CN114410505B - Clostridium thermocellum and its application in lignocellulose hydrolysis - Google Patents
Clostridium thermocellum and its application in lignocellulose hydrolysis Download PDFInfo
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- 241000193448 Ruminiclostridium thermocellum Species 0.000 title claims abstract description 71
- 230000007062 hydrolysis Effects 0.000 title claims abstract description 38
- 238000006460 hydrolysis reaction Methods 0.000 title claims abstract description 38
- 239000000835 fiber Substances 0.000 claims abstract description 51
- 239000010902 straw Substances 0.000 claims abstract description 45
- 238000004321 preservation Methods 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims description 56
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 40
- 238000000855 fermentation Methods 0.000 claims description 39
- 239000012528 membrane Substances 0.000 claims description 32
- 230000004151 fermentation Effects 0.000 claims description 23
- 239000000413 hydrolysate Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 18
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 14
- 238000001471 micro-filtration Methods 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 10
- 230000003301 hydrolyzing effect Effects 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 238000001728 nano-filtration Methods 0.000 claims description 9
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 8
- 241000209094 Oryza Species 0.000 claims description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- 235000009566 rice Nutrition 0.000 claims description 8
- 235000010265 sodium sulphite Nutrition 0.000 claims description 8
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 7
- 229920005610 lignin Polymers 0.000 claims description 7
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- 239000008108 microcrystalline cellulose Substances 0.000 claims description 7
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- 238000011218 seed culture Methods 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 6
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 claims description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical class SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 5
- 239000004743 Polypropylene Substances 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
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- 235000020958 biotin Nutrition 0.000 claims description 5
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- 229960002303 citric acid monohydrate Drugs 0.000 claims description 5
- -1 polypropylene Polymers 0.000 claims description 5
- 229920001155 polypropylene Polymers 0.000 claims description 5
- 239000001508 potassium citrate Substances 0.000 claims description 5
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims description 5
- HNWCOANXZNKMLR-UHFFFAOYSA-N pyridoxamine dihydrochloride Chemical compound Cl.Cl.CC1=NC=C(CO)C(CN)=C1O HNWCOANXZNKMLR-UHFFFAOYSA-N 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 235000015870 tripotassium citrate Nutrition 0.000 claims description 5
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 4
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- 238000005406 washing Methods 0.000 claims description 4
- 239000004695 Polyether sulfone Substances 0.000 claims description 2
- 229920006393 polyether sulfone Polymers 0.000 claims description 2
- 108010059892 Cellulase Proteins 0.000 abstract description 5
- 108010084185 Cellulases Proteins 0.000 abstract description 5
- 102000005575 Cellulases Human genes 0.000 abstract description 5
- 229940106157 cellulase Drugs 0.000 abstract description 5
- 241000499912 Trichoderma reesei Species 0.000 abstract description 3
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- 230000002708 enhancing effect Effects 0.000 abstract description 2
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- 241000193403 Clostridium Species 0.000 abstract 1
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- 229920002678 cellulose Polymers 0.000 description 9
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- 108090000623 proteins and genes Proteins 0.000 description 7
- 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 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
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- 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
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- 229920012266 Poly(ether sulfone) PES Polymers 0.000 description 2
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- 102000004157 Hydrolases Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- 239000003513 alkali 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
- 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
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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)
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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
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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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
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The application relates to the technical field of microorganism application, in particular to clostridium thermocellum and application thereof in lignocellulose hydrolysis. The application provides clostridium thermocellumClostridium thermocellumPN2101, 2021, 07 and 09, and the preservation number is CGMCC No.22868. The clostridium thermocellum provided by the application enhances endo-cellulase through metabolic modificationcelAExocellulasescelSAndβ-glucosidasebglThereby enhancing the enzymatic activity of clostridium thermocellum. The cellulase produced by the strain can obviously improve the hydrolysis rate of straw fibers in hydrolysis application; compared with the commercial Trichoderma reesei cellulase, the hydrolysis rate of the straw fiber can be improved by 20% under the same addition amount and hydrolysis conditions.
Description
Technical Field
The application belongs to the technical field of microorganism application, and particularly relates to clostridium thermocellum and application thereof in lignocellulose hydrolysis.
Background
Lignocellulose is a renewable resource with wide distribution, huge quantity and difficult treatment, and has important significance in converting the lignocellulose into renewable energy. Lignocellulose is mainly composed of lignin, hemicellulose and lignin, cellulose is a highly ordered, tightly bound crystal structure, it is tightly connected to hemicellulose, the periphery is surrounded by lignin, it is quite difficult to hydrolyze cellulose directly into usable glucose. At present, most of lignocellulose enzymatic hydrolysis uses cellulase derived from filamentous fungi, and the typical enzymes are trichoderma, aspergillus, penicillium and the like, but the enzyme consumption in the enzymolysis process is high, the activity is extremely easy to be inhibited by products, the hydrolysis efficiency is low, and the large-scale application of the lignocellulose enzymatic hydrolysis is restricted. Therefore, the construction of novel lignocellulose hydrolysis strains with high activity to improve the cellulose hydrolysis efficiency is a problem to be solved.
Disclosure of Invention
The application aims to provide clostridium thermocellum and application thereof in lignocellulose hydrolysis, so as to solve the problem of low hydrolysis efficiency of cellulose by adopting cellulose in the prior art and achieve the effect of improving the hydrolysis efficiency of cellulose.
The application provides clostridium thermocellum, which is Clostridium thermocellum PN2101, the preservation date is 2021, 07 and 09, the preservation unit is China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.22868, and the preservation address is China academy of sciences of China, national institute of sciences No. 3, the North Chen and West Lu No.1 of the Beijing area of the Chachiensis.
In one embodiment, the clostridium thermocellum comprises the nucleotide sequence shown in SEQ ID No.1 in the sequence listing.
The application provides application of the clostridium thermocellum in lignocellulose hydrolysis.
The application provides a method for hydrolyzing lignocellulose by adopting the clostridium thermocellum, which comprises the steps of straw fiber preparation, clostridium thermocellum seed liquid anaerobic fermentation, fiber hydrolysis and hydrolysate separation and concentration.
In one embodiment, the straw fiber preparation comprises the steps of: carrying out hydrothermal reaction on the washed and chopped rice straw raw materials and alkaline substances, fully removing lignin, extracting fibers, dehydrating and fully washing off residual lye to obtain straw fibers; wherein the alkaline substance comprises sodium hydroxide and sodium sulfite, and the ratio of the weight of the sodium hydroxide to the absolute dry weight of the rice straw raw material is 1: (4-6), the weight ratio of sodium sulfite to sodium hydroxide is 1: (3-5), the temperature of the hydrothermal reaction is 150-160 ℃, and the time of the hydrothermal reaction is 1-3h.
