CN109536565A - Method for producing succinic acid by utilizing mixed fermentation of high-temperature anaerobic bacteria for pyrolyzing sugar and actinobacillus succinogenes - Google Patents
Method for producing succinic acid by utilizing mixed fermentation of high-temperature anaerobic bacteria for pyrolyzing sugar and actinobacillus succinogenes Download PDFInfo
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- succinic acid
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 123
- 238000000855 fermentation Methods 0.000 title claims abstract description 105
- 230000004151 fermentation Effects 0.000 title claims abstract description 104
- 239000001384 succinic acid Substances 0.000 title claims abstract description 58
- 241000948980 Actinobacillus succinogenes Species 0.000 title claims abstract description 49
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 30
- 241001148471 unidentified anaerobic bacterium Species 0.000 title abstract 3
- 229920001221 xylan Polymers 0.000 claims abstract description 42
- 150000004823 xylans Chemical class 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000000197 pyrolysis Methods 0.000 claims abstract description 17
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 46
- 241000894006 Bacteria Species 0.000 claims description 40
- 230000004913 activation Effects 0.000 claims description 34
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 23
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 23
- 241000233866 Fungi Species 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 17
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 9
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 8
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 8
- 235000005822 corn Nutrition 0.000 claims description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 7
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 7
- 238000002255 vaccination Methods 0.000 claims description 7
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 241000193446 Thermoanaerobacterium thermosaccharolyticum Species 0.000 claims description 3
- 239000001095 magnesium carbonate Substances 0.000 claims description 3
- 229910000021 magnesium carbonate Inorganic materials 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 10
- 229910052799 carbon Inorganic materials 0.000 abstract description 10
- 239000000758 substrate Substances 0.000 abstract description 6
- 239000001963 growth medium Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000010802 sludge Substances 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 17
- 230000000694 effects Effects 0.000 description 14
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- 230000015556 catabolic process Effects 0.000 description 10
- 238000006731 degradation reaction Methods 0.000 description 10
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 6
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 6
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 229920002488 Hemicellulose Polymers 0.000 description 4
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229910001425 magnesium ion Inorganic materials 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 241000606750 Actinobacillus Species 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 241000588767 Proteus vulgaris Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000588902 Zymomonas mobilis Species 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940007042 proteus vulgaris Drugs 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- 241000193468 Clostridium perfringens Species 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 241000194033 Enterococcus Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229920006238 degradable plastic Polymers 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 235000011160 magnesium carbonates Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical class O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000010327 methods by industry Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000011846 petroleum-based material Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- -1 poly butylene succinate Polymers 0.000 description 1
- 239000004631 polybutylene succinate Substances 0.000 description 1
- 229920002961 polybutylene succinate Polymers 0.000 description 1
- 238000012643 polycondensation polymerization Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000010891 toxic waste Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- 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
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- 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
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Genetics & Genomics (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses a method for producing succinic acid by utilizing mixed fermentation of high-temperature anaerobic bacteria for pyrolyzing sugar and actinobacillus succinogenes, which comprises the following steps: (1) inoculating activated high-temperature anaerobic bacteria of the pyrolysis sugar into a fermentation culture medium containing xylan for fermentation to obtain fermentation liquor; (2) inoculating activated actinobacillus succinogenes into the fermentation liquor obtained in the step (1), and fermenting to produce succinic acid. When the sugar pyrolysis thermophilic anaerobe M5 is cultured for 48 hours, actinobacillus succinogenes sludge is added, the succinic acid yield is highest and reaches 43.10 g/L, the method is the highest succinic acid yield obtained by mixed fermentation when xylan is used as the only carbon source at present, the substrate cost of the industrial production of succinic acid is reduced, and the method has important application value.
Description
Technical field
The invention belongs to field of microbial fermentation, and in particular to a kind of to be put using the sugared high temperature anaerobic bacterium of pyrolysis and production succinic acid
The method of line bar bacterium mixed fungus fermentation production succinic acid.
