CN102174433A - Clostridium beijerinckii with high stress resistance and application thereof - Google Patents
Clostridium beijerinckii with high stress resistance and application thereof Download PDFInfo
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Abstract
The invention relates to Clostridium beijerinckii with high stress resistance which can directly utilize a virus-carrying wood fiber acid-hydrolyzed sugar solution for fermentation to produce butanol and an application thereof. The Clostridium beijerinckii disclosed by the invention has the category name of Clostridium beijerinckii IB4 and the collection number CCTCC No. is M2010310. Ion beam mutation is adopted, a resazurin flat plate containing inhibitors such as phenols and a shake flask are used for fermenting and screening, the obtained mutant strain uses a virus-carrying corncob acid-hydrolyzed sugar solution as a carbon source, the total solvent yield and butanol yield in a 2L fermentation tank are up to 10.3g/L and 7.1g/L respectively which are 4.3 times and 4.4 times of those obtained by adopting the starting strain cultivated under the same conditions, the sugar conversion rate is 0.35; and the Clostridium beijerinckii has the advantages of high stress resistance, high solvent yield, high sugar conversion rate and good repeatability, thereby being a good strain which is suitable for utilizing the wood fiber raw material to produce butanol.
Description
Technical field
The present invention relates to a strain and can utilize not the Bei Shi clostridium and the application thereof of detoxification wood fibre acidolysis liquid glucose, belong to technical field of biological fermentation.
Background technology
Butanols is a kind of important C4 hardware and software platform compound, has been widely used in the manufacturing of various fine chemicals; And come into one's own as a kind of important novel biological fuel with very big potentiality, United Nations's International Energy Agency is classified biological butanol as s-generation biofuel.Act as a fuel, butanols have energy density big, can be directly used in advantages such as oil engine, convenient transportation, in energy dilemma increasingly serious today, the butanols vast potential for future development that acted as a fuel.Because output in domestic can not be satisfied the demand, China has been maximum in the world butanols importer.
At present, butanols is mainly synthetic by chemical method, and along with petroleum resources acceleration exhaustion and price skyrocket, the fermentative Production butanols has been subjected to again paying attention to widely, has become one of research focus of bioenergy.Clostridium acetobutylicum and Bei Shi clostridium can directly effectively utilize raw materials such as corn and other starches material or molasses, but that the cost of these raw materials compares is higher, have the problem that disaccords with the people simultaneously.Under the double threat of food shortage and energy dilemma; Exploring cellulose raw material and produce the important composition that the fuel butanols becomes the biomass energy development strategy, also is one of focus of research.
In recent years, a lot of to the research of fibrous material fermentation product butanols both at home and abroad, mainly the fermentation such as preparation, optimization of fermentation condition and solvent extraction round induction mutation of bacterium seed selection, fibrous material liquid glucose carry out.
Chinese patent ZL95111733.5 has reported by chemomorphosis and has handled the acetone-butanol fusobacterium, utilizes Semen Maydis powder or Chinese sorghum to be substrate, and fermenting obtains total solvent output about 20g/L, and butanols is than the stable bacterial strain that is 70%; (Biotechnology and Bioengineering.1993 such as Mermelstein, 42:1053～1060) utilize genetic engineering technique to make up recombinant bacterial strain, butanols rate ratio starting strain Clostridiumn acetobutylicum ATCC 824 has improved 37%.(Biomass and Bioenergy.2008 such as NasibQureshi, 32:176-183) utilize Bei Shi clostridium P260, add cellulase, zytase etc. and carry out simultaneous saccharification and fermentation in wheat stalk, through 533 hours continuously ferment, the productive rate of its solvent was 0.41.(Appl.Environ.Microbiol.1991 57:2544-2548) utilizes chemomorphosis to Annous etc., has obtained super bacterial strain BA101, and the single batch fermentation total solvent reaches as high as more than the 25g/L; (Bioresource Technology.2008 such as Thaddeus Ezeji, 99:5915-5922) utilize mutant strain BA101, corncob acid hydrolysis and enzymolysis liquid glucose with the XAD-4resin detoxification are fermenting substrate, total solvent output is 9.30g/L, but it can not utilize the acidolysis of not detoxification and the fermentation of enzymolysis liquid glucose to produce butanols.Lignocellulose raw material can produce inhibitions such as organic acid, furfural, phenols after diluted acid is handled, these inhibitions to remove cost higher and microorganism growth had certain restraining effect; (Biotechnology﹠amp such as ThaddeusEzeji; Bioengineering.2007,97 (6): 1460-1469) discover that inhibitions such as organic acid, furfural do not influence the fermentation of butanols, and the phenols inhibition has the obvious suppression effect to butylic fermentation.So far, also there is not directly to utilize not detoxification fiber acidolysis liquid glucose fermentation to produce the patent and the bibliographical information of butanols.
