CN113122522A - Method for promoting biological fermentation to produce cellulase - Google Patents
Method for promoting biological fermentation to produce cellulase Download PDFInfo
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- CN113122522A CN113122522A CN201911402446.6A CN201911402446A CN113122522A CN 113122522 A CN113122522 A CN 113122522A CN 201911402446 A CN201911402446 A CN 201911402446A CN 113122522 A CN113122522 A CN 113122522A
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- fermentation
- cellulase
- butanol
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- acidolysis
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- 238000000855 fermentation Methods 0.000 title claims abstract description 83
- 230000004151 fermentation Effects 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 42
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 39
- 229940106157 cellulase Drugs 0.000 title claims abstract description 39
- 230000001737 promoting effect Effects 0.000 title claims abstract description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 88
- 239000000047 product Substances 0.000 claims abstract description 25
- 239000002244 precipitate Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 14
- 241000193454 Clostridium beijerinckii Species 0.000 claims abstract description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 241000223261 Trichoderma viride Species 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 241000228245 Aspergillus niger Species 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000015099 wheat brans Nutrition 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 239000011686 zinc sulphate Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 235000009529 zinc sulphate Nutrition 0.000 claims description 2
- 241000894007 species Species 0.000 claims 3
- 239000000203 mixture Substances 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 description 25
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 229940088598 enzyme Drugs 0.000 description 17
- 108010047754 beta-Glucosidase Proteins 0.000 description 12
- 102000006995 beta-Glucosidase Human genes 0.000 description 12
- 239000002994 raw material Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000001913 cellulose Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000002803 fossil fuel Substances 0.000 description 2
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 2
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000002921 fermentation waste Substances 0.000 description 1
- 239000003502 gasoline Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- CQRYARSYNCAZFO-UHFFFAOYSA-N salicyl alcohol Chemical compound OCC1=CC=CC=C1O CQRYARSYNCAZFO-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- 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
- C12P7/16—Butanols
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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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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- Life Sciences & Earth Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
A method for promoting the biological fermentation to produce cellulase comprises the steps of inoculating clostridium beijerinckii XH0906 disclosed by CN106554931A into a butanol fermentation culture medium, carrying out fermentation to produce butanol, removing the butanol from a fermentation product, adding ethanol, collecting precipitate, adding sulfuric acid or hydrochloric acid into the precipitate for acidolysis, adding an acidolysis product into the culture medium of a cellulase fermentation strain, and inoculating the cellulase fermentation strain to promote the fermentation production of the cellulase. The method of the invention effectively utilizes the special butanol fermentation product of the special strain, solves the problem of subsequent treatment of the fermentation liquor, promotes the production of the cellulase and generates greater economic benefit.
Description
Technical Field
The invention relates to the technical field of biomass energy, in particular to a method for promoting cellulase fermentation.
Background
Butanol is an important organic chemical raw material, has wide application in the industrial fields of chemical industry, medicine, petroleum and the like, and is more and more concerned by governments and many multinational companies all over the world as a potential renewable biological energy source capable of replacing gasoline. Butanol is mainly derived from chemical synthesis and microbial fermentation, wherein chemical synthesis currently has cost advantages but requires a large amount of fossil fuel as a raw material. The production of butanol by microbial fermentation represents a future development trend because fossil fuels are non-renewable, uncertain in price and non-uniform in world distribution and cause serious environmental pollution when used in large quantities. The production of biobutanol by fermentation was once the second to the mature industry of bioethanol and was later impacted by the petroleum industry, and declined gradually. The development of biobutanol has revealed new features since the new century. Firstly, the conversion of butanol production raw materials, the traditional raw materials are molasses and starch, the fermentation process and technology are quite mature, but the conversion is only suitable for the countries with few rich raw materials, and the problem that the starch is used for producing biofuel and has ' strive for grains with people ' and strive for land with grains ' is generated, so that the development of the anaerobic clostridium butanol fermentation process taking lignocellulose as the raw material becomes a research hotspot.
Lignocellulose is the most widely distributed and abundant renewable carbohydrate resource in nature. The cellulose raw materials such as crop straws and the like contain a large amount of cellulose and hemicellulose polysaccharide substances. These polysaccharides are stable in nature, and even after pretreatment, they are hydrolyzed into monosaccharides by the action of a catalyst, and then they are fermented to prepare cellulose butanol. The most commonly used catalysts for hydrolysis are mineral acids and cellulase preparations. The enzymolysis utilizes a microbial enzyme system to degrade natural cellulose and hemicellulose into fermentable monosaccharides, and compared with a chemical hydrolysis method, the method has the advantages of high yield of the fermentable monosaccharides, mild reaction conditions, low energy consumption and environmental friendliness. In the production process of the cellulose butanol, the cost of the cellulase accounts for about one fourth of the production cost of the butanol, and the development of the production process of the cellulase with low cost has important significance for reducing the cost of the cellulose butanol and improving the economy of the butanol.
