CN101864389A - Clostridium acetobutylicum strain and screening method and application thereof - Google Patents
Clostridium acetobutylicum strain and screening method and application thereof Download PDFInfo
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Abstract
The invention relates to a Clostridium acetobutylicum strain capable of quickly generating butanol by fermenting xylose and starch, and a screening method and application thereof. The invention discloses Clostridium acetobutylicum, which is classified and named Clostridium acetobutylicumXY16 with the preservation and registration number of CCTCC NO:M2010011. The strain obtained by ion beam mutation and screening of a xylose plate, a 2-deoxy-D-glucose plate, a bromocresol green plate and a resazurin plate can quickly and efficiently generate the butanol by fermenting xylose and starch, has the advantages of high yield of total solvent, high butanol ratio and good repeatability, and is a good strain suitable for industrial production.
Description
Technical field
The present invention relates to the high solvent output that a strain obtains by the ion beam mutagenesis seed selection and the acetone-butanol fusobacterium bacterial strain of high butanols ratio, and the screening method of this bacterial strain and its application in solvent fermentation industry, technical field of biological fermentation belonged to.
Background technology
Butanols is important Organic Chemicals, is widely used in fields such as paint, topcoating, leather processing, plastics.Act as a fuel, it is big that butanols has energy density, high to the stability of water, can be directly used in advantages such as oil engine, convenient transportation, in energy dilemma increasingly serious today, and the butanols vast potential for future development that acted as a fuel.The apparent consumption of China's butanols had reached 680,000 tons in 2005, estimated that domestic butanols demand in 2010 will reach 97.2 ten thousand tons.Because output in domestic can not be satisfied the demand, China has been maximum in the world butanols importer.
The production method of butanols mainly contains the acetaldehyde condensation method, propylene oxo synthesis and fermentation method.The technical process of acetaldehyde condensation method is long, and yield is low, and cost is higher, is eliminated abroad at present; The raw material that the propylene oxo synthesis is produced butanols is a petrochemical industry derived product propylene; Along with ballooning oil prices and resource are quickened exhaustion, the fermentative Production butanols has been subjected to paying attention to widely, becomes one of research focus of bioenergy gradually.
In recent years, domestic research to acetone butanol fermentation is a lot, mainly carries out round fermentation such as induction mutation of bacterium seed selection, genetic engineering modified, optimization of fermentation condition and solvent extractions.University Of Shanxi's face is chatted show (Shanxi foodstuffs industry .1995,2:26～28) and is adopted ultraviolet mutagenesis to handle, and screens that meta-bolites improves, the bacterial classification of good stability, and the total solvent ratio bacterium that sets out improves about 36%; Chinese patent application 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) made up the plasmid pFNK6 that contains E.C. 4.1.1.4, CoA transferring enzyme A, CoA transferring enzyme, after transforming Clostridiumnacetobutylicum ATCC 824, acetone, butanols and ethanol production have improved 95%, 37% and 90% respectively; (Applied Environment Microbiology.1994 such as Kim, 60:337～340) the endo glucanase gene eng B with Clostridium cellulorans imports among the Clostridium beijerinckii, its endoglucanase vigor has improved 4 times than original strain, this in acetone, the butylic fermentation cellulosic utilize significant; Tomas etc. (AppliedEnvironment Microbiology.2003,69:4951～4965) are with groESL operon gene overexpression in Clostridiumacetobutylicum ATCC 824, and solvent production is higher by 40% than wild-type.
As seen, strain improvement is one of key means that improves the acetone economic competitiveness, and the screening conditions of setting up at mutagenesis bacterial classification efficiently and accurately are one of important steps that obtain good acetone-butanol fusobacterium.The report that 2-deoxy-D-glucose plate screening acetone-strain of butyl alcohol producing is only arranged at present both at home and abroad.
Summary of the invention
One of the technical problem to be solved in the present invention is to cultivate the acetone-butanol bacterial strain with new performance, makes its enough wood sugar and starch of efficiently utilizing, and the solvent production of fermentation and butanols ratio height; Two of the technical problem to be solved in the present invention is to provide the novel screening methods of described acetone-butanol bacterial strain; Three of the technical problem to be solved in the present invention is to provide the purposes of described acetone-butanol bacterial strain.