As seen, the toxin inhibition severe inhibition in the lignocellulose raw material is produced the leavening property of Clostridium acetobutylicum; And strain improvement is to improve bacterial strain to one of the tolerance of inhibition, key means of fermentation economy.
Summary of the invention
The technical problem to be solved in the present invention provides the Bei Shi clostridium that a strain has high resistance to cold and diseases, and its tolerance to inhibition in the wood fibre acidolysis liquid glucose significantly improves, and solvent production height, the sugared transformation efficiency height of fermentation.
The technical problem that the present invention also will solve provides the application of above-mentioned high resistance to cold and diseases Bei Shi clostridium bacterial strain.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
One plant height resistance Bei Shi clostridium, its classification called after Bei Shi clostridium (Clostridium beijerinckii) IB4, be preserved in Chinese typical culture collection center C CTCC, address: Wuhan Wuhan University, postcode: 430072, deposit number: CCTCC NO:M 2010310, preservation date on November 23rd, 2010.
Above-mentioned high resistance to cold and diseases Bei Shi clostridium (Clostridium beijerinckii) IB4 is after being the starting strain ion beam mutagenesis with Bei Shi clostridium (NCIMB8052), the resazurin plate screening that utilization contains inhibitions such as phenols obtains inhibition tolerance height, bacterial strain that reducing power is strong, at last the corncob acid hydrolysis liquid glucose with not detoxification is a substrate, obtains solvent production height, Bei Shi clostridium aimed strain that sugared transformation efficiency is high through the anaerobically fermenting screening.
Its concrete mutagenesis screening step is as follows:
A) ion beam mutagenesis: with Bei Shi clostridium original strain activation culture, the bottled liquid measure 10～15mL of the Xiao Te anaerobism of 25mL, inflated with nitrogen 3min, 33～37 ℃ of culture temperature, incubation time 12～16h obtains being in the bacterium liquid of logarithmic phase, and cultured cells is diluted to OD
600=0.05～0.1, coat in the empty culture dish of sterilization, dry up with sterile air; With 5～15KeV as ion implantation energy, with 0.4～2.4 * 10
16Ions/cm
2As mutagenesis dosage bacterial strain is carried out ion beam mutagenesis;
B) the dull and stereotyped primary dcreening operation of resazurin: bacterial strain is after ion mutagenesis, wash out with physiological saline, being diluted to different concns coats on the conventional solid medium flat board that contains inhibition and resazurin (0.002%), anaerobism is cultivated 12～30h under 33～37 ℃ of temperature, picks out the bigger bacterium colony of transparent circle on this flat board;
C) the dull and stereotyped multiple sieve of corncob acid hydrolysis liquid glucose: with the inoculation of step b) screening in the bottled liquid measure 10～15mL of the Xiao Te of 25mL anaerobism, inflated with nitrogen 3min, 33～37 ℃ of anaerobism are cultivated 10～14h, stroke-physiological saline solution is made the bacteria suspension of concentration OD=0.1, drawing 2 μ L selects and drips on the corncob acid hydrolysis liquid glucose flat board that contains inhibition and resazurin (0.002%), anaerobism is cultivated 12～30h under 33～37 ℃ of temperature, picks out transparent circle obviously greater than the bacterium colony of the bacterium that sets out;
D) anaerobism bottle fermentation screening: the bacterium colony that step c) is sifted out inserts the seed culture medium enlarged culturing, 33～37 ℃ of culture temperature, anaerobism is cultivated incubation time 10～16h, in fermention medium, ferment then, inoculum size 5%～15% (v/v), inflated with nitrogen 3min, 33～37 ℃ of leavening temperatures, anaerobically fermenting fermentation time 60～80h; The output that butanols and total solvent are produced in the bacterium colony fermentation that investigation filters out is selected butanols and the high bacterial strain of total solvent output simultaneously.
