CN105255953B - Physical-chemical-Biological Pretreatment corn stover method - Google Patents
Physical-chemical-Biological Pretreatment corn stover method Download PDFInfo
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- CN105255953B CN105255953B CN201510647321.5A CN201510647321A CN105255953B CN 105255953 B CN105255953 B CN 105255953B CN 201510647321 A CN201510647321 A CN 201510647321A CN 105255953 B CN105255953 B CN 105255953B
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- corn stover
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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
- 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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- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The present invention relates to a kind of physical-chemical-Biological Pretreatment corn stover methods, and corn stover first carries out crushed material reason processing, then carries out diluted alkaline chemical treatment, finally using Phanerochaete chrysosporium (Phanerochaete chrysosporium) and cellulose-degrading bacteria to corn stover carry out biological treatment.The present invention facilitates the removal of lignin using diluted alkaline processing, facilitates fully degraded corn stover using mixed fungus fermentation, improves its utilization rate, and pretreated stalk accesses producing and ethanol micro-organisms ethyl alcohol, or access fodder yeast produces protein feed.Present invention process is relatively simple, convenient to carry out.
Description
Technical field
The present invention relates to the preprocess methods of stalk, and in particular to a kind of physical-chemical-Biological Pretreatment corn stover
Method belongs to technological field of biochemistry.
Background technique
Stalk is a kind of renewable energy very rich, is mainly made of cellulose, hemicellulose and lignin.Wherein
Lignin is a kind of macromolecule aromatic compound, cannot hydrolyze saccharogenesis, and cellulose and hemicellulose are all that can potentially send out
The source of ferment sugar.By straw be saccharified (enzyme hydrolysis), be converted into fermentable sugars, by microbial fermentation produce fuel alcohol,
Single cell protein and organic acid are considered as one of the effective way using straw, but cellulose, hemicellulose in stalk
Cell wall structure is formed together with being interleaved with each other with lignin, and cell is sticked together, and this structure being interweaved is determined
The degradation for having determined any kind ingredient will necessarily be made by the cladding of the restriction of other ingredients, especially lignin to cellulose
With.In addition, the fibre bundle of crystal structure is joined together to form between cellulose macromolecule by a large amount of hydrogen bond, this special knot
Structure makes grape sugar chain orderly and closely accumulation, forms the structure of highly crystalline.Therefore these features make many celluloses
Decomposer degrading straw inefficiency.So carrying out effectively pretreatment to stalk is mostly important one during stalk resource utilizes
A link.Common preprocess method is physics pretreatment (such as mechanical smashing, grinding, steam blasting, radiation treatment, microwave
Pretreatment, ultrasonic pretreatment etc.), chemical pretreatment (diluted acid, diluted alkaline, ammonium hydroxide etc.), Biological Pretreatment it is (composite bacteria agent, white
Rotten fungi, enzyme etc.) and combination pretreatment (such as ultrasonic wave and alkaline process combined pretreatment).Since each processing method has one
Determine defect, such as the problem that the processing of chemistry, physical method has environmental pollution, energy consumption high, and the Biochemical method period is long, efficiency
It is low.
Summary of the invention
The object of the present invention is to provide a kind of physical-chemical-Biological Pretreatment corn stover methods, using compound skill
Art, that is, physics, chemistry and biological method are used in mixed way, and make up respective deficiency, play maximum effect.
The method of the pretreatment corn stover that the technical solution of the invention is as follows the following steps are included:
(1) physical treatment: corn stover first passes through pulverizer and is crushed, and smashes it through 40 meshes；
(2) it is chemically treated: handling smashed corn stover with the NaOH solution of mass concentration 1.0%, solid-to-liquid ratio 1:
10,100 DEG C of 1h, filtering, filter residue are washed to neutrality, and drying to constant weight, as fermentation substrate；
(3) biological treatment: every 10mL is first prepared containing FeSO4·7H2O 0.0005g、MnSO4·7H2O 0.0016g、
ZnSO4·7H2O 0.0014g、CoCl2The micro-mixed liquor of 0.002g；Next prepares nutritive solution, each in nutritive solution
The mass percent concentration of component is: (NH4)2SO41%, KH2PO40.3%, MgSO40.05%, CaCl20.05%, remaining is
Water；Then mixed liquor is prepared, mixed liquor is made by adding micro-mixed liquor 1mL in every 100mL nutritive solution；To step
(2) add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2.25, adjust pH5.5 in 121 DEG C of sterilizing 20min and obtain solid state fermentation
Culture medium；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is connect with the inoculum concentration of quality 10%
Enter in solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%-14%6 The cellulose-degrading bacteria of a/mL
Spore suspension after 28 DEG C of culture 3-5 d, obtains pretreatment product.
