CN111334447A - Fermentation process of high-yield cellulase clostridium - Google Patents
Fermentation process of high-yield cellulase clostridium Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 95
- 230000004151 fermentation Effects 0.000 title claims abstract description 95
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 36
- 229940106157 cellulase Drugs 0.000 title claims abstract description 35
- 241000193403 Clostridium Species 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 35
- 238000011218 seed culture Methods 0.000 claims abstract description 22
- 238000011081 inoculation Methods 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 230000003213 activating effect Effects 0.000 claims abstract description 6
- 230000004913 activation Effects 0.000 claims abstract description 4
- 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 16
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 16
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 14
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 13
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- 241000193448 Ruminiclostridium thermocellum Species 0.000 claims description 9
- 235000000638 D-biotin Nutrition 0.000 claims description 8
- 239000011665 D-biotin Substances 0.000 claims description 8
- 229960004050 aminobenzoic acid Drugs 0.000 claims description 8
- HNWCOANXZNKMLR-UHFFFAOYSA-N pyridoxamine dihydrochloride Chemical compound Cl.Cl.CC1=NC=C(CO)C(CN)=C1O HNWCOANXZNKMLR-UHFFFAOYSA-N 0.000 claims description 8
- 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 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 7
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims description 7
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 7
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 7
- 239000004202 carbamide Substances 0.000 claims description 7
- 229960002303 citric acid monohydrate Drugs 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 239000001508 potassium citrate Substances 0.000 claims description 7
- 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 7
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 7
- 235000011152 sodium sulphate Nutrition 0.000 claims description 7
- 235000015870 tripotassium citrate Nutrition 0.000 claims description 7
- 239000011720 vitamin B Substances 0.000 claims description 7
- 239000011715 vitamin B12 Substances 0.000 claims description 7
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 229960002089 ferrous chloride Drugs 0.000 claims description 6
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 6
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 6
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 6
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 6
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 6
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 5
- RIPZAYPXOPSKEE-DKWTVANSSA-N (2r)-2-amino-3-sulfanylpropanoic acid;hydrate Chemical compound O.SC[C@H](N)C(O)=O RIPZAYPXOPSKEE-DKWTVANSSA-N 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 3
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 35
- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000013589 supplement Substances 0.000 abstract description 3
- 241000205062 Halobacterium Species 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 description 25
- 102000004190 Enzymes Human genes 0.000 description 25
- 229940088598 enzyme Drugs 0.000 description 25
- 239000000243 solution Substances 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 4
- 235000010980 cellulose Nutrition 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 101710112457 Exoglucanase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 241000499912 Trichoderma reesei Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229930010796 primary metabolite Natural products 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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- 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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- 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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Abstract
A fermentation process of high-yield cellulase of clostridium belongs to the technical field of fermentation engineering. Which comprises the following steps: inoculating thermobacter fuscus into a seed culture medium according to the inoculation amount of 1%, performing activation culture at 60 ℃, activating for 2-3 generations according to the same manner, and continuously culturing in the seed culture medium to obtain a seed solution of the thermobacter fuscus; inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2%, and performing fermentation culture at 40-60 ℃ for 48-96h to obtain a fermentation product. The invention optimizes the fermentation process of the clostridium thermobacter, optimizes the fermentation culture conditions, greatly improves the cellulase activity of the clostridium thermobacter, supplements the clostridium thermobacter with materials in due time according to the growth condition of thalli, greatly improves the cellulase activity of the clostridium thermobacter halobacter, saves the cost, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation process of high-yield cellulase of clostridium.
Background
The lignocellulose raw material has wide sources and is a renewable resource with abundant reserves. Cellulase is a general name of a group of enzymes capable of degrading cellulose into glucose, lignocellulose can be hydrolyzed by cellulase to generate sugar, the sugar can be directly used for fermentation of microorganisms, the cellulose can be converted into valuable primary metabolites, and the cellulase is often applied to industries such as food and feed industries, paper making industries, medicine aspects, biological energy sources and the like, and has good application prospect.
Microorganisms producing cellulase include bacteria, fungi, actinomycetes, etc., and among these microorganisms, a Clostridium (a kind of bacteria) is known for its high cellulolytic activity, and the cellulose degradation rate is comparable to that of Trichoderma reesei. The strain has the characteristics of wide available substrates, strong environmental adaptability, difficult pollution and the like, can generate a complex enzyme system for degrading cellulose, comprises endoglucanase, exoglucanase, hemicellulase and the like, and is a strain with the greatest potential of positive economic influence.
