CN103555693A - Culture method for improving activity of clostridium thermoceuum cellulase - Google Patents
Culture method for improving activity of clostridium thermoceuum cellulase Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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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Abstract
The invention discloses a culture method for improving the activity of clostridium thermoceuum cellulase, which comprises the following steps of (a) preparing a strain DSM1237T into a first-level strain liquid; (b) adding the first-level strain liquid into a base culture medium, wherein the volume ratio of the first-level strain liquid to the base culture medium is (1:10)-(1:20); performing fermentation culture for 48-72 hours to obtain a second-level strain liquid; (c) adding the cultured second-level strain liquid into the prepared fermentation culture medium, wherein the volume ratio of the second-level strain liquid to the fermentation culture medium is (1:30)-(1:10); (d) performing fermentation culture at a temperature of 50-70 DEG C under an anaerobic condition at a pressure of (-0.1)-0.1MPa, and standing, wherein the pH value is 6.5-8.0, and the culture time is 48-72 hours. According to the method disclosed by the invention, under the anaerobic condition, the energy consumption for introducing oxygen or compressed air in the fermentation process can be saved. Under the condition of same culture cost, the activity of the fermentation liquid cellulase is remarkably improved, and the economic benefits are remarkably increased.
Description
Technical field
The present invention relates to microorganism culturing and field of fermentation engineering, specifically a kind of cultural method that improves Clostridium thermocellum cellulase activity.
Background technology
The energy and ecocrisis are the great difficult problems that the whole world all faces, and are the hot issues of the research of current various countries.The energy of Biological resources develops for solving energy shortage, crisis in food and minimizing CO
2gaseous emission has extremely profound significance (Tilman et al, 2009; Regalbuto et al, 2009).Bio-ethanol is the promising alternative energy of carrying out at present, but the grain that adopts ferments more at present, and due to population expansion and crisis in food, starch ethanol is subject to many limitations.
In China, annual straw-stalk type cultural waste output more than 700,000,000 tons, is nearly that stock number is maximum, utilizes the class material that potentiality are the highest, has huge Wood Adhesives from Biomass potentiality.The process that is the energy by straw-stalk type cultural waste Wood Adhesives from Biomass, its rate-limiting step is cellulosic hydrolysis, the particularly insoluble fibrin of high-sequential crystallization hydrolysis.To mainly making good use of oxygen animalcule in the exploitation of Mierocrystalline cellulose renewable energy source, produce cellulase in early days, research both at home and abroad mainly concentrates on fungi (particularly Rui Shi wood is mould) etc.In recent years, the high-efficient characteristic of anaerobion degraded cellulose has received increasing attention (Tracy et al, 2011), and the cellulase being produced by anaerobion exists with the form of the outer complex body of born of the same parents, is referred to as " cellulosome ".Cellulosome mainly comprises: exoglucanase, endoglucanase, zytase and other hemicellulases.
Clostridium thermocellum is a kind of high temperature, anaerobism, cellulose-degrading bacteria.Growth temperature is at 50-70 ℃.Can utilize Mierocrystalline cellulose and hemicellulose to produce cellobiose, wood sugar and the poly-disaccharides of wood etc., also can produce ethanol, acetic acid, lactic acid, H
2and CO
2.Clostridium thermocellum is found at present research, and Clostridium thermocellum mainly utilizes hexose (glucose sugar) as energy derive, can not utilize five-carbon sugar (wood sugar), thereby the substrate of cultivating Clostridium thermocellum normally cellobiose, Microcrystalline Cellulose and cellulose powder etc.Clostridium thermocellum is the cellulose-degrading bacteria that the enzymic activity of every milligram of secretory protein in current acquired microorganism is the highest (Lynd, 2002), has huge researching value and practical application potentiality.
To the research of Clostridium thermocellum, be focus both domestic and external, the prozyme that at present research mainly concentrates on Clostridium thermocellum i.e. the aspect such as the structure of " cellulosome " and degradation mechanism.Also there is the fundamental research to the aspects such as change of component of the Physiology and biochemistry variation of microorganism under different carbon sources and cellulosome.But do not relate to the cultural method that utilizes the reasonably combined raising Clostridium thermocellum of several kinds of carbon source cellulase activity.