Wherein, cellulose-degrading bacteria be aspergillus niger, Trichoderma viride andTrichoderma asperelloidesOne of
Or it is several；Several mould mixed proportions are respectively as follows: aspergillus niger: Trichoderma viride=1:1, aspergillus niger:Trichoderma asperelloides=1:1, Trichoderma viride:Trichoderma asperelloides=1:1, aspergillus niger: Trichoderma viride:Trichoderma asperelloides=1:1:1, aspergillus niger: Trichoderma viride:Trichoderma asperelloides =
2:1:1, aspergillus niger: Trichoderma viride:Trichoderma asperelloides=1:2:1, aspergillus niger: Trichoderma viride:Trichoderma asperelloides =1:1:2。
Wherein, the aspergillus niger and Trichoderma viride are activated using PDA solid medium, the preparation of PDA culture medium
Cheng Shi: weighing 200g potato and be cut into small pieces, add boiling rotten after potato is cleaned peeling, then uses eight layers of filtered through gauze, addition
15g agar continues heating stirring and mixes, 20g glucose is added after agar has dissolved, stirs evenly, supplies again after slightly cooling down
Moisture is to 1000mL.
Wherein, describedTrichoderma asperelloidesIt is activated using brewer's wort solid medium, malt
The preparation process of juice solid medium is: weighing certain amount malt, crushes, add the water of 4 times of malt amounts, protects at 55-60 DEG C
Temperature saccharification, is stirred continuously, saccharification degree iodometric titration, after 3-4h, by 4-6 layers of filtered through gauze of saccharified liquid；Filtered fluid uses chicken
Egg white clarification, method is that an Egg-white is added water about 20mL, mixes well until generating foam, is subsequently poured into saccharified liquid and stirs
It mixes, is refiltered after boiling, obtain clear brewer's wort；It is diluted with water into the brewer's wort of 5-6 ° of B é；Add 16g agar in brewer's wort,
0.1MPa pressure, sterilize 15min, obtains brewer's wort solid medium.
Wherein, the Phanerochaete chrysosporium is activated using comprehensive PDA solid medium, comprehensive PDA culture medium
Preparation process be: KH is added on the basis of 1LPDA solid medium2PO43g、MgSO41.5g, vitamin B10.001g。
Wherein, involved microbial spore suspension the preparation method comprises the following steps: by the sterile washing of activated bacterial strain
Spore is washed, is placed in the triangular flask containing bead, 20 min are vibrated in 28 DEG C of 120 r/min shaking table, with 4 layers of sterilizing lens wiping paper
Filtering oscillation liquid, under the microscope counts filtrate miospore with blood counting chamber, adjusts its concentration to 106 A/mL is protected
There are 4 DEG C of refrigerators are spare.
The invention has the advantages that
1, the present invention greatly improves lignin in stalk using diluted alkaline pretreatment, Phanerochaete chrysosporium fermentation process
Degradation rate.
2, the present invention has studied influence of the mixed fungus fermentation to lignin, hemicellulose and cellulose degradation rate in stalk, sends out
Now using mixed fungus fermentation degradation crop material, different microorganisms secrete complementary enzyme in degradation, obtain stalk in metabolism
Fully degraded, to improve the degradation rate of stalk cellulose.
3, the present invention is to compare different pretreatments method to lignin, hemicellulose, content of cellulose change in stalk
It is screened after the influence of change, removal partial lignin is handled using diluted alkaline, stalk is carried out using efficient degradation microorganism
Biological Pretreatment, the degradation rate of lignin, hemicellulose and cellulose respectively reaches 95%, 75% and in stalk after pretreatment
35% or more, greatly improve stalk using efficiency, required equipment is simple, simple process, by this pretreated corn stalk
Stalk can be independent or be equipped with wheat bran, bean dregs, and access producing and ethanol micro-organisms ethyl alcohol or access fodder yeast production albumen is utilized to raise
Material etc..Solve the problems, such as that agricultural crop straw is difficult to degradation and recycles, it is very meaningful.
Detailed description of the invention
Fig. 1 is influence of the different pretreatments method to lignin, cellulose and hemicellulose level variation in corn stover.
Fig. 2 is the combination of different cellulose-degrading bacterias and inoculative proportion to lignin, cellulose and hemicellulose in corn stover
The influence of cellulose content variation.
Fig. 3 is cellulose-degrading bacteria inoculum concentration to lignin, cellulose and hemicellulose level variation in corn stover
It influences.