At present, the application of cellulase is often limited by high production cost, the high production cost of the cellulase is mainly focused on two aspects, firstly, the enzyme activity of a strain for producing the cellulase is not high and far meets the production requirement, so that the screening of the strain with high cellulase activity is a key point. Secondly, due to the characteristics, culture conditions and other factors of the currently used bacterial strain, the fermentation time of the cellulase is long, the efficiency is low, and the cost is overhigh.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a technical scheme of a fermentation process of high-yield cellulase-producing clostridium thermocellum.
The fermentation process of the clostridium thermocellum for highly producing the cellulase is characterized by comprising the following steps of:
1) inoculating thermobacter fuscus into a seed culture medium according to the inoculation amount of 1%, performing activation culture at 60 ℃, activating for 2-3 generations according to the same manner, and continuously culturing in the seed culture medium to obtain a seed solution of the thermobacter fuscus;
2) inoculating the seed liquid obtained in the step 1) into a fermentation culture medium according to the inoculation amount of 2%, and performing fermentation culture at 40-60 ℃ for 48-96h to obtain a fermentation product;
the seed culture medium contains the following components: 5-15g/L of cellobiose, 5-17g/L of corn steep liquor, 0.5-5g/L of tripotassium citrate, 0.5-2g/L of citric acid monohydrate, 0.5-1.5g/L of sodium sulfate, 0.5-1.5g/L of potassium dihydrogen phosphate, 1-5g/L of sodium bicarbonate, 1-3g/L of ammonium chloride, 2-8g/L of urea, 0.5-1.5g/L of yeast extract, 40-60g/L of magnesium chloride hexahydrate, 0.05-0.2g/L of ferrous chloride tetrahydrate, 0.1-0.3g/L of calcium chloride dihydrate, 0.5-2g/L of cysteine hydrochloride monohydrate, 0.01-0.05g/L of pyridoxamine dihydrochloride, 0.002-0.006g/L of p-aminobenzoic acid, 0.001-0.003g/L of D-biotin, vitamin B120.01-0.03g/L, vitamin Bl0.001-0.003g/L, and natural pH value;
the fermentation culture medium is prepared by adding microcrystalline cellulose 5-15g/L under basic condition of seed culture medium, and adjusting pH to 5.5-8.
The fermentation process of the clostridium thermocellum with high yield of cellulase is characterized in that a seed culture medium in the step 1) contains the following components: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, lg/L of sodium sulfate, lg/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, lg/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, lg/L of cysteine monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, and 0.002g/L of vitamin B120.02g/L, vitamin Bl0.002g/L, and natural pH value.
The fermentation process of the high-yield cellulase-producing clostridium is characterized in that the fermentation condition in the step 2) is fermentation culture at 55 ℃ for 60 hours.
The fermentation process of the high-yield cellulase-producing clostridium is characterized in that the fermentation medium in the step 2) is prepared by adding 10g/L of microcrystalline cellulose under the basic condition of a seed culture medium and adjusting the pH value to 7.
The fermentation process of the high-yield cellulase-producing clostridium is characterized in that the fermentation is carried out in the step 2) by adopting a fed-batch fermentation mode.
The fermentation process of the clostridium thermocellum for highly producing the cellulase is characterized in that the fed-batch fermentation specifically comprises the following steps: inoculating the seed solution into a fermentation medium according to the inoculation amount of 2% to obtain a first-stage seed solution, inoculating the first-stage seed solution into a fermentation tank filled with the fermentation medium according to the inoculation amount of 2.5%, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, intermittently stirring, slowly adding a cellobiose solution into the fermentation system within a 16-48 h period after the fermentation is started, wherein the flow rate is 1mL/min, and the concentration of the cellobiose solution is 200 g/L.
The invention optimizes the fermentation process of the clostridium thermobacter, optimizes the fermentation culture conditions, greatly improves the cellulase activity of the clostridium thermobacter, supplements the clostridium thermobacter with materials in due time according to the growth condition of thalli, greatly improves the cellulase activity of the clostridium thermobacter halobacter, saves the cost, and is suitable for industrial production.
Detailed Description
The present invention is further illustrated by the following examples.
Example 1:
1. seed culture medium: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, lg/L of sodium sulfate, lg/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, lg/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, lg/L of cysteine monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, and 0.002g/L of vitamin B120.02g/L, vitamin Bl0.002g/L, and natural pH value.
2. Strain activation: inoculating the laboratory preserved strain into a seed culture medium according to the inoculation amount of 1%, activating and culturing at 60 ℃, activating for 2-3 generations according to the same manner, and continuously culturing in the seed culture medium to obtain the bacterial liquid of the fusobacterium.
3. Fermentation medium: under the basic condition of a seed culture medium, 10g/L of microcrystalline cellulose is added.
4. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 2%, fermenting and culturing for 24-96h at 30-60 ℃, and determining the enzyme activity after the fermentation is finished. The enzyme activity was measured to be 3.41U/mL.
5. Enzyme activity assay (filter paper enzyme activity): 50mg of filter paper is taken, 1ml of citric acid buffer solution with the pH value of 60.1 mol/L is added, 0.5 ml of proper diluted enzyme solution is added, the reaction is carried out for 1 h at the temperature of 50 ℃, and 3 ml of DNS reagent is added for measuring sugar. The enzyme activity is calculated after reducing sugar in the fermentation liquor is deducted, the enzyme activity adopts international units, and the enzyme activity is defined as that the enzyme amount for generating 1 mu mol glucose by hydrolysis in each minute is 1 activity unit.
Example 2: fermentation culture condition optimization
Controlling other conditions to be unchanged, setting different fermentation temperatures of 30, 40, 50, 55 and 60 ℃ respectively, and measuring the cellulase activity after the fermentation is finished. As shown in Table 1, the enzyme activity of the strain was found to be highest at 55 ℃ and 55 ℃ was selected as the fermentation temperature. Controlling other conditions to be unchanged, setting different culture times of 24h, 48h, 60h, 72h, 84h and 96h respectively, and measuring the cellulase activity after the fermentation is finished. The results are shown in table 2, the enzyme activity is in an increasing trend along with the increase of the fermentation time, the peak value is reached when the fermentation time reaches 60 hours, and the enzyme activity begins to decrease after 96 hours. Therefore, the fermentation is stopped after 60 hours, and the fermentation cost can be greatly saved.
And (3) controlling other conditions to be unchanged, setting different initial pH values of the culture medium to be 5.5, 6, 6.5, 7, 7.5 and 8 respectively, and measuring the cellulase activity after the fermentation is finished. As a result, as shown in Table 3, the enzyme activity was highest when the initial pH of the medium was 7.
The enzyme activity is measured to be 5.12U/mL which is 1.5 times of the unoptimized enzyme activity after fermenting for 60 hours under the optimized conditions, namely under the conditions that the temperature is 55 ℃ and the pH of the initial culture medium is 7.
TABLE 1 Effect of different fermentation temperatures on enzyme Activity
TABLE 2 Effect of different fermentation times on enzyme Activity
TABLE 3 Effect of initial pH of different media on enzyme Activity
Example 3: process amplification
Sucking 200 mu L of glycerol tube storage strain, transferring to 25mL of seed culture medium, culturing at constant temperature of 55 ℃, activating the strain, transferring the activated strain to 50mL of fermentation culture medium according to the inoculum size of 2%, and culturing at constant temperature of 55 ℃ for 24h to obtain first-grade seed liquid.
Seed culture medium: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, lg/L of sodium sulfate, lg/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, lg/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, lg/L of cysteine monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, and 0.002g/L of vitamin B120.02g/L, vitamin Bl0.002g/L, and natural pH value. Sterilizing at 115 deg.C by wet heat.
Fermentation medium: 10g/L of cellobiose, 10g/L of microcrystalline cellulose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, 1.5g/L of sodium sulfate, 0.1g/L of potassium dihydrogen phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, 0.2g/L of calcium chloride dihydrate, 0.2g/L of cysteine hydrochloride monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin and vitamin B120.02g/L, vitamin Bl0.002g/L, and natural pH value. Sterilizing at 115 deg.C by wet heat.
After sterilization, nitrogen was introduced into the fermentor to remove the medium and oxygen from the fermentor. Adjusting the culture pH constant to 7, controlling the tank temperature to 55 ℃, inoculating the first-stage seed liquid, stopping ventilation, and intermittently stirring and fermenting. After the fermentation is finished, the enzyme activity is measured to be 12.37U/mL, which is 1.14 times of the enzyme activity in the state without controlling the pH value.
Example 4: comparison of different fermentation Processes
The fermentation mode mainly comprises the following steps: batch, fed-batch and continuous fermentation, and the continuous fed-batch fermentation is easy to pollute mixed bacteria, has high degeneration and small application range, so the experiment is used for batch and fed-batch research.
Batch fermentation: inoculating the preserved strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 2L of fermentation culture medium by 2.5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring.
Fed-batch fermentation: inoculating the preserved strain into a seed culture medium, then inoculating the strain into a fermentation culture medium by 2% of inoculation amount to obtain a first-stage seed solution, inoculating the seed solution into a 5L fermentation tank filled with 3L of fermentation culture medium by 2.5% of inoculation amount, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, and carrying out intermittent stirring. Slowly adding the cellobiose solution into the fermentation system within 16-48 h after the start of the fermentation at the flow rate of 1mL/min, wherein the concentration of the cellobiose solution is 200 g/L.