In industrial fermentation application, the cellulosome that mainly utilizes Clostridium thermocellum to produce is degraded to Mierocrystalline cellulose, thereby the cellulose degradation efficiency that improves hot line clostridium is for promoting that industrial application has great importance.The current general method that is applicable to culture presevation and fermentation culture, major function is as the growth of Clostridium thermocellum and goes down to posterity to provide good substratum.For example, China Patent No. " 201110189094.8 " discloses a kind of method of Clostridium thermocellum high-density culture, and open day is on 01 09th, 2013.But concerning industrial fermentation, for the cellulase activity of Clostridium thermocellum, there is no special effect.
Summary of the invention
The object of the invention is to overcome the problems referred to above that the cellulase activity of existing solution Clostridium thermocellum exists, a kind of cultural method that improves Clostridium thermocellum cellulase activity is provided.Adopt fermented liquid cellulase activity of the present invention to significantly improve.
For achieving the above object, the technical solution used in the present invention is as follows:
A cultural method that improves Clostridium thermocellum cellulase activity, is characterized in that, comprises the following steps:
A, by the preservation of bacterial classification (clostridium thermocellum) DSM1237T(lyophilized powder) be mixed with first class inoculum liquid;
B, first class inoculum liquid is joined in basic medium, the volume ratio of strain liquid and basic medium is 1:10 to 1:20, and fermentation culture 48-72 hour becomes second class inoculum liquid;
C, the second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of second class inoculum liquid and fermention medium is 1:30 ~ 1:10;
D, fermentation culture: 50 ~ 70 ℃ of temperature, anaerobism, pressure-0.1MPa ~ 0.1MPa, standing, pH value 6.5 ~ 8.0, incubation time is 48 ~ 72h.
Basic medium in described step b is: ammonium sulfate 1.30 g/L, magnesium chloride 2.60 g/L, potassium primary phosphate 1.43 g/L, dipotassium hydrogen phosphate 5.50 g/L, calcium chloride 0.13 g/L, ferric sulfate 1.10 mg/L, halfcystine 0.25 g/L, yeast extract paste 4.50 g/L, resazurin 1.0 mg/L, Microcrystalline Cellulose 10.00 g/L or cellobiose 5.00 g/L.
The fermention medium of preparing in described step c is: ammonium sulfate 1-2 g/L magnesium chloride 2-3 g/L, potassium primary phosphate 1-2 g/L, dipotassium hydrogen phosphate 5-6g/L, calcium chloride 0.1-0.3 g/L, ferric sulfate 1-2 mg/L, halfcystine 0.2-0.5 g/L, nitrogenous source 4-6 g/L, resazurin 1.0 mg/L, carbon source 10.0-12.0 g/L.
Described fermention medium, in when preparation, regulates pH to 7.0 before sterilizing, 121 ℃ of sterilizings 30 minutes.
Described carbon source is two kinds or three kinds of mass ratio combinations that carbon source is pressed 2:8 or 2:6:2 among Microcrystalline Cellulose (Avicel), xylan (xylan), chitosan, rice straw powder.
Described nitrogenous source is organic nitrogen source, comprises one or more combinations in any proportion in yeast powder, urea, vitamin H.
The mode of described fermentation comprises batch fermentation, feed supplement-batch fermentation or semicontinuous fermentation.
Employing the invention has the advantages that:
One,, in the present invention, anaerobic condition can save and in fermenting process, pass into oxygen or the required energy consumption of pressurized air; Thermophilic fermentation is difficult for polluting, and these all will further reduce costs.
Two, the fermention medium of preparing in the present invention is: ammonium sulfate 1-2 g/L magnesium chloride 2-3 g/L, potassium primary phosphate 1-2 g/L, dipotassium hydrogen phosphate 5-6g/L, calcium chloride 0.1-0.3 g/L, ferric sulfate 1-2 mg/L, halfcystine 0.2-0.5 g/L, nitrogenous source 4-6 g/L, resazurin 1.0 mg/L, carbon source 10.0-12.0 g/L; This fermention medium has uniqueness in the selection of carbon source and proportioning, is different from traditional similar substratum, by the allotment of carbon source, favourable stimulation the cellulase activity of microorganism, fermentation broth enzyme is lived significantly to be increased; And each component and content are interrelated, be impossible join in basic medium, the volume ratio of strain liquid and basic medium is 1:10 to 1:20, fermentation culture 48-72 hour; The advantage having is to have shortened first class inoculum liquid to enter into the lag phase that basic medium produces, and has promoted vegetative period, has guaranteed to cultivate the thalli growth concentration in 48-72 hour, is beneficial to as second class inoculum liquid and inoculates.