In the ion mutafacient system described in the step a), preferred 10KeV is as ion implantation energy, 1.6 * 10
16Ions/cm
2As mutagenesis dosage.
Step b) and c) the conventional solid medium, the carbon source that are adopted be one or more in glucose, starch and the wood fibre acidolysis liquid glucose; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast powder, extractum carnis and the corn steep liquor; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt, add agar in the solid medium.
In the seed culture medium and fermention medium that step d) adopted, carbon source is one or more in glucose, starch, the wood fibre acidolysis liquid glucose; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast powder, extractum carnis and the corn steep liquor; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt.Somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and the corn steep liquor.
The application of above-mentioned high resistance to cold and diseases Bei Shi clostridium (Clostridium beijerinckii) IB4 that filters out in producing butanols.
Concrete applying step is as follows:
1) the dull and stereotyped cultivation: Bei Shi clostridium CCTCC NO:M 2010310 is seeded to the plate culture medium anaerobism cultivates 33～37 ℃ of culture temperature, incubation time 12～24h;
2) seed culture: the Bei Shi clostridium CCTCC NO:M 2010310 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 40～60mL of the anaerobism of 100mL, inflated with nitrogen 3～5min, 33～37 ℃ of culture temperature, incubation time 12～24h;
3) butanols is produced in fermentation: seed culture fluid is inoculated in the fermention medium, and inoculum size 5～15% (v/v), inflated with nitrogen 3～5min, 33～37 ℃ of leavening temperatures, fermented incubation time are 60～80h.
Wherein, described plate culture medium comprises following component by mass percent: carbon source 0.3%～1%, nitrogenous source 0.5%～1%, inorganic salt 0.5%～0.8%, agar 1.5%～2%, all the other are water; Described carbon source be in glucose, starch and the fiber acidolysis liquid glucose any one or multiple; Described nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and the ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and the extractum carnis; Described inorganic salt be in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt any one or multiple.
Wherein, described seed culture medium comprises following component by mass percent: carbon source 0.5%～1%, nitrogenous source 0.5%～1%, inorganic salt 0.5%～0.8%, all the other are water; Described nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and the ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and the extractum carnis; Described inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt.
Wherein, described fermention medium comprises following component by mass percent: carbon source 3%～6%, nitrogenous source 0.1%～0.3%, inorganic salt 0.1%～0.2%, somatomedin 0.05～0.1%, all the other are water; Described carbon source is one or more in glucose, wood sugar, pectinose, wood fibre acidolysis liquid glucose and the wood fibre enzymolysis liquid glucose; Described nitrogenous source is one or more in ammonium acetate, ammonium chloride and the yeast powder; Described inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt; Described somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and the corn steep liquor.
Beneficial effect of the present invention is:
The present invention adopts ion beam mutagenesis Bei Shi clostridium, the resazurin plate screening that utilization contains the corncob acid hydrolysis liquid glucose of not detoxification has stronger tolerance, also can directly utilize wood fibre acidolysis liquid glucose fermentative production butanols, sugared transformation efficiency height, the bacterial strain that total solvent output is high the inhibitions such as phenols in the fiber acidolysis liquid glucose.Utilize bacterial strain of the present invention and technology to ferment, as carbon source, total solvent output and butanols output have reached 10.3g/L and 7.1g/L respectively in the 2L fermentor tank, are respectively 4.3 times and 4.4 times of starting strain with the corncob acid hydrolysis liquid glucose of not detoxification; Have important social meaning and economic worth.
