CN112522327A - Method for continuously preparing methanol by utilizing microorganisms with multi-substrate metabolic characteristics - Google Patents
Method for continuously preparing methanol by utilizing microorganisms with multi-substrate metabolic characteristics Download PDFInfo
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- CN112522327A CN112522327A CN202011084626.7A CN202011084626A CN112522327A CN 112522327 A CN112522327 A CN 112522327A CN 202011084626 A CN202011084626 A CN 202011084626A CN 112522327 A CN112522327 A CN 112522327A
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- methane
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- oxidizing bacteria
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 275
- 239000000758 substrate Substances 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 35
- 244000005700 microbiome Species 0.000 title claims abstract description 24
- 230000002503 metabolic effect Effects 0.000 title claims abstract description 19
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 94
- 241000894006 Bacteria Species 0.000 claims abstract description 54
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 44
- 230000001580 bacterial effect Effects 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 230000012010 growth Effects 0.000 claims abstract description 16
- 239000003112 inhibitor Substances 0.000 claims abstract description 15
- 230000004060 metabolic process Effects 0.000 claims abstract description 15
- 230000037353 metabolic pathway Effects 0.000 claims abstract description 8
- 239000002028 Biomass Substances 0.000 claims abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- 108010009977 methane monooxygenase Proteins 0.000 claims abstract description 6
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 5
- 230000001590 oxidative effect Effects 0.000 claims abstract description 5
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 38
- 108010048916 alcohol dehydrogenase (acceptor) Proteins 0.000 claims description 21
- 238000012258 culturing Methods 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 230000010261 cell growth Effects 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 230000003203 everyday effect Effects 0.000 claims description 7
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000011573 trace mineral Substances 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
- 235000013619 trace mineral Nutrition 0.000 claims description 4
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 3
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 3
- 238000007599 discharging Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 239000001630 malic acid Substances 0.000 claims description 3
- 235000011090 malic acid Nutrition 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 229940107700 pyruvic acid Drugs 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 229910021592 Copper(II) chloride Inorganic materials 0.000 claims description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 2
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 239000011565 manganese chloride Substances 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 abstract description 15
- 238000002360 preparation method Methods 0.000 abstract description 12
- 230000009471 action Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 239000007789 gas Substances 0.000 description 16
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- DSLRVRBSNLHVBH-UHFFFAOYSA-N 2,5-furandimethanol Chemical compound OCC1=CC=C(CO)O1 DSLRVRBSNLHVBH-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 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 5
- 239000008103 glucose Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 241000588697 Enterobacter cloacae Species 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- ZBFFNPODXBJBPW-VIFPVBQESA-N (R)-phenylacetylcarbinol Chemical compound CC(=O)[C@H](O)C1=CC=CC=C1 ZBFFNPODXBJBPW-VIFPVBQESA-N 0.000 description 2
- ZFUPOFQRQNJDNS-LBPRGKRZSA-N (s)-(4-chlorophenyl)-pyridin-2-ylmethanol Chemical compound C1([C@H](O)C=2N=CC=CC=2)=CC=C(Cl)C=C1 ZFUPOFQRQNJDNS-LBPRGKRZSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical compound COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- HFXDRFUDRMOUIC-UHFFFAOYSA-N ClC1=CC=C(C=C1)C=1C(=NC=CC1)C(=O)C1=NC=CC=C1C1=CC=C(C=C1)Cl Chemical compound ClC1=CC=C(C=C1)C=1C(=NC=CC1)C(=O)C1=NC=CC=C1C1=CC=C(C=C1)Cl HFXDRFUDRMOUIC-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000895244 Methylocella silvestris BL2 Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- NEHMKBQYUWJMIP-NJFSPNSNSA-N chloro(114C)methane Chemical compound [14CH3]Cl NEHMKBQYUWJMIP-NJFSPNSNSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000013527 degreasing agent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- IEJIGPNLZYLLBP-UHFFFAOYSA-N dimethyl carbonate Chemical compound COC(=O)OC IEJIGPNLZYLLBP-UHFFFAOYSA-N 0.000 description 1
- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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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
-
- 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
Abstract
The invention belongs to the technical field of methanol preparation, and relates to a method for continuously preparing methanol by utilizing multi-substrate metabolic characteristic microorganisms, wherein the methanol is prepared by using mixotrophic methane-oxidizing bacteria, and two substrates are provided for the mixotrophic methane-oxidizing bacteria during preparation: the methane in the methane sterile mixed gas enters microbial cells and is converted into methanol by methane monooxygenase, the polycarbon compound enters the microbial cells and is assimilated by a polycarbon metabolic pathway to maintain the normal growth metabolism and biomass synthesis of the mixotrophic methane oxidizing bacteria cells, and the test results of the OD value of the bacterial liquid and the yield of the methanol show that: when the methane sterile mixed gas and the acetic acid are used as double substrates, the growth speed of the bacteria is obviously improved, and the yield of the methanol is obviously improved, because the bacteria can oxidize the methane to produce the methanol under the action of the MDH inhibitor and can maintain the normal growth and metabolism of bacterial cells by utilizing the acetic acid in the presence of the acetic acid, so that the bacteria can continuously produce the methanol.
