CN107779478A - It is a kind of to eat cultivating system and cultural method of the gas anaerobic bacteria using synthesis gas fermentation production alcohol - Google Patents
It is a kind of to eat cultivating system and cultural method of the gas anaerobic bacteria using synthesis gas fermentation production alcohol Download PDFInfo
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- CN107779478A CN107779478A CN201610726560.4A CN201610726560A CN107779478A CN 107779478 A CN107779478 A CN 107779478A CN 201610726560 A CN201610726560 A CN 201610726560A CN 107779478 A CN107779478 A CN 107779478A
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- cultivating system
- synthesis gas
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- zinc ion
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- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 57
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 55
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 230000004151 fermentation Effects 0.000 title claims abstract description 42
- 238000000855 fermentation Methods 0.000 title claims abstract description 41
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 25
- 241001148471 unidentified anaerobic bacterium Species 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 14
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims abstract description 42
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims abstract description 41
- 239000001963 growth medium Substances 0.000 claims abstract description 38
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000002253 acid Substances 0.000 claims abstract description 32
- 239000011573 trace mineral Substances 0.000 claims abstract description 23
- 235000013619 trace mineral Nutrition 0.000 claims abstract description 23
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims abstract description 18
- 150000002500 ions Chemical class 0.000 claims abstract description 18
- 229910052750 molybdenum Inorganic materials 0.000 claims abstract description 18
- 239000011733 molybdenum Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 45
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- 241000894006 Bacteria Species 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 239000003638 chemical reducing agent Substances 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 19
- 235000016709 nutrition Nutrition 0.000 claims description 19
- 230000035764 nutrition Effects 0.000 claims description 19
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 15
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 15
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 12
- 239000011734 sodium Substances 0.000 claims description 12
- 229910052708 sodium Inorganic materials 0.000 claims description 12
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 229910002091 carbon monoxide Inorganic materials 0.000 claims description 11
- 238000006392 deoxygenation reaction Methods 0.000 claims description 11
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 9
- 235000019270 ammonium chloride Nutrition 0.000 claims description 9
- 239000001110 calcium chloride Substances 0.000 claims description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 9
- 239000001103 potassium chloride Substances 0.000 claims description 9
- 235000011164 potassium chloride Nutrition 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000011684 sodium molybdate Substances 0.000 claims description 9
- 235000015393 sodium molybdate Nutrition 0.000 claims description 9
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 5
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 5
- 239000011777 magnesium Substances 0.000 claims description 5
- 229910052749 magnesium Inorganic materials 0.000 claims description 5
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 claims description 5
- 239000011701 zinc Substances 0.000 claims description 5
- 229910052725 zinc Inorganic materials 0.000 claims description 5
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 4
- 239000010941 cobalt Substances 0.000 claims description 4
- 229910017052 cobalt Inorganic materials 0.000 claims description 4
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 4
- 229910052711 selenium Inorganic materials 0.000 claims description 4
- 239000011669 selenium Substances 0.000 claims description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 10
- 150000001298 alcohols Chemical class 0.000 abstract description 9
- 241001451494 Clostridium carboxidivorans P7 Species 0.000 abstract description 4
- 239000007789 gas Substances 0.000 description 70
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 46
- 229910052757 nitrogen Inorganic materials 0.000 description 23
- 210000002966 serum Anatomy 0.000 description 21
- 238000007789 sealing Methods 0.000 description 16
- 239000012452 mother liquor Substances 0.000 description 14
- 239000000463 material Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 241001611022 Clostridium carboxidivorans Species 0.000 description 5
- ZLXPLDLEBORRPT-UHFFFAOYSA-M [NH4+].[Fe+].[O-]S([O-])(=O)=O Chemical compound [NH4+].[Fe+].[O-]S([O-])(=O)=O ZLXPLDLEBORRPT-UHFFFAOYSA-M 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000031700 light absorption Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 5
- PMYDPQQPEAYXKD-UHFFFAOYSA-N 3-hydroxy-n-naphthalen-2-ylnaphthalene-2-carboxamide Chemical compound C1=CC=CC2=CC(NC(=O)C3=CC4=CC=CC=C4C=C3O)=CC=C21 PMYDPQQPEAYXKD-UHFFFAOYSA-N 0.000 description 4
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000011655 sodium selenate Substances 0.000 description 4
- 235000018716 sodium selenate Nutrition 0.000 description 4
- 229960001881 sodium selenate Drugs 0.000 description 4
- XMVONEAAOPAGAO-UHFFFAOYSA-N sodium tungstate Chemical compound [Na+].[Na+].[O-][W]([O-])(=O)=O XMVONEAAOPAGAO-UHFFFAOYSA-N 0.000 description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229940091258 selenium supplement Drugs 0.000 description 3
- 239000011781 sodium selenite Substances 0.000 description 3
- 235000015921 sodium selenite Nutrition 0.000 description 3
- 229960001471 sodium selenite Drugs 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical class [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 229910052721 tungsten Inorganic materials 0.000 description 2
- 239000010937 tungsten Substances 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- XMWRBQBLMFGWIX-UHFFFAOYSA-N C60 fullerene Chemical class C12=C3C(C4=C56)=C7C8=C5C5=C9C%10=C6C6=C4C1=C1C4=C6C6=C%10C%10=C9C9=C%11C5=C8C5=C8C7=C3C3=C7C2=C1C1=C2C4=C6C4=C%10C6=C9C9=C%11C5=C5C8=C3C3=C7C1=C1C2=C4C6=C2C9=C5C3=C12 XMWRBQBLMFGWIX-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910003472 fullerene Inorganic materials 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a kind of cultivating system and cultural method eaten gas anaerobic bacteria and utilize synthesis gas fermentation, the cultivating system includes following components：Trace element, zinc ion, molybdenum acid ion and culture medium；Concentration of the described zinc ion in cultivating system is 100~500 μM；The present invention is fermented using the cultivating system to food gas anaerobic bacteria, improves the ability of food gas anaerobe fermentation production alcohols, and by taking Clostridium carboxidivorans P7 as an example, the cell concentration of bacterial strain is maximum up to OD600Nm~1.543Abs, highest ethanol production are 2.531g/L, and n-butanol yield is up to 0.318g/L, and n-hexyl alcohol content is 0.034g/L.
