CN102174596B - Method for producing butanol by staged sugar supply anaerobic fermentation - Google Patents
Method for producing butanol by staged sugar supply anaerobic fermentation Download PDFInfo
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- CN102174596B CN102174596B CN201110069065.8A CN201110069065A CN102174596B CN 102174596 B CN102174596 B CN 102174596B CN 201110069065 A CN201110069065 A CN 201110069065A CN 102174596 B CN102174596 B CN 102174596B
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- clostridium acetobutylicum
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- 238000000855 fermentation Methods 0.000 title claims abstract description 60
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 235000000346 sugar Nutrition 0.000 title abstract description 16
- 238000004519 manufacturing process Methods 0.000 title abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 56
- 238000011218 seed culture Methods 0.000 claims abstract description 20
- 241000193401 Clostridium acetobutylicum Species 0.000 claims abstract description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 229960003487 xylose Drugs 0.000 claims description 12
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 239000002904 solvent Substances 0.000 abstract description 32
- 238000001994 activation Methods 0.000 abstract description 11
- 230000004913 activation Effects 0.000 abstract description 11
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 abstract description 2
- 238000010364 biochemical engineering Methods 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 19
- 239000001963 growth medium Substances 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 10
- 238000011081 inoculation Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000002028 Biomass Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- 239000008107 starch Substances 0.000 description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 238000005882 aldol condensation reaction Methods 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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Abstract
The invention relates to a method for producing butanol by staged sugar supply anaerobic fermentation, and belongs to the technical field of biochemical engineering. The invention adopts the technical scheme that the method comprises three steps of strain activation, seed culture and anaerobic fermentation for producing the butanol; according to the characteristic that the clostridium acetobutylicum shows different fermentation properties in different carbon-nitrogen ratios, the sugar supply strategy of fermentation is regulated under the same total sugar (concentration); when 10 to 20g/L of sugar is supplied when the fermentation starts, the rest sugar is complemented at one time after 8 to 12 hours; and compared with the sugar supply when the fermentation starts, the yield of the butanol is improved by 18.4 percent, the yield of the total solvent is improved by 19.3 percent, the production rate of the butanol is improved by 39.5 percent, and the production rate of the total solvent can reach 0.424gABE/g sugar and is improved by 35.9 percent. By the method, the yield and the production rate of the butanol are improved, the operation is simple and convenient, the carbon source is utilized more effectively, and the industrial yield of the butanol can be remarkably improved.
Description
Technical field
The present invention relates to a kind of method that sugared anaerobically fermenting is produced butanols of mending stage by stage, belong to technical field of biochemical industry.
Background technology
Butanols is important fine chemical material, also is novel renewable energy source, and very widely purposes is arranged.In world's energy structure in future, the recyclable organism energy will account for important proportion.In recent years, the scientific research personnel improves the butylic fermentation technology at improvement butylic fermentation bacterial classification, improve butanols output and productive rate, reduce the aspect such as butanols cost obtain remarkable progress [Zheng Haizhou etc. the progress of butanols biological fermentation. the Hebei chemical industry. 2008,31 (12), 36～37].Biological butanol has the advantages such as high-energy, miscibility, low volatility, pollution are few, can replace ethanol as a kind of reproducible fuel dope [Richard VN. Biobutanol enters battle of the alcohols[J] .Chemistry World, 2008,5 (2): 21～21].
The method of industrial production butanols has 3 kinds: oxo synthesis, aldol condensation method and fermentation method.Wherein oxo synthesis and aldol condensation method all belong to chemical synthesis, and the two is all take oil as raw material, and investment is large, and technical equipment requires high.And the butanols biological fermentation generally utilizes clostridium acetobutylicum to carry out under the strictly anaerobic condition, its primary product is butanols, acetone and ethanol, content is about 6:3:1, abbreviation AB or ABE fermentation [old Tao-sound. fermentation method acetone and production of butanol technology. Beijing: Chemical Industry Press, 1991].