Fig. 4 is that cellulose-degrading bacteria ferments number of days to lignin, cellulose and hemicellulose level variation in corn stover
Influence.
Specific embodiment
Technical solution of the invention is further illustrated combined with specific embodiments below, these embodiments should not be understood as
It is the limitation to technical solution.
Embodiment 1: corn stover is pre-processed according to following steps
(1) corn stover first passes through pulverizer and is crushed, and smashes it through 40 meshes；
(2) smashed corn stover is handled with the NaOH solution of mass concentration 1.0%, solid-to-liquid ratio 1:10,100 DEG C of 1h,
Filtering, filter residue are washed to neutrality, and drying to constant weight, as fermentation substrate；
(3) every 10mL is first prepared containing FeSO4·7H2O 0.0005g、MnSO4·7H2O 0.0016g、 ZnSO4·7H2O
0.0014g、CoCl2.0.002g micro-mixed liquor；Next prepares nutritive solution, the quality of each component in nutritive solution
Percent concentration is: (NH4)2SO41%, KH2PO40.3%, MgSO40.05%, CaCl20.05%, remaining is water；Then it prepares
Mixed liquor, mixed liquor are made by adding micro-mixed liquor 1mL in every 100mL nutritive solution；To the fermentation substrate of step (2)
According to solid-liquid ratio be that 1:2.25 adds mixed liquor, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid-state fermentation culture medium；It will match
It is 10 that bacteria concentration, which is made,6 The Phanerochaete chrysosporium spore suspension of a/mL is with the inoculum concentration access solid state fermentation training of quality 10%
It supports in base, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The aspergillus niger spore suspension of a/mL, 28 DEG C of culture 3d,
Obtain pretreatment product.
As shown in the 8th group of Fig. 1, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.7%,
81.6% and 43.2%.
Embodiment 2: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2.25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The Trichoderma viride spore suspension of a/mL,
28 DEG C of culture 4d, obtain pretreatment product.
As shown in the 1st group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.1%,
77.1% and 41.6%.
Embodiment 3: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2.25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 A/mL'sTrichoderma asperelloidesSpore suspension, 28 DEG C of culture 4d, obtains pretreatment product.
As shown in the 2nd group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.8%,
81.7% and 44.2%.
Embodiment 4: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d, obtains pretreatment product；Wherein, aspergillus niger, Trichoderma viride in cellulose-degrading bacteria spore suspension
Two bacterium inoculative proportions are 1:1.
As shown in the 3rd group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 99.1%,
78.1% and 45.9%.
Embodiment 5: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d, obtains pretreatment product；Wherein, aspergillus niger in cellulose-degrading bacteria spore suspension,Trichoderma asperelloidesTwo bacterium inoculative proportions are 1:1.
As shown in the 4th group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.7%,
81.3% and 47.8%.
Embodiment 6: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d, obtains pretreatment product；Wherein, cellulose-degrading bacteria spore suspension Green trichoderma,Trichoderma asperelloidesTwo bacterium inoculative proportions are 1:1.
As shown in the 5th group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 99.3%,
81.4% and 47.5%.
Embodiment 7: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d obtain the pretreatment product produced for protein feed；Wherein, cellulose-degrading bacteria spore suspension
Middle aspergillus niger, Trichoderma viride,Trichoderma asperelloidesThree bacterium inoculative proportions are 1:1:1.
As shown in the 6th group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.6%,
82.5% and 49.9%.
Embodiment 8: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d obtain the pretreatment product produced for protein feed；Wherein, cellulose-degrading bacteria spore suspension
Middle aspergillus niger, Trichoderma viride,Trichoderma asperelloidesThree bacterium inoculative proportions are 2:1:1.
As shown in the 7th group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 98.2%,
81.9% and 48.7%.
Embodiment 9: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Add mixed liquor according to solid-liquid ratio in fermentation substrate for 1:2. 25, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation training
Support base；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is accessed with the inoculum concentration of quality 10%
In solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 10%6 The cellulose-degrading bacteria spore of a/mL is outstanding
Supernatant liquid, 28 DEG C of culture 4d obtain the pretreatment product produced for protein feed；Wherein, cellulose-degrading bacteria spore suspension
Middle aspergillus niger, Trichoderma viride,Trichoderma asperelloidesThree bacterium inoculative proportions are 1:2:1.
As shown in the 8th group of Fig. 2, at this time in stalk the degradation rate of lignin, hemicellulose and cellulose be respectively 99.2%,
83.8% and 48.1%.