Description of drawings
Fig. 1 is the survival rate graphic representation of Bei Shi clostridium NCIMB 8052.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment is described only to be used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the method for the first step ion beam mutagenesis is carried out Bei Shi clostridium original strain in the present embodiment explanation.
Bei Shi clostridium NCIMB 8052 original strains are purchased in American Type Culture Collecti (ATCC); The method of carrying out the first step ion beam mutagenesis is as follows:
With with Bei Shi clostridium NCIMB 8052 original strain activation culture, 33～37 ℃ of culture temperature, 25mL shakes bottled liquid measure 10～15mL, inflated with nitrogen 3min, incubation time 12～18h obtains the bacterium liquid of growing vigorous, that thalline is sturdy; The cell dilution of getting fresh culture is to cell concn OD
600=0.05～0.1, to coat in the empty culture dish of sterilization, sterile air dries up; Inject various dose with 10KeV, pulse mode is injected 5s at every turn, at interval 15s.Behind the ion implantation mutagenesis, mycoderm is eluted, calculate survival rate.Experimental result as shown in Figure 1; As shown in Figure 1,1.6 * 10
16Ions/cm
2Be best mutagenesis dosage.
Embodiment 2: the explanation of this example is the good Bei Shi clostridial method of screening further.
Wherein, employed culture medium prescription (% is a mass percent):
(1) solid plate substratum: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar 1.5%, all the other are water, pH 6.
(2) resazurin plate culture medium: yeast powder 0.3%, peptone 0.5%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar 1.5%, resazurin 0.002%, the not detoxified corn core acidolysis liquid glucose of adding 1/2 (v/v), all the other are water, and pH 6.
(3) corncob acid hydrolysis liquid glucose plate culture medium: yeast powder 0.3%, peptone 0.5%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar 1.5%, resazurin 0.002% is with not detoxified corn core acidolysis liquid glucose configuration, all the other are water, and pH 6.
(4) shake flask fermentation screening culture medium I: glucose 3%, ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, bitter salt 0.02%, Manganous sulfate monohydrate 0.001%, ferrous sulfate 0.001%, sodium-chlor 0.001%, corn steep liquor 0.1%, all the other are water, and pH 6.6.
(5) shake flask fermentation screening culture medium II: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, bitter salt 0.02%, Manganous sulfate monohydrate 0.001%, ferrous sulfate 0.001%, sodium-chlor 0.001%, corn steep liquor 0.1%, not detoxified corn core acidolysis liquid glucose (total reducing sugars 3%), all the other are water, and pH 6.6.
The screening step:
1, resazurin plate screening
Wash out bacterial strain with physiological saline through ion beam mutagenesis, being diluted to different concns coats on the conventional solid medium flat board that contains phenols inhibition (0.02%) and resazurin (0.002%), anaerobism is cultivated 30h under 35 ℃ of temperature, selects and can screen big and obviously bigger bacterium colony 30 strains of variable color circle of growth, bacterium colony on the flat board.
2, the dull and stereotyped multiple sieve of corncob acid hydrolysis liquid glucose
With the screening inoculation in the bottled liquid measure 10～15mL of the Xiao Te of 25mL anaerobism, inflated with nitrogen 3min, 35 ℃ of anaerobism are cultivated 10～14h, stroke-physiological saline solution is made the bacteria suspension of concentration OD=0.1, absorption 2uL selects and drips on the corncob acid hydrolysis liquid glucose flat board that contains phenols inhibition (0.15%) and resazurin (0.002%), anaerobism is cultivated 12～24h under 35 ℃ of temperature, picks out transparent circle obviously greater than the bacterium colony of the bacterium that sets out.
Final bacterial strain IB4 and IB9 have shown stronger inhibition tolerance and reduction vigor.
