CN105838652B - Strain for enhancing glycerol metabolism and application thereof - Google Patents
Strain for enhancing glycerol metabolism and application thereof Download PDFInfo
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- CN105838652B CN105838652B CN201610382379.6A CN201610382379A CN105838652B CN 105838652 B CN105838652 B CN 105838652B CN 201610382379 A CN201610382379 A CN 201610382379A CN 105838652 B CN105838652 B CN 105838652B
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- bacterial strain
- organic acid
- strain
- actinobacillus succinogenes
- glycerol
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 92
- 230000004060 metabolic process Effects 0.000 title claims abstract description 10
- 230000002708 enhancing effect Effects 0.000 title abstract 2
- 238000000855 fermentation Methods 0.000 claims abstract description 67
- 241000948980 Actinobacillus succinogenes Species 0.000 claims abstract description 62
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 150000007524 organic acids Chemical class 0.000 claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims description 54
- 238000011218 seed culture Methods 0.000 claims description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000000843 powder Substances 0.000 claims description 29
- 239000001963 growth medium Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 27
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- 239000001110 calcium chloride Substances 0.000 claims description 14
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 14
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 14
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 14
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 14
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 13
- 210000002966 serum Anatomy 0.000 claims description 13
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 12
- 239000001632 sodium acetate Substances 0.000 claims description 12
- 235000017281 sodium acetate Nutrition 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 11
- 230000004913 activation Effects 0.000 claims description 9
- KVYRCBOUKXJXDK-UHFFFAOYSA-N 3,4-dimethylphenazine-1,2-diamine hydrochloride Chemical compound Cl.C1=CC=CC2=NC3=C(C)C(C)=C(N)C(N)=C3N=C21 KVYRCBOUKXJXDK-UHFFFAOYSA-N 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 241000196324 Embryophyta Species 0.000 claims description 7
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 2
- 230000005518 electrochemistry Effects 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 230000003014 reinforcing effect Effects 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 239000001384 succinic acid Substances 0.000 abstract description 5
- 235000005985 organic acids Nutrition 0.000 abstract description 2
- 238000002703 mutagenesis Methods 0.000 description 25
- 231100000350 mutagenesis Toxicity 0.000 description 23
- 239000000047 product Substances 0.000 description 14
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 239000012531 culture fluid Substances 0.000 description 8
- 230000035772 mutation Effects 0.000 description 6
- 239000001569 carbon dioxide Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 238000005138 cryopreservation Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 231100000225 lethality Toxicity 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000013028 medium composition Substances 0.000 description 5
- 231100000219 mutagenic Toxicity 0.000 description 5
- 230000003505 mutagenic effect Effects 0.000 description 5
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000606750 Actinobacillus Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 235000011044 succinic acid Nutrition 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- CRTGSPPMTACQBL-UHFFFAOYSA-N 2,3-dihydroxycyclopent-2-en-1-one Chemical compound OC1=C(O)C(=O)CC1 CRTGSPPMTACQBL-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000003225 biodiesel Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009466 transformation Effects 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
- 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
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
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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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- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a strain for enhancing glycerol metabolism, which is classified and named as actinobacillus succinogenes (A.succinogenes)Actinobacillussuccinogenes) JF1315, which has been deposited at the China center for type culture Collection at 31/3 in 2016, with the deposit numbers: CCTCC NO: m2016160. The invention also relates to application of the actinobacillus succinogenes JF1315 in fermentation production of organic acid. The strain can grow and metabolize under the condition of lower pH compared with a control strain under the anaerobic condition, and can efficiently utilize glycerol to ferment and synthesize reduced organic acid. Under the condition of pH4.8-6.8, the strain can grow normally and metabolize glucose to synthesize organic acids such as succinic acid and the like; in addition, under the condition, the strain can efficiently utilize the glycerol to carry out anaerobic fermentation, synthesize and accumulate the organic acid.
Description
Technical field
The bacterial strain for strengthening glycerol metabolism the present invention relates to one plant and its application belong to industrial microorganism and its fermentation technique neck
Domain.
Background technique
During traditional field of microbial fermentation engineering practice, generally mostly sent out by carbon source of glucide
Ferment, such as cornstarch hydrolyzate；Product is carried out by raw material of glucose for certain high value added products, or even directly
Synthesis.Although the product of higher concentration can be obtained when being fermented using raw materials such as glucose, which greatly increases
The economic cost of entire production process, causes the waste of resource.In order to solve this problem, it can attempt to cheap biomass
Material (such as cellulosic hydrolysate or glycerol) substitution glucose ferment, but in cellulosic hydrolysate noxious material presence
And concentration is lower greatly limits their application for sugar.