In one embodiment, the anaerobic fermentation of clostridium thermocellum seed solution comprises the steps of: inoculating clostridium thermocellum seed liquid into fermentation in the inoculation amount of 5-10%Culturing in culture medium at 50-65deg.C and 150-200rpm for 16-24 hr to obtain fermentation broth; wherein, the components of the fermentation medium comprise the following components in volume ratio of 40:2:1:1: liquid A, liquid B, liquid C, liquid D and liquid E of 1; the solution A comprises 5.00-10.00g/L straw fiber and 10.00 g/L3-morpholinopropane sulfonic acid; the solution B comprises 50.00g/L of tripotassium citrate, 31.25g/L of citric acid monohydrate and 25.00g/L of Na 2 SO 4 、25.00g/L KH 2 PO 4 、62.50g/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The solution C comprises 250.00g/L urea; the D solution comprises 50.00g/L MgCl 2 ·6H 2 O、10.00g/L CaCl 2 ·2H 2 O、5.00g/L FeCl 2 ·4H 2 O, 50.00g/L cysteine salt; the E solution comprises 1.00g/L pyridoxamine dihydrochloride, 0.20g/L para-aminobenzoic acid, 0.10g/L biotin and 0.10g/L VB 12 。
In one embodiment, the clostridium thermocellum seed solution is obtained by thawing clostridium thermocellum strains, culturing the clostridium thermocellum strains in a seed culture medium, wherein the inoculation amount of the clostridium thermocellum strains is 2.5%, the culturing temperature is 50-65 ℃, the shaking speed is 150-200rpm, and the time is 16-24 hours; wherein, the components of the seed culture medium comprise the following components in volume ratio of 40:2:1:1: liquid A, liquid B, liquid C, liquid D and liquid E of 1; the solution A comprises 5.00-10.00g/L microcrystalline cellulose and 10.00 g/L3-morpholinopropane sulfonic acid; the solution B comprises 50.00g/L of tripotassium citrate, 31.25g/L of citric acid monohydrate and 25.00g/L of Na 2 SO 4 、25.00g/L KH 2 PO 4 、62.50g/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The solution C comprises 250.00g/L urea; the D solution comprises 50.00g/L MgCl 2 ·6H 2 O、10.00g/L CaCl 2 ·2H 2 O、5.00g/L FeCl 2 ·4H 2 O, 50.00g/L cysteine salt; the E solution comprises 1.00g/L pyridoxamine dihydrochloride, 0.20g/L para-aminobenzoic acid, 0.10g/L biotin and 0.10g/L VB 12 。
In one embodiment, the fiber hydrolysis comprises the steps of: and soaking the straw fibers in a buffer solution, and then adding a fermentation liquor obtained by anaerobic fermentation of clostridium thermocellum seed liquid and xylanase for hydrolysis to obtain a hydrolysate.
In one embodiment, the pH of the hydrolysis is 4.5 to 5.5, and the solid to liquid ratio is 1: (10-30), the temperature is 45-55 ℃, the time is 12-48h, the oscillating speed is 150-250r/min, the added fermentation liquor per gram of straw fiber is 10-40 mu L, and the added xylanase per gram of straw fiber is 4mg.
In one embodiment, the hydrolysate separation and concentration comprises the steps of: carrying out solid-liquid separation on the hydrolysate, and concentrating the collected hydrolysate; wherein, the solid-liquid separation adopts a microfiltration membrane, and the microfiltration membrane is a polypropylene membrane; the concentration adopts nanofiltration membrane, and the nanofiltration membrane is polyether sulfone membrane.
The one or more technical schemes provided by the application have at least the following technical effects or advantages:
(1) The application provides a clostridium thermocellum engineering bacterium, which is subjected to metabolic transformation, so that the promoter expression of endo-cellulase celA, exo-cellulase celS and beta-glucosidase bgl is enhanced, the enzyme activity of clostridium thermocellum is enhanced, and the clostridium thermocellum engineering bacterium can be effectively applied to the hydrolysis of lignocellulose; under the same enzymolysis condition, compared with the traditional cellulose hydrolysis, the hydrolysis efficiency can be improved by 20 percent.
(2) The method for hydrolyzing lignocellulose by using the clostridium thermocellum provided by the application has high hydrolysis efficiency, the glucose concentration in the hydrolysate can reach 70.00g/L when the clostridium thermocellum is hydrolyzed for 48 hours, and the glucose concentration is about 48.00g/L when the clostridium thermocellum is hydrolyzed for 48 hours by using commercial Trichoderma reesei cellulase instead of clostridium thermocellum fermentation broth; the method can be used for industrial production and has good application prospect.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
FIG. 1 is a schematic diagram showing the structure of the inserted gene sequence of Clostridium thermocellum in example 1.
Detailed Description
The technical scheme of the application will be clearly and completely described in the following in connection with the embodiments of the application. The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.
The embodiment of the application provides clostridium thermocellum, which is Clostridium thermocellum PN2101, the preservation date is 2021, 07 and 09, the preservation unit is China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.22868, and the preservation address is China academy of sciences of China, national institute of microbiology, national institute No. 3, north Chen and West Lu 1, the Korean area of Beijing city.
The fibrous body secreted by clostridium thermocellum has extremely strong cellulose degradation capability, and consists of a plurality of catalytic units with cellulase activity and scaffold proteins for assembling the catalytic units. The ability of the fiber corpuscles to degrade cellulose is about 50 times greater than that of trichoderma, and this efficiency depends on the specific structure of the fiber corpuscles. Clostridium thermocellum is a strictly anaerobic thermophilic microorganism, and the special growth conditions enable the secreted lignocellulose hydrolase to have the advantages of better heat resistance, minimal risk of mixed bacteria pollution and the like.
The clostridium thermocellum provided by the embodiment of the application is an engineering strain which is obtained by genetically modifying wild clostridium thermocellum and is used for enhancing the expression of promoters of endo-cellulase celA, exo-cellulase celS and beta-glucosidase bgl, and comprises a nucleotide sequence shown as SEQ ID NO.1 in a sequence table. The wild clostridium thermocellum is separated from cotton heap, is a thermophilic and strictly anaerobic gram positive bacterium, and has a growth temperature of 50-65 ℃ and a fast growth speed.