Description
The technical field is as follows:
the invention belongs to the technical field of methanol preparation, and particularly relates to a method for continuously preparing methanol by using a multi-substrate metabolic characteristic microorganism.
Background art:
methanol (Methanol): saturated monohydric alcohol with the simplest structure, also called hydroxy methane, is an organic compound, belongs to important chemical raw materials and high-quality fuels, has wide application, is used for manufacturing various organic products such as methyl chloride, methylamine, dimethyl sulfate and the like, producing formaldehyde and acetic acid, manufacturing methyl formate, methylamine and growth promoters, synthesizing dimethyl carbonate, glycol and methanol protein, is used as a cleaning degreaser and an analytical reagent, dissolving inorganic salt, mixing gasoline as a substitute fuel, producing dimethyl ether and the like. In general, carbon monoxide is industrially hydrogenated under pressure to prepare methanol, but the reaction conditions are severe, the process is complicated and the cost is high. In addition, the method of preparing methanol by using methane-oxidizing bacteria is an effective method, and the ideal yield of methanol can be obtained under laboratory conditions. The methane-oxidizing bacteria are microorganisms which grow by using methane as a carbon source and an energy source, when the methane enters bacterial cells, methane monooxygenase catalyzes the methane to be converted into methanol by using molecular oxygen, the methanol is further converted into formaldehyde under the action of Methanol Dehydrogenase (MDH), and the formaldehyde enters a subsequent metabolic pathway to be converted into biomass or carbon dioxide. In this process, methanol is an intermediate product of the growth and metabolism of methane-oxidizing bacteria by the addition of MDH inhibitors (MgCl)2NaCl, EDTA, phosphoric acid, etc.) can block further oxidation of methanol by MDH, allowing methanol to accumulate. Compared with the traditional chemical substance preparation method, the microbial preparation method for the chemical substance has the advantages of mild reaction conditions, simplicity, feasibility, low cost, environmental friendliness and the like, and has a good application prospect. For example: chinese patent 201910333029.4 discloses a method for producing 2, 5-furandimethanol, which comprises the following steps: carrying out biocatalysis treatment on 5-hydroxymethylfurfural by utilizing enterobacter cloacae to obtain 2, 5-furandimethanol, carrying out fermentation culture on a culture medium containing 5-hydroxymethylfurfural by utilizing the enterobacter cloacae, wherein the content of 5-hydroxymethylfurfural in the culture medium is 5-10 g/L, and the raw material is obtainedThe object catalytic treatment comprises the following steps: inoculating the enterobacter cloacae into a culture medium, and performing pre-culture so as to obtain a pre-culture solution; adding 5-hydroxymethylfurfural into the pre-culture solution, continuing to culture to obtain 2, 5-furandimethanol, wherein the culture medium contains 10-30 g/L of glucose, the enterobacter cloacae is subjected to activation treatment in advance to obtain a seed solution, and inoculating 3-6 vol% of the seed solution into the culture medium; optionally, the pre-culturing time is 4-8 hours; optionally, the pre-culturing and the culturing are respectively and independently carried out at the temperature of 30-40 ℃ and the rotating speed of 100-200 rpm; optionally, the culture medium comprises: 1-5 g/L of (NH4)2SO4, 0.5-1 g/L of K2HPO4, 0.1-0.5 g/L of KH2PO4, 0.5-2 g/L of yeast extract powder, 0.1-0.5 g/L of MgSO4 & 7H2O, 0.5-1.5 mL/L of trace elements, 1-5 mL/L of FeSO4 solution, and the pH value is 6.5, in the culture process, 5-hydroxymethylfurfural is supplemented, in the culture process, 5-hydroxymethylfurfural is fed until the final concentration of 5-hydroxymethylfurfural in a culture