CN105838652A - Bacterial strain capable of enhancing glycerin metabolism and application thereof - Google Patents
Bacterial strain capable of enhancing glycerin metabolism and application thereof Download PDFInfo
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- CN105838652A CN105838652A CN201610382379.6A CN201610382379A CN105838652A CN 105838652 A CN105838652 A CN 105838652A CN 201610382379 A CN201610382379 A CN 201610382379A CN 105838652 A CN105838652 A CN 105838652A
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- bacterial strain
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 60
- 235000011187 glycerol Nutrition 0.000 title claims abstract description 31
- 230000004060 metabolic process Effects 0.000 title claims abstract description 12
- 230000002708 enhancing effect Effects 0.000 title abstract 2
- 238000000855 fermentation Methods 0.000 claims abstract description 62
- 241000948980 Actinobacillus succinogenes Species 0.000 claims abstract description 60
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 150000007524 organic acids Chemical class 0.000 claims abstract description 38
- 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 15
- 239000008103 glucose Substances 0.000 claims abstract description 15
- 238000011218 seed culture Methods 0.000 claims description 30
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 21
- 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
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 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
- 210000002966 serum Anatomy 0.000 claims description 14
- 239000011780 sodium chloride Substances 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
- 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
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 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
- 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
- 238000005728 strengthening Methods 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 3
- 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
- 230000005518 electrochemistry Effects 0.000 claims 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract description 13
- 230000002829 reductive effect Effects 0.000 abstract description 7
- 239000001384 succinic acid Substances 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 25
- 231100000350 mutagenesis Toxicity 0.000 description 25
- 238000002703 mutagenesis Methods 0.000 description 25
- 239000000047 product Substances 0.000 description 15
- 230000012010 growth Effects 0.000 description 12
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 239000012531 culture fluid Substances 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 7
- 230000006378 damage Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 241000606750 Actinobacillus Species 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000001569 carbon dioxide Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 238000005138 cryopreservation Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 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
- 241000894006 Bacteria Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 231100000219 mutagenic Toxicity 0.000 description 4
- 230000003505 mutagenic effect Effects 0.000 description 4
- 229960005137 succinic acid Drugs 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
- 238000003786 synthesis reaction Methods 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
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-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
- 239000003225 biodiesel Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 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
- 238000011081 inoculation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000008569 process Effects 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
- 235000011044 succinic acid Nutrition 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000012159 carrier gas 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
- 230000002950 deficient Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000007789 gas Substances 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
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 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
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- 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
-
- 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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- 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
-
- 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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Abstract
The invention discloses a bacterial strain capable of enhancing glycerin metabolism. The bacterial strain is classified and names as Actinobacillussuccinogenes JF1315, already collected at the China Center for Type Culture Collection on 31th, Mar. 2016 under CCTCC NO: M 2016160. The invention further relates to application of Actinobacillussuccinogenes JF1315 in producing organic acid through fermentation. Compared with control bacterial strains, under an anaerobic condition, the bacterial strain can grow and metabolizes at low pH, and reductive organic acid can be synthesized efficiently by utilizing glycerin fermentation. When pH is 4.8-6.8, the bacterial strain can normally grow and metabolize glucose to synthesize the organic acid like succinic acid; in addition, under the condition, the bacterial strain can efficiently utilize glycerin for anaerobic fermentation to synthesize and accumulate the organic acid.
Description
Technical field
The present invention relates to bacterial strain and the application thereof of a strain strengthening glycerol metabolism, belong to industrial microorganism and fermentation technique neck thereof
Territory.
Background technology
During traditional field of microbial fermentation engineering practice, the most how to carry out sending out with glucide for carbon source
Ferment, such as cornstarch hydrolyzate；For some high value added product, even directly carry out product with glucose for raw material
Synthesis.Although using the raw materials such as glucose can obtain the product of higher concentration when fermenting, but which greatly increases
The financial cost of whole production process, causes the waste of resource.In order to solve this problem, can attempt to use cheap living beings
Material (such as cellulosic hydrolysate or glycerine) substitutes glucose and ferments, but the existence of noxious material in cellulosic hydrolysate
And sugar concentration relatively low significantly limit its application.
Along with developing rapidly of field of biodiesel oil, the synthetic quantity of glycerine is huge, and (1 ton of biodiesel can produce 100,000
Gram glycerine), and for glycerine, the reducing power that intramolecular stores is higher, is conducive to improving yield and the receipts of reproducibility product
Rate.During reduced form Product formation, when with glucose for sole carbon source, glucose is when glycolytic pathway carries out metabolism
Producing 2 molecule reducing powers (NADH), the reducing power for strong reduced form Product formation can be the most not enough, and product yield reduces；And
When with glycerine for carbon source, glycerine can produce 2 molecule reducing powers (NADH) through metabolism, on this basis, during strain growth also
A part of NADH, substantial amounts of NADH can be synthesized and can increase the accumulation of reduced form product.But, if the reproducibility of synthetic product is relatively low
(such as succinic acid), can accumulate NADH in a large number in thalline, now superfluous reducing power can suppress the growth of microbial cells.In order to flat
Weighing apparatus intracellular coenzyme metabolism, ensures the synthesis of reduced form product, enters bacterial strain by the way of mutagenesis while recovering thalli growth
Row transformation, by the bacterial strain of the screening excellent of felicity condition.