Yet the productive rate that butanols transforms carbon source in the real attenuation process is than low many of ethanol, therefore limited the development [He Jingchang of butanols fuel, Zhang Zhengbo, Qiu Juanping. the progress of key enzyme and gene thereof in the biological butanol route of synthesis. food and fermentation industries. 2009,35 (2), 116～120].The factor of restriction acetone, butylic fermentation industry development mainly is solvent productive rate, production intensity, production cost, especially solvent productive rate (the total solvent quality that the raw material of unit mass produces) is low, take maize raw material as example, the total solvent productive rate generally only has about 30%, low like this solvent productive rate so that fermentation method when competing with chemical method, be in a disadvantageous position.The substrate of high density has stronger restraining effect to clostridium acetobutylicum, for preventing that substrate to the toxic action of organism, adding the substrate of high density with certain dilution ratio stream, can reduce the restraining effect of substrate.Different carbon-nitrogen ratios are specifically fermented period on butylic fermentation---and there is different impacts the product acid phase with the product alcohol phase, and the output of solvent is improved.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that sugared anaerobically fermenting is produced butanols of mending stage by stage, to improve output and the productive rate of butanols and total solvent, reaches the efficient utilization of carbon source.
In order to address the above problem, technical program of the present invention lies in:
A kind of method of mending stage by stage sugared anaerobically fermenting production butanols, comprise actication of culture, seed culture, anaerobically fermenting three steps, it is characterized in that: in the anaerobically fermenting step, the glucose, wood sugar or its combination that in the fermention medium of original not carbonaceous sources, add 10～20 g/L, the access seed liquor begins anaerobically fermenting, fermentation is carried out after 8～12 hours adding glucose, wood sugar or its combination into 40～50 g/L to fermentation system again, to finishing anaerobically fermenting.
Wherein, fermented bacterium of the present invention be clostridium acetobutylicum (
Clostridium acetobutylicum); Clostridium acetobutylicum of the present invention is
Clostridium acetobutylicumXY16(CCTCC NO:M 2010011), clostridium acetobutylicum
Clostridium acetobutylicumAS1.134(CGMCC 1.134), perhaps clostridium acetobutylicum
Clostridium acetobutylicumAS1.135(CGMCC 1.135).
Actication of culture step of the present invention is the actication of culture method that conventional clostridium acetobutylicum adopts.In the present invention, bacterial classification in 6.0～7.0 the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided that contains starch, passes into N by the pH that accesses 50 mL serum bottles in the frozen glycerine pipe
2Or CO
2, in constant incubator, to cultivate, temperature is 30～40 ℃, activation culture 12～24 h are used for seed culture medium inoculation and bacterial classification and preserve.
Seed culture step of the present invention is the seed culture method that conventional clostridium acetobutylicum adopts.In the present invention, the substratum of seed culture is the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided that contains carbohydrate of pH 6.0～7.0, with adding seed culture medium 100～400 mL in the 500 mL serum bottles, passes into N during cultivation
2Or CO
2, 115～121 ℃ of sterilization 15～30 min, the seed liquor of access activation after the cooling, culture temperature is 30～40 ℃, cultivates in constant incubator, incubation time is 8～16 h, is used for the fermentor tank inoculation.
Anaerobically fermenting of the present invention is produced in the step of butanols, and the substratum of fermentation culture is the conventional liq substratum that carbon source, nitrogenous source and inorganic salt can be provided that contains carbohydrate of pH 6.0～7.0, and the volume of 5 L fermentation cylinder for fermentation substratum is 2～4 L; And the operation of anaerobically fermenting and fermentation condition also are the conventional methods of utilizing clostridium acetobutylicum anaerobic fermentation butanols, and in the present invention, inoculum size is volume ratio 5%～10%, and temperature is 35～40 ℃, and fermentor tank passes into N
2Or CO2, to keep the anaerobic environment of fermentation system, mixing speed is at 100～200 rpm, and fermentation time is 48～72 h.
Beneficial effect of the present invention is:
The present invention mends sugar by the different steps control at microbial fermentation, reaches the efficient utilization of glucose, has improved the productive rate of butanols and total solvent.The present invention adopts in different steps and controls benefit sugar, and pure for product acid and the product of controlled fermentation, the butanols and the total solvent output that improve in batches and continuously ferment efficiently utilize carbon source that important theory significance and using value are arranged.The method equally can be for the butylic fermentation that utilizes inferior biomass to carry out.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, embodiment is described only to be used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1
Bacterial classification: clostridium acetobutylicum
Clostridium acetobutylicumXY16(CCTCC NO:M 2010011)
Seed culture medium: yeast powder 3g/L, peptone 5g/L, Zulkovsky starch 10g/L, ammonium acetate 2g/L, NaCl 2g/L, MgSO
43g/L, KH
2PO
41g/L, K
2HPO
41g/L, FeSO
47H
2O 0.1g/L, pH 6.0;
Fermention medium (P2): carbon source (glucose 60 g/L divide disappear), K
2HPO
40.5g/L, KH
2PO
40.5g/L, CH
3COONH
42.2g/L, MgSO
47H
2O 0.2g/L, MnSO
4H
2O 0.01g/L, NaCl 0.01g/L, FeSO
47H
2O 0.01g/L; Corn steep liquor 1g/L.