The embodiment of the application provides a method for hydrolyzing lignocellulose by clostridium thermocellum, which comprises the steps of straw fiber preparation, clostridium thermocellum seed liquid anaerobic fermentation, fiber hydrolysis and hydrolysate separation and concentration.
Step one: straw fiber preparation
In one embodiment, the straw fiber preparation comprises the steps of: carrying out hydrothermal reaction on the washed and chopped rice straw raw materials and alkaline substances, fully removing lignin, extracting fibers, dehydrating and fully washing off residual lye to obtain straw fibers.
Wherein the alkaline substance comprises sodium hydroxide and sodium sulfite, and the ratio of the weight of the sodium hydroxide to the absolute dry weight of the rice straw raw material is 1: (4-6), the weight ratio of sodium sulfite to sodium hydroxide is 1: (3-5), the temperature of the hydrothermal reaction is 150-160 ℃, and the time of the hydrothermal reaction is 1-3h.
Step two: anaerobic fermentation of clostridium thermocellum seed liquid
In one embodiment, the anaerobic fermentation of clostridium thermocellum seed liquid sequentially comprises the steps of preparing a culture medium, deoxidizing, preparing seed liquid and anaerobic fermentation.
Preparation of culture medium and deoxidization: the culture medium adopts MTC culture medium, and the culture medium comprises five types of ABCDE liquid. Wherein, the solution A is 5.00-10.00g/L carbon source (microcrystalline cellulose or straw fiber) and 10.00 g/L3-morpholinopropane sulfonic acid (MOPS); the solution B is 50.00g/L of tripotassium citrate, 31.25g/L of citric acid monohydrate and 25.00g/L of Na 2 SO 4 、25.00g/L KH 2 PO 4 、62.50g/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the 250.00g/L urea for solution C and 50.00g/L MgCl for solution D 2 ·6H 2 O、10.00g/L CaCl 2 ·2H 2 O、5.00g/L FeCl 2 ·4H 2 O, 50.00g/L cysteine salt; the E solution is 1.00g/L pyridoxamine dihydrochloride, 0.20g/L para-aminobenzoic acid, 0.10g/L biotin, 0.10g/L VB 12 . After the preparation of the solution A is finished, sterilizing at 121 ℃ for 20 min; B. c, D, E liquid is prepared according to the concentration, and is filled in an anaerobic bottle, and after being sealed by an aluminum cover with a rubber plug, the anaerobic bottle is repeatedly vacuumized and filled with high-purity nitrogen for 3 times, and finally the inside of the anaerobic bottle is kept at positive pressure. 5 culture solutions according to 40:2:1:1:1 (v/v) A sterile disposable syringe and 0.22 μm sterile filter were used in a super clean bench to fill the sterilized anaerobic jar with liquid A, the total volume not exceeding 40% of the anaerobic jar volume. The carbon source (microcrystalline cellulose or straw fiber) in the solution A was microcrystalline cellulose Avicel PH105 in the seed medium at a concentration of5.00-10.00g/L; straw fiber is used in the fermentation medium, and the concentration is 5.00-10.00g/L. In some embodiments, the straw fiber used in the fermentation medium is derived from the straw fiber produced in step one.
Seed liquid preparation: placing the clostridium thermocellum strain preserved at-80deg.C in a refrigerator at 4deg.C for thawing, sucking the strain under aseptic condition, injecting into seed culture medium with inoculum size of 2.5%, and culturing at 50-65deg.C and 150-200rpm for 16-24 hr.
Anaerobic fermentation: inoculating the seed solution into fermentation medium at 5% -10% inoculum size (v/v), and culturing at 50-65deg.C and 150-200rpm for 16-24 hr to obtain fermentation broth.
Step three: fiber hydrolysis
In one embodiment, the fiber hydrolysis comprises the steps of: and soaking the straw fibers in a buffer solution, and then adding a fermentation liquor obtained by anaerobic fermentation of clostridium thermocellum seed liquid and xylanase for hydrolysis to obtain a hydrolysate.
Wherein the pH value of the hydrolysis is 4.5-5.5, and the solid (absolute dry weight of straw fiber) liquid ratio is 1: (10-30), the temperature is 45-55 ℃, the time is 12-48h, the oscillating speed is 150-250r/min, the added fermentation liquor per gram of straw fiber is 10-40 mu L, and the added xylanase per gram of straw fiber is 4mg.
The buffer solution adopted in the embodiment of the application has a buffering effect, and the type of the buffer solution is not particularly limited, for example, acetic acid-sodium acetate buffer solution can be selected.
Step four: separating and concentrating hydrolysate
In one embodiment, the hydrolysate separation and concentration comprises the steps of: carrying out solid-liquid separation on the hydrolysate, and concentrating the collected hydrolysate; wherein, the solid-liquid separation adopts a microfiltration membrane, and the microfiltration membrane is a polypropylene membrane; the concentration adopts nanofiltration membrane, and the nanofiltration membrane is polyether sulfone (PES) membrane. In the embodiment of the application, a hollow fiber microfiltration membrane is adopted to remove thalli and residues in the hydrolysate.
The microfiltration membrane adopted in the embodiment of the application is a hollow fiber microfiltration membrane, a polypropylene membrane can be selected for removing thalli and residues in the hydrolysate, and the microfiltration membrane can be purchased from Shandong gold-bearing membrane Tian-liability companyModel MF1, pore diameter of 0.2 μm and effective area of 0.33m 2 The operating pressure was 0.1MPa and the cross-flow velocity was 0.052m/s. The nanofiltration membrane adopted by the embodiment of the application is a polyether sulfone (PES) membrane and is used for concentrating the hydrolysate at room temperature; can be purchased from the electric company of the United states, and has the model of nanofiltration membrane DK1812C-34D, the operating pressure is 17bar, the flow is 5L/min, the operating time is 3min/L, and the pH value is natural.
In order to better understand the above technical solutions, the following describes the above technical solutions in detail with reference to specific embodiments.
Example 1
The embodiment provides clostridium thermocellum, wherein the clostridium thermocellum is Clostridium thermocellum PN2101, the preservation date is 2021, 07 and 09, the preservation unit is China general microbiological culture collection center (CGMCC), the preservation number is CGMCC No.22868, and the preservation address is China academy of sciences of China, national institute of sciences of China, no.1, and No. 3 of the North Chen West road of the Beijing area.