In order to solve technical problem of the present invention, technical program of the present invention lies in:
One, the present invention has cultivated the new acetone-butanol fusobacterium bacterial strain of a strain, its classification called after acetone-butanol fusobacterium Clostridium acetobutylicum XY16, and the preserving number registration number is: CCTCC NO:M 2010011.
Two, the screening method of acetone-butanol fusobacterium Clostridium acetobutylicum XY16 of the present invention, it is characterized in that behind the acetone-butanol fusobacterium starting strain ion beam mutagenesis, utilize wood sugar flat board and 2-deoxy-D-glucose plate screening to obtain efficiently to utilize the acetone-butanol bacterial strain of wood sugar and starch, utilize tetrabromo-mcresolsulfonphthalein flat board, resazurin plate screening to obtain acid producing ability and the strong bacterial strain of reducing power again, after the shake flask fermentation screening obtains total solvent output and butanols than high acetone-butanol fusobacterium aimed strain.
Its concrete steps are as follows:
A) ion beam mutagenesis: with acetone-butanol fusobacterium original strain activation culture, 30～40 ℃ of culture temperature, 250mL shakes bottled liquid measure 100～150mL, paraffin fluid-tight 2～5cm, incubation time 12～18h obtains the bacterium liquid of growing vigorous, that thalline is sturdy, with 5～10 times of cultured cells dilutions, coat in the empty culture dish of sterilization, dry up with sterile air; With 5～15KeV as ion implantation energy, with 1.0～2.0 * 10
16Ions/cm
2As mutagenesis dosage bacterial strain is carried out ion beam mutagenesis;
B) wood sugar plate screening: bacterial strain is after ion mutagenesis, wash out with physiological saline, be diluted to different concns and coat with on 0.5～2% the flat board of wood sugar as sole carbon source, anaerobism is cultivated 24～72h under 30～40 ℃ of temperature, picks out the bacterium colony that can grow on this flat board;
C) 2-deoxy-D-glucose plate screening: the mutant strain point that step b) is filtered out is planted in the dull and stereotyped enterprising row filter of conventional solid medium that contains 0.05～0.2% 2-deoxy-D-glucose, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, selects the bacterium colony that growing state significantly is better than the bacterium that sets out;
D) tetrabromo-mcresolsulfonphthalein plate screening: with the inoculation of step c) screening on conventional solid medium flat board, 30～40 ℃ of anaerobism are cultivated 12～36h, stroke-physiological saline solution is made the identical bacteria suspension of concentration, join in the hole of the conventional solid medium flat board that contains 0.001%～0.01% tetrabromo-mcresolsulfonphthalein, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
E) resazurin plate screening: with the inoculation of step d) screening on conventional solid medium flat board, 30～40 ℃ of anaerobism are cultivated 12～36h, stroke-physiological saline solution is made the identical bacteria suspension of concentration, join in the hole that contains the flat conventional solid medium flat board of 0.001%～0.01% resazurin, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
F) shake flask fermentation screening: the bacterium colony that step e) is sifted out inserts the seed culture medium enlarged culturing, 30～40 ℃ of culture temperature, anaerobism is cultivated incubation time 12～36h, in fermention medium, ferment then, inoculum size 5%～15% (v/v), 30～40 ℃ of leavening temperatures, anaerobically fermenting fermentation time 30～60h; Investigate step d) and e) the bacterium colony fermentation that filters out produces the amount and the ratio of the butanols in the solvent of total solvent, selects total solvent output and butanols simultaneously than the highest bacterium colony.
In above-mentioned screening method: 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.
In above-mentioned screening method: step c), d) and the conventional solid medium, the carbon source that e) are adopted be in wood sugar, the starch one or more; 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 above-mentioned screening method: in the seed culture medium and fermention medium that step f) adopted, 1) carbon source is wood sugar; 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.Perhaps 2) seed culture medium and fermention medium are, 20～80g/L Semen Maydis powder boils back gelatinization 0.5～2h, supplies evaporable moisture, natural pH.