Background technique
Succinic acid (also known as succinic acid) is a kind of four important carbon platform chemicals, is widely used in food, plastics, doctor
The industry such as medicine, fragrance.In addition, the primary raw material of succinic acid or synthesized degradable plastics, it can with butanediol, ethylene glycol,
The condensation polymerizations such as propylene glycol production poly butylene succinate, polyethylene glycol succinate, polypropylene glycol succinate etc. have
Good characteristic and the high molecular material that can be biodegradable completely are the potential fields most with prospects of succinic acid.The U.S.
In Ministry of Energy's 12 kinds of most potential bulk products announced in 2004, succinic acid makes number one.
The succinic acid being commercialized at present is mainly logical by raw material of petroleum based materials such as maleic acid, maleic anhydrides
Catalytic hydrogenation or electrochemistry formated are crossed, in the process with the discharge of a large amount of greenhouse gases and toxic waste, environmental pollution
Seriously.As petroleum resources supply being increasingly enhanced for growing tension and environmental consciousness, chemical method synthesis is gradually restricted, no
Conducive to the development of succinic acid related industry.And it is original that Production by Microorganism Fermentation succinic acid, which has with reproducible biomass resource,
Material, fermentation condition are mild, can fix CO2Etc. series of advantages, become one of research hotspot of recent domestic.
In the microorganism of numerous succinic acid-producings, Actinobacillus succinogenes (Actinobacillus succinogenes) high with its yield (reaching as high as 110 g/L), tolerance (highest is resistant to 160 g/L glucose) the advantages that,
It is one of production strain most with prospects at present.But the production cost of current Production by Microorganism Fermentation succinic acid is higher,
Affect the process of its industrialization.Succinic acid production is carried out using cheap or discarded non-grain biomass resource substitution glucose,
It is not only to recycle discarded biomass resource, and succinic acid raw materials for production cost can be effectively reduced, to promotion fourth two
Sour biological fermentation process production has great importance.Main component in grain straw is cellulose and hemicellulose, wherein half
The main component of cellulose is xylan, and can produce succinic acid as sole carbon source using xylan can substantially reduce production cost,
And it can also turn waste into wealth.
Thermophilic anaerobic bacillus is can directly to be sent out by integral biological process engineering using hemicellulose for carbon source
The thermophilic type wild strain of ferment.CN106995790A, which discloses one plant, can directly utilize the pyrolysis sugar that lignocellulosic is carbon source
High temperature anaerobic bacterial strain M5(Thermoanaerobacterium thermosaccharolyticum), it can be with efficient degradation wood
Matter cellulose.
Mixed fungus fermentation refers to that the one kind for completing certain fermentation process jointly using the synergistic effect of two or more microorganisms is new
Type fermentation technique.It is that the new development of pure-blood ferment technology and a kind of DNA vitro recombination for not needing to carry out complexity can but obtain
The novel fermentation technology of similar effect.Advantage is that fermentation efficiency can be improved can even to form new product.According to interbiotic combination
Mode can divide following four type.(1) combined ferment: being inoculated with simultaneously with two or more microorganisms and culture, such as China's invention
Sorbose is converted into the mixed fungus fermentation during diketo 2-KLG in production of vitamin C.(2) sequential fermentation: first with first bacterium into
Row normal fermentation, then fermented in order by second bacterium etc., to complete several biochemical reactions, such as Rhizopus arrhizus jointly
(Rhizopus arhizus) glucose is first converted into fumaric acid, then again by clostridium perfringen (Enterococcus aerogenes) or proteus vulgaris (Proteus vulgaris) it is reduced to tunning succinic acid.(3) fixed altogether
Change cell mixed fungus fermentation: two or more microbial cells being embedded or be adsorbed in simultaneously the mixed bacterium carried out in identical carrier and send out
Ferment, such as aspergillus niger (Aspergillus niger) and zymomonas mobilis (Zymomonas mobilis) jointly starch
It is converted into alcohol etc.；(4) mixed immobilization cell mixed fungus fermentation: after two or more microbial cells are distinguished immobilization, then
They are mixed and carries out mixed fungus fermentation.