On the other hand, the change of raw materials brings a series of scientific problems to butanol fermentation. For example, the lower product concentration and yield results in higher unit consumption of raw material. The main products of traditional butanol biofermentation are acetone, butanol and ethanol, the content of which is about 6:3:1, abbe for short. Butanol selectivity was low, accounting for only 60% of the total ABE solvent. However, a large amount of byproducts are often generated in the butanol fermentation process, and how to fully utilize the byproducts to improve the added value is also the key to reduce the production cost of butanol.
Disclosure of Invention
Aiming at the high production cost of cellulase in the prior art, the invention provides a method for promoting the production of cellulase by biological fermentation, which takes the fermentation product of butanol produced by fermentation of clostridium beijerinckii XH0906 as an accelerant, is used for the biological fermentation of the cellulase, effectively utilizes the special butanol fermentation product of a special strain, solves the problem of subsequent treatment of fermentation liquor, plays a role in promoting the production of the cellulase and generates greater economic benefit.
The technical purpose of the invention is realized by the following technical scheme:
a method for promoting the biological fermentation to produce cellulase comprises the steps of inoculating clostridium beijerinckii XH0906 disclosed by CN106554931A into a butanol fermentation culture medium, carrying out fermentation to produce butanol, removing the butanol from a fermentation product, adding ethanol, collecting precipitate, adding sulfuric acid or hydrochloric acid into the precipitate for acidolysis, adding an acidolysis product into the culture medium of a cellulase fermentation strain, and inoculating the cellulase fermentation strain to promote the fermentation production of the cellulase.
Further, in the above method, the carbon source in the butanol fermentation medium is 50 to 150g/L of glucose or lignocellulose saccharification liquid having a sugar content equivalent thereto. As a more specific technical scheme, the butanol fermentation medium also comprises 5-10g/L of peptone, 4-8g/L of beef extract, 0.5-1.0g/L of ammonium sulfate, 0.05-0.2g/L of ferric sulfate and 0.1-0.5g/L of magnesium sulfate.
Furthermore, in the method, the fermentation temperature of the clostridium beijerinckii XH0906 for producing the butanol is 30-38 ℃, the pH is controlled to be 5.0-8.0, and the fermentation time is 72-120 h.
Further, among the above-mentioned methods, the Clostridium beijerinckii XH0906, the classification of which has been disclosed in CN106554931AIs named as Clostridium beijerinckii (Clostridium beijerinckii) The microbial inoculum is preserved in China general microbiological culture Collection center in 04 th month 05 in 2014, and the preservation number is CGMCC No. 9124.
Further, in the above method, the method for removing butanol from the fermentation product is distillation.
Further, in the above method, butanol is removed from the fermentation product, ethanol is added according to the volume of 3-8 times, after uniform mixing reaction, the precipitate is collected by centrifugation or filtration, and dried for 2-4h at 40-60 ℃.
Further, in the above method, the mass concentration of the sulfuric acid or hydrochloric acid is 0.5% to 5%.
Further, in the above method, the acidolysis treatment is followed by sterilization treatment, and the pH is adjusted to 5.5 to 7.0 after the sterilization treatment.
Further, in the above method, the cellulase-fermenting strain can be selected from all strains available in the prior art for producing cellulase, preferably Trichoderma viride (Trichoderma viride) (CN 105647813A)Trichodermaviride) Aspergillus niger F4, CN101899398A (A)Aspergillusniger) T2, more preferably Trichoderma viride (Trichoderma viride) ((III))Trichodermaviride）F4。
Further, in the above method, the addition ratio of the acidolysis product to the culture medium of the cellulase-fermenting strains is 20-100g solid/L culture medium by mass of the solid precipitate before acidolysis.