Four, in the present invention, second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of second class inoculum liquid and fermention medium is 1:30 ~ 1:10, this mode has shortened second class inoculum liquid and has entered into the growthing lag phase after fermention medium, promote vegetative period, guaranteed to cultivate fermentation concentration in 48-72 hour and the generation speed of cellulase.
Five, in the present invention, fermentation culture: 50 ~ 70 ℃ of temperature, anaerobism, pressure-0.1MPa ~ 0.1MPa, standing, pH value 6.5 ~ 8.0, incubation time is 48 ~ 72h, and this mode is that culture temperature is the optimum temperuture of Clostridium thermocellum, is conducive to the growth of thalline and the generation of cellulase.
Six, in the present invention, basic medium in described step b is: ammonium sulfate 1.30 g/L, magnesium chloride 2.60 g/L, potassium primary phosphate 1.43 g/L, dipotassium hydrogen phosphate 5.50 g/L, calcium chloride 0.13 g/L, ferric sulfate 1.10 mg/L, halfcystine 0.25 g/L, yeast extract paste 4.50 g/L, resazurin 1.0 mg/L, Microcrystalline Cellulose 10.00 g/L or cellobiose 5.00 g/L, use Clostridium thermocellum bacterial classification that this substratum is conducive to activate lyophilized powder state for first class inoculum liquid and enlarged culturing be second class inoculum liquid.
Seven,, in the present invention, described fermention medium, when preparation, regulates pH to 7.0 before sterilizing, 121 ℃ of sterilizings 30 minutes, not only consider the thorough sterilizing of substratum but also consider the destruction of sterilization time to medium nutrient content, select 121 ℃ of the most frequently used sterilising conditions, 30min; Selecting to regulate pH before sterilizing, is in order to reduce microbiological contamination probability.
Eight,, in the present invention, described carbon source is two kinds or three kinds of mass ratio combinations that carbon source is pressed 2:8 or 2:6:2 among Microcrystalline Cellulose (Avicel), xylan (xylan), chitosan, rice straw powder; Several kinds of carbon source has induction and hormesis to the growth of Clostridium thermocellum and product enzyme, and this proportioning induction and hormesis are best.
Nine, in the present invention, described nitrogenous source is organic nitrogen source, comprise one or more combinations in any proportion in yeast powder, urea, vitamin H, organic nitrogen source has vital role to the growth of Clostridium thermocellum, and these organic nitrogen sources are the most favourable to the growth of Clostridium thermocellum.
Ten, compare with China Patent No. " 201110189094.8 ", the bacterial classification not only using is different, in China Patent No. " 201110189094.8 ", using bacterial classification is clostridium thermocellum JYT01, in this patent, use bacterial classification for clostridium thermocellumJ DSM1237T, and technique is different, the carbon-nitrogen ratio of taking on zymotechnique is also different, China Patent No. " 201110189094.8 " is by cultivating the proliferation of high-density that object and emphasis are fermentation thalline, and object in the present invention and focus on improving the vigor of fermented cellulose enzyme, in this patent, in fermentation culture, carbon source consumption is few, save fermentation costs, under equal carbon source drops into, have advantages of that enzyme raising alive and economic benefit increase.
Embodiment
embodiment 1
The present invention is usingd several kinds of carbon source as the moiety of substratum, and the stimulation by carbon source and add the raising of induction cellulase activity is not additionally being added under the condition of carbon source, improves fermentation broth enzyme and lives.Present method mainly comprises the following steps:
1. basal liquid substratum is: ammonium sulfate 1.30 g/L, magnesium chloride 2.60 g/L, potassium primary phosphate 1.43 g/L, dipotassium hydrogen phosphate 5.50 g/L, calcium chloride 0.13 g/L, ferric sulfate 1.10 mg/L, halfcystine 0.25 g/L, yeast extract paste 4.50 g/L, resazurin 1.0 mg/L, Microcrystalline Cellulose 10.00 g/L, or cellobiose 5.00 g/L.First class inoculum liquid is joined in basic medium, and the volume ratio of first class inoculum liquid and basic medium is 1:10 to 1:20, fermentation culture 48-72 hour.