solution is maintained at 0-3 g/L; or in the culture process, when the concentration of the 5-hydroxymethylfurfural in the culture solution is 0-3 g/L, supplementing a 5-hydroxymethylfurfural solution with the final concentration of 5-6 g/L; chinese patent 201110419992.8 discloses a method for preparing (S) - (4-chlorophenyl) - (pyridin-2-yl) -methanol by microorganism catalysis, which comprises the following steps: (1) and (3) culturing wet thalli: the composition of the culture medium in g/L is as follows: 5-50 parts of glucose, 3-30 parts of yeast extract, 1-10 parts of potassium dihydrogen phosphate, 0.2-2 parts of magnesium sulfate heptahydrate, and the pH value of 4-9, fermenting and culturing for 1-5 days, and filtering the fermentation liquor to obtain wet thalli; (2) preparing a reaction system: 4-chlorphenyl- (pyridine-2-yl) -ketone is used as a substrate, PEG with different molecular weights and different inorganic salt solutions are used for preparing various aqueous two-phase reaction systems, the concentration of the substrate is 0.5-20 g/L, and the concentration of glucose is 10-100 g/L; (3) and (3) enzyme conversion reaction: adding CCTCC NO: carrying out enzyme reaction on the M2011385 wet thallus at 50-250g/L at 25-45 ℃ for 3-72 h; (4) and (3) post-treatment of the conversion solution: extracting the conversion solution with ethyl acetate, evaporating the organic phase obtained by extraction to recover the solvent, further separating and purifying by silica gel column chromatography, drying and dehydrating, evaporating to recover the solvent to obtain colorless oily liquid product (S) - (4-Chlorophenyl) - (pyridin-2-yl) -methanol; chinese patent 200710046918.X discloses a method for synthesizing L-phenylacetylcarbinol by microbial conversion in a cloud point system, which comprises the following steps: the first step, microbial fermentation is utilized in a culture medium to obtain microbial cells with biological activity as a biocatalyst for microbial transformation; secondly, constructing a cloud point system by using the microbial cells obtained in the first step, setting operation parameters of microbial transformation in the cloud point system, including acetaldehyde, benzaldehyde as a substrate, the amounts of the microbial cells and glucose and the pH value, and then realizing the microbial transformation in the cloud point system under the set operation conditions to obtain L-phenylacetylcarbinol; the cloud point system is characterized in that nonionic surfactants TritonX-114 and TritonX-45 are mixed according to a volume ratio of 1: 0.1-0.4, and then mixing with an aqueous solution to form a cloud point system, wherein the volume fraction of the mixed surfactant is 10-14%; or mixing surfactant TritonX-100 and polyethylene glycol, and mixing with 100ml of water solution to form a cloud point system, wherein the volume fraction of the surfactant and the polyethylene glycol is 10-20%; the aqueous solution, 100ml of which contains: 2g of peptone, 2g of yeast powder, 0.4-1 ml of acetaldehyde, 2-11g of microbial cells, 0.3-1.2ml of benzaldehyde and 1.2-6g of glucose, wherein the pH is controlled between 4 and 6. In the prior art, the technology for preparing methanol by microorganisms can only carry out batch tests under laboratory conditions, but in actual production, large-scale continuous preparation cannot be realized because: the methanol production and the assimilation of microbial cells share one path, and when the MDH inhibitor inhibits the activity of MDH, the intermediate product methanol can be accumulated in a short time, but the further assimilation of the methanol by the microbes is greatly interfered, so that the microbes cannot complete the subsequent metabolic path, the growth and metabolism of the microbes are influenced, the long-term, stable and continuous production of the methanol cannot be realized, and the large-scale industrial application of the technology for preparing the methanol by the microbes is greatly limited.