The substratum liquid amount is 3L during the 5L fermentor cultivation.Bacterial classification in the frozen glycerine pipe is accessed in the seed culture medium of 30 mL in the 50 mL serum bottles, pass into N
2, in constant incubator, to cultivate, temperature is 37 ℃, activation culture.After cultivating 12h, contain in the 500mL serum bottle of 300 mL seed culture mediums in the access of 5% ratio with the bacterial classification of aseptic liquid-transfering gun with activation, enlarged culturing, be used for above-mentioned fermention medium inoculation (5L fermentor tank) behind the 12h, inoculum size is 10%(v/v), mixing speed is at 120rpm, 37 ℃ of fermentation culture.Pass into N after the inoculation
2, air flow is 0.25 vvm, stops ventilation behind the 10min, closes ventage, guarantees the anaerobic environment of fermentation.Mend sugared mode 1: total carbon source demand of fermention medium is for dividing the glucose that disappears, and its total concn is 60 g/L; Begin to access the seed liquor fermentation after in the fermention medium of original not carbonaceous sources, adding first the glucose of 10 g/L, fermentation is carried out behind 12 h glucose of 50 g/L of remainder is filled in the fermentor tank, and with the contrast of the fermentation results of control group, fermenting to total solvent output no longer changes.Result such as table 1.
Table 1 is mended the fermentation results contrast of sugared mode 1 and control group
| ? | Maximum biomass | Butanols (g/L) | Total solvent (g/L) | The butanols productive rate | The total solvent productive rate |
| Control group | 8.4 | 11.4 | 18.1 | 0.190 | 0.312 |
| Mend sugared mode 1 | 5.1 | 13.5 | 21.6 | 0.265 | 0.424 |
Can be found out by the result in the table 1, when adopting this kind benefit sugar mode 1, the effect of producing butanols is better than the effect of disposable benefit sugar, maximum biomass has reduced by 39.3%, the output of butanols and total solvent has improved respectively 18.4%, 19.3%, and the productive rate of butanols and total solvent has improved respectively 39.5%, 35.9%.Mend the productive rate that sugared mode 1 has improved butanols and total solvent significantly.
Embodiment 2
Bacterial classification: clostridium acetobutylicum
Clostridium acetobutylicumXY16(CCTCC NO:M 2010011)
Seed culture medium: yeast powder 3g/L, peptone 5g/L, Zulkovsky starch 10g/L, ammonium acetate 2g/L, NaCl 2g/L, MgSO
43g/L, KH
2PO
41g/L, K
2HPO
41g/L, FeSO
47H
2O 0.1g/L, pH 6.0;
Fermention medium (P2): carbon source (wood sugar 60g/L divide disappear), K
2HPO
40.5g/L, KH
2PO
40.5g/L, CH
3COONH
42.2g/L, MgSO
47H
2O 0.2g/L, MnSO
4H
2O 0.01g/L, NaCl 0.01g/L, FeSO
47H
2O 0.01g/L; Corn steep liquor 1g/L.
The substratum liquid amount is 3L during the 5L fermentor cultivation.In the seed culture medium with 30mL in the access of the bacterial classification in the frozen glycerine pipe 50mL serum bottle, pass into N
2, in constant incubator, to cultivate, temperature is 37 ℃, activation culture.After cultivating 12h, contain in the 500mL serum bottle of 300mL seed culture medium in the access of 5% ratio with the bacterial classification of aseptic liquid-transfering gun with activation, enlarged culturing, be used for above-mentioned fermention medium inoculation (5L fermentor tank) behind the 12h, inoculum size is 10%(v/v), mixing speed is at 120rpm, 37 ℃ of fermentation culture.Pass into N after the inoculation
2, air flow is 0.25vvm, stops ventilation behind the 10min, closes ventage, guarantees the anaerobic environment of fermentation.Mend sugared mode 2: total carbon source demand of fermention medium is for dividing the wood sugar that disappears, and its total concn is 60 g/L; Begin to access the seed liquor fermentation after in the fermention medium of original not carbonaceous sources, adding first the wood sugar of 20 g/L, fermentation is carried out behind 12 h wood sugar of 40 g/L of remainder is filled in the fermentor tank, and with the contrast of the fermentation results of control group, fermenting to total solvent output no longer changes.Result such as table 2.