3, shake flask fermentation screening
Bacterial strain IB4, IB9 and original strain are inserted seed culture medium enlarged culturing, 35 ℃ of culture temperature, the bottled liquid measure 100mL of Xiao Te anaerobism of 250mL, inflated with nitrogen 3min, incubation time 12h.In with fermention medium, ferment then, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, the bottled liquid measure 60mL of 100mL Xiao Te anaerobism, inflated with nitrogen 3min, the total solvent output and the butanols output that detect each bacterial strain behind the fermentation time 72h are as shown in table 1:
IB4, IB9 and original bacterium fermentation result in table 1 screening culture medium
Total solvent output and butanols output are all apparently higher than starting strain during the fermentation in the two plant mutant strains that obtain through dull and stereotyped combined sorting, and wherein IB4 has the highest total solvent output, sugared transformation efficiency is also the highest.This is consistent with the assembled flat results of screening.
Embodiment 3: the mitotic stability of present embodiment explanation mutant strain IB4 and IB9.
Be in the fermention medium of carbon source with glucose, detecting the mitotic stability of mutant strain IB4 and IB9.Bacterial strain IB4 and the IB9 fermentation test result that goes down to posterity is as shown in table 2:
Table 2 bacterial strain IB4 and the IB9 fermentation test result that goes down to posterity
From experimental result as can be known, through 7 continuous passages, the total solvent output and the butanols output of two plant mutant strains are more stable, have mitotic stability preferably, can be used as the production bacterial strain of further research and development.
Embodiment 4: the technology of present embodiment explanation Bei Shi clostridium C.beijerinckii IB4 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar powder 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: glucose 3%; Ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, all the other are water, pH 6.6.
Bei Shi clostridium C.beijerinckii IB4 is seeded to the plate culture medium anaerobism cultivates 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 100mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in the fermention medium, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, the bottled liquid measure 60mL of 100mL Xiao Te anaerobism, inflated with nitrogen 3min behind the fermentation culture 72h, detects total solvent output and butanols output and has reached 12.6g/L and 9.2g/L respectively, the starting strain of cultivating than equal conditions has improved 16.7% and 24.3%, and sugared transformation efficiency reaches 0.42.
Embodiment 5: the technology of present embodiment explanation Bei Shi clostridium C.beijerinckii IB4 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar powder 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: mixing sugar (glucose: wood sugar: pectinose mass ratio=5: 4: 1) 3%; Ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, all the other are water, pH 6.6.
Bei Shi clostridium C.beijerinckii IB4 is seeded to the plate culture medium anaerobism cultivates 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 100mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in the fermention medium, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, the bottled liquid measure 60mL of 100mL Xiao Te anaerobism, inflated with nitrogen 3min, fermentation culture 12h, behind the fermentation culture 72h, detect total solvent output and butanols output and reached 12.1g/L and 9.0g/L respectively, the starting strain of cultivating than equal conditions has improved 19.4% and 28.6%, and sugared transformation efficiency reaches 0.40.
Embodiment 6: the technology of present embodiment explanation Bei Shi clostridium C.beijerinckii IB4 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar powder 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1%, the corncob acid hydrolysis of gac detoxification and the liquid glucose of enzymolysis (total reducing sugars is 3%) configuration, all the other are water, and pH 6.6.
Bei Shi clostridium C.beijerinckii IB4 is seeded to the plate culture medium anaerobism cultivates 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 150mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in the 2L fermentor tank that the 1L fermention medium is housed, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, feed nitrogen continuously, flow velocity is 0.3L/min, behind the fermentation culture 72h, detects total solvent output and butanols output and has reached 11.6g/L and 8.3g/L respectively, the starting strain of cultivating than equal conditions has improved 22.1% and 32.6%, and sugared transformation efficiency reaches 0.38.
Embodiment 7: the technology of present embodiment explanation Bei Shi clostridium C.beijerinckii IB4 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar powder 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1% is with the not corncob acid hydrolysis liquid glucose of detoxification (total reducing sugars is 3%) configuration, all the other are water, and pH 6.6.