The clostridium thermocellum is obtained by genetically modifying wild clostridium thermocellum, and the celA, the celS and the bgl are over-expressed by a method of increasing the gene copy number on the clostridium thermocellum genome and adding a strong promoter, and the celA, the celS and the bgl are over-expressed by a method of increasing the gene copy number on the genome and adding a strong promoter. An expressed gene sequence was inserted downstream of the L-lactate dehydrogenase gene on the genome. The plasmid is transferred into clostridium thermocellum by electrotransformation, and the target gene sequence is inserted into genome by homologous recombination, the structure of the inserted gene sequence is shown in figure 1, and the inserted gene sequence is shown in SEQ ID NO.1 in the sequence table, namely GTGGGAATAGGCATGGAAAAGATTTTTTTGCCCGAGGATGCATTGATAAAGTATTTCTTTTCCGAAAGAGAGGAAAAGATTCTAAAGAGTTTTGGAAATACTGATGAATATTGTGTGCAGAGTACAATTCTATGGACAAGAAAAGAGGCTTTGTCAAAACTTTTTCGTCTGGGAATGAGGATGGATTTTAAAAAGCTGGATACTTTGGAGGGCGAGGTGGTTTTTCAGGAAACAAACAGGGCGCGTCTGTTTTCTTTTATATGCAATAATTACTGTATCTCTCTGGCATTGCCAGGTTTTAATAAAGATTAAAATTATTGACTAGAAATAAAAAAATTGTCCATAATATTAATGGACAAAAAAACAAAGAATTACATCAAAGGAAGATAAAAATACTTTGTTAAAAAATTAATTATTTTTTATCTAAACTATTGAAAATGAAAATAAAATAATATAAAATGAATCATAGTGCAAGAGATACTTGCCAGAGGATGAATATTTTACTGCATTCATGCTTTATGGCAGCTAATAGAGGCATTAAATTAAATTTTAATTTACAATAGGAGGCGATATTAGTGAAGAACGTAAAAAAAAGAGTAGGTGTGGTTTTGCTGATTCTTGCAGTGTTGGGGGTTTATATGTTGGCAATGCCGGCAAACACTGTGTCAGCGGCAGGTGTGCCTTTTAACACAAAATACCCCTATGGTCCTACTTCTATTGCCGATAATCAGTCGGAAGTAACTGCAATGCTCAAAGCAGAATGGGAAGACTGGAAGAGCAAGAGAATTACCTCGAACGGTGCAGGAGGATACAAGAGAGTACAGCGTGATGCTTCCACCAATTATGATACGGTATCCGAAGGTATGGGATACGGACTTCTTTTGGCGGTTTGCTTTAACGAACAGGCTTTGTTTGACGATTTATACCGTTACGTAAAATCTCATTTCAATGGAAACGGACTTATGCACTGGCACATTGATGCCAACAACAATGTTACAAGTCATGACGGCGGCGACGGTGCGGCAACCGATGCTGATGAGGATATTGCACTTGCGCTCATATTTGCGGACAAGTTATGGGGTTCTTCCGGTGCAATAAACTACGGGCAGGAAGCAAGGACATTGATAAACAATCTTTACAACCATTGTGTAGAGCATGGATCCTATGTATTAAAGCCCGGTGACAGATGGGGAGGTTCATCAGTAACAAACCCGTCATATTTTGCGCCTGCATGGTACAAAGTGTATGCTCAATATACAGGAGACACAAGATGGAATCAAGTGGCGGACAAGTGTTACCAAATTGTTGAAGAAGTTAAGAAATACAACAACGGAACCGGCCTTGTTCCTGACTGGTGTACTGCAAGCGGAACTCCGGCAAGCGGTCAGAGTTACGACTACAAATATGATGCTACACGTTACGGCTGGAGAACTGCCGTGGACTATTCATGGTTTGGTGACCAGAGAGCAAAGGCAAACTGCGATATGCTGACCAAATTCTTTGCCAGAGACGGGGCAAAAGGAATCGTTGACGGATACACAATTCAAGGTTCAAAAATTAGCAACAATCACAACGCATCATTTATAGGACCTGTTGCGGCAGCAAGTATGACAGGTTACGATTTGAACTTTGCAAAGGAACTTTATAGGGAGACTGTTGCTGTAAAGGACAGTGAATATTACGGATATTACGGAAACAGCTTGAGACTGCTCACTTTGTTGTACATAACAGGAAACTTCCCGAATCCTTTGAGTGACCTTTCCGGCCAACCGACACCACCGTCGAATCCGACACCTTCATTGCCTCCTCAGGTTGTTTACGGTGATGTAAATGGCGACGGTAATGTTAACTCCACTGATTTGACTATGTTAAAAAGATATCTGCTGAAGAGTGTTACCAATATAAACAGAGAGGCTGCAGACGTTAATCGTGACGGTGCGATTAACTCCTCTGACATGACTATATTAAAGAGATATCTGATAAAGAGCATACCCCACCTACCTTATTAGTTAAAAAGAATAAAAAAGTATATTTGAAAGGGGAAGATGGAGAGAATGGTAAAAAGCAGAAAGATTTCTATTCTGTTGGCAGTTGCAATGCTGGTATCCATAATGATACCCACAACTGCATTCGCAGGTCCTACAAAGGCACCTACAAAAGATGGGACATCTTATAAGGATCTTTTCCTTGAACTCTACGGAAAAATTAAAGATCCTAAGAACGGATATTTCAGCCCAGACGAGGGAATTCCTTATCACTCAATTGAAACATTGATCGTTGAAGCGCCGGACTACGGTCACGTTACTACCAGTGAGGCTTTCAGCTATTATGTATGGCTTGAAGCAATGTATGGAAATCTCACAGGCAACTGGTCCGGAGTAGAAACAGCATGGAAAGTTATGGAGGATTGGATAATTCCTGACAGCACAGAGCAGCCGGGTATGTCTTCTTACAATCCAAACAGCCCTGCCACATATGCTGACGAATATGAGGATCCTTCATACTATCCTTCAGAGTTGAAGTTTGATACCGTAAGAGTTGGATCCGACCCTGTACACAACGACCTTGTATCCGCATACGGTCCTAACATGTACCTCATGCACTGGTTGATGGACGTTGACAACTGGTACGGTTTTGGTACAGGAACACAGGCAACATTCATAAACACCTTCCAAAGAGGTGAACAGGAATCCACATGGGAAACCATTCCTCATCCGTCAATAGAAGAGTTCAAATACGGCGGACCGAACGGATTCCTTGATTTGTTTACAAAGGACAGATCATATGCAAGACAGTGGCGTTATACAAACGCTCCTGACGCAGAAGGCCGTGCTATACAGGCTGTTTACTGGGCAAACAAATGGGCAAAGGAGCAGGGTAAAGGTTCTGCCGTTGCTTCCGTTGTATCCAAGGCTGCAAAGATGGGTGACTTCTTGAGAAACGACATGTTCGACAAATACTTCATGAAGATCGGTGCACAGGACAAGACTCCTGCTACCGGTTATGACAGTGCACACTACCTTATGGCCTGGTATACTTCATGGGGTGGTGGAATTGGTGCATCCTGGGCATGGAAGATCGGATGCAGCCACGCACACTTCGGATATCAGAACCCATTCCAGGGATGGGTAAGTGCAACACAGAGCGACTTTGCTCCTAAATCATCCAACGGTAAGAGAGACTGGACAACAAGCTACAAGAGACAGCTTGAATTCTATCAGTGGTTGCAGTCGGCTGAAGGTGCTATTGCCGGTGGAGCAACCAACTCCTGGAACGGTAGATATGAGAAATATCCTGCTGGTACGTCAACGTTCTATGGTATGGCATATGTTCCGCATCCTGTATACGCTGACCCGGGTAGTAACCAGTGGTTCGGATTCCAGGCATGGTCAATGCAGCGTGTAATGGAGTACTACCTCGAAACAGGAGATTCATCAGTTAAGAATTTGATTAAGAAGTGGGTCGACTGGGTAATGAGCGAAATTAAGCTCTATGACGATGGAACATTTGCAATTCCTAGCGACCTCGAGTGGTCAGGTCAGCCTGATACATGGACCGGAACATACACAGGCAACCCGAACCTCCATGTAAGAGTAACTTCTTACGGTACTGACCTTGGTGTTGCAGGTTCACTTGCAAATGCTCTTGCAACTTATGCCGCAGCTACAGAAAGATGGGAAGGAAAACTTGATACAAAAGCAAGAGACATGGCTGCTGAACTGGTTAACCGTGCATGGTACAACTTCTACTGCTCTGAAGGAAAAGGTGTTGTTACTGAGGAAGCACGTGCTGACTACAAACGTTTCTTTGAGCAGGAAGTATACGTTCCGGCAGGTTGGAGCGGTACTATGCCGAACGGTGACAAGATTCAGCCTGGTATTAAGTTCATAGACATCCGTACAAAATATAGACAAGATCCTTACTACGATATAGTATATCAGGCATACTTGAGAGGCGAAGCTCCTGTATTGAATTATCACCGCTTCTGGCATGAAGTTGACCTTGCAGTTGCAATGGGTGTATTGGCTACATACTTCCCGGATATGACATATAAAGTACCTGGTACTCCTTCTACTAAATTATACGGCGACGTCAATGATGACGGAAAAGTTAACTCAACTGACGCTGTAGCATTGAAGAGATATGTTTTGAGATCAGGTATAAGCATCAACACTGACAATGCCGATTTGAATGAAGACGGCAGAGTTAATTCAACTGACTTAGGAATTTTGAAGAGATATATTCTCAAAGAAATAGATACATTGCCGTACAAGAACTAATATAAACGTGTAAAGGGGGTGGCGATAATGGTTACTTTTAAGCTTAATGATGAATTCATGTTTGGAACCGCTACCGCAAGTACTCAGATTGAGGGTGGGGATACGGGAAATACATGGTATAAGTGGTGCCAGGAAGGACGTATCAAGGACTCCAGCAGCTGTATCACTGCATGTGACCATTGGAACAGGGTGGAGGAGGATACGGAGCTGTTGAAGAACTTGGGAGTTCAAACCCACAGAATGAGTCTTGAGTGGAGCAGAATAGAGCCTTCCAGGGGCAAATTTTCCGATGACGCAATGAAACATTACAGAGATGAGATTAAGCTTTTGGTGGAGAACAACATAAAGCCTCTGGTTACGCTTCATCACTTTTCCGAGCCCATTTGGTTTCATGAAATGGGGGGATGGAAAAAAACGGGCAATGCAGATATTTTTATAGAATATGTGAAGTATGTGGTTGAAAATTTGGGTGACCTTGTAAGCGACTGGGTAACCTTTAACGAGCCCAATGTCTATGTTGATTTTGGTTATGTAATCGGCATTTTCCCTCCGGGGGAAAGAAGCCTGTCTGAAGGGTTAAAGGTTACGGCAGAGCTTATAAACACCCATGTAAAACTATACCGGCTGATACATAGGATAAGAAGAGAGCGCAAATTTGCAGGCAGGACAATGGTAGGAACGGCAATGCACCTTCGCATCTTTGACGGGATAAGTTCTACCGGAAAAATGATAGCCAAAGTTGTAGATTATCTGTTTAACGAAATGTTTATGGAAGGCATGACGACAGGGCACATGATGTTTCCTCTTTCCAAAAAGGGTTCAAGCCATAAAAAAGGCAGGTATGCGGATTTTTTGGGAATTAATTATTATACAAGAAATATTGTTGAGTTCGTATTTGACCCGTCCCTTTATTTTCACGAGCTTGTATGTGACAAGGATTTGACCAAATCGGACCTCGGGTGGGACATATATCCGGAAGGCATATACAAAGTATGCAAGAGGTACTATAAGAAATATAAACTTCCCATTTATATAACCGAAAACGGAATAAGCGATAAAAATGACACCAAACGGCCGAGCTTTATTGCCAGCCATCTTGCTTATATTGCAAAAGCCATAAAAGAAGGGATTCCGATAGAACGGTATTATTACTGGACGCTGATGGATAACTTCGAATGGCTTGAAGGTGAGTCAACGGATTTCGGCCTTTACGACTGCAATTTCCGCACGCAGGAGAGGATACCGAGAAAAAGCGTCCGGCTTTATGAGCAAATATGCAGAAGAAAAGAATTAACCGCGGAGATGATTGAGGATTTTAAGAAGTACAGCGGGATTACTATAGAAACAATCCGGTGA.
Example 2
The embodiment provides a method for hydrolyzing lignocellulose by clostridium thermocellum, which comprises the following steps.
Step one: straw fiber preparation
Cleaning and cutting rice straw raw materials, performing hydrothermal treatment by adopting sodium hydroxide and sodium sulfite, wherein the ratio of the weight of the sodium hydroxide to the absolute dry weight of the rice straw raw materials is 1:6, the weight ratio of sodium sulfite to sodium hydroxide is 1: and 3, the hydrothermal treatment conditions are as follows: the reaction temperature was 160℃and the time was 1h. And after lignin is fully removed, extracting fibers, dehydrating and fully washing off residual alkali liquor to obtain straw fibers.