Embodiment 10: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Fermentation substrate according to solid-liquid ratio be 1:2. 25 plus mixed liquor, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation
Culture medium；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is connect with the inoculum concentration of quality 10%
Enter in solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 12%6 The cellulose-degrading bacteria spore of a/mL
Suspension, 28 DEG C of culture 4d obtain the pretreatment product produced for protein feed；Wherein, cellulose-degrading bacteria spore suspension
Aspergillus niger in liquid, Trichoderma viride,Trichoderma asperelloidesThree bacterium inoculative proportions are 1:1:2.
As shown in figure 3, the degradation rate of lignin, hemicellulose and cellulose is respectively 98%, 84.3% and in stalk at this time
53.2%。
Embodiment 11: step (1), (2) are the same as embodiment 1；The preparation of step (3) mixed liquor is the same as embodiment 1；To step (2)
Fermentation substrate according to solid-liquid ratio be 1:2. 25 plus mixed liquor, adjust pH5.5, sterilize 20min at 121 DEG C, obtains solid state fermentation
Culture medium；To be configured to bacteria concentration is 106 The Phanerochaete chrysosporium spore suspension of a/mL is connect with the inoculum concentration of quality 10%
Enter in solid-state fermentation culture medium, ferment 10d；10 are accessed again with the inoculum concentration of quality 14%6 The cellulose-degrading bacteria spore of a/mL
Suspension, 28 DEG C of culture 5d obtain the pretreatment product produced for protein feed；Wherein, cellulose-degrading bacteria spore suspension
Aspergillus niger in liquid, Trichoderma viride,Trichoderma asperelloidesThree bacterium inoculative proportions are 1:1:2.
As shown in figure 4, the degradation rate of lignin, hemicellulose and cellulose is respectively 98.6%, 86.7% in stalk at this time
With 58.3%.
Fig. 1 is influence of the different pretreatments method to lignin, cellulose and hemicellulose level variation in corn stover；
1 indicates crushing+diluted acid group, and 2 indicate crushing+diluted alkaline group, and 3 indicate crushing+ammonium hydroxide group, and 4 indicate crushing+microwave+diluted alkaline group, 5 tables
Show crushing+microwave+ammonium hydroxide group, 6 indicate crushing+whiterot fungi+aspergillus niger groups, and 7 indicate crushing+diluted acid+whiterot fungi+aspergillus niger groups, 8
Expression crushing+diluted alkaline+whiterot fungi+aspergillus niger group, 9 expression crushing+ammonium hydroxide+whiterot fungi+aspergillus niger groups, 10 expression crushing+microwaves+
Diluted alkaline+whiterot fungi+aspergillus niger group, 11 indicate crushing+microwave+ammonium hydroxide+whiterot fungi+aspergillus niger group, the original that the 8th group of final choice
Cause, although the 10th group of cellulose degradation rate is higher than the 8th group, the degradation of the 10th group of cellulose some be to occur in powder
Broken+microwave+diluted alkaline pretreatment stage, and the degradation of this stage fiber element can not provide carbon source for saccharomycete, because of physics-change
It after learning pretreatment, needs to filter, filter residue is washed to neutrality, and drying to constant weight is just used as fermentation substrate.Therefore it compares, the
8 groups of pretreating scheme is preferable.
Fig. 2 is the combination of different cellulose-degrading bacterias and inoculative proportion to lignin, cellulose and hemicellulose in corn stover
The influence of cellulose content variation；Experimental group 1 is Trichoderma viride, and experimental group 2 isTrichoderma asperelloides, experimental group 3
For aspergillus niger: Trichoderma viride=1:1, experimental group 4 are aspergillus niger:Trichoderma asperelloides=1:1, experimental group 5
For Trichoderma viride:Trichoderma asperelloides=1:1, experimental group 6 are aspergillus niger: Trichoderma viride:Trichoderma asperelloides=1:1:1, experimental group 7 are aspergillus niger: Trichoderma viride:Trichoderma asperelloides=2:1:1, experimental group 8 are aspergillus niger: Trichoderma viride:Trichoderma asperelloides =1:2:
1, experimental group 9 is aspergillus niger: Trichoderma viride:Trichoderma asperelloides =1:1:2。
Fig. 3 is cellulose-degrading bacteria inoculum concentration to lignin, cellulose and hemicellulose level variation in corn stover
It influences.
Fig. 4 cellulose-degrading bacteria ferments what number of days changed lignin, cellulose and hemicellulose level in corn stover
It influences.