Description
Technical field
The present invention relates to technical field of microbial fermentation, more particularly to a kind of food gas anaerobic bacteria to utilize synthesis gas fermentation production alcohol
Cultivating system and cultural method.
Background technology
Synthesis gas is that one kind mainly includes CO, H2And a small amount of CO2Etc. the mixed gas of other components, the source of synthesis gas
It is relatively broad, such as coal, oil, natural gas, industrial waste gas (such as coke-stove gas, refinery gas), municipal solid wastes and biology
Matter etc..At present, the excessive use of fossil energy and the burning of municipal refuse, stalk etc. cause CO, CO in air2、CH4Deng gas
Constantly riseing for body content, damages health, causes the greenhouse effects in global range, exacerbate EI Nino phenomenon,
Cause many serious social environments and economic problems such as unusual weather conditions, sea level rise, crop yield decline.Above-mentioned synthesis
The methods of processing of gas is typically using absorbing or burning, treatment effeciency is low, high energy consumption.
The research of prior art finds a kind of anaerobism food gas microorganism in nature be present, can be with synthesis gas (CO, CO2With
And H2) grown as unique carbon source and energy source, and a variety of organic acids and alcohols material are translated into, including second
The bio-fuel such as the high valuable chemicals such as acid, butyric acid, 2,3-butanediol and ethanol, butanol, n-hexyl alcohol.Therefore, will synthesize
Gas, into the industrial fermentation processes of microorganism, can not only form the energy of a sustainable development as carbon source and energy source use
With the green channel of chemicals exploitation, the operating cost of enterprise is saved, is increased economic efficiency, carbonaceous gas row can also be reduced
Put, post fullerenes circulate, and mitigate greenhouse effects, create society and environmental benefit.
But at present, although there is document (Phillips et al., 2015) to be reported in Clostridium
The culture medium prescription developed in carboxidivorans P7 using the laboratory oneself, Mo concentration is improved 10 on this basis
Promote the synthesis of acetic acid, butyric acid and hexanol again, Mo concentration is promoted to from 1.05, the 0.12 of original culture medium and 0.09g/L
2.05th, 0.64 and 0.88g/L, but Product formation ability is weaker during the fermentation for food gas anaerobic bacteria, the yield of alcohols material
Lowly, the especially yield of the higher alcohol such as butanol, hexanol, limit microbial fermentation technology industrial production in practice should
With.
The content of the invention
In view of this, side of the gas anaerobic bacteria using synthesis gas fermentation production alcohol ability is eaten it is an object of the invention to provide a kind of
Method, come solve in the prior art utilize synthesis gas fermentation process in Product formation ability it is weaker, alcohols yield poorly under the problem of.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of cultivating system eaten gas anaerobic bacteria and utilize synthesis gas fermentation, including following components：It is micro
Element, zinc ion, molybdenum acid ion and culture medium；
Described trace element includes following element：Magnesium elements, ferro element, cobalt element, selenium element and wolfram element；Described
The volume of trace element is the 0.3~0.4% of culture volume；
Concentration of the described zinc ion in cultivating system is 100~500 μM；
Described culture medium includes the component of volumes below fraction：1~5% a great number of elements solution, 0.2~0.8%
Reductant solution, the nutrition aqueous solution of surplus.
Preferably, concentration of the described zinc ion in cultivating system is 150~350 μM.
Preferably, concentration of the described zinc ion in cultivating system is 250~300 μM.
Preferably, described zinc ion source is in zinc sulfate or zinc chloride.
Preferably, mass concentration of the described molybdenum acid ion in cultivating system is 0.005~0.02%, described molybdenum
Acid ion derives from sodium molybdate.
Preferably, described a great number of elements solution includes the component of following content：6~10wt% sodium chloride, 8~12wt%
Ammonium chloride, 0.5~1.5wt% potassium chloride, 0.5~1.5wt% potassium dihydrogen phosphates, 1~3wt% magnesium sulfate, 0.2~0.6wt%
The water of calcium chloride and surplus.