Further, in the above method, as a more specific technical solution, the culture medium of the cellulase zymocyte further comprises the following components: wheat bran 10-60g/L, potassium dihydrogen phosphate 0.1-10g/L, ammonium sulfate 1-10g/L, calcium chloride 0.01-3g/L, magnesium sulfate 0.01-3g/L, ZnSO4 & 7H2O 0.01-50mg/L，MnSO4·4H2O 0.01-20mg/mL，CoCl20.01-50mg/L and FeSO4 0.1-20mg/L。
Further, in the method, the cellulase fermentation adopts batch fermentation, the inoculation volume ratio of the strains is 5-15%, the fermentation temperature is 23-35 ℃, the pH value is 4.0-7.0, and the fermentation time is 48-168 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) polysaccharide byproducts in the process of fermenting butanol by clostridium beijerinckii are used as an induced carbon source for partial cellulase fermentation through a series of pretreatment, so that the fermentation of the cellulase is promoted, the production cost of the cellulase is effectively reduced, the product of butanol produced by fermentation is effectively utilized, and the economy of the product is improved.
(2) The residual insoluble nitrogen source, residual sugar and the like in the fermentation liquor for producing the butanol by fermenting the clostridium beijerinckii can be used as the components of the culture medium for fermenting the cellulase, so that the fermentation cost of the cellulase is saved, and the emission and treatment cost of the fermentation waste liquid is also reduced.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The invention determines the enzyme activities of FPA and beta-glucosidase, and the substrates for determining the enzyme activities respectively adopt filter paper and saligenin. The determination of the filter paper enzyme activity by the DNS method is defined as that a reducing sugar which catalyzes the hydrolysis of a substrate to generate 1 mu mol of glucose in 1min at 50 ℃ and pH5.0 is defined as an international enzyme activity unit. The enzyme activity unit of the beta-glucosidase is defined as the amount of enzyme required for generating 1 mu mol of p-nitrophenol by hydrolysis per minute under standard reaction conditions.
Glucose, xylose and butanol were all assayed using an Agilent 1200 liquid chromatograph (HPLC). The chromatographic column is Aminex HPX-87H (300 mm. times.7.8 mm) from Bio-Rad, and the detector is differential refractometric detectionDetector, mobile phase 0.005M H2SO4The flow rate of the aqueous solution was 0.7 mL/min.
Example 1
(1) The preparation components are 100g/L glucose, 8g/L peptone, 5g/L beef extract, 0.5g/L ammonium sulfate, 0.1g/L ferric sulfate and 0.25g/L magnesium sulfate, and the culture medium is inoculated with the Clostridium beijerinckii XH0906 strain for butanol fermentation. In the fermentation process, the stirring speed is 60rpm, the fermentation temperature is controlled to be 34 ℃, the pH value is controlled to be 7.0, and the fermentation time is 72 hours.
(2) And (2) after removing butanol from the fermentation liquor obtained in the step (1) by using a distillation method, adding 6 times of volume of absolute ethyl alcohol into the residual solution, uniformly mixing for 10min by using a shaker at room temperature, centrifuging for 10min at 4000rpm, removing supernatant, collecting precipitate, and drying for 2h at 40 ℃. The results show that the fermentation under the condition produces 13.5g/L of butanol, consumes 82g/L of glucose, and collects 28.9g/L of precipitate; adding the precipitate into 2% sulfuric acid at a concentration of 40g/L, performing acidolysis, reacting in a sterilizing pot at 121 deg.C for 60min, cooling, and adjusting pH to 6.0;
(3) preparing a cellulase fermentation culture medium according to the following component proportion: 30g/L of wheat bran, 0.5g/L of monopotassium phosphate, 4.8g/L of ammonium sulfate, 0.3g/L of calcium chloride, 0.3g/L of magnesium sulfate and ZnSO4•7H2O 6.3mg/L，MnSO4•4H2O 1.6mg/mL，CoCl24mg/L，FeSO42mg/L and the product obtained in the step (2), wherein the mass of solid precipitates in the culture medium is 40g of solid/L, and the Trichoderma viride provided by CN105647813A is inoculated (the culture medium is inoculated with the solid precipitates before acidolysis)Trichodermaviride) F4, fermenting in batches, wherein the inoculation volume ratio of the strain is 10%, the fermentation temperature is 30.5 ℃, the pH value is 6.0, the stirring speed is 400r/min, and sampling is carried out after 144 hours of fermentation to analyze the filter paper enzyme activity and the beta-glucosidase enzyme activity of the fermentation liquid. Analysis results show that the filter paper enzyme activity reaches 15.8 IU/mL, and the average beta-glucosidase activity is 3.7 IU/mL.
Example 2
The procedure of example 1 was repeated, except that the product of step (2) was added to the cellulase fermentation medium in step (3) in an amount of 20g solid/L based on the mass of the solid precipitate before acidolysis. After 144h of fermentation, sampling and analyzing the filter paper enzyme activity and the beta-glucosidase activity of the fermentation liquor. Analysis results show that the filter paper enzyme activity reaches 12.4 IU/mL, and the average beta-glucosidase activity is 2.2 IU/mL.
