CN103667147A - High ferulic acid stress resistance clostridium beijerinckii and application thereof - Google Patents
High ferulic acid stress resistance clostridium beijerinckii and application thereof Download PDFInfo
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Abstract
The invention discloses high ferulic acid stress resistance clostridium beijerinckii and an application thereof. The high ferulic acid stress resistance clostridium beijerinckii is classified and named Clostridium beijerinckii M11 with the collection number of CCTCC No:M2013423, and the collection date of the high ferulic acid stress resistance clostridium beijerinckii is December 14, 2013. A mutant strain obtained by adopting plasmas for mutating the clostridium beijerinckii and a ferulic acid containing resazurin flat plate and a shake flask for fermentation and screening can normally grow in a fermentation medium containing 0.8g/L of ferulic acid, the yield of butanol reaches 6.1g/L, and an original strain cultured under equal conditions hardly grows. Moreover, by using virus-carrying corncob acidolysis sugar liquid as a carbon source, the yield of butanol in a 2L fermentation tank reaches 6.4g/L, and the original strain cultured under equal conditions hardly grows. The high ferulic acid stress resistance clostridium beijerinckii has strong stress resistance, high butanol yield and good repeatability, and is an excellent strain suitable for the fermentation production of butanol by using wood fiber raw materials such as corncobs and the like.
Description
Technical field
The present invention relates to a strain and forulic acid there is to the Bai Shi clostridium of high resistance to cold and diseases, with and application in acetone butanol fermentation industry, belong to technical field of biological fermentation.
Background technology
Butanols, as novel reproducible liquid energy, is paid attention to by the increasing country in the world; Butanols have energy density large, can be with gasoline arbitrarily than mixing, can being directly used in the advantages such as oil engine, convenient transportation.At present; the production method of butanols is mainly synthesized by chemical method; along with energy dilemma is increasingly serious; petroleum resources accelerate exhaustion and price skyrockets; biological process is produced butanols; as a kind of effectively, utilize biomass resource to produce the conversion technology of Important Platform compound and fuel, start again to be again taken seriously.Since two thousand eight, China has set up many biological butanol production lines, and annual capacity reaches 100,000 tons; But due to constantly riseing of starchiness, molasses raw material, make biological butanol manufacturer mostly in end-of-life state.Under the double threat of food shortage and energy dilemma; Exploring reproducible wood fibre raw material and produce the important composition that fuel butanols becomes biomass energy development strategy, is also 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.(the Biomass and Bioenergy.2008 such as Nasib Qureshi, 32:176-183) utilize Bai Shi clostridium P260, in wheat stalk, add cellulase, zytase etc. to carry out simultaneous saccharification and fermentation, through 533 hours continuously ferment, the productive rate of its solvent was 0.41.Annous etc. (Appl.Environ.Microbiol.1991,57:2544-2548) utilize chemomorphosis, have obtained super bacterial strain BA101, more than single batch fermentation total solvent reaches as high as 25g/L; Thaddeus Ezeji etc. (Bioresource Technology.2008,99:5915-5922) utilize mutant strain BA101, and corncob acid hydrolysis and the enzymolysis liquid glucose of XAD-4resin detoxification of take is fermenting substrate, and total solvent output is 9.30g/L; But mutant strain BA101 can not utilize the acidolysis of not detoxification and the fermentation of enzymolysis liquid glucose to produce butanols, and especially forulic acid concentration, when 0.5g/L is above, is not grown substantially.Lignocellulose raw material, after dilute acid pretreatment, can produce the inhibitions such as organic acid, furfural, phenols, these inhibitions to remove cost higher and microorganism growth is had to certain restraining effect; (the Biotechnology & Bioengineering.2007 such as Thaddeus Ezeji, 97 (6): 1460-1469) research finds that the inhibitions such as organic acid, furfural do not affect the fermentation of butanols, and phenols inhibition (especially forulic acid) has obvious retarding effect to butylic fermentation.Chinese patent ZL201110020102.6 report: the Clostridium beijerinckii IB4 obtaining by particle beam selection by mutation has higher resistance to phenolic compound, it can using the corncob acid hydrolysis liquid glucose of not detoxification as carbon source, and in 2L fermentor tank, total solvent output and butanols output have reached respectively 10.3g/L and 7.1g/L; (the J Ind Microbiol Biotechnol. such as Guo Ting, 2012,39 (3), 401 – 407) research is found, the concentration of the phenolic compound in Corncob hydrolysate rises to 1.5g/L when above, and Clostridium beijerinckii IB4 does not grow substantially.