Preferably, described reductant solution includes the component of following content：0.8~1.0wt% sodium hydroxide, 2~
6wt% L-cysteine hydrochloride and 2~6wt% nine water vulcanized sodium, the water of surplus.
Preferably, the nutrition aqueous solution includes the yeast extract and quality point that mass fraction is 0.05~0.15%
Number is 1.0~4.0% morpholino b acid.
Method of the gas anaerobic bacteria using synthesis gas fermentation production alcohol is eaten present invention also offers a kind of, is comprised the following steps：
1) culture medium after trace element, zinc ion, molybdenum acid ion and sterilizing deoxygenation is mixed, obtains cultivating system；
2) fermented bacterium is accessed in the cultivating system obtained to the step 1) and has obtained bacterium cultivating system；
3) sealed after being passed through synthesis gas in bacterium cultivating system to having of obtaining of the step 2), shaken cultivation, including
The tunning of ethanol, n-butanol and n-hexyl alcohol.
Preferably, synthesis gas described in step 3) includes the component of volumes below content：45~55% CO, 30~40%
CO2With 10~20% H2；The speed for being passed through synthesis gas is 1.5~2.5L/min.
Beneficial effects of the present invention：The present invention includes culture medium, trace element, zinc ion and molybdenum acid ion by structure
Cultivating system, and using the cultivating system to food gas anaerobic bacteria ferment, zinc ion adds especially in cultivating system
Add, the thalline maximum concentration of food gas anaerobe fermentation and the ability of fermentation production alcohols are improved, with Clostridium
Exemplified by carboxidivorans P7, the cell concentration of bacterial strain is maximum to be up to OD600nm~1.543Abs, highest ethanol production
2.531g/L, n-butanol yield are up to 0.318g/L, and n-hexyl alcohol content is 0.034g/L.
Brief description of the drawings
Fig. 1 is the OD of strain fermentation in comparative example 1600Value changes schematic diagram；
Fig. 2 is the yield of strain fermentation ethanol and n-butanol in comparative example 1；
Fig. 3 is the OD of strain fermentation in embodiment 1600Value changes schematic diagram；
Fig. 4 is the yield of the 4th day ethanol and n-butanol of being fermented in embodiment 1；
Fig. 5 is the OD of strain fermentation in embodiment 2600Value changes schematic diagram；
Fig. 6 is the yield of strain fermentation ethanol and n-butanol in embodiment 2；
Fig. 7 is the OD of strain fermentation in embodiment 3600Value changes schematic diagram；
Fig. 8 is the yield of fermenting alcohol, n-butanol and n-hexyl alcohol in embodiment 3；
Fig. 9 is the OD of strain fermentation in embodiment 4600Value changes schematic diagram；
Figure 10 is the yield of strain fermentation ethanol, n-butanol and n-hexyl alcohol in embodiment 4.
Embodiment
The invention provides a kind of cultivating system eaten gas anaerobic bacteria and utilize synthesis gas fermentation, including following components：It is micro
Element, zinc ion, molybdenum acid ion and culture medium；Described trace element includes following element：Magnesium elements, ferro element, cobalt member
Element, selenium element and wolfram element；Described micro- volume is the 0.3~0.4% of culture volume；Described zinc ion exists
Concentration in cultivating system is 100~500 μM；Described culture medium includes the component of volumes below fraction：1~5% it is a large amount of
Element Solution, 0.2~0.8% reductant solution, the nutrition aqueous solution of surplus.
In the present invention, described trace element includes following element：Magnesium elements, ferro element, cobalt element, selenium element and tungsten
Element, preferably also include nitrogen.The present invention is not limited described micro- source, as long as it can provide described
The micro- material of species, the material described in the specific embodiment of the present invention can be nitrilotriacetic acid, magnesium sulfate, sulphur
Sour ferrous ammonium, sodium selenate/sodium selenite, cobalt chloride and sodium tungstate；It is commercially available that the micro- material is provided in the present invention
Analyze pure.In the present invention, the micro- addition volume is preferably the 0.1~0.5% of culture volume, more preferably
0.2~0.4, most preferably 0.33%.
In the present invention, concentration of the described zinc ion in cultivating system be 100~500 μM, preferably 150~
350 μM, more preferably 250~300 μM, most preferably 280 μM；The present invention does not have special want to the source of the zinc ion
Ask, as long as zinc ion can be dissociateed under dissolved state, specifically in embodiments of the present invention zinc ion source in zinc salt, example
Such as zinc sulfate or zinc chloride, preferably zinc sulfate.
Mass concentration of the heretofore described molybdenum acid ion in cultivating system is preferably 0.005~0.02%,
More preferably 0.08~0.012%, most preferably 0.01%；The present invention is not limited the source of the molybdenum acid ion
It is fixed, as long as the material of molybdenum acid ion can be provided, sodium molybdate is specifically preferably in embodiments of the present invention, it is described
Sodium molybdate for it is commercially available analysis it is pure.
In the present invention, described culture medium includes the component of volumes below fraction：1~5% a great number of elements solution,
0.2~0.8% reductant solution, the nutrition aqueous solution of surplus.