Actinobacillus succinogenes can not be using the polysaccharose substances such as starch and cellulose, but it is using monosaccharide conduct
Carbon source, such as xylose carry out the efficient production of succinic acid.And the sugared high temperature anaerobic bacterial strain of pyrolysis can be by the hemicelluloses such as xylan height
Effect is degraded into xylose.Sugared high temperature anaerobic bacterial strain will be pyrolyzed and Actinobacillus succinogenes carry out mixed fungus fermentation, may be implemented to utilize
Xylan efficiently produces the target of succinic acid.There is presently no the reports that bacterium production succinic acid is mixed using lignocellulosic.
Summary of the invention
Goal of the invention: it is produced to solve traditional succinic acid fermentation process using grain or other starchiness byproducts as raw material
The excessively high problem of cost utilizes the sugared high temperature anaerobic bacterium of pyrolysis and Actinobacillus succinogenes mixed fungus fermentation the present invention provides a kind of
The method for producing succinic acid.
Technical solution: a kind of utilize of the present invention is pyrolyzed sugared high temperature anaerobic bacterium and Actinobacillus succinogenes mixed fungus fermentation
The method for producing succinic acid, comprising the following steps:
(1) the sugared high temperature anaerobic bacterium of the pyrolysis of activation is inoculated into the fermentation medium containing xylan and is fermented, fermentation 24 ~
120 hours, obtain fermentation liquid；
(2) Actinobacillus succinogenes of activation are inoculated into the fermentation liquid that step (1) obtains, mixed fungus fermentation produces fourth two
Acid.
Preferably, step (1) described fermentation time is preferably 24 ~ 72h, further preferably fermentation 48 hours, will
The Actinobacillus succinogenes of activation are inoculated into the fermentation liquid that step (1) obtains.There is a certain amount of xylose in fermentation liquid at this time, and
The active highest of zytase and xylobiose enzyme, can continuous degradation xylan be xylose, and Actinobacillus succinogenes can be with
The xylose production succinic acid obtained using degradation.Too early (M5 fermentation is less than 24 h), the enzyme activity of zytase is lower for inoculation time,
The degradation of subsequent xylan is not utilized；Inoculation time too late (72 h-120 h of M5 fermentation), then will lead to bacterial strain M5 incubation time
Too long, zytase and xylobiose enzyme secretory volume are reduced, and enzyme activity decline of the enzyme previously secreted out of at 55 DEG C, to lead
The enzyme activity decline of enzyme system is caused, and the xylose that Partial digestion obtains also is utilized by bacterial strain M5, produces succinic acid unwrapping wire bar to reduce
The carbon flow of bacterium eventually leads to the reduction of succinic acid yield.Therefore, the inoculation time of Actinobacillus succinogenes is that mixed fungus fermentation is raw
The extremely crucial step of succinic acid-producing.
The inoculum concentration of the sugared high temperature anaerobic bacterium of the pyrolysis of step (1) described activation is the 1-10% of fermentation medium volume；It is described
Fermentative medium formula containing xylan are as follows: 0.5-2.0 g/L NaCl, 0.5-2.0 g/L K2HPO4, 0.5-2.0 g/L
KH2PO4, 1.0-5.0 g/L yeast powder, 1.0-3.0 g/L Dried Corn Steep Liquor Powder, 0.2-1.0 g/L MgCl2·6H2O, 0.1-
0.6 g/L NH4Cl, 0.01-0.05 g/L CaCl2·2H2O, 0.5-2.0 g/L FeCl2·4H2O, 0.1-0.5 g/L
KCl, xylan 30-90 g/L, solvent is water, adjusts pH to 6.0-6.5, leads to carbon dioxide 10-20 min, 121 DEG C of sterilizings 15
min；The fermentation condition are as follows: 50-65 DEG C of fermentation temperature, fermentation time 36-72h, fermentation pH is 5.5-8.5(every 12-
PH is adjusted for 24 hours), revolving speed 0-120 rpm can be left to ferment, can also stir fermentation.