Visible, the toxin inhibition in lignocellulose raw material (especially forulic acid) severe inhibition is produced the leavening property of Clostridium acetobutylicum; And strain improvement is to improve bacterial strain to one of key means of the tolerance of inhibition, fermentation economy.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide a strain forulic acid to be had to the Bai Shi clostridium of high resistance to cold and diseases, makes it under high density forulic acid exists, and still can grow and fermentation.
Two of the technical problem to be solved in the present invention is to provide the application of described high resistance against Bai Shi clostridium.
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 forulic acid resistance Bai Shi clostridium, its Classification And Nomenclature is Bai Shi clostridium (Clostridium beijerinckii) M11, be preserved in Chinese Typical Representative culture collection center, its deposit number is CCTCC NO:M2013423, and preservation date is on September 14th, 2013.
The screening method of Bai Shi clostridium Clostridium beijerinckii M11 of the present invention, Bai Shi clostridium starting strain NCIMB8052(is purchased to American Type Culture Collecti (ATCC)) after plasma body mutagenesis, utilize resazurin plate screening to obtain the bacterial strain that reducing power is strong, then obtain the high Bai Shi clostridium aimed strain of total solvent output through anaerobism bottle fermentation screening.
Its concrete steps are as follows:
A) plasma body mutagenesis: by Bai Shi clostridium original strain activation culture, 33～37 ℃ of culture temperature, the bottled liquid measure of Xiao Te anaerobism of 25mL is 10～15mL, incubation time 12～18h obtains the bacterium liquid in logarithmic phase, and cultured cells is diluted to OD
600=0.1～1.0, drip on the cooled slide glass of sterilizing, with sterile air, dry up; Take helium as discharge gas, using 80～120W as radio frequency power, using 10～30SLM as gas flow, the 10～1800s of usining carries out plasma body mutagenesis as irradiation time to bacterial strain;
B) the dull and stereotyped primary dcreening operation of resazurin: the slide glass after mutagenesis is placed in to the tool plug test tube that 1～2mL physiological saline is housed, concuss, by the bacterial strain wash-out on slide glass, being diluted to different concns coats on the culture medium flat plate containing resazurin (0.002%) and forulic acid (0.5g/L), 33～37 ℃ of anaerobism are cultivated 24～36h, pick out the bacterium colony that variable color circle is obviously greater than the bacterium that sets out;
C) anaerobism bottle fermentation screening: the bacterium colony access seed culture medium enlarged culturing that step b) is sifted out, 33～37 ℃ of culture temperature, anaerobism is cultivated incubation time 10～18h, then in fermention medium, ferment, inoculum size 5%～10%(v/v), 33～37 ℃ of leavening temperatures, anaerobically fermenting fermentation time 60～90h; The output that butanols and total solvent are produced in the bacterium colony fermentation that investigation step b) filters out is selected butanols and the highest bacterial strain of total solvent output simultaneously.
D) in above-mentioned screening method: in the plasma body mutagenesis method described in step a), preferably 100W is as radio frequency power, and 10SLM is as gas flow, and 180s is as irradiation time.
In above-mentioned screening method: the conventional solid medium that step b) adopts, carbon source are glucose or starch; Nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is ammonium acetate, and nitrogen-containing organic compound is one or more in peptone, yeast powder and corn steep liquor; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, ferrous salt, add agar in solid medium.
In above-mentioned screening method: in seed culture medium and fermention medium that step c) adopts, carbon source is in glucose or corncob acid hydrolysis liquid glucose; Nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast powder, extractum carnis and corn steep liquor; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, ferrous salt; Somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and corn steep liquor.
The application of above-mentioned high forulic acid resistance Bai Shi clostridium in fermentative production butanols.
Wherein, the method for described fermentative production butanols comprises following steps:
1) dull and stereotyped cultivation: Bai Shi clostridium Clostridium beijerinckii M11 is seeded to plate culture medium anaerobism and cultivates, 33～37 ℃ of culture temperature, incubation time 12～18h;
2) seed culture: the Bai Shi clostridium Clostridium beijerinckii M11 that flat board is cultivated is inoculated in seed culture medium, the bottled liquid measure 40～60mL of 100mL anaerobism, inflated with nitrogen 3～5min, 33～37 ℃ of culture temperature, incubation time 12～18h;
3) butanols is produced in fermentation: seed culture fluid is inoculated in fermention medium, and inoculum size 5～10 (v/v) %, inflated with nitrogen 3～5min, 33～37 ℃ of leavening temperatures, fermented incubation time is 60～90h.