In the present invention, the culture medium preferably includes a great number of elements solution that volume fraction is 1~5%, more preferably
For 2~4%, most preferably 3%.In the present invention, a great number of elements preferably includes sodium element, nitrogen, potassium element, phosphorus
Element, element sulphur, magnesium elements, chlorine element and calcium constituent.The present invention is not particularly limited to the source of a great number of elements, as long as
Above-mentioned a great number of elements can be provided, specifically in embodiments of the present invention, a great number of elements solution preferably includes following
The component of mass content：6~10wt% sodium chloride, 8~12wt% ammonium chlorides, 0.5~1.5wt% potassium chloride, 0.5~
The water of 1.5wt% potassium dihydrogen phosphates, 1~3wt% magnesium sulfate, 0.2~0.6wt% calcium chloride and surplus.
In the present invention, the content of the sodium chloride is preferably 6~10wt%, more preferably 7~9%, most preferably
For 8%；The content of the ammonium chloride is preferably 8~12wt%, more preferably 9~11%, most preferably 10%；Institute
The content for stating potassium chloride is preferably 0.5~1.5wt%, more preferably 0.8~1.2%, most preferably 1.0%；It is described
The content of potassium dihydrogen phosphate is preferably 0.5~1.5wt%, more preferably 0.8~1.2%, most preferably 1.0%；Institute
The content for stating magnesium sulfate is preferably 1~3wt%, more preferably 1.5~2.5%, most preferably 2%；The calcium chloride
Content be preferably 0.2~0.6wt%, more preferably 0.3~0.5%, most preferably 0.4%；A large amount of members of the invention
Also include the water of surplus in plain solution, the present invention is not particularly limited to the water in a great number of elements mother liquor, conventional using this area
Water, can be specifically sterilized water, pure water, distilled water or distilled water in embodiments of the present invention, a large amount of members of the present invention
Water in plain mother liquor dissolves above-mentioned each component as solvent；Group in the present invention in above-mentioned a great number of elements mother liquor than water
Divide is that commercially available analysis is pure.
In the present invention, described culture medium includes reductant solution, and the volume fraction of the reductant solution is preferable
For 0.2~0.8%, more preferably 0.4~0.6%, most preferably 0.5%.In the present invention, described reducing agent includes
Sodium hydroxide, L-cysteine hydrochloride and nine water vulcanized sodium；In the reductant solution, the content of described sodium hydroxide
Preferably 0.8~1.0wt%, more preferably 0.9wt%；The content of the L-cysteine hydrochloride is preferably 2~
6wt%, more preferably 3~5wt%, most preferably 4wt%；The content of the nine water vulcanized sodium is preferably 2~
6wt%, more preferably 3~5wt%, most preferably 4wt%；Also include the water of surplus in reducing agent of the present invention, this
Invention is not particularly limited to the water in reducing agent, using the conventional water in this area, specifically in embodiments of the present invention
Can be sterilized water, pure water, distilled water or distilled water, the water in a great number of elements mother liquor of the present invention dissolves above-mentioned each as solvent
Individual component；Component in the present invention in above-mentioned reducing agent than water is that commercially available analysis is pure.
Also include the nutrition aqueous solution of residual volume in culture medium of the present invention, the described nutrition aqueous solution includes
Yeast extract and morpholino b acid, on the basis of the quality of the nutrition aqueous solution, the quality of the yeast extract contains the present invention
Amount preferably 0.05~0.15%, more preferably 0.08~0.12, most preferably 0.1%；The morpholino b acid
Mass content is preferably 1.0~4.0%, more preferably 2.0~3.0%, most preferably 2.5%.The present invention is to described
The source of yeast extract and morpholino b acid does not have special restriction, using yeast extract well known to those skilled in the art
With the commercially available prod of morpholino b acid.
The invention provides a kind of method that hexanol is produced using synthesis gas cold fermentation, comprise the following steps：1) will be micro-
Culture medium mixing after secondary element, zinc ion, molybdenum acid ion and sterilizing deoxygenation, obtains cultivating system；2) to the step 1)
Fermented bacterium is accessed in obtained cultivating system and has obtained bacterium cultivating system；3) there is bacterium cultivating system to what the step 2) obtained
In be passed through synthesis gas after seal, shaken cultivation, obtain the tunning for including ethanol and n-butanol.
The present invention carries out deoxygenation and sterilizing to the culture medium, obtains the culture medium after sterilizing and deoxygenation.For the ease of training
The deoxygenation of base is supported, culture medium described in preferred pair above-mentioned technical proposal of the present invention is sealed, and the present invention is to described seal means
There is no special restriction with method, as long as sealing effectiveness can be ensured, preferably will in a particular embodiment of the present invention
Culture medium is placed in serum bottle, is sealed to obtain sealing culture medium using rubber stopper, the preferable volume of serum bottle is
100ml, the volume of culture medium is preferably 20~30ml in the serum bottle.
The present invention is preferably filled with nitrogen into the culture medium of the sealing and carries out deoxygenation to culture medium, obtains anaerobic culture
Base.It is currently preferred to provide source nitrogen using the nitrogen cylinder for being connected with syringe needle, in discharge culture medium during inflated with nitrogen
Air, the described speed for being filled with nitrogen is preferably 1.5~2.5L/min, more preferably 2.0L/min, the nitrogen
Purity be 99.999%.