Three, the application of acetone-butanol fusobacterium Clostridium acetobutylicum XY16 of the present invention in the fermentative production butanols, its detailed process is as follows:
The dull and stereotyped cultivation: acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 30～40 ℃ of culture temperature, incubation time 12～36h;
Seed culture: the acetone-butanol fusobacterium Clostridium acetobutylicum XY16 that flat board is cultivated is inoculated in the seed culture medium, 30～40 ℃ of culture temperature, 250mL shakes bottled liquid measure 100～150mL, paraffin fluid-tight 1～3cm, incubation time 12～36h;
Butanols is produced in fermentation: with seed culture fluid heat shock 1～5min, is inoculated in the fermention medium, and inoculum size 5%～15% (v/v), 30～40 ℃ of leavening temperatures, 250mL shake bottled liquid measure 100～150mL, and paraffin fluid-tight 1～3cm, fermented incubation time are 48～80h.
Wherein, consisting of of plate culture medium, seed culture medium and fermention medium: carbon source is a wood sugar; 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 plate culture medium;
Perhaps, the component of plate culture medium, seed culture medium and fermention medium is: seed culture medium and fermention medium be, 20～80g/L Semen Maydis powder boils back gelatinization 0.5～2h, supplies evaporable moisture, and natural pH adds agar in the plate culture medium.
Beneficial effect of the present invention is:
Solvent is produced in the acid back and high vigor anaerobically fermenting cell has the characteristics of strong reducing power according to producing earlier in the acetone butanol fermentation pathways metabolism in the present invention, adopt ion beam mutagenesis acetone-butanol fusobacterium, utilize wood sugar flat board, tetrabromo-mcresolsulfonphthalein flat board and resazurin flat board to unite screening first and efficiently utilize wood sugar and starch, the fermentation high yield bacterium of high solvent output and high butanols ratio.Utilize bacterial strain of the present invention and technology to ferment, as sole carbon source, total solvent output and butanols output have reached 17.8g/L and 12.9g/L respectively in the 5L fermentor tank with wood sugar, and starting strain can't be sole carbon source fermentative production butanols with the wood sugar; With the Semen Maydis powder is raw material, and total solvent output and butanols output have reached 20.2g/L and 15.9g/L respectively in the 5L fermentor tank, have improved 66.9% and 114.9% than starting strain, and the butanols ratio has reached 78.7%, has important social meaning and economic worth.
Description of drawings
The ion implantation survival rate curve of Fig. 1 acetone-butanol fusobacterium
Microorganism classification called after acetone-butanol fusobacterium Clostridium acetobutylicum XY16 of the present invention, its preservation date is on January 15th, 2010, depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, and deposit number is CCTCC NO:M 2010011.
Embodiment
Embodiment one
The method of the first step ion beam mutagenesis is carried out acetone-butanol fusobacterium original strain in the present embodiment explanation.
Acetone-butanol fusobacterium original strain derives from from row filter, and concrete grammar is as follows:
1. with the fermentation screening culture medium that contains the 25g/L butanols the anti-butanols microorganism in the pedotheque of area, Nanjing is carried out enrichment culture, filter out the bacterial strain that can tolerate the 25g/L butanols.Bacterium liquid in the sample hose that can grow is rule on the solid plate substratum, cultivates 48h, obtains single bacterium colony for 37 ℃.
Wherein, fermentation screening culture medium: butanols 1%, yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
2. single bacterium colony that primary dcreening operation is obtained is chosen to the fermentation screening culture medium, cultivates 48h for 37 ℃, and the output of total solvent and butanols is chosen total solvent and the highest bacterial strain of butanols output, as mutagenic strain in the detection tunning.
Wherein, shake flask fermentation screening culture medium: Semen Maydis powder 5% (solid content 40～50%), mixing boils back gelatinization 1h, supplies volatilization moisture, the pH nature.
The method that acetone-butanol fusobacterium original strain carries out the first step ion beam mutagenesis is as follows:
With the acetone-butanol fusobacterium original strain activation culture from the row filter acquisition with preservation, 30～40 ℃ of culture temperature, 250mL shakes bottled liquid measure 100～150mL, paraffin fluid-tight 2～5cm, and 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 two
The explanation of this example is the method for the good acetone-butanol fusobacterium of screening further.