Claims (3)
1. physical-chemical-Biological Pretreatment corn stover method, the method for the pretreatment corn stover the following steps are included:
(1) physical treatment: corn stover first passes through pulverizer and is crushed, and smashes it through 40 meshes；
(2) it is chemically treated: handling smashed corn stover with the NaOH solution of mass concentration 1.0%, solid-liquid mass volume ratio is
1:10,100 DEG C of 1h, filtering, filter residue are washed to neutrality, and drying to constant weight, as fermentation substrate；
(3) biological treatment: every 10mL is first prepared containing FeSO4·7H2O 0.0005g、MnSO4·7H2O 0.0016g、 ZnSO4·
7H2O 0.0014g、CoCl2The micro-mixed liquor of 0.002g；Next prepares nutritive solution, each component in nutritive solution
Mass percent concentration is: (NH4)2SO41%, KH2PO40.3%, MgSO40.05%, CaCl20.05%, remaining is distilled water；
Then mixed liquor is prepared, mixed liquor is made by adding micro-mixed liquor 1mL in every 100mL nutritive solution；To step (2)
Add mixed liquor according to feed liquid mass volume ratio in fermentation substrate for 1:2.25, adjusts pH5.5 in 121 DEG C of sterilizing 20min and obtain solid-state
Fermentation medium；To be configured to bacteria concentration is 106Phanerochaete chrysosporium (the Phanerochaete of a/mL
Chrysosporium) spore suspension is to send out in the inoculum concentration access solid-state fermentation culture medium of volume mass percentage 10%
Ferment 10d；10 are accessed again with the inoculum concentration of volume mass percentage 10%-14%6The cellulose-degrading bacteria spore suspension of a/mL,
28 DEG C of culture 3-5 d, obtain pretreatment product；The cellulose-degrading bacteria is aspergillus niger (Aspergillusniger), green
One or more of color trichoderma (Trichodermaviride) and Trichodermaasperelloides；Several moulds
Mixed volume ratio is respectively as follows: aspergillus niger: Trichoderma viride=1:1, aspergillus niger: Trichodermaasperelloides=1:1, green
Color trichoderma: Trichodermaasperelloides=1:1, aspergillus niger: Trichoderma viride: Trichodermaasperelloides
=1:1:1, aspergillus niger: Trichoderma viride: Trichodermaasperelloides=2:1:1, aspergillus niger: Trichoderma viride:
Trichodermaasperelloides=1:2:1, aspergillus niger: Trichoderma viride: Trichodermaasperelloides=1:1:
2；The Phanerochaete chrysosporium is activated using comprehensive PDA solid medium, the preparation process of comprehensive PDA culture medium
It is: KH is added on the basis of 1L PDA solid medium2PO4 3g、MgSO41.5g, vitamin B10.001g；It is involved
Microbial spore suspension the preparation method comprises the following steps: by the sterile water washing spore of activated bacterial strain, be placed in containing bead
In triangular flask, 20 min are vibrated in 28 DEG C of 120 r/min shaking table, vibrate liquid with 4 layers of sterilizing lens wiping paper filtering, under the microscope
Filtrate miospore is counted with blood counting chamber, adjusts its concentration to 106It is spare to be stored in 4 DEG C of refrigerators by a/mL.
2. physical-chemical according to claim 1-Biological Pretreatment corn stover method, it is characterized in that: the black song
Mould and Trichoderma viride is activated using PDA solid medium, and the preparation process of PDA culture medium is: potato is cleaned peeling
After weigh 200g potato and be cut into small pieces, add boiling rotten, then with eight layers of filtered through gauze, 15g agar is added, it is mixed to continue heating stirring
It is even, 20g glucose is added after agar has dissolved, stirs evenly, supplies moisture again to 1000mL after slightly cooling down.
3. physical-chemical according to claim 1-Biological Pretreatment corn stover method, it is characterized in that: described
Trichodermaasperelloides is activated using brewer's wort solid medium, the preparation of brewer's wort solid medium
Cheng Shi: weighing certain mass malt, crushes, and adds the water of 4 times of malt qualities, and 55-60 DEG C of heat preservation saccharification is stirred continuously, is saccharified
Degree iodometric titration, after 3-4h, by 4-6 layers of filtered through gauze of saccharified liquid；Filtered fluid is clarified with egg white；Method is by one
Egg resets and add water about 20mL, mixes well until generating foam, is subsequently poured into saccharified liquid and stirs, refilters after boiling to get clear
Clear brewer's wort；It is diluted with water into the brewer's wort of 5-6 ° of B é；In brewer's wort plus 16g agar, 0.1MPa pressure, sterilizing 15min are obtained
Brewer's wort solid medium.
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