With the rapid development of field of biodiesel oil, the synthetic quantity of glycerol is huge, and (1 ton of biodiesel can produce 100,000
Gram glycerol), and for glycerol, the reducing power of intramolecular storage is higher, is conducive to the yield and receipts that improve reproducibility product
Rate.During reduced form Product formation, when using glucose as sole carbon source, when glucose is metabolized through glycolytic pathway
2 molecule reducing powers (NADH) are generated, the reducing power for strong reduced form Product formation can be obvious insufficient, and product yield reduces；And
When using glycerol as carbon source, glycerol can generate 2 molecule reducing powers (NADH) through metabolism, herein on basis, when strain growth
A part of NADH can be synthesized, a large amount of NADH will increase the accumulation of reduced form product.But if the reproducibility of synthetic product is lower
(such as succinic acid) can largely accumulate NADH in thallus, and superfluous reducing power can inhibit the growth of microbial cells at this time.In order to flat
Weigh coenzyme intracellular metabolism, restore the synthesis for guaranteeing reduced form product while thalli growth, by way of mutagenesis to bacterial strain into
Row transformation, the bacterial strain of function admirable is obtained by the screening of felicity condition.
Summary of the invention
One of technical purpose of the invention is to provide one plant of production succinic acid that anaerobic fermentation can be efficiently carried out using glycerol
Actinobacillus.
To realize the above-mentioned technical purpose, the present invention adopts the following technical scheme that:
One plant reinforcing glycerol metabolism bacterial strain, classification naming be Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315, China typical culture collection center, deposit number are preserved on March 31st, 2016
Are as follows: CCTCC NO:M 2016160.
Actinobacillus succinogenes JF1315 of the present invention is by starting strain Actinobacillus succinogenes
(Actinobacillus succinogenes) NJ113 (has been disclosed in the patent of same inventor's earlier application, the patent No.
ZL200610085415.9, saving number is CGMCC No.1716) after ARTP mutagenesis, pass through o- nitrobenzene-β-d- gala
It is obtained after glucosides (ONPG) and fluorescent dye NPN screening.Bacterial strain verifies its glycerol through anaerobic fermentation again and utilizes and produce acid after screening
Ability.
Steps are as follows for specific mutagenesis screening:
ARTP mutagenesis: starting strain Actinobacillus succinogenes NJ113 is inoculated in the anaerobism blood equipped with seed culture medium
The 6-12 hours bacterial strains to obtain logarithmic growth phase are cultivated in clear bottle.Seed culture fluid is carried out suitably to be diluted to OD660=0.5-
It is coated on after 1.5 through high-temperature sterilization and on ice on loading iron plate to the cold and carries out mutagenesis.Mutagenic condition is chosen are as follows: is made with helium
For carrier gas, throughput 10SLM, power 80-120W, mutation time 0-300s, and measures lethality and draw the lethal song of mutagenesis
Line.On the basis of destruction curve, the time of lethality larger (90% or more) is chosen for mutation time, to bacterial strain under the same terms
Carry out mutagenic treatment.
Single colonie separates after mutagenesis: the micro slide for being coated with seed culture fluid after mutagenesis is put into sterile saline,
It is coated on after mixing in solid plate culture medium, 35-37 DEG C separates single colonie after culture 8-15 hours.Single colonie is inoculated in and is contained
Have in the anaerobism serum bottle of seed culture medium, 35-37 DEG C obtains seed culture fluid after culture 8-15 hours.
O- nitrobenzene-beta-d-galactopyranoside glycosides (ONPG) screening: seed culture fluid is diluted to OD660=0.5-2.0, takes 50-
After 200 microlitres of dilutions are mixed with 5-15 microlitres of ONPG, absorbance is detected under 405nm after reacting 2 hours at 35-37 DEG C
Value, and the higher bacterial strain of absorbance value is saved.
Fluorescent dye NPN screening: the above-mentioned bacterial strain that screens is carried out after Anaerobic culturel obtains seed liquor, to seed liquor into
Row is diluted to OD660=0.5-2.0 after taking 1-3 mL dilution to mix with 10-30 microlitres of NPN, reacts 2-10 points at 35-37 DEG C
The size of fluorescent value is detected after clock, the lower bacterial strain of picking fluorescence angle value is saved, and bacterial strain as provided by the invention produces amber
Amber acid Actinobacillus JF1315.