Example 3
The procedure of example 1 was repeated, except that the product of step (2) was added to the cellulase fermentation medium in step (3) in an amount of 80g solid/L based on the mass of the solid precipitate before acidolysis. After 144h of fermentation, sampling and analyzing the filter paper enzyme activity and the beta-glucosidase activity of the fermentation liquor. Analysis results show that the filter paper enzyme activity reaches 14.1 IU/mL, and the average beta-glucosidase activity is 2.8 IU/mL.
Example 4
The procedure of example 1 was repeated, except that Aspergillus niger T2 supplied in CN101899398A was used as the fermentative strain in the cellulase fermentation of step (3). Analysis results show that the filter paper enzyme activity reaches 9.3 IU/mL, and the average beta-glucosidase activity is 6.5 IU/mL.
Comparative example 1
The procedure of example 1 was followed in the same manner as in step (3) except that the product obtained in step (2) was not added to the cellulase fermentation medium in accordance with step (3) of example 1. Analysis results show that the filter paper enzyme activity reaches 6.5 IU/mL, and the average beta-glucosidase activity is 1.8 IU/mL.
Comparative example 2
The same procedure as in example 1 was repeated, except that the same amount of acid-treated corncobs was used in step (3) instead of the product of step (2). Analysis results show that the filter paper enzyme activity reaches 12.5 IU/mL, and the average beta-glucosidase activity is 2.5 IU/mL.
Comparative example 3
The procedure of example 1 was repeated, except that the acid treatment was not carried out in step (2) using sulfuric acid having a mass concentration of 2%. The analysis result shows that the filter paper enzyme activity reaches 7.3 IU/mL, and the average beta-glucosidase activity is 2.0 IU/mL.
Claims (11)
1. A method for promoting the biological fermentation to produce cellulase comprises the steps of inoculating clostridium beijerinckii XH0906 disclosed by CN106554931A into a butanol fermentation culture medium, carrying out fermentation to produce butanol, removing the butanol from a fermentation product, adding ethanol, collecting precipitate, adding sulfuric acid or hydrochloric acid into the precipitate for acidolysis, adding an acidolysis product into the culture medium of a cellulase fermentation strain, and inoculating the cellulase fermentation strain to promote the fermentation production of the cellulase.
2. The method of claim 1, wherein the carbon source in the butanol fermentation medium is 50-150g/L glucose or lignocellulose saccharification solution of comparable sugar content.
3. The method of claim 1, wherein the clostridium beijerinckii XH0906 fermentation to produce butanol is performed at a fermentation temperature of 30-38 ℃, a pH of 5.0-8.0, and a fermentation time of 72-120 h.
4. The method of claim 1, wherein the butanol is removed from the fermentation product by distillation.
5. The method of claim 1, wherein the butanol is removed from the fermentation product, ethanol is added in an amount of 3 to 8 times the volume of the fermentation product, the mixture is mixed and reacted, and the precipitate is collected by centrifugation or filtration and dried at 40 to 60 ℃ for 2 to 4 hours.
6. The method according to claim 1, wherein the sulfuric acid or hydrochloric acid has a mass concentration of 0.5% to 5%.
7. The method as claimed in claim 1, wherein the acidolysis treatment is followed by a sterilization treatment, and the pH is adjusted to 5.5 to 7.0 after the sterilization treatment.
8. The method of claim 1, wherein the cellulase fermenting species is Trichoderma viride (Trichoderma viride) provided in CN105647813ATrichodermaviride) Aspergillus niger (A.niger) provided in F4 or CN101899398AAspergillusniger）T2。
9. The method as claimed in claim 1, wherein the acidolysis product is added to the medium of the cellulase-fermenting species in a proportion of 20 to 100g solid/L medium based on the mass of the solid precipitate before acidolysis.
10. The method of claim 1, wherein the culture medium of the cellulase fermenting species further comprises the following components: wheat bran 10-60g/L, potassium dihydrogen phosphate 0.1-10g/L, ammonium sulfate 1-10g/L, calcium chloride 0.01-3g/L, magnesium sulfate 0.01-3g/L, ZnSO4 & 7H2O 0.01-50mg/L，MnSO4·4H2O 0.01-20mg/mL，CoCl20.01-50mg/L and FeSO4 0.1-20mg/L。
11. The method as claimed in claim 1, wherein the cellulase fermentation is a batch fermentation, the inoculation volume ratio of the strain is 5% -15%, the fermentation temperature is 23-35 ℃, the pH is 4.0-7.0, and the fermentation time is 48-168 h.
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