The component that described plate culture medium comprises following 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; Wherein, described carbon source is starch or glucose; Described nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is ammonium acetate, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and corn steep liquor; Described inorganic salt are one or more the mixing in sodium salt, sylvite and magnesium salts.
The component that described seed culture medium comprises following mass percent: carbon source 0.5%～1%, nitrogenous source 0.2%～1%, inorganic salt 0.5%～0.8%, all the other are water; Wherein, described carbon source is one or both the mixing in grape sugar and starch; Described nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and corn steep liquor; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and calcium salt.
The component that described fermention medium comprises following 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; Wherein, described carbon source is one or more the mixing in glucose, wood sugar, sucrose, pectinose and molasses; Described nitrogenous source is one or more the mixing in ammonium acetate, ammonium chloride and yeast powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and calcium salt; Described somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and corn steep liquor.
Beneficial effect of the present invention is:
Using plasma mutagenesis Bai Shi clostridium of the present invention, utilize containing the resazurin flat screen of forulic acid and select the bacterial strain that reducing power is stronger, this bacterial strain can be containing normal growth in the fermention medium of 0.8g/L forulic acid, butanols output reaches 6.1g/L, and the starting strain that equal conditions is cultivated is not grown substantially.And, using the corncob acid hydrolysis liquid glucose of not detoxification as carbon source, in 2L fermentor tank, butanols output has reached 6.4g/L, and the starting strain that equal conditions is cultivated is not grown substantially.Its strong stress resistance, butanols output are high, reproducible, are to be a kind ofly applicable to utilizing the lignocellulose raw material fermentations such as corn cob to produce the excellent species of butanols.
Accompanying drawing explanation
Microorganism classification called after Bai Shi clostridium Clostridium beijerinckii M11 of the present invention, depositary institution's full name is Chinese Typical Representative culture collection center, be called for short CCTCC, preservation address is China. Wuhan. and Wuhan University, postcode 430072, deposit number is CCTCC NO:M2013423, and preservation date is on September 14th, 2013.
Fig. 1 is the plasma body mutagenesis survival rate curve of Bai Shi clostridium.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, processing condition and result thereof be only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1
The method that the present embodiment explanation is carried out the mutagenesis of the first step plasma body by Bai Shi clostridium original strain.
The method that Bai Shi clostridium original strain carries out the mutagenesis of the first step plasma body is as follows:
By Bai Shi clostridium NCIMB8052 original strain activation culture, 33～37 ℃ of culture temperature, the bottled liquid measure of 50ml Xiao Te anaerobism is 15～20ml, inflated with nitrogen 3min, incubation time 12～18h, obtains eugonic bacterium liquid; Get the cell dilution of fresh culture to cell concn OD
600=1～1.5, drip on the cooled slide glass of sterilizing, with sterile air, dry up; Take helium as discharge gas, using 100W as radio frequency power, using 10SLM as gas flow, the 10～1800s of usining carries out plasma body mutagenesis as irradiation time to bacterial strain, after mutagenesis, the mycoderm on carrier is eluted, and calculates survival rate.Experimental result as shown in Figure 1; As shown in Figure 1,180s is best mutagenesis irradiation time.
Embodiment 2
The method of the good Bai Shi clostridium of this example explanation screening.
Wherein, the culture medium prescription using (% is 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%, pH6.
(2) resazurin 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 1.5%, resazurin 0.002%, forulic acid 0.5%, pH6.
(3) 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%, pH6.
(4) shake flask fermentation screening culture 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%, pH6.6.
Screening step:
1, the dull and stereotyped primary dcreening operation of resazurin
Slide glass after mutagenesis is placed in to the tool plug test tube that 1～2ml physiological saline is housed, concuss, by the bacterial strain wash-out on slide glass, being diluted to different concns coats on the culture medium flat plate containing resazurin (0.002%), 33～37 ℃ of anaerobism are cultivated 12～36h, pick out bacterium colony 50 strains that variable color circle is obviously greater than the bacterium that sets out.