The formula of above-mentioned solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/ of Dried Corn Steep Liquor Powder
L, 10 g/L of yeast powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L.
Another technical purpose of the invention is application of the bacterial strain JF1315 in production organic acid.
The present invention provides a kind of methods using organic acids such as bacterial strain anaerobic fermentation provided by the invention production succinic acid.
The anaerobic fermentation method is as follows:
Actinobacillus succinogenes (Actinobacillus succinogenes) experiment of YJ1315 anaerobic fermentation: this hair
The bright actication of culture, seed culture step are conventional Actinobacillus actication of culture method and seed culture method, this hair
It is bright it is middle by Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 bacterial strain is through solid plate culture medium
After activation, under the conditions of 37 DEG C, switching is in seed culture medium after cultivating 12-14 hours in anaerobism serum bottle, at 37 DEG C, 200 turns/
It is cultivated 6-8 hours under conditions of minute and obtains seed liquor.
By the seed liquor according to 6-10 %(v/v) inoculum concentration be inoculated in the anaerobism serum containing the fermentation medium
In bottle.
Above-mentioned fermentative medium formula are as follows: glycerol 10-60 g/L, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, second
Sour 1.36 g/L of sodium, dimethyl diaminophenazine chloride 0.1-2.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L,
NaH2PO4 1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L。
The formula of above-mentioned solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/ of Dried Corn Steep Liquor Powder
L, 10 g/L of yeast powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L.
For the present invention by carrying out ARTP mutagenesis to starting strain, screening obtains one plant of bacterial strain that can efficiently utilize glycerol
JF1315, compared with starting strain, the beneficial effect is that:
The present invention is obtained one plant and in the fermentation medium can by ARTP mutagenesis means and reasonable bacterial strain screening method
The bacterial strain for efficiently growing and being metabolized using glycerol.When using glycerol as primary carbon source, bacterial strain can use glycerol under anaerobic condition
Grow and synthesize reduced form metabolite: after fermenting 48 hours in complex medium under anaerobic condition, glycerol consumption is reachable
60 g/L, total reductic acid cumulative amount is up to 97 g/L.Demonstrate the growth and metabolism that can change thallus by ARTP mutagenesis means
Performance reduces reducing power level intracellular, restores the growth ability of cell, while being conducive to reduced form product on basis herein
Accumulation and electric energy generation.
Detailed description of the invention
Starting strain destruction curve when Fig. 1 is ARTP mutagenesis.
Biomaterial of the present invention, classification naming be Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315, be preserved in on March 31st, 2016 China typical culture collection center (abbreviation CCTCC,
Address: the Chinese Wuhan Wuhan University), deposit number are as follows: CCTCC NO:M 2016160.
Specific embodiment
According to following embodiment, the present invention can be better understood.Specific material proportion described in case study on implementation,
Process conditions and its result are merely to illustrate the present invention, without that should will not limit sheet described in detail in claims
Invention.
Embodiment 1
This example demonstrates that the construction method of Actinobacillus succinogenes JF1315 bacterial strain of the present invention.
The present invention screen JF1315 bacterial strain use starting strain Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 strain has been disclosed in the patent of same inventor's earlier application, the patent No.
ZL200610085415.9, saving number is CGMCC No.1716.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 bacterial strain is by production succinic acid unwrapping wire
Bacillus (Actinobacillus succinogenes) NJ113 is through ARTP mutagenesis screening.
Steps are as follows for specific mutagenesis screening:
ARTP mutagenesis: starting strain Actinobacillus succinogenes NJ113 is inoculated in the anaerobism blood equipped with seed culture medium
The 6-12 hours bacterial strains to obtain logarithmic growth phase are cultivated in clear bottle.Seed culture fluid is carried out suitably to be diluted to OD660=0.5-
It is coated on after 1.5 on micro slide to the cold and carries out mutagenesis.Mutagenic condition is chosen are as follows: using helium as carrier gas, throughput 10SLM,
Power 80-120W, mutation time 0-300s, and measure lethality and draw mutagenesis destruction curve.On the basis of destruction curve,
The time for choosing lethality larger (90% or more) is mutation time, carries out mutagenic treatment to bacterial strain under the same terms.