The culture medium after sterilizing deoxygenation is obtained after obtained anaerobic culture medium is sterilized in the present invention, the present invention is preferably
Using the conventional high-temperature heat sterilization method of the art, the temperature of the sterilizing is preferably 115~125 DEG C, more preferably
For 118~123 DEG C, most preferably 121 DEG C；The time of the sterilizing is preferably 15~40min, more preferably 20min.
Culture medium of the present invention after sterilizing deoxygenation is obtained, by the culture medium and trace element, molybdenum after the sterilizing deoxygenation
Acid ion and zinc ion mixing, obtain cultivating system.It is currently preferred to make its nature after sterile anaerobic culture medium is obtained
Be cooled to less than 40 DEG C, more preferably 40 DEG C~10 DEG C, then by the culture medium for the deoxygenation that sterilized after cooling and trace element, zinc from
Son and molybdenum acid ion mixing.
The present invention accesses fermented bacterium into the cultivating system, has obtained bacterium cultivating system after cultivating system is obtained.
The present invention preferably accesses fermented bacterium under sterile oxygen free condition into the cultivating system, in embodiments of the present invention, preferably
Described be seeded in sterile anaerobic operation case carry out.In the present invention, the inoculation volume of the fermented bacterium is preferably trained
The 8~12% of the system of supporting volume, more preferably 10%.In the present invention, the OD of the inoculation fermented bacterium600Extinction
Value preferably 0.6~1.5, more preferably 0.8~1.2, most preferably 1.0.Zymophyte is used in heretofore described inoculation
Kind is the cell in exponential phase.Heretofore described fermented bacterium is food gas anaerobic bacteria, preferably
Clostridium carboxidivorans P7, described Clostridium carboxidivorans P7 purchases are certainly German
The German microorganism of the Leibnitz research institute of Brunswick and Cell Culture Collection, numbering DSM15243.
The present invention after bacterium cultivating system has been obtained, to it is described have in bacterium cultivating system be passed through synthesis gas, then seal,
25~35 DEG C of shaken cultivations, obtain the product containing hexanol.In the present invention, described synthesis gas preferably includes volumes below
The component of content：45~55% CO, 30~40% CO2With 10~20% H2, it is preferred including 50% CO, 35%
CO2With 15% H2；The present invention does not have special restriction to the source of the synthesis gas, specifically in embodiments of the present invention
Using commercially available synthesis gas.In the present invention, the speed that is passed through of the synthesis gas is preferably 1.5~2.5L/min, more preferably
For 1.8~2.2L/min, most preferably 2.0L/min；The heretofore described pressure for being passed through synthesis gas is preferably 0.15
~0.25MPa, more preferably 0.20MPa, present invention control after synthesis gas is passed through have the pressure of bacterium cultivating system 0.15
~0.25MPa；The present invention has been again sealed off bacterium cultivating system after completing to be passed through synthesis gas.
The present invention has bacterium cultivating system to carry out vibration training to after having bacterium cultivating system to be passed through synthesis hermetic seal to described
Support.The sealing for having thalline system is not particularly limited in the present invention, sealing is realized using the conventional seal means in this area
, specifically described sealing is carried out using rubber stopper in embodiments of the present invention；The temperature of the shaken cultivation is preferable
For 28~38 DEG C, more preferably 30~37 DEG C；The rotating speed of the shaken cultivation is preferably 100~250rpm, more preferably
For 150~200rpm, most preferably 180rpm；The time of the shaken cultivation is preferably 1~9 day, more preferably 3~
6 days, most preferably 4~5 days.
For the present invention in having bacterium cultivating system incubation, preferable timing sampling carries out microorganism growing state and micro- life
The detection of thing tunning, preferably to determine the time of fermentation ends；Tunning described in the present invention include ethanol,
N-butanol and n-hexyl alcohol.The detection method of the microbial fermentation product is the conventional gas chromatography in this area.
With reference to embodiment side of the food gas anaerobic bacteria using synthesis gas fermentation production alcohol ability is improved to provided by the invention
Method is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Comparative example 1
By 20ml a great number of elements mother liquor, the 477.5ml nutrition aqueous solution and 2.5ml reducing agent are configured to 500ml cultures
Base, wherein a great number of elements mother liquor include the component of following mass content：8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1%
Potassium dihydrogen phosphate, the water of 2% magnesium sulfate, 0.4% calcium chloride and surplus；The nutrition aqueous solution includes the group of following quality
Point：0.5g yeast extract, 2.5g morpholino b acid and the water of surplus；Reducing agent includes the component of following mass content：
0.9% sodium hydroxide, 4% L-cysteine hydrochloride, 4% nine water vulcanized sodium and the water of surplus.
The culture medium prepared is dispensed into the serum bottle of 100mL capacity, liquid amount 20mL.With rubber stopper by bottleneck
Sealing, 99.999% nitrogen is filled with into serum bottle to remove blood with 2L/min speed using the nitrogen cylinder for being connected to syringe needle
The oxygen remained in clear bottle, inflation duration terminates inflation after reaching 5min, in 121 DEG C of high-temperature sterilization 20min.After sterilizing, cooling
To 40 DEG C, the trace element and the mass concentration that add 0.1ml are 0.01% sodium molybdate, do not add zinc ion, complete culture
System construction, trace element are to include nitrilotriacetic acid, magnesium sulfate, iron ammonium sulfate, cobalt chloride, sodium selenate and sodium tungstate it is molten
Liquid.