Preferably, xylan concentration is 60 g/L in the fermentation medium containing xylan.
Preferably, step (1) fermentation condition are as follows: 55 DEG C of fermentation temperature, 48 h of fermentation time, fermentation pH is 7.5,
120 rpm of revolving speed.
The activation culture based formulas for being pyrolyzed sugared high temperature anaerobic bacterium is 0.5-2.0 g/L NaCl, 0.5-2.0 g/L
K2HPO4, 0.5-2.0 g/L KH2PO4, 1.0-5.0 g/L yeast powder, 1.0-3.0 g/L Dried Corn Steep Liquor Powder, 0.2-1.0 g/
L MgCl2·6H2O, 0.1-0.6 g/L NH4Cl, 0.01-0.05 g/L CaCl2·2H2O, 0.5-2.0 g/L FeCl2·
4H2O, 0.1-0.5 g/L KCl, xylan 30-90 g/L, solvent is water, adjusts pH to 6.0-6.5；Activation condition are as follows: will be hot
It solves sugared high temperature anaerobic bacterium to be inoculated into activation medium with 5 % v/v of inoculum concentration, 50-65 DEG C, 0-120 rpm activation 48-96
H adjusts pH to 5.5-8.5 every 12 h.
Wherein, be pyrolyzed sugared high temperature anaerobic bacterium (Thermoanaerobacterium thermosaccharolyticum), bacterium
Strain number is M5, has been stored in China typical culture collection center, the deposit date is on 2 27th, 2017, deposit number CCTCC
NO:M 2017072, preservation address are as follows: the Chinese Wuhan Wuhan University, it has been disclosed that Yu Zhongguo applies for a patent CN 106995790A
In.The Actinobacillus succinogenes classification naming be Actinobacillus succinogenes (Actinobacillus succinogenes), bacterial strain number is 130Z, and it is existing bacterial strain which discloses in the literature.
The vaccination ways of the Actinobacillus succinogenes of step (2) described activation are as follows: by the production succinic acid unwrapping wire of activation
The precipitating that centrifugation obtains is inoculated into the fermentation liquid that step (1) obtains, wherein the production succinic acid activated by bacillus medium centrifugal
Actinobacillus culture solution is identical as the fermentating liquid volume that step (1) obtains；The fermentation condition are as follows: 35-39 DEG C of fermentation temperature,
Fermentation time is 24-96 h, and fermentation pH is 6.0-7.5, and revolving speed is 100-200 rpm.
Preferably, step (2) fermentation condition are as follows: 37 DEG C of fermentation temperature, fermentation time is 72 h, and fermentation pH is
6.8, revolving speed is 150 rpm.
The activation culture based formulas of the Actinobacillus succinogenes is 0.5-2.0 g/L NaCl, 10-20 g/L
K2HPO4, 5-15 g/L NaH2PO4, 5-15 g/L NaHCO3, 1.0-5.0 g/L yeast powder, 1.0-5.0 g/L corn pulp is dry
40-70 g/L basic magnesium carbonate is added in powder, xylose 30-90 g/L, and solvent is water；Activation condition is as follows: will produce succinic acid unwrapping wire
Bacillus is inoculated into activation medium with 5 % v/v of inoculum concentration, 37 DEG C, the production amber that is activated of 150 rpm activation 12-24 h
Sour Actinobacillus culture solution.