Starting strain destruction curve when Fig. 1 is ARTP mutagenesis.Under identical mutagenesis intensity, mutation time has been investigated to bacterial strain
The influence of growth.It will be seen from figure 1 that mutation time is longer, Strain survival rate is lower, and mutagenesis lethality is higher.
Single colonie separates after mutagenesis: the micro slide for being coated with seed culture fluid after mutagenesis is put into sterile saline,
It is coated in solid plate after mixing, 35-37 DEG C separates single colonie after culture 8-15 hours.Single colonie is inoculated in containing seed
In the anaerobism serum bottle of culture medium, 35-37 DEG C obtains seed culture fluid after culture 8-15 hours.
O- nitrobenzene-beta-d-galactopyranoside glycosides (ONPG) screening: seed culture fluid is diluted to OD660=0.5-2.0, takes 50-
After 200 microlitres of dilutions are mixed with 5-15 microlitres of ONPG, absorbance is detected under 405nm after reacting 2 hours at 35-37 DEG C
Value, and the higher bacterial strain of absorbance value is saved.
Fluorescent dye NPN screening: the above-mentioned bacterial strain that screens is carried out after Anaerobic culturel obtains seed liquor, to seed liquor into
Row is diluted to OD660=0.5-2.0 after taking 1-3 mL dilution to mix with 10-30 microlitres of NPN, reacts 2-10 points at 35-37 DEG C
The size of fluorescent value is detected after clock, the lower bacterial strain of picking fluorescence angle value is saved, and bacterial strain as provided by the invention produces amber
Amber acid Actinobacillus JF1315.
The formula of above-mentioned solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/ of Dried Corn Steep Liquor Powder
L, 10 g/L of yeast powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L.
Embodiment 2
This example demonstrates that the physiological and biochemical property for the Actinobacillus succinogenes JF1315 that above-described embodiment 1 obtains, tool
Body is as follows:
Bacterial strain and original bacteria after mutagenesisActinobacillussuccinogenesNJ113 colonial morphology and growth performance
And no significant difference: bacterial strain Gram-negative, flat-plate bacterial colony is rounded, and neat in edge is smooth, metabolizable grape under anaerobic condition
Sugar, xylose synthesize organic acid, wherein the group of main acid becomes succinic acid, acetic acid, lactic acid and formic acid.Bacterial strain is glutamic acid deficiency
Bacterial strain need to add glutamic acid when growing on synthetic media.
The genetic stability of the Actinobacillus succinogenes JF1315 of embodiment 1 is tested, and strain passage fermenting experiment is as follows
It is shown.
Bacterial strain after mutagenesisActinobacillus succinogenesJF1315 under the conditions of low pH (5.0-6.0) into
Row continuous passage culture, the period sampling measuring difference generation constantly cell growth of cell and product acid activity at low ph conditions, knot
Fruit is as shown in table 1 below.After cell continuous passage culture to 10 generation, growth and product acid activity are uninfluenced in acid condition.
The growth of passage cell and product acid activity under 1. low ph condition of table
Embodiment 3
This example demonstrates that bacterial strain after mutagenesis in the present inventionActinobacillus succinogenesJF1315 is compared
The superiority of starting strain.
By Actinobacillus succinogenes NJ113 and JF1315 bacterial strain by being inoculated with after the culture of solid plate culture medium in the present invention
Into seed culture medium, culture obtains seed liquor；Then seed liquor is inoculated into low pH fermentation medium and carries out anaerobic fermentation.
The method may include following steps:
(1) Actinobacillus succinogenes NJ113 and JF1315 bacterial strain are forwarded to anaerobism after the activation of solid plate culture medium
Serum bottle, 37 DEG C, switching is in seed culture medium after cultivating 12-14 hours under anaerobic condition, at 37 DEG C, 200 revs/min of condition
Lower culture obtains seed liquor in 6-8 hours；
(2) above-mentioned seed liquor is inoculated according to the inoculum concentration of 6-10 % (v/v) containing low pH fermentation medium (pH5.8)
Serum bottle in, as a child sampled in 37 DEG C of progress anaerobic fermentations 48, and measured cell concentration and organic acid content.
The formula of above-mentioned solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/ of Dried Corn Steep Liquor Powder
L, 10 g/L of yeast powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L.
The formula of above-mentioned fermentation medium are as follows: 10 g/L of glucose, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, second
Sour 1.36 g/L of sodium, dimethyl diaminophenazine chloride 0.1-2.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L,
NaH2PO4 1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L
The organic acid concentration of detection is as shown in table 2:
Table 2
It, can be within the scope of pH=4.8-6.8 according to table 2, either original strain NJ113 or mutagenic strain JF1315
Survival and fermentation production of organic acid, and reach optimal growth and metabolism performance in pH=6.8.