Access is cultivated to the Clostridium carboxidivorans bacterial strain P7 nutrient solution 5ml of exponential phase,
(OD600Nm light absorption values are 0.7, then with 2L/min flow velocitys, 0.2MPa pressure is continually fed into the analog synthesis gas 5min (groups of synthesis gas
Into：50%CO, 35%CO2, 15%H2) to remove the nitrogen in serum bottle, bacterium is completely in the gaseous environment of synthesis gas
Under fermented, finally by synthesis gas be forced into 0.2MPa sealing.Under 37 DEG C of temperature conditionss, shaken cultivation 4 days under 180rpm,
Detection cell concentration and fermentation product production are sampled every 24h.As a result as depicted in figs. 1 and 2, in whole fermentation period, bacterium
The cell concentration OD of strain600Nm is maximum up to 1.163Abs, and highest ethanol production is 1.356g/L, and highest n-butanol yield is
0.181g/L, it is substantially not detectable the yield of n-hexyl alcohol.
Embodiment 1
By 15ml a great number of elements mother liquor, the 482.5ml nutrition aqueous solution and 2.5ml reducing agent are configured to 500ml cultures
Base, wherein a great number of elements mother liquor include the component of following mass content：8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1%
Potassium dihydrogen phosphate, the water of 2% magnesium sulfate, 0.4% calcium chloride and surplus；The nutrition aqueous solution includes the group of following quality
Point：0.5g yeast extract, 2.5g morpholino b acid and the water of surplus；Reducing agent includes the component of following mass content：
0.9% sodium hydroxide, 4% L-cysteine hydrochloride, 4% nine water vulcanized sodium and the water of surplus.
The culture medium prepared is dispensed into the serum bottle of 100mL capacity, liquid amount 30mL.With rubber stopper by bottleneck
Sealing, 99.999% nitrogen is filled with into serum bottle to remove with 2.5L/min speed using the nitrogen cylinder for being connected to syringe needle
The oxygen remained in serum bottle, inflation duration terminates inflation after reaching 5min, in 121 DEG C of high-temperature sterilization 20min.It is cold after sterilizing
But to 35 DEG C, 0.1ml trace element is added, the sodium molybdate and final concentration of 7 μM of zinc sulfate that mass concentration is 0.01%,
Cultivating system structure is completed, trace element is to include nitrilotriacetic acid, magnesium sulfate, iron ammonium sulfate, cobalt chloride, sodium selenite and tungsten
The solution of the solution of sour sodium.
Access is cultivated to the Clostridium carboxidivorans bacterial strain P7 nutrient solution 4ml of exponential phase,
(OD600nm light absorption values are 1.1, then with 2L/min flow velocitys, 0.2MPa pressure be continually fed into analog synthesis gas 5min (synthesis gas
Composition：50%CO, 35%CO2, 15%H2) to remove the nitrogen in serum bottle, bacterium is completely in the Ring of synthesis gas
Fermented under border, synthesis gas is finally forced into 0.2MPa sealings.Under 37 DEG C of temperature conditionss, shaken cultivation 5 under 190rpm
My god, sample detection cell concentration and fermentation product production every 24h.As shown in Figure 3 and Figure 4, in whole fermentation period, bacterial strain
Cell concentration OD600Nm is maximum up to 1.213Abs, the highest ethanol production 1.265g/L detected, highest n-butanol yield
0.178g/L, it is substantially not detectable the yield of n-hexyl alcohol.
Embodiment 2
By 10ml a great number of elements mother liquor, the 487.5ml nutrition aqueous solution and 2.5ml reducing agent are configured to 500ml cultures
Base, wherein a great number of elements mother liquor include the component of following mass content：8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1%
Potassium dihydrogen phosphate, the water of 2% magnesium sulfate, 0.4% calcium chloride and surplus；The nutrition aqueous solution includes the group of following quality
Point：0.5g yeast extract, 2.5g morpholino b acid and the water of surplus；Reducing agent includes the component of following mass content：
0.9% sodium hydroxide, 4% L-cysteine hydrochloride, 4% nine water vulcanized sodium and the water of surplus.
The culture medium prepared is dispensed into the serum bottle of 100mL capacity, liquid amount 30mL.With rubber stopper by bottleneck
Sealing, 99.999% nitrogen is filled with into serum bottle to remove with 2.5L/min speed using the nitrogen cylinder for being connected to syringe needle
The oxygen remained in serum bottle, inflation duration terminates inflation after reaching 5min, in 121 DEG C of high-temperature sterilization 30min.It is cold after sterilizing
But to 45 DEG C, 0.15ml trace element is added, the sodium molybdate and final concentration of 70 μM of sulfuric acid that mass concentration is 0.011%
Zinc, cultivating system structure is completed, trace element is to include nitrilotriacetic acid, magnesium sulfate, iron ammonium sulfate, cobalt chloride, sodium selenite
With the solution of the solution of sodium tungstate.