Table 2 is mended the fermentation results contrast of sugared mode 2 and control group
| ? | Maximum biomass | Butanols (g/L) | Total solvent (g/L) | The butanols productive rate | The total solvent productive rate |
| Control group | 6.1 | 9.2 | 14.9 | 0.157 | 0.271 |
| Mend sugared mode 2 | 3.2 | 10.5 | 16.9 | 0.191 | 0.307 |
Result from table 2 finds out, adopt mend sugared mode 2 after, maximum biomass has reduced by 47.5%, the output of butanols and total solvent has improved respectively 12.4% and 13.4%, the productive rate of butanols and total solvent has improved respectively 21.7% and 13.3%.
Embodiment 3
Bacterial classification: clostridium acetobutylicum
Clostridium acetobutylicumAS1.135(CGMCC 1.135)
Seed culture medium: yeast powder 3g/L, peptone 5g/L, Zulkovsky starch 10g/L, ammonium acetate 2g/L, NaCl 2g/L, MgSO
43g/L, KH
2PO
41g/L, K
2HPO
41g/L, FeSO
47H
2O 0.1g/L, pH 6.0;
Fermention medium (P2): carbon source (glucose and xylose of mass ratio 2:1,60g/L divide disappear), K
2HPO
40.5g/L, KH
2PO
40.5g/L, CH
3COONH
42.2g/L, MgSO
47H
2O 0.2g/L, MnSO
4H
2O 0.01g/L, NaCl 0.01g/L, FeSO
47H
2O 0.01g/L; Corn steep liquor 1g/L.
The substratum liquid amount is 3L during the 5L fermentor cultivation.In the seed culture medium with 30mL in the access of the bacterial classification in the frozen glycerine pipe 50mL serum bottle, pass into N
2, in constant incubator, to cultivate, temperature is 37 ℃, activation culture.After cultivating 12h, contain in the 500mL serum bottle of 300mL seed culture medium in the access of 5% ratio with the bacterial classification of aseptic liquid-transfering gun with activation, enlarged culturing, be used for above-mentioned fermention medium inoculation (5L fermentor tank) behind the 12h, inoculum size is 10%(v/v), mixing speed is at 120rpm, 37 ℃ of fermentation culture.Pass into N after the inoculation
2, air flow is 0.25vvm, stops ventilation behind the 10min, closes ventage, guarantees the anaerobic environment of fermentation.Mend sugared mode 3: total carbon source demand of fermention medium is to divide the mixture of the glucose and xylose of the mass ratio 2:1 that disappears, and its total concn is 60 g/L; Begin to access the seed liquor fermentation after in the fermention medium of original not carbonaceous sources, adding first the mixing sugar of 15 g/L, fermentation is carried out behind 12 h mixing sugar of 45 g/L of remainder is filled in the fermentor tank, and with the contrast of the fermentation results of control group, fermenting to total solvent output no longer changes.Result such as table 3.
Table 3 is mended the fermentation results contrast of sugared mode 3 and control group
| ? | Maximum biomass | Butanols (g/L) | Total solvent (g/L) | The butanols productive rate | The total solvent productive rate |
| Control group | 7.2 | 10.7 | 16.5 | 0.188 | 0.289 |
| Mend sugared mode 3 | 4.3 | 12.5 | 18.5 | 0.220 | 0.324 |
From the results shown in Table 3, adopt maximum biomass to reduce by 40.3%, the output of butanols and total solvent has improved respectively 16.8% and 12.1%, and the productive rate of butanols and total solvent has improved respectively 17.0% and 12.1%.