Embodiment 4 is this example demonstrates that the method that bacterial strain anaerobic fermentation produces organic acid in the present invention.
By Actinobacillus succinogenes bacterial strain by being seeded to seed culture after the culture of solid plate culture medium in the present invention
Culture obtains seed liquor in base；Then seed liquor is inoculated into fermentation medium and carries out anaerobic fermentation.The method can wrap
Include following steps:
(1) Actinobacillus succinogenes bacterial strain is forwarded to anaerobism serum bottle after the activation of solid plate culture medium, 37 DEG C, detests
Switching is cultivated 6-8 hours under conditions of 37 DEG C, 200 revs/min in seed culture medium after cultivating 12-14 hours under the conditions of oxygen
Obtain seed liquor；
(2) above-mentioned seed liquor is inoculated in the dress of the electrochemistry containing fermentation medium according to the inoculum concentration of 6-10 % (v/v)
In setting, in 37 DEG C of progress anaerobic fermentations.
(3) it carries out sterile sampling at regular intervals during the fermentation, to measurement carbon source after sample centrifugal treating and has
Machine acid concentration.
The formula of above-mentioned solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/ of Dried Corn Steep Liquor Powder
L, 10 g/L of yeast powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L.
Embodiment 5
This example demonstrates that by Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 progress
The method of anaerobic fermentation production organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 anaerobic fermentation method is as follows:
By in cryopreservation tube Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 by implement
4 the method for example is activated, after activation and after seed culture 12 hours, secondary seed culture 10 hours, will be planted
It is inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control is 6.8) after sub- liquid, while being passed through carbon dioxide 2 and dividing
Clock is to guarantee anaerobic environment.Fermentation retains supernatant after being centrifuged fermentation broth sample after 48 hours, passes through high performance liquid chromatography detection
Organic acid content.
The fermentation medium composition are as follows: 10 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L。
The organic acid concentration of detection is as shown in table 3:
Organic acid content after 3 anaerobic fermentation 48h of table
Embodiment 6
This example demonstrates that by Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 progress
The method of anaerobic fermentation production organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By in cryopreservation tube Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 by implement
4 the method for example is activated, after activation and after seed culture 12 hours, secondary seed culture 10 hours, will be planted
It is inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control is 6.8) after sub- liquid, while being passed through carbon dioxide 2-3
Minute is to guarantee anaerobic environment.Fermentation retains supernatant after being centrifuged fermentation broth sample after 48 hours, is examined by high performance liquid chromatography
Survey organic acid content.
The fermentation medium composition are as follows: 10 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L。
The organic acid concentration of detection is as shown in table 4:
Organic acid content after 4 anaerobic fermentation 48h of table
Embodiment 7
This example demonstrates that by Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 progress
The method of anaerobic fermentation production organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By in cryopreservation tube Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 by implement
4 the method for example is activated, after activation and after seed culture 12 hours, secondary seed culture 10 hours, will be planted
Be inoculated in after sub- liquid in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), at the same be passed through carbon dioxide with
Guarantee anaerobic environment.Fermentation retains supernatant after being centrifuged fermentation broth sample after 48 hours, organic by high performance liquid chromatography detection
Acid content.
The fermentation medium composition are as follows: 20 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO43 g/L.The organic acid concentration of detection is as shown in table 5:
Organic acid content after 5 anaerobic fermentation 48h of table
Embodiment 8
This example demonstrates that by Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 progress
The method of anaerobic fermentation production organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By in cryopreservation tube Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 by implement
4 the method for example is activated, after activation and after seed culture 12 hours, secondary seed culture 10 hours, will be planted
Be inoculated in after sub- liquid in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), at the same be passed through carbon dioxide with
Guarantee anaerobic environment.Fermentation retains supernatant after being centrifuged fermentation broth sample after 48 hours, organic by high performance liquid chromatography detection
Acid content.
The fermentation medium composition are as follows: 30 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L。
The organic acid concentration of detection is as shown in table 6:
Organic acid content after 6 anaerobic fermentation 48h of table
Embodiment 9
This example demonstrates that by Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 progress
The method of anaerobic fermentation production organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By in cryopreservation tube Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 by implement
4 the method for example is activated, after activation and after seed culture 12 hours, secondary seed culture 10 hours, will be planted
Be inoculated in after sub- liquid in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), at the same be passed through carbon dioxide with
Guarantee anaerobic environment.Fermentation retains supernatant after being centrifuged fermentation broth sample after 48 hours, organic by high performance liquid chromatography detection
Acid content.