2. fermention medium: ammonium sulfate 1-2 g/L magnesium chloride 2-3 g/L, potassium primary phosphate 1-2 g/L, dipotassium hydrogen phosphate 5-6g/L, calcium chloride 0.1-0.3 g/L, ferric sulfate 1-2 mg/L, halfcystine 0.2-0.5 g/L, yeast extract paste 4-6 g/L, resazurin 1.0 mg/L, carbon source 10.0-12.0 g/L.Preparation fermention medium, regulates pH7.0 before sterilizing, 121 ℃ of sterilizings 30 minutes.
3. inoculation: cultured strain liquid is joined in fermention medium by a certain percentage.
4. cultivate
50 ~ 70 ℃ of temperature, anaerobism, tank or bottle pressure-0.1MPa ~ 0.1MPa, standing.PH value 6.5 ~ 8.0.Incubation time is 48 ~ 72h.
5. after 48h-72h, cell concentration reaches 2.5-2.8(OD600), it is maximum that fermentation broth enzyme work reaches, and the work of CMC enzyme reaches 0.223IU; Xylanase activity reaches 0.205 IU, avicelase 0.142 IU alive, and filter paper enzyme activity 0.025 IU, respectively increases by 60%, 36%, 0.5% and 25% under cultivating than initial medium.
| Enzyme is lived | Initial incubation method | Cultural method of the present invention | Improve per-cent |
| CMC enzyme is lived | 0.139IU | 0.223IU | 60% |
| Xylanase activity | 0.150IU | 0.205IU | 36% |
| Avicelase is lived | 0.135IU | 0.142IU | 0.5% |
| Filter paper enzyme activity | 0.010IU | 0.025IU | 25% |
According to above step, it is that those skilled in the art can realize that Clostridium thermocellum improves enzyme cultural method alive.
The present invention has significant advantage: anaerobic condition can save and in fermenting process, pass into oxygen or the required energy consumption of pressurized air; Thermophilic fermentation is difficult for polluting, and these all will further reduce costs.Equal cultivation under cost condition, fermented liquid cellulase activity of the present invention significantly improves, and will significantly improve economic benefit.
Bacterial classification in the present invention (clostridium thermocellum) DSM1237
tsource is the ，You Methane Scientific Research Inst., Ministry of Agriculture microorganism center preservation of German DSMZ.
embodiment 2
The fermentation of 50ml anaerobism bottle
1.by C.thermocellum DSM1237
tlyophilized powder bacterium is configured to first class inoculum liquid.
2.by the C.thermocellum DSM1237 having activated
tfirst class inoculum liquid 5ml is inoculated in 50ml basic medium,, 60 ℃ of constant temperature culture 48h, obtain second class inoculum liquid.Second class inoculum liquid is inoculated into fermention medium, and inoculative proportion is 1:10.
3.by recipe configuration substratum, fermention medium: ammonium sulfate 1-2 g/L magnesium chloride 2-3 g/L, potassium primary phosphate 1-2 g/L, dipotassium hydrogen phosphate 5-6g/L, calcium chloride 0.1-0.3 g/L, ferric sulfate 1-2 mg/L, halfcystine 0.2-0.5 g/L, yeast extract paste 4-6 g/L, resazurin 1.0 mg/L, carbon source 10.0-12.0 g/L.Boil, be chilled to normal temperature and regulate pH6.5-7.5, air in not open close nitrogen emptying bottle, packing 50ml, in 150ml culturing bottle, with poly-butyl ester plug sealing, guarantees anaerobic environment in bottle.121 ℃ of sterilizings 30 minutes.Wherein Ca, Mg and Fe salt is made into respectively 100 mother liquors, after sterilizing, adds.