In recent years, mixotrophic methane-oxidizing bacteria have been discovered and known, which subverts the concept that methane-oxidizing bacteria can only utilize methane as a sole carbon source. Compared with the heterotrophic methane-oxidizing bacteria, the mixotrophic methane-oxidizing bacteria have wider substrate range, can utilize methane and also can utilize multi-carbon compounds (acetic acid, pyruvic acid, succinic acid, malic acid, ethanol and the like) as carbon sources and energy sources. Because the mixotrophic methane-oxidizing bacteria have the capability of utilizing various substrates, although the MDH inhibitor can inhibit a methane oxidation channel, the utilization channel of the multi-carbon compound is not influenced, so that the microorganism can utilize the multi-carbon compound to maintain normal growth and metabolism while oxidizing methane to prepare methanol, and the methanol preparation and cell growth are hopeful to be considered. At present, the research on the preparation of methanol by using the mixotrophic methane-oxidizing bacteria is not reported, and the potential application value of the multi-substrate utilization characteristic of the mixotrophic methane-oxidizing bacteria in the process of preparing methanol is deeply excavated, so that the stable and continuous preparation of methanol by using microorganisms is realized, the large-scale industrial application of the technology for preparing methanol by using microorganisms is promoted, and the method has high economic benefit and environmental protection significance.
The invention content is as follows:
the invention aims to overcome the defects of the prior art and seek to design a method for continuously preparing methanol by utilizing a microorganism with multiple substrate metabolic characteristics, wherein the microorganism is utilized to prepare the methanol by oxidizing methane and maintain the cell growth by multi-carbon compounds at the same time, thereby realizing the aim of stably and continuously preparing the methanol.
In order to achieve the above object, the present invention relates to a method for continuously preparing methanol by using microorganisms with multiple substrate metabolic characteristics, which comprises the following steps:
firstly, two substrates are provided for the mixotrophic methane-oxidizing bacteria: methane sterile mixed gas and multi-carbon compounds are added, an MDH inhibitor is added, and NMS (nitrate mineral salt) culture solution is adopted for culture to obtain bacterial solution;
secondly, after methane in the methane sterile mixed gas enters cells of the mixotrophic methane oxidizing bacteria, the methane is converted into methanol by methane monooxygenase, and the MDH activity of the methanol is inhibited and cannot be further oxidized, so that the methanol is accumulated; meanwhile, the multi-carbon compound enters a multi-carbon metabolic pathway in the cells of the mixotrophic methane-oxidizing bacteria to be oxidized and utilized so as to maintain the normal growth metabolism and biomass synthesis of the cells of the mixotrophic methane-oxidizing bacteria;
thirdly, detecting the mass percent concentration of the methanol in the bacterial liquid every day at the initial stage of culturing the mixotrophic methane-oxidizing bacteria, determining that the cells of the mixotrophic methane-oxidizing bacteria start to accumulate the methanol when the mass percent concentration of the methanol continuously rises for three days, and then performing feeding and discharging operations every day: taking out 15-20% of the total volume of the bacterial liquid, adding NMS culture solution with the same volume, and determining that the fermentation period is finished when the mass percentage concentration value of methanol in the bacterial liquid does not rise any more and the concentration values of methane at the gas inlet and the gas outlet are not changed;
fourthly, separating and purifying the methanol.
The mixotrophic methane-oxidizing bacteria have the characteristic of multi-substrate metabolism, and can prepare methanol by using methane and maintain cell growth by using multi-carbon compounds.
The methane sterile mixed gas is prepared by mixing methane and air according to the volume ratio of 1: 4.
The molar concentration of the polycarbon compound related to the invention is 5-50mmol L-1The method can provide carbon source and energy source for the mixotrophic methane-oxidizing bacteria, and maintain cell growth metabolism, and comprises the following steps: acetic acid, pyruvic acid, succinic acid, malic acid and ethanol.