After the fermentation is finished, the enzyme activity of the fermentation liquor is measured, the enzyme activity is measured to be 16.56U/mL, the condition that no control is carried out in the fermentation process is taken as a reference, and through comparison, the enzyme activity measured by adopting the fermentation process is improved by 52.62 percent relative to the reference.
In conclusion, the invention optimizes the fermentation culture conditions, greatly improves the cellulase activity, supplements and adds the bacteria timely according to the growth condition of the bacteria, greatly improves the cellulase activity, saves the cost and is suitable for industrial production.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. The fermentation process of the clostridium thermocellum for highly producing the cellulase is characterized by comprising the following steps of:
1) inoculating thermobacter fuscus into a seed culture medium according to the inoculation amount of 1%, performing activation culture at 60 ℃, activating for 2-3 generations according to the same manner, and continuously culturing in the seed culture medium to obtain a seed solution of the thermobacter fuscus;
2) inoculating the seed liquid obtained in the step 1) into a fermentation culture medium according to the inoculation amount of 2%, and performing fermentation culture at 40-60 ℃ for 48-96h to obtain a fermentation product;
the seed culture medium contains the following components: 5-15g/L of cellobiose, 5-17g/L of corn steep liquor, 0.5-5g/L of tripotassium citrate, 0.5-2g/L of citric acid monohydrate, 0.5-1.5g/L of sodium sulfate, 0.5-1.5g/L of potassium dihydrogen phosphate, 1-5g/L of sodium bicarbonate, 1-3g/L of ammonium chloride, 2-8g/L of urea, 0.5-1.5g/L of yeast extract, 40-60g/L of magnesium chloride hexahydrate, 0.05-0.2g/L of ferrous chloride tetrahydrate, 0.1-0.3g/L of calcium chloride dihydrate, 0.5-2g/L of cysteine hydrochloride monohydrate, 0.01-0.05g/L of pyridoxamine dihydrochloride, 0.002-0.006g/L of p-aminobenzoic acid, 0.001-0.003g/L of D-biotin, vitamin B120.01-0.03g/L, vitamin Bl0.001-0.003g/L, and natural pH value;
the fermentation culture medium is prepared by adding microcrystalline cellulose 5-15g/L under basic condition of seed culture medium, and adjusting pH to 5.5-8.
2. The process for fermenting Clostridium thermocellum with high yield of cellulase as claimed in claim 1, wherein said seed culture medium in step 1) comprises the following components: 10g/L of cellobiose, 12g/L of corn steep liquor, 2g/L of tripotassium citrate, 1.25g/L of citric acid monohydrate, lg/L of sodium sulfate, lg/L of monopotassium phosphate, 2.5g/L of sodium bicarbonate, 1.5g/L of ammonium chloride, 5g/L of urea, lg/L of yeast extract, 50g/L of magnesium chloride hexahydrate, 0.1g/L of ferrous chloride tetrahydrate, 0.2g/L of calcium chloride dihydrate, lg/L of cysteine monohydrate, 0.02g/L of pyridoxamine dihydrochloride, 0.004g/L of p-aminobenzoic acid, 0.002g/L of D-biotin, and 0.002g/L of vitamin B120.02g/L, vitamin Bl0.002g/L, and natural pH value.
3. The fermentation process of Clostridium thermocellum for highly producing cellulase as claimed in claim 1, wherein the fermentation condition in step 2) is 55 ℃ fermentation culture for 60 h.
4. The fermentation process of Clostridium thermocellum for highly producing cellulase according to claim 1 or 2, wherein the fermentation medium in step 2) is prepared by adding 10g/L microcrystalline cellulose under the basic conditions of seed culture medium and adjusting pH to 7.
5. The fermentation process of Clostridium thermocellum for highly producing cellulase according to claim 1, wherein the fermentation in step 2) is performed by fed-batch fermentation.
6. The fermentation process of Clostridium thermobacter with high cellulase yield as claimed in claim 5, wherein said fed-batch fermentation specifically comprises: inoculating the seed solution into a fermentation medium according to the inoculation amount of 2% to obtain a first-stage seed solution, inoculating the first-stage seed solution into a fermentation tank filled with the fermentation medium according to the inoculation amount of 2.5%, carrying out anaerobic culture, controlling the tank temperature to be 55 ℃, adjusting the pH to be 7, intermittently stirring, slowly adding a cellobiose solution into the fermentation system within a 16-48 h period after the fermentation is started, wherein the flow rate is 1mL/min, and the concentration of the cellobiose solution is 200 g/L.
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