Access is cultivated to the Clostridium carboxidivorans bacterial strain P7 nutrient solution 2ml of exponential phase,
(OD600nm light absorption values are 1.5, then with 2L/min flow velocitys, 0.2MPa pressure be continually fed into analog synthesis gas 5min (synthesis gas
Composition：50%CO, 35%CO2, 15%H2) to remove the nitrogen in serum bottle, bacterium is completely in the Ring of synthesis gas
Fermented under border, synthesis gas is finally forced into 0.2MPa sealings.Under 37 DEG C of temperature conditionss, shaken cultivation 4 under 170rpm
My god, sample detection cell concentration and fermentation product production every 24h.As it can be seen in figures 5 and 6, in whole fermentation period, bacterial strain
Cell concentration OD600Nm is maximum up to 1.125Abs (Fig. 5), and highest ethanol production is 1.498g/L, and n-butanol yield is up to
0.18g/L (Fig. 6), it is substantially not detectable the yield of n-hexyl alcohol.
Embodiment 3
By 15ml a great number of elements mother liquor, the 484ml nutrition aqueous solution and 1.0ml reducing agent are configured to 500ml culture mediums,
Wherein a great number of elements mother liquor includes the component of following mass content：8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1% phosphorus
Acid dihydride potassium, the water of 2% magnesium sulfate, 0.4% calcium chloride and surplus；The nutrition aqueous solution includes the component of following quality：
0.5g yeast extract, 2.5g morpholino b acid and the water of surplus；Reducing agent includes the component of following mass content：
0.9% sodium hydroxide, 4% L-cysteine hydrochloride, 4% nine water vulcanized sodium and the water of surplus.
The culture medium prepared is dispensed into the serum bottle of 100mL capacity, liquid amount 20mL.With rubber stopper by bottleneck
Sealing, 99.999% nitrogen is filled with into serum bottle to remove with 2.0L/min speed using the nitrogen cylinder for being connected to syringe needle
The oxygen remained in serum bottle, inflation duration terminates inflation after reaching 5min, in 121 DEG C of high-temperature sterilization 20min.It is cold after sterilizing
But to 35 DEG C, 0.1ml trace element is added, the sodium molybdate and final concentration of 140 μM of sulfuric acid that mass concentration is 0.009%
Zinc, complete cultivating system structure, trace element is to include nitrilotriacetic acid, magnesium sulfate, iron ammonium sulfate, cobalt chloride, sodium selenate and
The solution of the solution of sodium tungstate.
Access is cultivated to the Clostridium carboxidivorans bacterial strain P7 nutrient solution 4ml of exponential phase,
(OD600nm light absorption values are 1.0, then with 3L/min flow velocitys, 0.2MPa pressure be continually fed into analog synthesis gas 5min (synthesis gas
Composition：50%CO, 35%CO2, 15%H2) to remove the nitrogen in serum bottle, bacterium is completely in the Ring of synthesis gas
Fermented under border, synthesis gas is finally forced into 0.2MPa sealings.Under 37 DEG C of temperature conditionss, shaken cultivation 5 under 180rpm
My god, sample detection cell concentration and fermentation product production every 24h.As a result as shown in Figure 7 and Figure 8, in whole fermentation period,
The cell concentration OD of bacterial strain600Nm is maximum up to 1.303Abs (Fig. 7), and highest ethanol production is 1.425g/L, and n-butanol yield is most
A height of 0.248g/L, n-hexyl alcohol maximum output are 0.024g/L (Fig. 8).
Embodiment 4
By 20ml a great number of elements mother liquor, the 477.5ml nutrition aqueous solution and 2.5ml reducing agent are configured to 500ml cultures
Base, wherein a great number of elements mother liquor include the component of following mass content：8% sodium chloride, 10% ammonium chloride, 1% potassium chloride, 1%
Potassium dihydrogen phosphate, the water of 2% magnesium sulfate, 0.4% calcium chloride and surplus；The nutrition aqueous solution includes the group of following quality
Point：0.5g yeast extract, 2.5g morpholino b acid and the water of surplus；Reducing agent includes the component of following mass content：
0.9% sodium hydroxide, 4% L-cysteine hydrochloride, 4% nine water vulcanized sodium and the water of surplus.
The culture medium prepared is dispensed into the serum bottle of 100mL capacity, liquid amount 20mL.With rubber stopper by bottleneck
Sealing, 99.999% nitrogen is filled with into serum bottle to remove blood with 2L/min speed using the nitrogen cylinder for being connected to syringe needle
The oxygen remained in clear bottle, inflation duration terminates inflation after reaching 5min, in 121 DEG C of high-temperature sterilization 20min.After sterilizing, cooling
To 40 DEG C, 0.1ml trace element is added, the sodium molybdate and final concentration of 280 μM of zinc sulfate that mass concentration is 0.01%,
Cultivating system structure is completed, trace element is to include nitrilotriacetic acid, magnesium sulfate, iron ammonium sulfate, cobalt chloride, sodium selenate and wolframic acid
The solution of sodium.