The molar concentration of the MDH inhibitor related by the invention is 10-100mmol L-1Comprising MgCl2NaCl, EDTA and phosphoric acid can inhibit the activity of methanol dehydrogenase in the cells of the mixotrophic methane-oxidizing bacteria, so that methanol is difficult to be assimilated by the cells, and the methanol is accumulated.
The NMS culture solution is distilled water and MgSO4x7H2O、CaCl2x6H2O、Fe(III)NH4-EDTA、NaNO3Trace element solution, phosphoric acid buffer solution and KH2PO4And Na2HPO4x12H2And O.
Compared with the prior art, the method for preparing the methanol by using the mixotrophic methane-oxidizing bacteria has the advantages that two substrates are provided for the mixotrophic methane-oxidizing bacteria during preparation: methane in the methane sterile mixed gas enters microbial cells and is converted into methanol by methane monooxygenase; the polycarbon compound enters the microbial cells and is assimilated by a polycarbon metabolic pathway to maintain the normal growth metabolism and biomass synthesis of the cells of the mixotrophic methane-oxidizing bacteria, so that the problems that the cell growth is inhibited and the continuous preparation cannot be carried out when the methanol is prepared by the heterotrophic methane-oxidizing bacteria are solved, and the method has high economic benefit and environmental protection significance for large-scale industrial application of the technology for preparing the methanol by the microbes.
Description of the drawings:
FIG. 1 is a schematic view of a curve showing changes of OD values of a bacterial liquid according to the present invention with time.
FIG. 2 is a schematic diagram of a methanol concentration of a bacterial liquid according to the present invention changing with time.
The specific implementation mode is as follows:
the invention is further described below by way of an embodiment example in conjunction with the accompanying drawings.
Example 1:
the specific process of the method for continuously preparing methanol by utilizing the microorganisms with the multi-substrate metabolic characteristics comprises the following steps:
firstly, two substrates are provided for the mixotrophic methane-oxidizing bacteria: methane sterile mixed gas and multi-carbon compounds are added, an MDH inhibitor is added, and NMS culture solution is adopted for culture to obtain bacterial solution;
1) methylocella silvestris BL2 purchased from a DSMZ website in Germany is selected as the mixotrophic methane-oxidizing bacteria;
2) preparing an NMS culture solution: 0.2g MgSO 2 dissolved in 1000mL distilled water in that order4x7H2O、0.006g CaCl2x6H2O、4mg Fe(III)NH4-EDTA、0.2g NaNO30.5mL of trace element solution, 5mL of phosphate buffer, 0.272g of KH2PO4、0.717g Na2HPO4x12H2O, completing the preparation of NMS culture solution;
wherein the microelement solution is prepared by dissolving 10mg of ZnSO in 1000mL of distilled water in sequence4x7H2O、3mg MnCl2x4H2O、30mg H3BO3、1mg CuCl2x6H2O、20mg CaCl2x2H2O、3mg Na2MoO4x2H2O is prepared; the phosphate buffer solution was prepared by dissolving 28.71g of NaH in 1000mL of distilled water in this order2PO4x2H2O、5.73g Na2HPO4x12H2O is prepared into solution with pH value of 5.8;
3) pre-culturing: 50mL of sterile NMS culture solution was placed in a 250mL fermenter, and 5mL of Methylcella silvestris BL2 bacterial solution (dry cell weight 2-3g L) was added by injecting sterile mixed gas-1) Inoculating into a culture bottle, placing into a shaking table, and culturing at 140rpm and 25 ℃ for a week to obtain pre-cultured bacterial liquid;
4) inoculation: inoculating 50mL of the bacterial liquid pre-cultured in the step 3) into 500mL of the bacterial liquid containing the MDH inhibitor (the molar concentration is 20mmol L)-1MgCl of2) And polycarbon compound (molar concentration 10mmol L)-1Acetic acid) in NMS culture solution, continuously introducing sterile mixed gas, and culturing at 25 deg.C;
secondly, after methane in the methane sterile mixed gas enters Methocella silvestris BL2 cells, the methane is converted into methanol by methane monooxygenase, the MDH activity of the methanol is inhibited and cannot be further oxidized, so that the methanol is accumulated; meanwhile, the polycarbon compound enters a polycarbon metabolic pathway in Methocella silvestris BL2 cells and is oxidized and utilized so as to maintain the normal growth metabolism and biomass synthesis of Methocella silvestris BL2 cells;
thirdly, detecting the mass percent concentration of methanol in the bacterial liquid every day in the initial stage of culturing Methocella silvestris BL2, determining that the cells of the mixotrophic methane-oxidizing bacteria start to accumulate methanol when the mass percent concentration of methanol continuously rises for three days, and then performing feeding and discharging operations every day: taking out 15-20% of the total volume of the bacterial liquid, adding a culture solution with the same volume, and determining that the fermentation period is finished when the mass percentage concentration value of methanol in the bacterial liquid does not rise any more and the volume percentage concentration value of methane at the gas inlet and the gas outlet is not changed;
fourthly, separating and purifying the methanol.