4.cultivate after approximately 48 hours for 60 ℃, the work of bacterium liquid enzyme reaches maximum value, and the work of CMC enzyme reaches 0.223IU; Xylanase activity reaches 0.205 IU, improves 60% and 36% respectively compared with initial incubation method.
5.detection method:
1) work of CMC enzyme is that Xylo-Mucine (CMC-Na) solution and enzyme liquid are mixed, at 50 ℃, react 30 min, then by DNS method, measure the growing amount of reducing sugar, and with per minute hydrolysis substrate, produce the required enzyme amount of 1 μ mol reducing sugar and be defined as 1 unit of activity (IU).
2) xylanase activity is that xylan solution and enzyme liquid are mixed, at 50 ℃, react 30 min, then by DNS method, measure the growing amount of reducing sugar, and with per minute hydrolysis substrate, produce the required enzyme amount of 1 μ mol reducing sugar and be defined as 1 unit of activity (IU).
3) filter paper enzyme activity is that filter paper solution and enzyme liquid are mixed, and reacts 30 min at 50 ℃, then by DNS method, measures the growing amount of reducing sugar, and with per minute hydrolysis substrate, produces the required enzyme amount of 1 μ mol reducing sugar and be defined as 1 unit of activity (IU).
embodiment 3
The fermentation of 1L anaerobism bottle
1.according to method described in case study on implementation 2, prepare C.thermocellum DSM1237
tsecond class inoculum liquid 100ml, inoculum size is 1:10, volume required separately when fermented liquid cumulative volume is 1L.
2.by recipe configuration substratum, (NH
4)
2sO
41.30 g/L MgCl
2x 6 H2O 2.60 g/L, KH
2pO
41.43 g/L, K
2hPO
45.50 g/L, CaCl
2x 2 H
2o 0.13 g/L, FeSO
4x 7 H
2o 1.10 mg/L, Cysteien hydrochloride 0.5 g/L, Yeast extract 2.50 g/L, Resazurin 1.0 mg/L, carbon source 10.0 g/L.Boil, be chilled to normal temperature and regulate pH6.5-7.5, air in not open close nitrogen emptying bottle, packing 50ml, in 150ml culturing bottle, with poly-butyl ester plug sealing, guarantees anaerobic environment in bottle.121 ℃ of sterilizings 30 minutes.Calcium, magnesium, molysite are made into respectively 100 mother liquors, after sterilizing, add.
3.cultivate static cultivation after 48 hours for 60 ℃, the work of bacterium liquid enzyme reaches maximum value, and the work of CMC enzyme reaches 0.223IU; Xylanase activity reaches 0.205 IU, avicelase 0.142 IU alive, filter paper enzyme activity 0.025 IU.
embodiment 4
A cultural method that improves Clostridium thermocellum cellulase activity, comprises the following steps:
A, by bacterial classification (clostridium thermocellum) DSM1237
t(lyophilized powder preservation) is mixed with first class inoculum liquid, and specific practice is that 1ml basic medium is joined in lyophilized powder, mixes, and is inoculated in the anaerobism pipe that 5ml basic medium is housed, and 60 ℃ of constant temperature culture, activate as first class inoculum liquid.
B, first class inoculum liquid is joined in basic medium, the volume ratio of first class inoculum liquid and basic medium is 1:10, and fermentation culture 48 hours is second class inoculum liquid;
C, the second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of strain liquid and fermention medium is 1:30;
D, fermentation culture: temperature 50 C, anaerobism, pressure-0.1MPa, standing, pH value 6.5, incubation time is 72h.
Basic medium in described step b is: ammonium sulfate 1.30 g/L, magnesium chloride 2.60 g/L, potassium primary phosphate 1.43 g/L, dipotassium hydrogen phosphate 5.50 g/L, calcium chloride 0.13 g/L, ferric sulfate 1.10 mg/L, halfcystine 0.25 g/L, yeast extract paste 4.50 g/L, resazurin 1.0 mg/L, Microcrystalline Cellulose 10.00 g/L or cellobiose 5.00 g/L.