The utility model has the advantages that compared with prior art, technical advantage of the invention is as follows:
(1) present invention can use the pyrolysis sugar high temperature anaerobic bacterium M5 that xylan is sole carbon source growth by first culture, in height
In the case where warm anaerobism by xylan degrading be xylose, Actinobacillus succinogenes cannot directly using hemicellulose but can benefit
The production of succinic acid is carried out with xylose；It is all at present to utilize acidolysis, alkali by the report that substrate produces succinic acid of lignocellulosic
Lignocellulosic is hydrolyzed for solution and enzymatic hydrolysis, is fermented using its hydrolyzate, at high cost.And the present invention is using mixed
Sequential fermentation method in bacterium fermentation, the zytase and xylobiose enzyme secreted when being fermented by pyrolysis sugar high temperature anaerobic bacterium M5,
Xylan is gradually decomposed into xylose, Actinobacillus succinogenes are accessed when enzyme activity is higher, using made of having decomposed
Xylose and the xylose being gradually decomposed ferment, and produce succinic acid.
(2) Actinobacillus succinogenes bacterium mud is added when being pyrolyzed sugar high temperature anaerobic bacterium M5 and having cultivated 48 h, succinic acid produces
Highest is measured, 43.10 g/L are reached, this is also currently with xylan mixed fungus fermentation obtained highest succinic acid when being sole carbon source
Yield.When M5 cultivates 48 h, at this time in fermentation liquid zytase enzyme activity highest, zytase effectively degrades xylan,
And obtain the xylose of about 20 g/L；Hereafter, in mixed thallus system, zytase and xylobiose enzyme enzyme activity still with higher, energy
Enough continually degradation of xylan are xylose, and Actinobacillus succinogenes can use the xylose production fourth two that degradation obtains
Acid realizes simultaneous saccharification and fermentation.
(3) this method is a kind of novel fermentation that does not need the complicated DNA vitro recombination of progress and can but obtain similar to effect
Technology.This method reduce the costs of industrial production succinic acid, have important application value.
Detailed description of the invention
Fig. 1 is the product that 60 g/L xylans are substrate under the conditions of optimizing mixed fungus fermentation；
Fig. 2 is that 60 g/L xylans are substrate under the conditions of optimization mixed fungus fermentation, and in the fermentation liquid that is mixed of M5 fermentation 48h
Production concentration variation diagram.
Specific embodiment
Influence of the different xylan concentration of embodiment 1 to final succinic acid yield
(1) sugared high temperature anaerobic bacterium will be pyrolyzed to be inoculated into activation medium with 5 % v/v of inoculum concentration, 55 DEG C, 120 rpm activation
48 h adjust pH to 7.5 every 12 h；
Activation culture based formulas are as follows: 1 g/L NaCl, 0.75 g/L K2HPO4, 0.75 g/L KH2PO4, 5 g/L yeast powders,
2.5g/L Dried Corn Steep Liquor Powder, 0.5 g/L MgCl2•6H2O, 0.3 g/L NH4Cl, 0.015 g/L CaCl2•2H2O, 1.5 g/L
FeCl2·4H2O, 0.3 g/L KCl, 60 g/L of xylan, solvent is water, adjusts pH to 7.5；
(2) the pyrolysis sugar high temperature anaerobic bacterium M5 of activation is inoculated into fermentation medium with 5 % v/v of inoculum concentration, 55 DEG C, 120
Rpm 48 h of fermentation, adjust pH to 7.5 every 12 h, obtain fermentation liquid；
Fermentative medium formula are as follows: 1 g/L NaCl, 0.75 g/L K2HPO4, 0.75 g/L KH2PO4, 5g/L yeast powder,
2.5g/L Dried Corn Steep Liquor Powder, 0.5 g/L MgCl2·6H2O, 0.3 g/L NH4Cl, 0.3 g/L KCl, 0.015 g/L
CaCl2·2H2O, 1.5 g/L FeCl2·4H2O, solvent are water, and pH7.5 leads to 10~20 min of nitrogen, 121 DEG C of sterilizings 15
min；Three groups of fermentation mediums are set, and wherein xylan concentration is respectively 30 g/L, 60 g/L, 90 g/L；
(3) Actinobacillus succinogenes are inoculated into the activation medium of Actinobacillus succinogenes with 5 % v/v of inoculum concentration,
37 DEG C, 150 rpm activation 12-24 h；
Activation culture based formulas are as follows: 1 g/L NaCl, 0.75 g/L K2HPO4, 0.75 g/L KH2PO4, 3 g/L yeast powders,
0.5 g/L MgCl2·6H2O, 0.3 g/L NH4Cl, 0.015 g/L CaCl2·2H2O, 1.5 g/L FeCl2·4H2O, 0.3
50 g/L basic magnesium carbonates are added in g/L KCl, 60 g/L of xylose, and solvent is water；
(4) by taking incorporation time is 72 h as an example, activated Actinobacillus succinogenes are inoculated into step (2) with 10 % v/v
In obtained fermentation liquid, 37 DEG C, 150 rpm fermentation, 72 h.