Embodiment 4
Bacterial classification: clostridium acetobutylicum
Clostridium acetobutylicumAS1.134(CGMCC 1.134)
Seed culture medium: yeast powder 3g/L, peptone 5g/L, Zulkovsky starch 10g/L, ammonium acetate 2g/L, NaCl 2g/L, MgSO
43g/L, KH
2PO
41g/L, K
2HPO
41g/L, FeSO
47H
2O 0.1g/L, pH 6.0;
Fermention medium (P2): carbon source (glucose 60g/L divide disappear), K
2HPO
40.5g/L, KH
2PO
40.5g/L, CH
3COONH
42.2g/L, MgSO
47H
2O 0.2g/L, MnSO
4H
2O 0.01g/L, NaCl 0.01g/L, FeSO
47H
2O 0.01g/L; Corn steep liquor 1g/L.
The substratum liquid amount is 3L during the 5L fermentor cultivation.In the seed culture medium with 30mL in the access of the bacterial classification in the frozen glycerine pipe 50mL serum bottle, pass into N
2, in constant incubator, to cultivate, temperature is 37 ℃, activation culture.After cultivating 12h, contain in the 500mL serum bottle of 300mL seed culture medium in the access of 5% ratio with the bacterial classification of aseptic liquid-transfering gun with activation, enlarged culturing, be used for above-mentioned fermention medium inoculation (5L fermentor tank) behind the 12h, inoculum size is 10%(v/v), mixing speed is at 120rpm, 37 ℃ of fermentation culture.Pass into N after the inoculation
2, air flow is 0.25vvm, stops ventilation behind the 10min, closes ventage, guarantees the anaerobic environment of fermentation.Mend sugared mode 4: total carbon source demand of fermention medium is for dividing the glucose that disappears, and its total concn is 60 g/L; Begin to access the seed liquor fermentation after in the fermention medium of original not carbonaceous sources, adding first the glucose of 10 g/L, fermentation is carried out behind 8 h glucose of 50 g/L of remainder is filled in the fermentor tank, and with the contrast of the fermentation results of control group, fermenting to total solvent output no longer changes.Result such as table 4.
Table 4 is mended the fermentation results contrast of sugared mode 4 and control group
| ? | Maximum biomass | Butanols (g/L) | Total solvent (g/L) | The butanols productive rate | The total solvent productive rate |
| Control group | 7.9 | 11.2 | 17.9 | 0.188 | 0.305 |
| Mend sugared mode 1 | 4.8 | 13.2 | 21.5 | 0.240 | 0.415 |
Can be found out by the experimental result in the table 4, when adopting this kind benefit sugar mode 1, the effect of producing butanols is better than the effect of disposable benefit sugar, maximum biomass has reduced by 39.2%, the output of butanols and total solvent has improved respectively 17.9%, 20.1%, and the productive rate of butanols and total solvent has improved respectively 27.6%, 36.1%.
Claims (1)
1. mend stage by stage the method that sugared anaerobically fermenting is produced butanols for one kind, comprise actication of culture, seed culture, anaerobically fermenting three steps, it is characterized in that: in the anaerobically fermenting step, the glucose, wood sugar or its combination that in the fermention medium of original not carbonaceous sources, add 10～20 g/L, the access seed liquor begins anaerobically fermenting, fermentation is carried out after 8～12 hours adding glucose, wood sugar or its combination into 40～50 g/L to fermentation system again, to finishing anaerobically fermenting;
Wherein, bacterial classification is clostridium acetobutylicum
Clostridium acetobutylicumXY16, clostridium acetobutylicum
Clostridium acetobutylicumAS1.134, perhaps clostridium acetobutylicum
Clostridium acetobutylicumAS1.135;
Described fermention medium is: carbon source is: the glucose of 10～20 g/L, wood sugar or its combination, fermentation are carried out after 8～12 hours adding glucose, wood sugar or its combination into 40～50 g/L to fermentation system again; Other prescriptions: K
2HPO
40.5g/L, KH
2PO
40.5g/L, CH
3COONH
42.2g/L, MgSO
47H
2O 0.2g/L, MnSO
4H
2O 0.01g/L, NaCl 0.01g/L, FeSO
47H
2O 0.01g/L; Corn steep liquor 1g/L; Dividing the carbon source total concn that disappears is 60 g/L;
Fermentation condition is: inoculum size is volume ratio 5%～10%, and temperature is 35～40 ℃, and fermentor tank passes into N
2Or CO2, to keep the anaerobic environment of fermentation system, mixing speed is at 100～200 rpm, and fermentation time is 48～72 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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