The fermention medium of preparing in described step c is: ammonium sulfate 1 g/L magnesium chloride 2 g/L, potassium primary phosphate 1g/L, dipotassium hydrogen phosphate 5g/L, calcium chloride 0.1g/L, ferric sulfate 1mg/L, halfcystine 0.2g/L, nitrogenous source 4g/L, resazurin 1.0 mg/L, carbon source 10.0g/L.
In the present invention, described fermention medium, in when preparation, regulates pH to 7.0 before sterilizing, 121 ℃ of sterilizings 30 minutes.
In the present invention, described carbon source is the mass ratio combination that any two among Microcrystalline Cellulose (Avicel), xylan (xylan), chitosan, rice straw powder pressed 2:8.
In the present invention, described nitrogenous source is organic nitrogen source, comprises one or more combinations in any proportion in yeast powder, urea, vitamin H.
In the present invention, the mode of described fermentation comprises batch fermentation, feed supplement-batch fermentation or semicontinuous fermentation.
embodiment 5
The present embodiment and above-described embodiment are basic identical, and the key distinction is:
A cultural method that improves Clostridium thermocellum cellulase activity, comprises the following steps:
A, by bacterial classification (clostridium thermocellum) DSM1237
t(lyophilized powder preservation) is mixed with first class inoculum liquid;
B, first class inoculum liquid is joined in basic medium, the volume ratio of first class inoculum liquid and basic medium is 1:20, and fermentation culture 72 hours is second class inoculum liquid;
C, the second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of strain liquid and fermention medium is 1:10;
D, fermentation culture: temperature 70 C, anaerobism, pressure 0.1MPa, standing, pH value 7.5, incubation time is 72h.
The fermention medium of preparing in described step c is: ammonium sulfate 2 g/L magnesium chloride 3 g/L, potassium primary phosphate 2 g/L, dipotassium hydrogen phosphate 6g/L, calcium chloride 0.3 g/L, ferric sulfate 2 mg/L, halfcystine 0.5 g/L, nitrogenous source 6 g/L, resazurin 1.0 mg/L, carbon source 12.0 g/L.
In the present invention, described carbon source is three kinds of mass ratio combinations that any carbon source is pressed 2:6:2 among Microcrystalline Cellulose (Avicel), xylan (xylan), chitosan, rice straw powder.
embodiment 6
The present embodiment and above-described embodiment are basic identical, and the key distinction is:
A cultural method that improves Clostridium thermocellum cellulase activity, comprises the following steps:
A, by bacterial classification (clostridium thermocellum) DSM1237
t(lyophilized powder preservation) is mixed with first class inoculum liquid;
B, first class inoculum liquid is joined in basic medium, the volume ratio of first class inoculum liquid and basic medium is 1:15, and fermentation culture 60 hours is second class inoculum liquid;
C, the second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of second class inoculum liquid and fermention medium is 1:20;
D, fermentation culture: temperature 60 C, anaerobism, pressure 0.01MPa, standing, pH value 8.0, incubation time is 60h.
The fermention medium of preparing in described step c is: ammonium sulfate 1.5g/L magnesium chloride 2.5g/L, potassium primary phosphate 1.5g/L, dipotassium hydrogen phosphate 5.5g/L, calcium chloride 0.2g/L, ferric sulfate 1.5mg/L, halfcystine 0.3g/L, nitrogenous source 5g/L, resazurin 1.0 mg/L, carbon source 11.0g/L.
Be more than in conjunction with specific embodiments son the present invention is done further describe.The technician of the industry should understand, and the present invention is not appealed the restriction of embodiment, and without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.
Claims (7)
1. a cultural method that improves Clostridium thermocellum cellulase activity, is characterized in that, comprises the following steps:
A, by bacterial classification (clostridium thermocellum) DSM1237
tbe mixed with first class inoculum liquid;
B, first class inoculum liquid is joined in basic medium, the volume ratio of first class inoculum liquid and basic medium is 1:10 to 1:20, within fermentation culture 48-72 hour, is second class inoculum liquid;
C, the second class inoculum liquid after cultivating is joined in the fermention medium of preparation, the volume ratio of second class inoculum liquid and fermention medium is 1:30 ~ 1:10;
D, fermentation culture: 50 ~ 70 ℃ of temperature, anaerobism, pressure-0.1MPa ~ 0.1MPa, standing, pH value 6.5 ~ 8.0, incubation time is 48 ~ 72h.