Example 2:
the embodiment relates to a method for testing the OD value of a bacterial liquid and the yield of methanol when the methanol is continuously prepared by utilizing multi-substrate metabolic characteristic microorganisms:
using a 250mL fermentor, 5mL of the broth (dry cell weight: 2-3g L) pre-cultured in step 3) of example 1 was placed in a flask-1) The mixture was inoculated into 45mL of a mixture containing MDH inhibitor (molar concentration 20mmol L)-1MgCl of2) The NMS culture solution of (2) is prepared by adding 200mL of sterile methane mixture as substrate to the top of one group, adding 200mL of sterile methane mixture to the top of the other group, and adding 10mmol L of sterile methane mixture to the NMS culture solution-1The acetic acid is used as a double substrate, the fermentation bottle is sealed and put into a shaking table to be cultured under the conditions of 140rpm and 25 ℃, and three parallel substances are arranged in each group;
an ultraviolet-visible spectrophotometer is adopted to monitor the OD (optical density) value of the bacteria liquid at 600nm to obtain a curve graph of the change of the OD value of the bacteria liquid along with time as shown in figure 1, and the graph shows that when the methane sterile mixed gas is used as a single substrate, the growth rate of bacteria is slow, and the change curve of the OD value of the bacteria liquid is fast and smooth, because the MDH inhibitor inhibits the activity of MDH enzyme, the metabolic pathway of the bacteria to the methane is blocked, and the growth of the bacteria is adversely affected; when the methane sterile mixed gas and the acetic acid are used as double substrates, the growth speed of the bacteria is obviously improved, because when the acetic acid exists, the MDH inhibitor cannot block the metabolic pathway of the bacteria to the acetic acid, so that the normal growth and metabolism of bacterial cells can be maintained;
taking out 1mL of bacterial liquid every day, and detecting the mass percent concentration of methanol in the bacterial liquid by using a gas chromatograph (Agilent 6890N) with a hydrogen flame detector to obtain a curve graph of the change of the methanol concentration of the bacterial liquid along with time as shown in figure 2, wherein the graph shows that when the methane sterile mixed gas is used as a single substrate, the yield of the methanol is extremely low because the MDH inhibitor seriously inhibits the normal growth and metabolism of bacteria, so that the bacteria can not continuously produce the methanol; when the methane sterile mixed gas and the acetic acid are used as double substrates, the yield of the methanol is obviously improved, because under the condition of the existence of the acetic acid, the bacteria can oxidize the methane to produce the methanol under the action of an MDH inhibitor, and can also utilize the acetic acid to maintain the normal growth and metabolism of bacterial cells, so that the bacteria can continuously produce the methanol.