Step two: anaerobic fermentation of clostridium thermocellum seed liquid
In one embodiment, the anaerobic fermentation of clostridium thermocellum seed liquid sequentially comprises the steps of preparing a culture medium, deoxidizing, preparing seed liquid and anaerobic fermentation.
Preparation of culture medium and deoxidization: the culture medium adopts MTC culture medium, and the culture medium comprises five ABCDE solutions, and is specifically shown in table 1.
TABLE 1 Medium components and content
After the preparation of the solution A is finished, sterilizing at 121 ℃ for 20 min; B. c, D, E liquid is prepared according to the concentration, and is filled in an anaerobic bottle, and after being sealed by an aluminum cover with a rubber plug, the anaerobic bottle is repeatedly vacuumized and filled with high-purity nitrogen for 3 times, and finally the inside of the anaerobic bottle is kept at positive pressure. 5 culture solutions according to 40:2:1:1:1 (v/v) A sterile disposable syringe and 0.22 μm sterile filter were used in a super clean bench to fill the sterilized anaerobic jar with liquid A, the total volume not exceeding 40% of the anaerobic jar volume.
The carbon source (microcrystalline cellulose or straw fiber) in the solution A, microcrystalline cellulose Avicel PH105 is used in the seed culture medium, and the concentration is 5.00g/L; the straw fiber prepared in the first step is used in a fermentation medium, and the concentration is 5.00g/L.
Seed liquid preparation: placing the clostridium thermocellum strain preserved at-80deg.C in a refrigerator at 4deg.C for thawing, sucking the strain under aseptic condition, injecting into seed culture medium with inoculum size of 2.5%, and culturing at 55deg.C and 200rpm for 24 hr.
Anaerobic fermentation: the seed solution was inoculated into a fermentation medium at an inoculum size (v/v) of 10%, and cultured at 55℃and 200rpm for 16 hours to give a fermentation broth.
Step three: fiber hydrolysis
Soaking the straw fibers in an acetic acid-sodium acetate buffer solution, and adding clostridium thermocellum fermentation liquor and xylanase to hydrolyze the straw fibers. The hydrolysis conditions were as follows: the pH value is 5, and the solid (absolute dry weight of straw fiber) liquid ratio is 1:10, for each gram of straw fiber, the adding amount of clostridium thermocellum fermentation liquor is 40 mu L, the adding amount of xylanase is 4mg, the reaction temperature is 50 ℃, the reaction time is 48h, and the oscillation rate is 200r/min.
At the end of 48 hours of hydrolysis, the glucose concentration in the hydrolysate was about 70.00g/L. Under the same conditions, when the commercial Trichoderma reesei cellulase is used for hydrolysis instead of the clostridium thermocellum fermentation broth, the glucose concentration is about 48g/L when the hydrolysis is carried out for 48 hours.
Step four: separating and concentrating hydrolysate
Solid-liquid separation of the hydrolysate: removing thallus and residue in the hydrolysate by using hollow fiber microfiltration membrane, wherein the microfiltration membrane is purchased from Shandong gold membrane Tian-Limited liability company, and is made of MF1, polypropylene material, and has an effective membrane area of 0.33m 2 The pore diameter is 0.2 μm, the operating pressure is 0.1MPa, and the cross flow speed is 0.052m/s. The cell removal rate was 100%, and the total sugar recovery rate was 98%.
Concentrating: the concentration of the hydrolyzate was carried out at room temperature. Concentrating the hydrolysate by adopting a nanofiltration membrane DK1812C-34D of the United states electric company, wherein the operating pressure is 17bar, the flow is 5L/min, the operating time is 3min/L, and the pH value is natural. The concentration of glucose and xylose in the hydrolysate after concentration is 6.1 times and 6.3 times that before concentration.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application, and are intended to be included within the scope of the appended claims and description.
SEQUENCE LISTING
<110> su state poverty creature biotechnology limited
<120> Clostridium thermocellum and its use in lignocellulose hydrolysis
<130> 2
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 5636
<212> DNA
<213> artificial sequence
<400> 1
gtgggaatag gcatggaaaa gatttttttg cccgaggatg cattgataaa gtatttcttt 60
tccgaaagag aggaaaagat tctaaagagt tttggaaata ctgatgaata ttgtgtgcag 120
agtacaattc tatggacaag aaaagaggct ttgtcaaaac tttttcgtct gggaatgagg 180
atggatttta aaaagctgga tactttggag ggcgaggtgg tttttcagga aacaaacagg 240
gcgcgtctgt tttcttttat atgcaataat tactgtatct ctctggcatt gccaggtttt 300
aataaagatt aaaattattg actagaaata aaaaaattgt ccataatatt aatggacaaa 360
aaaacaaaga attacatcaa aggaagataa aaatactttg ttaaaaaatt aattattttt 420
tatctaaact attgaaaatg aaaataaaat aatataaaat gaatcatagt gcaagagata 480
cttgccagag gatgaatatt ttactgcatt catgctttat ggcagctaat agaggcatta 540
aattaaattt taatttacaa taggaggcga tattagtgaa gaacgtaaaa aaaagagtag 600
gtgtggtttt gctgattctt gcagtgttgg gggtttatat gttggcaatg ccggcaaaca 660
ctgtgtcagc ggcaggtgtg ccttttaaca caaaataccc ctatggtcct acttctattg 720
ccgataatca gtcggaagta actgcaatgc tcaaagcaga atgggaagac tggaagagca 780