The fermentation medium composition are as follows: 60 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl2 0.2 g/L, NaH2PO4
1.6 g/L, Na2HPO4 0.3 g/L, K2HPO4 3 g/L。
The organic acid concentration of detection is as shown in table 7:
Organic acid content after 7 anaerobic fermentation 48h of table
Claims (8)
1. the bacterial strain of one plant of reinforcing glycerol metabolism, which is characterized in that the strain classification is named as Actinobacillus succinogenes
(Actinobacillus succinogenes), from JF1315 is named, Chinese Typical Representative training has been preserved on March 31st, 2016
Support object collection, deposit number are as follows: CCTCC NO:M 2016160.
2. application of the bacterial strain described in claim 1 in production organic acid, which is characterized in that the organic acid is succinic acid, second
Acid, lactic acid and formic acid.
3. application of the bacterial strain in production organic acid according to claim 2, which is characterized in that the fermented and cultured of the bacterial strain
PH range is 4.8-6.8.
4. application of the bacterial strain according to Claims 2 or 3 in production organic acid, which is characterized in that the fermentation of the bacterial strain
Cultivating pH is 6.8.
5. application of the bacterial strain in production organic acid according to claim 2, which is characterized in that the fermented and cultured of the bacterial strain
In the process, using glycerol or glucose as fermented and cultured carbon source.
6. application of the bacterial strain in production organic acid according to claim 2, which is characterized in that the fermented and cultured of the bacterial strain
In the process, using high concentration glycerine as carbon source, the glycerol concentration is the g/L of 10 g/L ~ 60.
7. application of the bacterial strain in production organic acid according to claim 2, which comprises the steps of:
(1) Actinobacillus succinogenes bacterial strain is forwarded to anaerobism serum bottle after the activation of solid plate culture medium, and 37 DEG C, anaerobism item
Switching is cultivated 6-8 hours under conditions of 37 DEG C, 200 revs/min and is obtained in seed culture medium after cultivating 12-14 hours under part
Seed liquor；
(2) step (1) seed liquor is inoculated in the electrochemistry containing fermentation medium according to the inoculum concentration of 6-10% (v/v)
In device, in 37 DEG C of progress anaerobic fermentations；
(3) sterile sampling is carried out at regular intervals during the fermentation, to measurement carbon source and organic acid after sample centrifugal treating
Concentration.
8. application of the bacterial strain in production organic acid according to claim 7, which is characterized in that
The formula of the solid plate culture medium and seed culture medium are as follows: 20 g/L of glucose, 7.5 g/L of Dried Corn Steep Liquor Powder, ferment
10 g/L of female powder, sodium acetate 1.36 g/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl20.2 g/L, NaH2PO4 1.6
G/L, Na2HPO40.3 g/L, K2HPO43 g/L, agar powder 15-20 g/L；
The fermentative medium formula are as follows: 10 ~ 60 g/L of glycerol, 7.5 g/L of Dried Corn Steep Liquor Powder, 10 g/L of yeast powder, sodium acetate
1.36 g/L, dimethyl diaminophenazine chloride 1.0 mmol/L, NaCl 1 g/L, CaCl20.2 g/L, MgCl20.2 g/L, NaH2PO4 1.6
G/L, Na2HPO40.3 g/L, K2HPO4 3 g/L。
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| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Bacterial strain for producing succinic acid and screening method and application thereof |
| CN102154387A (en) * | 2010-12-27 | 2011-08-17 | 山东大学 | Method for producing succinic acid and polyhydroxyalkanoate (PHA) by using biodiesel byproducts |
| CN102732449A (en) * | 2012-04-25 | 2012-10-17 | 南京工业大学 | Actinobacillus succinogenes strain YH123 and application thereof |
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| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Bacterial strain for producing succinic acid and screening method and application thereof |
| CN102154387A (en) * | 2010-12-27 | 2011-08-17 | 山东大学 | Method for producing succinic acid and polyhydroxyalkanoate (PHA) by using biodiesel byproducts |
| CN102732449A (en) * | 2012-04-25 | 2012-10-17 | 南京工业大学 | Actinobacillus succinogenes strain YH123 and application thereof |
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