2, shake flask fermentation screening
By bacterial strain M5, M11 and original strain access seed culture medium enlarged culturing, 35 ℃ of culture temperature, the bottled liquid measure 100mL of 250mL Xiao Te anaerobism, inflated with nitrogen 3min, incubation time 12h.Then in fermention medium, ferment, inoculum size 10%(v/v), 35 ℃ of leavening temperatures, the bottled liquid measure 50mL of 100mL Xiao Te anaerobism, the total solvent output and the butanols output that after fermentation time 72h, detect each bacterial strain 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 combined sorting, and wherein the total solvent output of M11 and butanols output are also the highest.This is consistent with the result of plate screening.
Embodiment 3
The mitotic stability of the present embodiment explanation mutant strain M11.
Take in the fermention medium that glucose is carbon source, detecting the mitotic stability of mutant strain M11, the bacterial strain M11 fermentation test result that goes down to posterity is as shown in table 2:
Table 2
From experimental result, through 7 continuous passages, total solvent output and the butanols output of two plant mutant strains are more stable, have good mitotic stability, can be used as the production bacterial strain of further research and development.
Embodiment 4
The high resistance to cold and diseases of the present embodiment explanation Bai Shi clostridium Clostridium beijerinckii M11 to forulic acid.
Culture medium prescription described in the present embodiment (% is 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 1.5%, all the other are water, pH6.
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, pH6.
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%, forulic acid 0.08%, all the other are water, pH6.6.
Bai Shi clostridium Clostridium beijerinckii M11 is seeded to plate culture medium anaerobism and cultivates, 35 ℃ of culture temperature, incubation time 12h.The M11 that flat board is cultivated is inoculated in seed culture medium, 35 ℃ of culture temperature, the bottled liquid measure 30mL of 50mL Xiao Te anaerobism, inflated with nitrogen 3min, 35 ℃ of culture temperature, incubation time 12h; Seed is inoculated in fermention medium, inoculum size 10% (v/v), 35 ℃ of leavening temperatures, the bottled liquid measure 50mL of 100mL Xiao Te anaerobism, inflated with nitrogen 3min, after fermentation culture 72h, detect butanols output and reached 6.1g/L, and the C.beijerinckii IB4(CCTCC NO:M2010310 that equal conditions is cultivated) and starting strain substantially do not grow.
Embodiment 5
The present embodiment explanation Bai Shi clostridium Clostridium beijerinckii M11 utilizes corncob acid hydrolysis liquid glucose, produces the technique of butanols in 2L fermentation cylinder for fermentation.
Culture medium prescription described in the present embodiment (% is 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, pH6.
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, pH6.
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%, with the not corncob acid hydrolysis liquid glucose of detoxification (total reducing sugars is 3%) configuration, all the other are water, pH6.6.
Bai Shi clostridium C.beijerinckii M11 is seeded to plate culture medium anaerobism and cultivates, 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in 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 1L fermention medium is housed to inoculum size 10%(v/v), 35 ℃ of leavening temperatures, pass into continuously nitrogen, flow velocity is 0.3L/min, after fermentation culture 72h, detect butanols output and reached 6.4g/L, and the starting strain that equal conditions is cultivated is not grown substantially.
Embodiment 6
The high resistance to cold and diseases of the present embodiment explanation Bai Shi clostridium Clostridium beijerinckii M11 to corncob acid hydrolysis liquid glucose.
Culture medium prescription described in the present embodiment (% is 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, pH6.
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, pH6.
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%, with the not corncob acid hydrolysis liquid glucose of detoxification (total reducing sugars is 4%) configuration, all the other are water, pH6.6.
Bai Shi clostridium C.beijerinckii M11 is seeded to plate culture medium anaerobism and cultivates, 35 ℃ of culture temperature, incubation time 12h.The IB4 that flat board is cultivated is inoculated in 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 1L fermention medium is housed, inoculum size 10%(v/v), 35 ℃ of leavening temperatures, pass into continuously nitrogen, flow velocity is 0.3L/min, after fermentation culture 72h, detect butanols output and reached 3.2g/L, and the C.beijerinckii IB4(CCTCC NO:M2010310 that equal conditions is cultivated) and starting strain all substantially do not grow.
Claims (6)
1. a plant height forulic acid resistance Bai Shi clostridium, its Classification And Nomenclature is Bai Shi clostridium (Clostridium beijerinckii) M11, be preserved in Chinese Typical Representative culture collection center, its deposit number is CCTCC NO:M2013423, and preservation date is on September 14th, 2013.