Bei Shi clostridium C.beijerinckii IB4 is seeded to the plate culture medium anaerobism cultivates 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 150mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in the 2L fermentor tank that the 1L fermention medium is housed, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, feed nitrogen continuously, flow velocity is 0.3L/min, behind the fermentation culture 72h, detects total solvent output and butanols output and has reached 10.3g/L and 7.1g/L respectively, be 4.3 times and 4.4 times of the starting strain cultivated of equal conditions, sugared transformation efficiency reaches 0.35.
Embodiment 8: the technology of present embodiment explanation Bei Shi clostridium C.beijerinckii IB4 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, agar powder 1.5%, all the other are water, pH 6.
Seed culture medium: yeast powder 0.3%, peptone 0.5%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, bitter salt 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, ferrous sulfate 0.01%, all the other are water, pH 6.
Fermention medium: ammonium acetate 0.22%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.05%, sodium-chlor 0.001%, bitter salt 0.02%, ferrous sulfate 0.001%, Manganous sulfate monohydrate 0.001%, corn steep liquor 0.1% is with the not bagasse acidolysis liquid glucose of detoxification (total reducing sugars is 3%) configuration, all the other are water, and pH 6.6.
Bei Shi clostridium C.beijerinckii IB4 is seeded to the plate culture medium anaerobism cultivates 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 150mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in the 2L fermentor tank that the 1L fermention medium is housed, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, feed nitrogen continuously, flow velocity is 0.3L/min, behind the fermentation culture 72h, detects total solvent output and butanols output and has reached 10.6g/L and 7.3g/L respectively, be 3.8 times and 3.6 times of the starting strain cultivated of equal conditions, sugared transformation efficiency reaches 0.36.
Claims (6)
1. a plant height resistance Bei Shi clostridium, its classification called after Bei Shi clostridium (Clostridium beijerinckii) IB4 has been preserved in Chinese typical culture collection center C CTCC, deposit number: CCTCC NO:M 2010310.
2. the application of the described high resistance to cold and diseases Bei Shi of claim 1 clostridium in producing butanols.
3. application according to claim 2 is characterized in that concrete applying step is as follows:
1) the dull and stereotyped cultivation: Bei Shi clostridium CCTCC NO:M 2010310 is seeded to the plate culture medium anaerobism cultivates 33～37 ℃ of culture temperature, incubation time 12～24h;
2) seed culture: the Bei Shi clostridium CCTCC NO:M 2010310 that flat board is cultivated is inoculated in the seed culture medium the bottled liquid measure 40～60mL of the anaerobism of 100mL, inflated with nitrogen 3～5min, 33～37 ℃ of culture temperature, incubation time 12～24h;
3) butanols is produced in fermentation: seed culture fluid is inoculated in the fermention medium, and inoculum size 5～15% (v/v), inflated with nitrogen 3～5min, 33～37 ℃ of leavening temperatures, fermented incubation time are 60～80h.
4. application according to claim 3 is characterized in that described plate culture medium comprises following component by mass percent: carbon source 0.3%～1%, nitrogenous source 0.5%～1%, inorganic salt 0.5%～0.8%, agar 1.5%～2%, all the other are water; Described carbon source is one or more in glucose, starch and the fiber acidolysis liquid glucose; Described nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and the ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder, extractum carnis and the corn steep liquor; Described inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt.
5. application according to claim 3 is characterized in that described seed culture medium comprises following component by mass percent: carbon source 0.5%～1%, nitrogenous source 0.5%～1%, inorganic salt 0.5%～0.8%, all the other are water; Described carbon source is one or both mixing of starch and glucose; Described nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and the ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder, extractum carnis and the corn steep liquor; Described inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt.
6. application according to claim 3 is characterized in that described fermention medium comprises following component by mass percent: carbon source 3%～6%, nitrogenous source 0.1%～0.3%, inorganic salt 0.1%～0.2%, somatomedin 0.05%～0.1%, all the other are water; Described carbon source is one or more in glucose, wood sugar, pectinose, wood fibre acidolysis liquid glucose and the wood fibre enzymolysis liquid glucose; Described nitrogenous source is one or more in ammonium acetate, ammonium chloride and the yeast powder; Described inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, the ferrous salt; Described somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and the corn steep liquor.
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