In incubation, its succinic acid yield is measured by sampling every 12 h.As xylan 60 g/L of concentration, mixed fungus fermentation
Obtained succinic acid concentration highest, has reached 15.80 g/L；When xylan concentration is 30 g/L, fourth that mixed fungus fermentation obtains
Two acid concentrations only have 6.28 g/L；Succinic acid concentration when 90 g/L xylan concentration is 17.69 g/L, but comes from economy
Consider, 60 g/L xylan of final choice is as concentration of substrate.
2 Actinobacillus succinogenes of embodimentActinobacillus succinogenes 130Z different vaccination method
Influence to succinic acid yield
Method is with embodiment 1, the difference is that the concentration of xylan is 60 g/L, step (3) activated production in fermentation medium
Actinobacillus succinogenes are put into 4 DEG C of centrifuge centrifugations, and revolving speed is 6000 rpm, 10 min, then outwell supernatant, by bacterium mud
It is added in the fermentation liquid of step (2), wherein the volume of the volume of activated Actinobacillus succinogenes culture solution and fermentation liquid
It is identical；Then continue to cultivate for 37 DEG C, 150 rpm of revolving speed, adjust pH to 6.8 every 12 h, ferment 72 h；
In incubation, is sampled every 12 h and measure its succinic acid yield.When the vaccination ways mixing with embodiment 1, fourth
Diacid maximum output only has 15.80 g/L；When the vaccination ways mixing with the present embodiment, succinic acid yield has reached 30.28
g/L；Therefore use the vaccination ways of the present embodiment more excellent.
Embodiment 3 mixes influence of the basic carbonate magnesium density different in bacterium system culture medium to final succinic acid yield
Method is with embodiment 2, and wherein xylan concentration is 60 g/L, and inoculation method is centrifugation, the difference is that setting in step (3)
7 groups of experiments, every group of basic carbonate magnesium density are respectively 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L,
70g/L。
In incubation, is sampled every 12 h and measure its succinic acid yield.When basic carbonate magnesium density reaches 65 g/L
When, succinic acid yield highest reaches 36.28 g/L.When basic carbonate magnesium density improves, magnesium ion concentration and culture medium
PH, which mixes thallus system tool to it, to have a significant impact.Being pyrolyzed sugared high temperature anaerobic bacterium optimal pH is 7.5, Actinobacillus succinogenes optimal pH
It is 6.8, optimal regulation can be carried out to mixed thallus system pH by optimizing basic carbonate magnesium density.And magnesium ion is as micro gold
Belong to ion, the metabolism of Actinobacillus succinogenes to succinic acid can also be improved to a certain extent.And be pyrolyzed sugared high temperature anaerobic bacterium by
In could be used that xylose obtained from its degradation of xylan, so (sugared high temperature anaerobic bacterium can be pyrolyzed by improving magnesium ion concentration
Can hardly be grown after improving magnesium ion concentration) and reduction fermentation temperature (access Actinobacillus succinogenes post-fermentation temperature
It is 37 DEG C), grow it cannot after mixed bacterium, and the zytase that it is secreted can still be stablized in fermentation liquid and deposit
?.
