bacterial strain is the thermotolerant ethanol fermentation yeast that laboratory is screened by evolution engineering
Saccharomyces cerevisiae H5.The composition of bacterial strain seed synthetic media is (g/L)：Sucrose 0.8, glucose 20,
Soy peptone 5, dusty yeast 3, running water, and pH to 5.5 is adjusted with 1mol/L HCl.Inoculation to seed is synthesized and is cultivated
In base, in Shaking culture 20h under 35 DEG C, 80rpm, seed liquor is obtained.
(5) fermentative production of ethanol：Seed liquor is linked into zymotic fluid with 10% inoculum concentration, at the same time, is added
The compounding cellulase solution of 20FPU/g degradation treatment fiber residues；In the 72h that fermented at rotating speed 180rpm, 35 DEG C, in fermentation ends
Afterwards, fermentation liquid is taken in centrifugation 10 minutes under 10000rpm；The supernatant after centrifugation is taken with residual in HPLC methods measure supernatant
Sugar, with ethanol production in gas chromatography measure supernatant, measurement result is as shown in table 3.
The blue or green fresh and sweet broomcorn straw fermenting alcohol result of table 3
| Detection project | Fermentation results |
| Remaining sugar concentration (g/L) | 0.3 (being fructose, glucose, sucrose total concentration, disregard xylose) |
| Concentration of alcohol (g/L) | 88.4 |
| Alcohol yied (g/L h) | 1.23 |
| Blue or green fresh and sweet sorghum straw is to ethanol conversion (g/kg) | 57.5 |
Embodiment 2：Ethanol is produced by fermenting raw materials of blue or green fresh and sweet broomcorn straw.
Step (1), (2) are with embodiment 1.
(3) preparation of zymotic fluid：Fiber residue after 118.5g is processed through alkaline degradation is crushed to 60 mesh, is divided into 4 parts,
Take a copy of it to be sufficiently mixed with obtained liquid containing syrup in step (1), insert in fermentation tank；Additionally, to extra in fermentation tank
1.7g/L urea, 1g/L potassium dihydrogen phosphates are added, 2g/L anhydrous magnesium sulfates, in the 25min that sterilized at 121 DEG C, obtain zymotic fluid, standby
With.
(4) preparation of seed liquor：Producing and ethanol bacterial strain is the thermotolerant ethanol fermentation yeast that laboratory is screened by evolution engineering
Saccharomyces cerevisiae H5.The composition of bacterial strain seed synthetic media is (g/L)：Sucrose 0.8, glucose 20,
Soy peptone 5, dusty yeast 3, running water, and pH to 5.5 is adjusted with 1mol/L HCl.Inoculation to seed is synthesized and is cultivated
In base, in Shaking culture 20h under 37 DEG C, 200rpm, seed liquor is obtained.
(5) fermentative production of ethanol：Seed liquor is linked into zymotic fluid with 5% inoculum concentration, at the same time, is added
The compounding cellulase solution of 25FPU/g degradation treatment fiber residues；It is pending in alcohol fermentation is carried out at rotating speed 200rpm, 37 DEG C
When ferment is to 12,24,36h, other 3 equal portions for having dispensed in step (3) are sequentially added through the fiber residue after alkali process, with
The enzyme addition of 25FPU/g degradation treatment fiber residues, adds corresponding compounding cellulase solution, is fermented to 72h and terminated, and takes hair
Zymotic fluid is in centrifugation 10 minutes under 10000rpm；The residual sugar that the supernatant after centrifugation is determined in liquid with HPLC methods is taken, with gas phase color
Spectrometry determines ethanol production in liquid, and measurement result is as shown in table 4：
The blue or green fresh and sweet broomcorn straw fermenting alcohol result of table 4
Embodiment 3：Ethanol is produced by fermenting raw materials of blue or green stalk of fresh corn.