Claims (10)
1. A method for continuously preparing methanol by utilizing microorganisms with multiple substrate metabolic characteristics is characterized in that the specific technological process comprises the following steps:
firstly, two substrates are provided for the mixotrophic methane-oxidizing bacteria: methane sterile mixed gas and multi-carbon compounds are added, an MDH inhibitor is added, and NMS culture solution is adopted for culture to obtain bacterial solution;
secondly, after methane in the methane sterile mixed gas enters cells of the mixotrophic methane oxidizing bacteria, the methane is converted into methanol by methane monooxygenase, and the MDH activity of the methanol is inhibited and cannot be further oxidized, so that the methanol is accumulated; meanwhile, the multi-carbon compound enters a multi-carbon metabolic pathway in the cells of the mixotrophic methane-oxidizing bacteria to be oxidized and utilized so as to maintain the normal growth metabolism and biomass synthesis of the cells of the mixotrophic methane-oxidizing bacteria;
thirdly, detecting the mass percent concentration of the methanol in the bacterial liquid every day at the initial stage of culturing the mixotrophic methane-oxidizing bacteria, determining that the cells of the mixotrophic methane-oxidizing bacteria start to accumulate the methanol when the mass percent concentration of the methanol continuously rises for three days, and then performing feeding and discharging operations every day: taking out 15-20% of the total volume of the bacterial liquid, adding NMS culture solution with the same volume, and determining that the fermentation period is finished when the mass percentage concentration value of methanol in the bacterial liquid does not rise any more and the concentration values of methane at the gas inlet and the gas outlet are not changed;
fourthly, separating and purifying the methanol.
2. The method according to claim 1, wherein the mixotrophic methane-oxidizing bacteria has a multi-substrate metabolic property, and can produce methanol from methane while maintaining cell growth by using a multi-carbon compound.
3. The method for continuously preparing methanol by utilizing microorganisms with multiple substrate metabolic characteristics according to claim 2, wherein the sterile methane mixed gas is a sterile mixed gas formed by mixing methane and air according to a volume ratio of 1: 4.
4. The method for continuously preparing methanol by using microorganisms with multiple substrate metabolic properties according to claim 3, wherein the molar concentration of the polycarbon compound is 5-50mmol L-1The method can provide carbon source and energy source for the mixotrophic methane-oxidizing bacteria, and maintain cell growth metabolism, and comprises the following steps: acetic acid, pyruvic acid, succinic acid, malic acid and ethanol.
5. The method for continuously preparing methanol by using microorganisms with multiple substrate metabolic properties according to claim 4, wherein the molar concentration of the MDH inhibitor is 10-100mmol L-1Comprising MgCl2NaCl, EDTA and phosphoric acid, can inhibit the activity of methanol dehydrogenase in cells of the mixotrophic methane-oxidizing bacteria.
6. The method for continuously preparing methanol by using microorganisms with multiple substrate metabolic characteristics according to claim 5, wherein NMS culture solution is distilled water, MgSO4x7H2O、CaCl2x6H2O、Fe(III)NH4-EDTA、NaNO3Trace element solution, phosphoric acid buffer solution and KH2PO4And Na2HPO4x12H2And O.
7. The method for continuously preparing methanol using microorganisms with multi-substrate metabolic characteristics according to claim 1 or 2, wherein the mixotrophic methane-oxidizing bacteria is a dry cell with a weight of 2-3g L-1Methylocella silvestris BL2 bacterial liquid.
8. The method for continuously preparing methanol using microorganisms with multi-substrate metabolic characteristics according to claim 1 or 6, wherein NMS culture solution is 0.2g MgSO 2 dissolved in 1000mL distilled water sequentially4x7H2O、0.006g CaCl2x6H2O、4mg Fe(III)NH4-EDTA、0.2g NaNO30.5mL of trace element solution, 5mL of phosphate buffer, 0.272g of KH2PO4、0.717g Na2HPO4x12H2And O prepared culture solution.
9. The method for continuously preparing methanol by using microorganisms with multi-substrate metabolic characteristics according to claim 8, wherein the solution of trace elements is prepared by sequentially dissolving 10mg of ZnSO in 1000mL of distilled water4x7H2O、3mg MnCl2x4H2O、30mg H3BO3、1mg CuCl2x6H2O、20mg CaCl2x2H2O、3mg Na2MoO4x2H2And O is configured.
10. The method for continuously preparing methanol using microorganisms with multiple substrate metabolic properties according to claim 8, wherein the phosphate buffer is prepared by sequentially dissolving 28.71g NaH in 1000mL of distilled water2PO4x2H2O、5.73g Na2HPO4x12H2O is prepared into a solution with the pH value of 5.8.
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