Influence of the different inoculation time of 4 Actinobacillus succinogenes of embodiment to final succinic acid yield
Method is with embodiment 3, and wherein xylan concentration is 60 g/L, and vaccination ways are centrifugation, and basic carbonate magnesium density is 65 g/
L；The difference is that 5 groups of experiments are arranged in step (2), fermentation time is respectively 24 h, 48 h, 72 h, 96 h, 120 h.
In incubation, is sampled every 12 h and measure its succinic acid yield.When pyrolysis sugar high temperature anaerobic bacterium M5 is cultivated
Actinobacillus succinogenes bacterium mud is added when 48 h, succinic acid yield highest reaches 43.10 g/L, sees Fig. 1, Fig. 2.In this body
In system, the zytase of bacterial strain M5 secretion effectively degrades xylan, and obtains the xylose of about 20 g/L, hereafter, in mixed bacterium
In system, zytase and xylobiose enzyme enzyme activity still with higher can degradation of xylan be continually xylose, and produce amber
Amber acid Actinobacillus can use the xylose production succinic acid that degradation obtains.Inoculation time too early (24 h), xylanase activity compared with
It is low, do not utilize the degradation of subsequent xylan, and be inoculated with too late (72-120 h), then it is too long to will lead to bacterial strain M5 incubation time, wood
Dextranase and xylobiose enzyme secretory volume are reduced, and enzyme activity decline of the enzyme previously secreted out of at 55 DEG C, so as to cause enzyme system
Enzyme activity decline, and the xylose that Partial digestion obtains also is utilized by bacterial strain M5, to reduce the carbon flow of Actinobacillus succinogenes
Amount eventually leads to the reduction of succinic acid yield.
Experiments have shown that Actinobacillus succinogenes single bacterium can not be fermented using xylan, and it directly utilizes 60 g/L
When xylose carries out single bacterium fermentation, succinic acid yield is 39.03 g/L, yield 65%；And when using mixed thallus system, 60 g/L
Xylan can produce 43.10 g/L succinic acid, yield 72%.Mixed thallus system is that a kind of DNA for not needing to carry out complexity is external
Recombination can but obtain the novel fermentation technology of similar effect, and which not only reduces the substrate costs of industrial production succinic acid, and
Succinic acid yield can be improved to a certain extent.
Claims (10)
1. it is a kind of using the method for being pyrolyzed sugared high temperature anaerobic bacterium and Actinobacillus succinogenes mixed fungus fermentation production succinic acid, it is special
Sign is, comprising the following steps:
(1) the sugared high temperature anaerobic bacterium of the pyrolysis of activation is inoculated into the fermentation medium containing xylan and is fermented, fermentation 24 ~
120 hours；
(2) Actinobacillus succinogenes of activation are inoculated into the fermentation liquid of step (1), fermentation production of succinic acid.
2. the method according to claim 1, wherein the sugared high temperature anaerobic bacterium classification naming of step (1) pyrolysis
For be pyrolyzed sugared high temperature anaerobic bacterium (Thermoanaerobacterium thermosaccharolyticum), bacterial strain number is M5, step
Suddenly (2) described Actinobacillus succinogenes classification naming be Actinobacillus succinogenes (Actinobacillus succinogenes), bacterial strain number is 130Z.
3. the method according to claim 1, wherein the sugared high temperature anaerobic bacterium fermentation 48 of step (1) pyrolysis is small
When, the Actinobacillus succinogenes that step (2) activate are inoculated into the fermentation liquid that step (1) obtains, fermentation production of succinic acid.
4. the method according to claim 1, wherein the pyrolysis of step (1) described activation sugared high temperature anaerobic bacterium
Inoculum concentration is the 1-10% of fermentation medium volume.