2. the application of high forulic acid resistance Bai Shi clostridium claimed in claim 1 in fermentative production butanols.
3. application according to claim 2, is characterized in that, the method for described fermentative production butanols comprises following steps:
1) dull and stereotyped cultivation: Bai Shi clostridium Clostridium beijerinckii M11 is seeded to plate culture medium anaerobism and cultivates, 33～37 ℃ of culture temperature, incubation time 12～18h;
2) seed culture: the Bai Shi clostridium Clostridium beijerinckii M11 that flat board is cultivated is inoculated in seed culture medium, the bottled liquid measure 40～60mL of 100mL anaerobism, inflated with nitrogen 3～5min, 33～37 ℃ of culture temperature, incubation time 12～18h;
3) butanols is produced in fermentation: seed culture fluid is inoculated in fermention medium, and inoculum size 5～10 (v/v) %, inflated with nitrogen 3～5min, 33～37 ℃ of leavening temperatures, fermented incubation time is 60～90h.
4. application according to claim 3, is characterized in that, described plate culture medium comprises the component of following 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; Wherein, described carbon source is starch or glucose; Described nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is ammonium acetate, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and corn steep liquor; Described inorganic salt are one or more the mixing in sodium salt, sylvite and magnesium salts.
5. application according to claim 3, is characterized in that, described seed culture medium comprises the component of following mass percent: carbon source 0.5%～1%, nitrogenous source 0.2%～1%, inorganic salt 0.5%～0.8%, all the other are water; Wherein, described carbon source is one or both the mixing in grape sugar and starch; Described nitrogenous source is organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or both the mixing in ammonium acetate and ammonium chloride, and nitrogen-containing organic compound is one or more the mixing in peptone, yeast powder and corn steep liquor; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and calcium salt.
6. application according to claim 3, is characterized in that, described fermention medium comprises the component of following 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; Wherein, described carbon source is one or more the mixing in glucose, wood sugar, sucrose, pectinose and molasses; Described nitrogenous source is one or more the mixing in ammonium acetate, ammonium chloride and yeast powder; Described inorganic salt are one or more the mixing in sodium salt, sylvite, magnesium salts and calcium salt; Described somatomedin is one or more the mixing in para-amino benzoic acid, VITMAIN B1, vitamin H and corn steep liquor.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898031A (en) * | 2014-04-21 | 2014-07-02 | 南京工业大学 | Clostridium beijerinckii with high electricity yield and application thereof |
| CN103911334A (en) * | 2014-04-16 | 2014-07-09 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174433A (en) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | Clostridium beijerinckii with high stress resistance and application thereof |
| CN102533612A (en) * | 2012-02-16 | 2012-07-04 | 广西科学院 | Clostridium beijerinckii strain and screening method and use thereof |
| CN102703523A (en) * | 2012-07-03 | 2012-10-03 | 广西科学院 | Method for producing butanol by mixed fermentation of bagasse and molasses serving as raw materials |
-
2013
- 2013-12-11 CN CN201310676856.6A patent/CN103667147B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174433A (en) * | 2011-01-18 | 2011-09-07 | 南京工业大学 | Clostridium beijerinckii with high stress resistance and application thereof |
| CN102533612A (en) * | 2012-02-16 | 2012-07-04 | 广西科学院 | Clostridium beijerinckii strain and screening method and use thereof |
| CN102703523A (en) * | 2012-07-03 | 2012-10-03 | 广西科学院 | Method for producing butanol by mixed fermentation of bagasse and molasses serving as raw materials |
Non-Patent Citations (2)
| Title |
|---|
| 庞浩等: "拜氏梭菌13 -2发酵甘蔗渣水解液生产丁醇的研究", 《生物技术》 * |
| 张婉璐等: "诱变选育拜氏梭菌利用碱处理木糖渣水解液生产丁醇", 《可再生能源》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911334A (en) * | 2014-04-16 | 2014-07-09 | 广州甘蔗糖业研究所 | Clostridium beijerinckii with high stress resistance and application thereof |
| CN103911334B (en) * | 2014-04-16 | 2016-04-20 | 广州甘蔗糖业研究所 | A kind of high resistance to cold and diseases Bai Shi clostridium and application thereof |
| CN103898031A (en) * | 2014-04-21 | 2014-07-02 | 南京工业大学 | Clostridium beijerinckii with high electricity yield and application thereof |
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