agagaattac ctcgaacggt gcaggaggat acaagagagt acagcgtgat gcttccacca 840
attatgatac ggtatccgaa ggtatgggat acggacttct tttggcggtt tgctttaacg 900
aacaggcttt gtttgacgat ttataccgtt acgtaaaatc tcatttcaat ggaaacggac 960
ttatgcactg gcacattgat gccaacaaca atgttacaag tcatgacggc ggcgacggtg 1020
cggcaaccga tgctgatgag gatattgcac ttgcgctcat atttgcggac aagttatggg 1080
gttcttccgg tgcaataaac tacgggcagg aagcaaggac attgataaac aatctttaca 1140
accattgtgt agagcatgga tcctatgtat taaagcccgg tgacagatgg ggaggttcat 1200
cagtaacaaa cccgtcatat tttgcgcctg catggtacaa agtgtatgct caatatacag 1260
gagacacaag atggaatcaa gtggcggaca agtgttacca aattgttgaa gaagttaaga 1320
aatacaacaa cggaaccggc cttgttcctg actggtgtac tgcaagcgga actccggcaa 1380
gcggtcagag ttacgactac aaatatgatg ctacacgtta cggctggaga actgccgtgg 1440
actattcatg gtttggtgac cagagagcaa aggcaaactg cgatatgctg accaaattct 1500
ttgccagaga cggggcaaaa ggaatcgttg acggatacac aattcaaggt tcaaaaatta 1560
gcaacaatca caacgcatca tttataggac ctgttgcggc agcaagtatg acaggttacg 1620
atttgaactt tgcaaaggaa ctttataggg agactgttgc tgtaaaggac agtgaatatt 1680
acggatatta cggaaacagc ttgagactgc tcactttgtt gtacataaca ggaaacttcc 1740
cgaatccttt gagtgacctt tccggccaac cgacaccacc gtcgaatccg acaccttcat 1800
tgcctcctca ggttgtttac ggtgatgtaa atggcgacgg taatgttaac tccactgatt 1860
tgactatgtt aaaaagatat ctgctgaaga gtgttaccaa tataaacaga gaggctgcag 1920
acgttaatcg tgacggtgcg attaactcct ctgacatgac tatattaaag agatatctga 1980
taaagagcat accccaccta ccttattagt taaaaagaat aaaaaagtat atttgaaagg 2040
ggaagatgga gagaatggta aaaagcagaa agatttctat tctgttggca gttgcaatgc 2100
tggtatccat aatgataccc acaactgcat tcgcaggtcc tacaaaggca cctacaaaag 2160
atgggacatc ttataaggat cttttccttg aactctacgg aaaaattaaa gatcctaaga 2220
acggatattt cagcccagac gagggaattc cttatcactc aattgaaaca ttgatcgttg 2280
aagcgccgga ctacggtcac gttactacca gtgaggcttt cagctattat gtatggcttg 2340
aagcaatgta tggaaatctc acaggcaact ggtccggagt agaaacagca tggaaagtta 2400
tggaggattg gataattcct gacagcacag agcagccggg tatgtcttct tacaatccaa 2460
acagccctgc cacatatgct gacgaatatg aggatccttc atactatcct tcagagttga 2520
agtttgatac cgtaagagtt ggatccgacc ctgtacacaa cgaccttgta tccgcatacg 2580
gtcctaacat gtacctcatg cactggttga tggacgttga caactggtac ggttttggta 2640
caggaacaca ggcaacattc ataaacacct tccaaagagg tgaacaggaa tccacatggg 2700
aaaccattcc tcatccgtca atagaagagt tcaaatacgg cggaccgaac ggattccttg 2760
atttgtttac aaaggacaga tcatatgcaa gacagtggcg ttatacaaac gctcctgacg 2820
cagaaggccg tgctatacag gctgtttact gggcaaacaa atgggcaaag gagcagggta 2880
aaggttctgc cgttgcttcc gttgtatcca aggctgcaaa gatgggtgac ttcttgagaa 2940
acgacatgtt cgacaaatac ttcatgaaga tcggtgcaca ggacaagact cctgctaccg 3000
gttatgacag tgcacactac cttatggcct ggtatacttc atggggtggt ggaattggtg 3060
catcctgggc atggaagatc ggatgcagcc acgcacactt cggatatcag aacccattcc 3120
agggatgggt aagtgcaaca cagagcgact ttgctcctaa atcatccaac ggtaagagag 3180
actggacaac aagctacaag agacagcttg aattctatca gtggttgcag tcggctgaag 3240
gtgctattgc cggtggagca accaactcct ggaacggtag atatgagaaa tatcctgctg 3300
gtacgtcaac gttctatggt atggcatatg ttccgcatcc tgtatacgct gacccgggta 3360
gtaaccagtg gttcggattc caggcatggt caatgcagcg tgtaatggag tactacctcg 3420
aaacaggaga ttcatcagtt aagaatttga ttaagaagtg ggtcgactgg gtaatgagcg 3480
aaattaagct ctatgacgat ggaacatttg caattcctag cgacctcgag tggtcaggtc 3540
agcctgatac atggaccgga acatacacag gcaacccgaa cctccatgta agagtaactt 3600
cttacggtac tgaccttggt gttgcaggtt cacttgcaaa tgctcttgca acttatgccg 3660
cagctacaga aagatgggaa ggaaaacttg atacaaaagc aagagacatg gctgctgaac 3720
tggttaaccg tgcatggtac aacttctact gctctgaagg aaaaggtgtt gttactgagg 3780
aagcacgtgc tgactacaaa cgtttctttg agcaggaagt atacgttccg gcaggttgga 3840
gcggtactat gccgaacggt gacaagattc agcctggtat taagttcata gacatccgta 3900
caaaatatag acaagatcct tactacgata tagtatatca ggcatacttg agaggcgaag 3960
ctcctgtatt gaattatcac cgcttctggc atgaagttga ccttgcagtt gcaatgggtg 4020
tattggctac atacttcccg gatatgacat ataaagtacc tggtactcct tctactaaat 4080
tatacggcga cgtcaatgat gacggaaaag ttaactcaac tgacgctgta gcattgaaga 4140
gatatgtttt gagatcaggt ataagcatca acactgacaa tgccgatttg aatgaagacg 4200
gcagagttaa ttcaactgac ttaggaattt tgaagagata tattctcaaa gaaatagata 4260
cattgccgta caagaactaa tataaacgtg taaagggggt ggcgataatg gttactttta 4320
agcttaatga tgaattcatg tttggaaccg ctaccgcaag tactcagatt gagggtgggg 4380
atacgggaaa tacatggtat aagtggtgcc aggaaggacg tatcaaggac tccagcagct 4440
gtatcactgc atgtgaccat tggaacaggg tggaggagga tacggagctg ttgaagaact 4500
tgggagttca aacccacaga atgagtcttg agtggagcag aatagagcct tccaggggca 4560
aattttccga tgacgcaatg aaacattaca gagatgagat taagcttttg gtggagaaca 4620
acataaagcc tctggttacg cttcatcact tttccgagcc catttggttt catgaaatgg 4680
ggggatggaa aaaaacgggc aatgcagata tttttataga atatgtgaag tatgtggttg 4740
aaaatttggg tgaccttgta agcgactggg taacctttaa cgagcccaat gtctatgttg 4800
attttggtta tgtaatcggc attttccctc cgggggaaag aagcctgtct gaagggttaa 4860
aggttacggc agagcttata aacacccatg taaaactata ccggctgata cataggataa 4920
gaagagagcg caaatttgca ggcaggacaa tggtaggaac ggcaatgcac cttcgcatct 4980
ttgacgggat aagttctacc ggaaaaatga tagccaaagt tgtagattat ctgtttaacg 5040
aaatgtttat ggaaggcatg acgacagggc acatgatgtt tcctctttcc aaaaagggtt 5100
caagccataa aaaaggcagg tatgcggatt ttttgggaat taattattat acaagaaata 5160
ttgttgagtt cgtatttgac ccgtcccttt attttcacga gcttgtatgt gacaaggatt 5220
tgaccaaatc ggacctcggg tgggacatat atccggaagg catatacaaa gtatgcaaga 5280