Wherein, employed culture medium prescription (% is a mass percent):
(1) solid plate substratum: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
(2) wood sugar plate culture medium: corn steep liquor 0.1% (solid content is 40～50%), wood sugar 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfates 0.01%, agar 1.5%, pH5～7.
(3) 2-deoxy-D-glucose plate culture medium: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, iron vitriol 0.01%, agar 1.5%, 2-deoxy-D-glucose 0.1%, pH5～7.
(4) tetrabromo-mcresolsulfonphthalein plate culture medium: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, iron vitriol 0.01%, agar 1.5%, tetrabromo-mcresolsulfonphthalein 0.002%, pH5～7.
(5) resazurin plate culture medium: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, iron vitriol 0.01%, agar 1.5%, resazurin 0.002%, pH5～7.
(6) seed culture medium: yeast powder 1%, peptone 1%, Zulkovsky starch 4%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
(7) shake flask fermentation screening culture medium I: Semen Maydis powder 5% (solid content 40～50%), mixing boils back gelatinization 1h, supplies volatilization moisture, the pH nature.
(8) shake flask fermentation screening culture medium II: corn steep liquor 0.8% (solid content 40～50%), wood sugar 5%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfates 0.01%, agar 1.5%, pH5～7.
The screening step:
1, wood sugar plate screening
Wash out bacterial strain through ion beam mutagenesis with physiological saline, be diluted to different concns and coat on the wood sugar flat board, anaerobism is cultivated 48h and pick out the bacterial strain that 15 strains can be grown on the wood sugar flat board under 37 ℃ of temperature.
2,2-deoxy-D-glucose plate screening
The 15 plant mutant strain points that the wood sugar flat screen is selected are planted in the 2-deoxy-D-glucose flat board, put into anaerobism cultivation 24h under 37 ℃ of temperature, wherein 4 strain growing states significantly are better than the bacterium that sets out.
3, tetrabromo-mcresolsulfonphthalein flat board and resazurin plate screening
Tetrabromo-mcresolsulfonphthalein plate screening: the mutant strain point with takadiastase vigor that 2-deoxidation-D glucose flat screen is selected is planted on the solid medium flat board, 37 ℃ of anaerobism are cultivated 12h, stroke-physiological saline solution is made bacteria suspension, join in the hole of the conventional solid medium flat board that contains 0.01% tetrabromo-mcresolsulfonphthalein, anaerobism is cultivated 24h under 37 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
The resazurin plate screening: the inoculation that above-mentioned screening is obtained is on the solid medium flat board, 37 ℃ of anaerobism are cultivated 12h, stroke-physiological saline solution is made and the identical bacteria suspension of the rapid concentration of previous step, join in the hole that contains the flat conventional solid medium flat board of 0.01% resazurin, anaerobism is cultivated 12h under 37 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
Final strain X Y16 and XY10 have shown stronger wood sugar and starch utilising efficiency, acid producing ability and reduction vigor.
4, fermentation shake flask screening
Strain X Y16, XY10 and original strain are inserted the seed culture medium enlarged culturing, 37 ℃ of culture temperature, 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h.In fermention medium, ferment then, inoculum size 10% (v/v), 37 ℃ of leavening temperatures, 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, the total solvent output and the butanols output that detect each bacterial strain behind the fermentation time 48h are as shown in table 1:
Table 1
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 XY16 has the highest solvent and butanols output, and butanols is than also the highest.This is consistent with the assembled flat results of screening.
Embodiment three
The mitotic stability of present embodiment explanation mutant strain XY16 and XY10.
Strain X Y16 and the XY10 fermentation test result that goes down to posterity is as shown in table 2:
Table 2
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 four
The technology of present embodiment explanation acetone-butanol fusobacterium Clostridium acetobutylicum XY16 fermentative production butanols.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Seed culture medium: Semen Maydis powder 4%, boil back gelatinization 1.5h, supply evaporable moisture, the pH nature.
Fermention medium: Semen Maydis powder 6%, boil back gelatinization 1.5h, supply evaporable moisture, the pH nature.
Acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 37 ℃ of culture temperature, incubation time 12h.The XY16 that flat board is cultivated is inoculated in the seed culture medium, 37 ℃ of culture temperature, and 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h; With seed culture fluid heat shock 1min, be inoculated in the fermention medium, inoculum size 10% (v/v), 37 ℃ of leavening temperatures, 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, fermentation culture 12h, the acetate of interpolation 0.2% and 0.3% butyric acid detect total solvent output behind the fermentation culture 72h and butanols output has reached 18.6g/L and 13.7g/L respectively, improved 53.7% and 85.1% than starting strain, the butanols ratio has reached 73.6%.
Embodiment five
The technology of present embodiment explanation acetone-butanol fusobacterium Clostridium acetobutyicum XY16 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%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH 6.
Seed culture medium: yeast powder 1%, peptone 1%, Zulkovsky starch 4%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Fermention medium: corn steep liquor 1.6%, wood sugar 8%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 37 ℃ of culture temperature, incubation time 12h.The XY16 that flat board is cultivated is inoculated in the seed culture medium, 37 ℃ of culture temperature, and 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h.With seed culture fluid heat shock 1min, be inoculated in the fermention medium, inoculum size 10% (v/v), 37 ℃ of leavening temperatures, 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, fermentation culture 12h, the acetate of interpolation 0.2% and 0.3% butyric acid detect total solvent output behind the fermentation culture 72h and butanols output has reached 16.6g/L and 11.7g/L respectively, and the butanols ratio has reached 70.5%.
Embodiment six
The technology of present embodiment explanation acetone-butanol fusobacterium Clostridium acetobutylicum XY16 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%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Seed culture medium: yeast powder 1%, peptone 1%, Zulkovsky starch 4%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Fermention medium: wood sugar 5%, Semen Maydis powder 3.5% boils back gelatinization 1.5h, supplies evaporable moisture, the pH nature.
Acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 37 ℃ of culture temperature, incubation time 12h.The XY16 that flat board is cultivated is inoculated in the seed culture medium, 37 ℃ of culture temperature, and 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h.With seed culture fluid heat shock 1min, be inoculated in the fermention medium, inoculum size 10% (v/v), 37 ℃ of leavening temperatures, 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, fermentation culture 12h, the acetate of interpolation 0.2% and 0.3% butyric acid detect total solvent output behind the fermentation culture 72h and butanols output has reached 17.2g/L and 12.3g/L respectively, and the butanols ratio has reached 71.5%.
Embodiment seven
Present embodiment explanation acetone-butanol fusobacterium Clostridium acetobutylicum XY16 produces butanols in the 5L fermentation cylinder for fermentation technology.
The described culture medium prescription of present embodiment (% is a mass percent):
Plate culture medium: yeast powder 0.2%, peptone 0.3%, Zulkovsky starch 1%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH 6.
Seed culture medium: Semen Maydis powder 4%, boil back gelatinization 1.5h, supply evaporable moisture, the pH nature.
Fermention medium: Semen Maydis powder 6%, boil back gelatinization 1.5h, supply evaporable moisture, the pH nature.
Acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 37 ℃ of culture temperature, incubation time 12h.The XY16 that flat board is cultivated is inoculated in the seed culture medium, 37 ℃ of culture temperature, and 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h; With seed culture fluid heat shock 1min, be inoculated in the 5L fermentor tank that the 3L fermention medium is housed, inoculum size 10% (v/v), 37 ℃ of leavening temperatures feed nitrogen continuously in the fermenting process, flow velocity is 0.8L/min, fermentation culture 12h, the acetate of interpolation 0.2% and 0.3% butyric acid detect total solvent output behind the fermentation culture 72h and butanols output has reached 20.2g/L and 15.9g/L respectively, improved 66.9% and 114.9% than starting strain, the butanols ratio has reached 78.7%.
Embodiment eight
Present embodiment explanation acetone-butanol fusobacterium Clostridium acetobutylicum XY16 produces butanols in the 5L fermentation cylinder for fermentation technology.