2. the cultural method of raising Clostridium thermocellum cellulase activity according to claim 1, it is characterized in that: the basic medium in described step b is: ammonium sulfate 1.30 g/L, magnesium chloride 2.60 g/L, potassium primary phosphate 1.43 g/L, dipotassium hydrogen phosphate 5.50 g/L, calcium chloride 0.13 g/L, ferric sulfate 1.10 mg/L, halfcystine 0.25 g/L, yeast extract paste 4.50 g/L, resazurin 1.0 mg/L, Microcrystalline Cellulose 10.00 g/L or cellobiose 5.00 g/L.
3. the cultural method of raising Clostridium thermocellum cellulase activity according to claim 1, it is characterized in that: the fermention medium of preparing in described step c is: ammonium sulfate 1-2 g/L magnesium chloride 2-3 g/L, potassium primary phosphate 1-2 g/L, dipotassium hydrogen phosphate 5-6g/L, calcium chloride 0.1-0.3 g/L, ferric sulfate 1-2 mg/L, halfcystine 0.2-0.5 g/L, nitrogenous source 4-6 g/L, resazurin 1.0 mg/L, carbon source 10.0-12.0 g/L.
4. the cultural method of raising Clostridium thermocellum cellulase activity according to claim 3, is characterized in that: described fermention medium, when preparation, regulates pH to 7.0 before sterilizing, 121 ℃ of sterilizings 30 minutes.
5. according to the cultural method of the raising Clostridium thermocellum cellulase activity described in claim 3 or 4, it is characterized in that: described carbon source is two kinds or three kinds of mass ratio combinations that carbon source is pressed 2:8 or 2:6:2 among Microcrystalline Cellulose (Avicel), xylan (xylan), chitosan, rice straw powder.
6. according to the cultural method of the raising Clostridium thermocellum cellulase activity described in claim 3 or 4, it is characterized in that: described nitrogenous source is organic nitrogen source, comprise one or more combinations in any proportion in yeast powder, urea, vitamin H.
7. according to the cultural method of the raising Clostridium thermocellum cellulase activity described in claim 3 or 4, it is characterized in that: the mode of described fermentation comprises batch fermentation, feed supplement-batch fermentation or semicontinuous fermentation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310535035.0A CN103555693A (en) | 2013-11-04 | 2013-11-04 | Culture method for improving activity of clostridium thermoceuum cellulase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109392831A (en) * | 2018-10-30 | 2019-03-01 | 中国科学院青岛生物能源与过程研究所 | For producing nutrition fortifier and the application of the DHA-ARA egg of suitable infant |
| CN110540982A (en) * | 2019-09-30 | 2019-12-06 | 江南大学 | Fermentation method for increasing yield of Thermobacteroides cellulase |
| CN111334490A (en) * | 2018-12-18 | 2020-06-26 | 浙江科峰生物技术有限公司 | Preparation method of high-temperature-resistant cellulase preparation |
| CN111334447A (en) * | 2018-12-18 | 2020-06-26 | 浙江科峰生物技术有限公司 | Fermentation process of high-yield cellulase clostridium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864089A (en) * | 2011-07-07 | 2013-01-09 | 中国科学院青岛生物能源与过程研究所 | High-density culture method of clostridium thermocellum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102864089A (en) * | 2011-07-07 | 2013-01-09 | 中国科学院青岛生物能源与过程研究所 | High-density culture method of clostridium thermocellum |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109392831A (en) * | 2018-10-30 | 2019-03-01 | 中国科学院青岛生物能源与过程研究所 | For producing nutrition fortifier and the application of the DHA-ARA egg of suitable infant |
| CN111334490A (en) * | 2018-12-18 | 2020-06-26 | 浙江科峰生物技术有限公司 | Preparation method of high-temperature-resistant cellulase preparation |
| CN111334447A (en) * | 2018-12-18 | 2020-06-26 | 浙江科峰生物技术有限公司 | Fermentation process of high-yield cellulase clostridium |
| CN110540982A (en) * | 2019-09-30 | 2019-12-06 | 江南大学 | Fermentation method for increasing yield of Thermobacteroides cellulase |
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