ggtactataa gaaatataaa cttcccattt atataaccga aaacggaata agcgataaaa 5340
atgacaccaa acggccgagc tttattgcca gccatcttgc ttatattgca aaagccataa 5400
aagaagggat tccgatagaa cggtattatt actggacgct gatggataac ttcgaatggc 5460
ttgaaggtga gtcaacggat ttcggccttt acgactgcaa tttccgcacg caggagagga 5520
taccgagaaa aagcgtccgg ctttatgagc aaatatgcag aagaaaagaa ttaaccgcgg 5580
agatgattga ggattttaag aagtacagcg ggattactat agaaacaatc cggtga 5636
Claims (6)
1. A method for hydrolyzing lignocellulose using clostridium thermocellum, comprising: the method comprises the steps of straw fiber preparation, clostridium thermocellum seed liquid anaerobic fermentation, fiber hydrolysis and hydrolysate separation and concentration, wherein the straw fiber preparation comprises the following steps: carrying out hydrothermal reaction on the washed and chopped rice straw raw materials and alkaline substances, fully removing lignin, extracting fibers, dehydrating and fully washing off residual lye to obtain straw fibers; wherein the alkaline substance comprises sodium hydroxide and sodium sulfite, and the ratio of the weight of the sodium hydroxide to the absolute dry weight of the rice straw raw material is 1: (4-6), wherein the weight ratio of the sodium sulfite to the sodium hydroxide is 1: (3-5), wherein the temperature of the hydrothermal reaction is 150-160 ℃, the time of the hydrothermal reaction is 1-3h, and the clostridium thermocellum isClostridium thermocellumPN2101, 2021, 07 and 09, and the preservation number is CGMCC No.22868.
2. The method of hydrolyzing lignocellulose by clostridium thermocellum according to claim 1, wherein the anaerobic fermentation of clostridium thermocellum seed liquid comprises the steps of: inoculating clostridium thermocellum seed liquid into fermentation medium in the inoculation amount of 5-10%, and culturing at 50-65 deg.c and 150-200rpm for 16-24h to obtain fermentation liquid; wherein, the components of the fermentation medium comprise the following components in volume ratio of 40:2:1:1: liquid A, liquid B, liquid C, liquid D and liquid E of 1; the solution A comprises 5.00-10.00g/L straw fiber and 10.00 g/L3-morpholinopropane sulfonic acid; the solution B comprises 50.00g/L tripotassium citrate, 31.25g/L citric acid monohydrate and 25.00g/L Na 2 SO 4 、25.00 g/L KH 2 PO 4 、62.50 g/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The solution C comprises 250.00g/L urea; the D solution comprises 50.00g/L MgCl 2 ·6H 2 O、10.00 g/L CaCl 2 ·2H 2 O、5.00 g/L FeCl 2 ·4H 2 O, 50.00g/L cysteine salt; the E solution comprises 1.00g/L pyridoxamine dihydrochloride, 0.20g/L para-aminobenzoic acid, 0.10g/L biotin, 0.10g/L VB 12 。
3. The method for hydrolyzing lignocellulose by clostridium thermocellum according to claim 2, wherein: the clostridium thermocellum seed liquid is obtained by thawing clostridium thermocellum strains, culturing the clostridium thermocellum strains in a seed culture medium, wherein the inoculation amount of the clostridium thermocellum strains is 2.5%, the culturing temperature is 50-65 ℃, the oscillating speed is 150-200rpm, and the time is 16-24h; wherein, the components of the seed culture medium comprise the following components in volume ratio of 40:2:1:1: liquid A, liquid B, liquid C, liquid D and liquid E of 1; the solution A comprises 5.00-10.00g/L microcrystalline cellulose and 10.00 g/L3-morpholinopropane sulfonic acid; the liquid B comprises50.00g/L tripotassium citrate, 31. 31.25g/L citric acid monohydrate, 25.00g/L Na 2 SO 4 、25.00 g/L KH 2 PO 4 、62.50 g/L NaHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The solution C comprises 250.00g/L urea; the D solution comprises 50.00g/L MgCl 2 ·6H 2 O、10.00 g/L CaCl 2 ·2H 2 O、5.00 g/L FeCl 2 ·4H 2 O, 50.00g/L cysteine salt; the E solution comprises 1.00g/L pyridoxamine dihydrochloride, 0.20g/L para-aminobenzoic acid, 0.10g/L biotin, 0.10g/L VB 12 。
4. The method of hydrolyzing lignocellulose by clostridium thermocellum according to claim 1, wherein the fiber hydrolysis comprises the steps of: and soaking the straw fibers in a buffer solution, and then adding a fermentation liquor obtained by anaerobic fermentation of the clostridium thermocellum seed liquid and xylanase for hydrolysis to obtain a hydrolysate.
5. The method for hydrolyzing lignocellulose by clostridium thermocellum according to claim 4, wherein: the pH value of the hydrolysis is 4.5-5.5, and the solid-liquid ratio is 1: (10-30), the temperature is 45-55 ℃, the time is 12-48h, the oscillating rate is 150-250r/min, the added fermentation liquor is 10-40 mu L per gram of straw fiber, and the added xylanase is 4mg per gram of straw fiber.
6. The method of hydrolyzing lignocellulose by clostridium thermocellum according to claim 1, wherein the hydrolysate separating and concentrating comprises the steps of: carrying out solid-liquid separation on the hydrolysate, and concentrating the collected hydrolysate; wherein, the solid-liquid separation adopts a microfiltration membrane, and the microfiltration membrane is a polypropylene membrane; the concentration adopts a nanofiltration membrane, wherein the nanofiltration membrane is a polyethersulfone membrane.
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