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%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Seed culture medium: yeast powder 1%, peptone 1%, Zulkovsky starch 4%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Fermention medium: corn steep liquor 1.6%, wood sugar 8%, ammonium acetate 0.2%, sodium-chlor 0.2%, sal epsom 0.3%, potassium primary phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, 7 aqueous ferrous sulfate 0.01%, pH6.
Acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 37 ℃ of culture temperature, incubation time 12h.The XY16 that flat board is cultivated is inoculated in the seed culture medium, 37 ℃ of culture temperature, and 250mL shakes bottled liquid measure 100mL, paraffin fluid-tight 2～5cm, incubation time 12h.With seed culture fluid heat shock 1min, be inoculated in the 5L fermentor tank that the 3L fermention medium is housed, inoculum size 10% (v/v), 37 ℃ of leavening temperatures feed nitrogen continuously in the fermenting process, flow velocity is 0.8L/min, fermentation culture 12h, the acetate of interpolation 0.2% and 0.3% butyric acid detect total solvent output behind the fermentation culture 72h and butanols output has reached 17.8g/L and 12.9g/L respectively, and the butanols ratio has reached 72.5%.
Claims (7)
1. a clostridium acetobutylicum strain, its classification called after Clostridium acetobutylicum XY16, its preserving number registration number is: CCTCC NO:M 2010011.
2. the screening method of acetone-butanol fusobacterium bacterial strain according to claim 1, it is characterized in that behind the acetone-butanol fusobacterium starting strain ion beam mutagenesis, utilize wood sugar flat board and 2-deoxy-D-glucose plate screening to obtain efficiently to utilize the acetone-butanol bacterial strain of wood sugar and starch, utilize tetrabromo-mcresolsulfonphthalein flat board, resazurin plate screening to obtain acid producing ability and the strong bacterial strain of reducing power again, after the shake flask fermentation screening obtains total solvent output and butanols than high acetone-butanol fusobacterium aimed strain.
3. screening method according to claim 2 is characterized in that, concrete screening step is as follows:
A) ion beam mutagenesis: with acetone-butanol fusobacterium original strain activation culture, 30～40 ℃ of culture temperature, 250mL shakes bottled liquid measure 100～150mL, paraffin fluid-tight 2～5cm, incubation time 12～18h obtains the bacterium liquid of growing vigorous, that thalline is sturdy, with 5～10 times of cultured cells dilutions, coat in the empty culture dish of sterilization, dry up with sterile air; With 5～15KeV as ion implantation energy, with 1.0～2.0 * 10
16Ions/cm
2As mutagenesis dosage bacterial strain is carried out ion beam mutagenesis;
B) wood sugar plate screening: bacterial strain is after ion mutagenesis, wash out with physiological saline, be diluted to different concns and coat with on 0.5～2% the flat board of wood sugar as sole carbon source, anaerobism is cultivated 24～72h under 30～40 ℃ of temperature, picks out the bacterium colony that can grow on this flat board;
C) 2-deoxy-D-glucose plate screening: the mutant strain point that step b) is filtered out is planted in the dull and stereotyped enterprising row filter of conventional solid medium that contains 0.05～0.2% 2-deoxy-D-glucose, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, selects the bacterium colony that growing state significantly is better than the bacterium that sets out;
D) tetrabromo-mcresolsulfonphthalein plate screening: with the inoculation of step c) screening on conventional solid medium flat board, 30～40 ℃ of anaerobism are cultivated 12～36h, stroke-physiological saline solution is made the identical bacteria suspension of concentration, join in the hole of the conventional solid medium flat board that contains 0.001%～0.01% tetrabromo-mcresolsulfonphthalein, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
E) resazurin plate screening: with the inoculation of step d) screening on conventional solid medium flat board, 30～40 ℃ of anaerobism are cultivated 12～36h, stroke-physiological saline solution is made the identical bacteria suspension of concentration, join in the hole that contains the flat conventional solid medium flat board of 0.001%～0.01% resazurin, anaerobism is cultivated 12～36h under 30～40 ℃ of temperature, picks out the variable color circle obviously greater than the bacterium colony of the bacterium that sets out;
F) shake flask fermentation screening: the bacterium colony that step e) is sifted out inserts the seed culture medium enlarged culturing, 30～40 ℃ of culture temperature, anaerobism is cultivated incubation time 12～36h, in fermention medium, ferment then, inoculum size 5%～15% (v/v), 30～40 ℃ of leavening temperatures, anaerobically fermenting fermentation time 30～60h; Investigate step d) and e) the bacterium colony fermentation that filters out produces the amount and the ratio of the butanols in the solvent of total solvent, selects total solvent output and butanols simultaneously than the highest bacterium colony.