5. the method according to claim 1, wherein step (1) described fermentation condition are as follows: fermentation temperature 50-65
DEG C, fermentation pH is 5.5-8.5, revolving speed 0-120rpm.
6. the method according to claim 1, wherein the fermented and cultured basigamy containing xylan described in step (1)
Side are as follows: 0.5-2.0 g/L NaCl, 0.5-2.0 g/L K2HPO4, 0.5-2.0 g/L KH2PO4, 1.0-5.0 g/L yeast
Powder, 1.0-3.0 g/L Dried Corn Steep Liquor Powder, 0.2-1.0 g/L MgCl2·6H2O, 0.1-0.6 g/L NH4Cl, 0.01-0.05
g/L CaCl2·2H2O, 0.5-2.0 g/L FeCl2·4H2O, 0.1-0.5 g/L KCl, xylan 30-90 g/L, solvent are
Water adjusts pH to 5.5-8.5.
7. the method according to claim 1, wherein step (1) is pyrolyzed sugared high temperature anaerobic bacterium activation condition are as follows: will
It is pyrolyzed sugared high temperature anaerobic bacterium to be inoculated into activation medium with 5 % v/v of inoculum concentration, 50-65 DEG C, 0-120 rpm activation 48-96
H adjusts pH to 5.5-8.5 every 12 h；Activation culture based formulas is 0.5-2.0 g/L NaCl, 0.5-2.0 g/L K2HPO4,
0.5-2.0 g/L KH2PO4, 1.0-5.0 g/L yeast powder, 1.0-3.0 g/L Dried Corn Steep Liquor Powder, 0.2-1.0 g/L
MgCl2·6H2O, 0.1-0.6 g/L NH4Cl, 0.01-0.05 g/L CaCl2·2H2O, 0.5-2.0 g/L FeCl2·
4H2O, 0.1-0.5 g/L KCl, xylan 30-90 g/L, solvent is water, adjusts pH to 5.5-8.5.
8. the method according to claim 1, wherein the Actinobacillus succinogenes of step (2) described activation
Vaccination ways are as follows: by the Actinobacillus succinogenes medium centrifugal of activation, the precipitating that centrifugation obtains being inoculated into step (1)
In obtained fermentation liquid, wherein the Actinobacillus succinogenes culture solution activated is identical as the fermentating liquid volume that step (1) obtains.
9. the method according to claim 1, wherein step (2) described fermentation condition are as follows: fermentation temperature 35-39
DEG C, fermentation time is 48 h, and fermentation pH is 6.0-7.5, and revolving speed is 100-200 rpm.
10. the method according to claim 1, wherein the activation item of step (2) described Actinobacillus succinogenes
Part is as follows: Actinobacillus succinogenes being inoculated into activation medium with 5 % v/v of inoculum concentration, 37 DEG C, 150 rpm activation
The Actinobacillus succinogenes culture solution that 12-24 h is activated；Activation culture based formulas is 0.5-2.0 g/L NaCl, 10-
20 g/L K2HPO4, 5-15 g/L NaH2PO4, 5-15 g/L NaHCO3, 1.0-5.0 g/L yeast powder, 1.0-5.0 g/L
40-70 g/L basic magnesium carbonate is added in Dried Corn Steep Liquor Powder, xylose 30-90 g/L, and solvent is water.
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| CN110964754A (en) * | 2019-12-31 | 2020-04-07 | 广西科学院 | Method for reducing proportion of succinic acid fermentation by-products of actinobacillus succinogenes |
| CN111073877A (en) * | 2019-05-20 | 2020-04-28 | 南京工业大学 | Xylanase with excellent temperature stability and pH tolerance and application thereof |
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| CN111073877A (en) * | 2019-05-20 | 2020-04-28 | 南京工业大学 | Xylanase with excellent temperature stability and pH tolerance and application thereof |
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