(1) squeezing of blue or green stalk of fresh corn：1kg grown in field is taken, the blue or green fresh cornstalk of ripe corn kernel has been harvested, gone
About 574ml liquid containing syrup and fiber residue are obtained after being squeezed repeatedly by three times after skin；Determined through HPLC methods, this contains in syrup liquid
Free sugar containing about 97g/L.Additionally, measured by ICP-MS and Kjeldahl's method, this contains contain in syrup liquid inorganic
Ion and nitrogen and phosphorus content are as shown in table 5：
Inorganic ions and nitrogen and phosphorus content in the blue or green stalk of fresh corn liquid containing syrup of table 5
| Element | Content (mg/L) |
| K | 362.48 |
| Na | 11.04 |
| Ca | 238.68 |
| Mg | 468.10 |
| Fe | 3.30 |
| Mn | 1.14 |
| P | 154.86 |
| N | 1128.20 |
(2) degradation treatment of fiber residue：By remaining about 380g fiber residues in over dry is overnight dried at 105 DEG C, obtain
Obtain 256g bone dry fiber residues；Bone dry fiber residue is crushed to 40 mesh, is 1 with solid-to-liquid ratio:10 ratio be added to 2% it is dilute
Soaked in sulfuric acid, in being incubated 60 minutes at 121 DEG C, separation of solid and liquid is realized in filtration under diminished pressure mode afterwards；Filter residue is taken through running water
It is washed till pH neutral, over dry is overnight dried at 105 DEG C, obtain the fiber residue after being processed through acid degradation, its main component is shown in Table
6。
The composition of the blue or green stalk of fresh corn fiber residue after the acid degradation of table 6 treatment
(3) preparation of zymotic fluid：Fiber residue after 256g is processed through acid degradation is crushed to below 80 mesh, is taken wherein
57.4g is 1 according to solid-to-liquid ratio:10 ratio is sufficiently mixed with obtained liquid containing syrup in step (1), inserts in fermentation tank；This
Outward, 2g/L urea, 1.5g/L potassium dihydrogen phosphates are added to extra in fermentation tank, 1g/L anhydrous magnesium sulfates, in sterilizing 25 at 121 DEG C
Minute, obtain zymotic fluid standby.
(4) preparation of seed liquor：Producing and ethanol bacterial strain is the thermotolerant ethanol fermentation yeast that laboratory is screened by evolution engineering
Saccharomyces cerevisiae H5.The composition of the seed synthetic media of bacterial strain is (g/L)：Sucrose 0.8, glucose
20th, soy peptone 5, dusty yeast 3, running water, and pH to 5.5 is adjusted with 1mol/L HCl.Inoculation to seed is synthesized
In culture medium, in Shaking culture 20h under 35 DEG C, 200rpm, seed liquor is obtained.
(5) fermentative production of ethanol：Seed liquor is linked into zymotic fluid with 10% inoculum concentration, in rotating speed 200rpm, 35
Alcohol fermentation is carried out at DEG C, monitor in real time is carried out to the residual sugar in zymotic fluid in fermentation process, treat that zymotic fluid total residual sugar concentration is low
When 5g/L, the compounding cellulase solution of 15FPU/g degradation treatment fiber residues is added, continues to ferment, fermented to 72h and terminated,
Zymotic fluid is taken in centrifugation 10 minutes under 10000rpm；The residual sugar that the supernatant after centrifugation is determined in liquid with HPLC methods is taken, with gas
Phase chromatography determines ethanol production in liquid, and measurement result is as shown in table 7：
The blue or green stalk of fresh corn fermenting alcohol result of table 7
| Detection project | Fermentation results |
| Remaining sugar concentration (g/L) | 0.2 (being fructose, glucose, sucrose total concentration, disregard xylose) |
| Concentration of alcohol (g/L) | 68.83 |
| Alcohol yied (g/L h) | 0.96 |
It should be noted that embodiment described above is only used for explaining the present invention, do not constitute to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word wherein used is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it is related to
And specific method, material and embodiment, it is not intended that the present invention is limited to wherein disclosed particular case, conversely, this hair
It is bright to can be extended to other all methods and applications with identical function.
Claims (10)
1. a kind of using the blue or green fresh method containing sugared Ethanol Production by Biomass Fermentation, it includes containing syrup containing sugared biomass sources to green grass or young crops is fresh
The blue or green fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content, cellulase and producing and ethanol bacterial strain are added in liquid to be carried out
The step of fermentation obtains the fermentation liquid rich in ethanol；Wherein, it is blue or green fresh containing sugared biomass sources liquid containing syrup and blue or green fresh containing sugared biological
Matter source wood fibre Solid content is obtained by blue or green fresh squeezing containing sugared biomass material.
2. method according to claim 1, it is characterised in that the green grass or young crops it is fresh containing sugared biomass material contain liquid containing syrup and
Wood fibre Solid content；It is preferred that the wood fibre Solid content includes cellulose and/or hemicellulose；It is preferred that the green grass or young crops is fresh containing sugar
Biomass material includes blue or green stalk of fresh corn of the water content in more than 40wt%, blue or green fresh broomcorn straw, blue or green fresh sugarcane bar and green grass or young crops
One or more in fresh and sweet dish stem tuber.