4. screening method according to claim 3 is characterized in that described step a) intermediate ion mutagenesis employing 10KeV is as ion implantation energy, 1.6 * 10
16Ions/cm
2As mutagenesis dosage.
5. the application of acetone-butanol fusobacterium bacterial strain according to claim 1 in the fermentative production butanols.
6. application according to claim 5 is characterized in that described concrete applying step is as follows:
1) the dull and stereotyped cultivation: acetone-butanol fusobacterium Clostridium acetobutylicum XY16 is seeded to the plate culture medium anaerobism cultivates 30～40 ℃ of culture temperature, incubation time 12～36h;
2) seed culture: the acetone-butanol fusobacterium Clostridium acetobutylicum XY16 that flat board is cultivated is inoculated in the seed culture medium, 30～40 ℃ of culture temperature, 250mL shakes bottled liquid measure 100～150mL, paraffin fluid-tight 1～3cm, incubation time 12～36h;
3) butanols is produced in fermentation: with seed culture fluid heat shock 1～5min, be inoculated in the fermention medium, and inoculum size 5%～15% (v/v), 30～40 ℃ of leavening temperatures, fermented incubation time are 36～80h.
7. the application of the described acetone-butanol bacterial strain of claim 1 in producing butanols, it is characterized in that: utilize acetone-butanol bacterial strain XY16 fermentation, total solvent output reaches 16～23g/L, and the butanols ratio has reached 64～75%.
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| CN102226163A (en) * | 2011-06-16 | 2011-10-26 | 南京工业大学 | Clostridium acetobutylicum strain and application thereof |
| CN102417888A (en) * | 2011-10-17 | 2012-04-18 | 广西科学院 | Clostridium acetobutylicum for producing butanol by utilizing manihot as raw materials and application thereof |
| WO2012159571A1 (en) * | 2011-05-25 | 2012-11-29 | 中国科学院上海生命科学研究院 | Method for improving sugar utilization rate of clostridium acetobutylicum in mixed sugar fermentation |
| CN103980092A (en) * | 2014-05-21 | 2014-08-13 | 南京工业大学 | Method for extracting butanol from fermentation broth |
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| CN101250496A (en) * | 2008-03-25 | 2008-08-27 | 中国科学院微生物研究所 | Acetone-butanol clostridium strain and uses thereof |
| CN101353632A (en) * | 2008-01-11 | 2009-01-28 | 上海凯赛生物技术研发中心有限公司 | A strain of Clostridium acetobutylicum, screening method and use thereof |
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| CN101353632A (en) * | 2008-01-11 | 2009-01-28 | 上海凯赛生物技术研发中心有限公司 | A strain of Clostridium acetobutylicum, screening method and use thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012159571A1 (en) * | 2011-05-25 | 2012-11-29 | 中国科学院上海生命科学研究院 | Method for improving sugar utilization rate of clostridium acetobutylicum in mixed sugar fermentation |
| CN102226163A (en) * | 2011-06-16 | 2011-10-26 | 南京工业大学 | Clostridium acetobutylicum strain and application thereof |
| CN102226163B (en) * | 2011-06-16 | 2012-08-29 | 南京工业大学 | Clostridium acetobutylicum strain and application thereof |
| CN102417888A (en) * | 2011-10-17 | 2012-04-18 | 广西科学院 | Clostridium acetobutylicum for producing butanol by utilizing manihot as raw materials and application thereof |
| CN102417888B (en) * | 2011-10-17 | 2013-06-19 | 广西科学院 | Clostridium acetobutylicum for producing butanol by utilizing manihot as raw materials and application thereof |
| CN103980092A (en) * | 2014-05-21 | 2014-08-13 | 南京工业大学 | Method for extracting butanol from fermentation broth |
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