Access is cultivated to the Clostridium carboxidivorans bacterial strain P7 nutrient solution 5ml of exponential phase,
(OD600nm light absorption values are 0.7, then with 2L/min flow velocitys, 0.2MPa pressure be continually fed into analog synthesis gas 5min (synthesis gas
Composition：50%CO, 35%CO2, 15%H2) to remove the nitrogen in serum bottle, bacterium is completely in the Ring of synthesis gas
Fermented under border, synthesis gas is finally forced into 0.2MPa sealings.Under 37 DEG C of temperature conditionss, shaken cultivation 4 under 180rpm
My god, sample detection cell concentration and fermentation product production every 24h.As shown in Figure 9 and Figure 10, in whole fermentation period, bacterium
The cell concentration OD of strain600Nm is maximum up to 1.543Abs (Fig. 9), and highest ethanol production is 2.531g/L, n-butanol yield highest
For 0.318g/L, n-hexyl alcohol content is 0.034g/L (Figure 10).
As seen from the above embodiment, the present invention includes culture medium, trace element, molybdenum acid ion and zinc ion by structure
Cultivating system, and using the cultivating system to food gas anaerobic bacteria ferment, zinc ion adds especially in cultivating system
Add, improve the ability of food gas anaerobe fermentation production alcohols, by taking Clostridium carboxidivorans P7 as an example, adding
In the fermentation system for adding higher concentration zinc ion, not only the upgrowth situation of microorganism is preferable, OD600Nm is up to 1.543, bright
Aobvious is respectively the system of 0 μM and 7 μM higher than zinc ion concentration, while the yield of ethanol, n-butanol and n-hexyl alcohol also has and substantially carried
Rise.Final concentration of 280 μM in cultivating system of zinc ion, product ethanol concentration has reached 2.531g/L, without add zinc from
Ethanol production is 1.356g/L in the system of son；Similarly, n-hexyl alcohol is more there is also analogue and in tunning for n-butanol
It is due to the raising for adding zinc ion concentration, qualitative change from scratch is realized, when addition concentration of the zinc ion in cultivating system
It is that the yield of n-hexyl alcohol has reached 0.034g/L as can be seen here for 280 μM, being added with for zinc ion is beneficial to improve in cultivating system
The ability of the production alcohols material of gas anaerobic bacteria is eaten, improves the yield of ethanol and n-butanol in tunning, and enrich hair
The species of alcohols material in ferment product so that food gas anaerobic bacteria can produce n-hexyl alcohol.In summary, method provided by the invention
Improve ability of the food gas anaerobic bacteria using synthesis gas production alcohols material.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of cultivating system eaten gas anaerobic bacteria and utilize synthesis gas fermentation, it is characterised in that including following components：Micro member
Element, zinc ion, molybdenum acid ion and culture medium；
Described trace element includes following element：Magnesium elements, ferro element, cobalt element, selenium element and wolfram element；Described
The volume of trace element is the 0.3~0.4% of culture volume；
Concentration of the described zinc ion in cultivating system is 100~500 μM；
Described culture medium includes the component of volumes below fraction：1~5% a great number of elements solution, 0.2~0.8% reduction
Agent solution, the nutrition aqueous solution of surplus.
2. cultivating system according to claim 1, it is characterised in that concentration of the described zinc ion in cultivating system is
150~350 μM.
3. cultivating system according to claim 1, it is characterised in that concentration of the described zinc ion in cultivating system is
250~300 μM.
4. cultivating system according to claim 1, it is characterised in that described zinc ion source is in zinc sulfate or chlorination
Zinc.
5. cultivating system according to claim 1, it is characterised in that matter of the described molybdenum acid ion in cultivating system
It is 0.005~0.02% to measure concentration, and described molybdenum acid ion derives from sodium molybdate.
6. cultivating system according to claim 1, it is characterised in that described a great number of elements solution includes following content
Component：6~10wt% sodium chloride, 8~12wt% ammonium chlorides, 0.5~1.5wt% potassium chloride, 0.5~1.5wt% biphosphates
The water of potassium, 1~3wt% magnesium sulfate, 0.2~0.6wt% calcium chloride and surplus.
7. cultivating system according to claim 1, it is characterised in that described reductant solution includes the group of following content
Point：0.8~1.0wt% sodium hydroxide, 2~6wt% L-cysteine hydrochloride and 2~6wt% nine water vulcanized sodium, it is remaining
The water of amount.
8. cultivating system according to claim 1, it is characterised in that the nutrition aqueous solution includes mass fraction and is
0.05~0.15% yeast extract and mass fraction is 1.0~4.0% morpholino b acid.
9. a kind of eat method of the gas anaerobic bacteria using synthesis gas fermentation production alcohol, comprise the following steps：
1) culture medium after trace element, zinc ion, molybdenum acid ion and sterilizing deoxygenation is mixed, obtains cultivating system；
2) fermented bacterium is accessed in the cultivating system obtained to the step 1) and has obtained bacterium cultivating system；
3) sealed after being passed through synthesis gas in bacterium cultivating system to having of obtaining of the step 2), shaken cultivation, obtain including ethanol,
The tunning of n-butanol and n-hexyl alcohol.
10. according to the method for claim 9, it is characterised in that synthesis gas described in step 3) includes volumes below content
Component：45~55% CO, 30~40% CO2With 10~20% H2；The speed for being passed through synthesis gas be 1.5~
2.5L/min。
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