3. method according to claim 1 and 2, it is characterised in that the green grass or young crops is fresh to be contained admittedly containing sugared biomass sources wood fibre
The degradation treatment residue of thing is added in the way of mixing containing sugared biomass sources liquid containing syrup with green grass or young crops is fresh, or fresh containing sugared biomass in green grass or young crops
Sugar in the liquid containing syrup of source is added before being fermented degraded completely in the way of fed-batch or continuous feeding；It is preferred that the green grass or young crops is fresh
Degradation treatment residue containing sugared biomass sources wood fibre Solid content is sent out in the fresh sugar containing in sugared biomass sources liquid containing syrup of green grass or young crops
Added in the way of fed-batch before ferment degraded completely.
4. the method according to claim any one of 1-3, it is characterised in that the producing and ethanol bacterial strain is can be metabolized generation
The bacterium of ethanol, the one kind or many that the fungi of generation ethanol can be metabolized and can be metabolized in the genetic engineering bacterium for producing ethanol
Kind；It is preferred that the producing and ethanol bacterial strain is that can be metabolized the fungi for producing ethanol.
5. the method according to claim any one of 1-4, it is characterised in that the cellulase includes endo-glucanase glycosides
One or more in enzyme, exoglucanase, beta-glucosidase and zytase.
6. the method according to claim any one of 1-5, it is characterised in that the green grass or young crops is fresh containing sugared biomass sources liquid containing syrup
In total sugar content ＜ 300g/L.
7. the method according to claim any one of 1-6, it is characterised in that the producing and ethanol bacterial strain is in the form of seed liquor
Add, or added in the form of active dry bacterium powder；
When the producing and ethanol bacterial strain in the form of seed liquor when to add, the addition of the seed liquor is fresh mixed containing sugared biomass with green grass or young crops
The total volume meter of compound is 1v%-20v%, and the addition of preferably described seed liquor is with the fresh totality containing sugared biomass mixture of green grass or young crops
Product is calculated as 5v%-10v%；
When the producing and ethanol bacterial strain in the form of active dry bacterium powder when to add, the addition of the active dry bacterium powder is fresh containing sugar with green grass or young crops
The gross weight meter of biomass mixture is 0.005w%-0.1w%.
8. the method according to claim any one of 1-7, it is characterised in that the addition of the cellulase contains so that green grass or young crops is fresh
The gross weight meter of the degradation treatment residue of sugared biomass sources wood fibre Solid content is 1-60FPU/g；Preferred cellulose enzyme plus
It is 5-40FPU/g to enter amount with the gross weight meter of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of green grass or young crops, enters one
The addition of preferred cellulose enzyme is walked with the gross weight of the fresh degradation treatment residue containing sugared biomass sources wood fibre Solid content of green grass or young crops
It is calculated as 5-20FPU/g.
9. the method according to claim any one of 1-8, it is characterised in that the green grass or young crops is fresh containing sugared biomass sources wood fibre
Solid content carries out degradation treatment in the way of powder；It is preferred that the mesh of particle diameter ＞ 30 of the powder；The further preferred powder
Particle diameter>60 mesh；It is preferred that the method for degradation treatment is alkaline process, acid system, hydro-thermal method, organic solvent method, ionic liquid, steam blasting
One or more in method, ultrasonic method, ball-milling method, high-pressure homogenization and bioanalysis, the method for further preferred degradation treatment is
Acid system and/or alkaline process.
10. the method according to claim any one of 1-9, it is characterised in that the temperature of the fermentation is 20-60 DEG C, excellent
The temperature of ferment is published for 30-50 DEG C, further preferably the temperature of fermentation is 35-45 DEG C.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008045506A2 (en) * | 2006-10-10 | 2008-04-17 | President And Fellows Of Harvard College | Biopolymer structures |
| CN101784668A (en) * | 2007-07-04 | 2010-07-21 | 丽格诺新创有限公司 | concurrent saccharification and fermentation of fibrous biomass |
| CN101798584A (en) * | 2005-07-19 | 2010-08-11 | 因比肯公司 | Method and apparatus for conversion of cellulosic material to ethanol |
| CN102321679A (en) * | 2011-07-29 | 2012-01-18 | 温志明 | Comprehensive utilization method of sweet sorghum straw and juice thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798584A (en) * | 2005-07-19 | 2010-08-11 | 因比肯公司 | Method and apparatus for conversion of cellulosic material to ethanol |
| WO2008045506A2 (en) * | 2006-10-10 | 2008-04-17 | President And Fellows Of Harvard College | Biopolymer structures |
| CN101784668A (en) * | 2007-07-04 | 2010-07-21 | 丽格诺新创有限公司 | concurrent saccharification and fermentation of fibrous biomass |
| CN102321679A (en) * | 2011-07-29 | 2012-01-18 | 温志明 | Comprehensive utilization method of sweet sorghum straw and juice thereof |
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