Summary of the invention
One of technical purpose of the present invention is to provide a strain and can efficiently utilize glycerine to carry out the product butanedioic acid of anaerobic fermentation
Actinobacillus.
For realizing above-mentioned technical purpose, the present invention adopts the following technical scheme that:
The bacterial strain of one strain strengthening glycerol metabolism, its Classification And Nomenclature is Actinobacillus succinogenes (Actinobacillus
Succinogenes) JF1315, is preserved in China typical culture collection center on March 31st, 2016, its deposit number
For: 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, preserves numbered CGMCC No.1716) after ARTP mutagenesis, by o-nitrobenzene-β-d-gala
Obtain after glucosides (ONPG) and fluorescent dye NPN screening.After screening, through anaerobic fermentation, bacterial strain verifies that its glycerine utilizes and produces acid again
Ability.
Concrete mutagenesis screening step is as follows:
ARTP mutagenesis: starting strain Actinobacillus succinogenes NJ113 is inoculated in the anaerobism serum bottle equipped with seed culture medium
Middle cultivate 6-12 hour with obtain exponential phase bacterial strain.Seed culture fluid is suitably diluted to OD660After=0.5-1.5
Coat through high-temperature sterilization and meet on ice and carry out mutagenesis on cold loading iron plate.Mutagenic condition is chosen for: using helium as load
Gas, throughput 10SLM, power 80-120W, mutation time 0-300s, and measure fatal rate drafting mutagenesis destruction curve.With
On the basis of destruction curve, the time choosing fatal rate relatively big (more than 90%) is mutation time, under the same terms lures bacterial strain
Change processes.
After mutagenesis, single bacterium colony separates: after being coated with mutagenesis, the micro slide of seed culture fluid is put in SPSS,
Coat in solid plate culture medium after mixing, cultivate for 35-37 DEG C and separate single bacterium colony after 8-15 hour.By list colony inoculation in containing
Have in the anaerobism serum bottle of seed culture medium, cultivate for 35-37 DEG C and obtain seed culture fluid after 8-15 hour.
O-nitrobenzene-beta-d-galactopyranoside glycosides (ONPG) screening: seed culture fluid is diluted to OD660=0.5-2.0, takes 50-
After 200 j diluent mix with 5-15 microlitre ONPG, under 405nm, after reacting 2 hours at 35-37 DEG C, detect absorbance
Value, and bacterial strain higher for absorbance is preserved.
Fluorescent dye NPN screens: after the above-mentioned bacterial strain screened carries out Anaerobic culturel acquisition seed liquor, enter seed liquor
Row is diluted to OD660=0.5-2.0, takes after 1-3 mL dilution mixes with 10-30 microlitre NPN, reacts 2-10 and divide at 35-37 DEG C
Detecting the size of fluorescent value after clock, the relatively low bacterial strain of picking fluorescence angle value preserves, and the bacterial strain that being the present invention provides produces amber
Amber acid Actinobacillus JF1315.
The formula of above-mentioned solid plate culture medium and seed culture medium is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/
L, dusty yeast 10 g/L, 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 present invention is that bacterial strain JF1315 is in the application producing organic acid.
The method that the bacterial strain anaerobic fermentation that the invention provides a kind of present invention of utilization provides produces the organic acids such as succinic acid.
Described anaerobic fermentation method is as follows:
Actinobacillus succinogenes (Actinobacillus succinogenes) YJ1315 anaerobic fermentation is tested: institute of the present invention
Actication of culture, the seed culture step stated are conventional Actinobacillus actication of culture method and seed culture method, in the present invention
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 bacterial strain is activated through solid plate culture medium
After, under the conditions of 37 DEG C, transfer in seed culture medium, at 37 DEG C, 200 revs/min after cultivating 12-14 hour in anaerobism serum bottle
Under conditions of cultivate within 6-8 hour, obtain seed liquor.
By described seed liquor according to 6-10 %(v/v) inoculum concentration be inoculated in the anaerobism serum containing described fermentation medium
In Ping.
Above-mentioned fermentative medium formula is: glycerine 10-60 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, second
Acid sodium 1.36 g/L, 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 is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/
L, dusty yeast 10 g/L, 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 present invention by carrying out ARTP mutagenesis to starting strain, and screening obtains a strain can efficiently utilize the bacterial strain of glycerine
JF1315, compared with starting strain, it has the beneficial effects that:
The present invention is by ARTP mutagenesis means and rational bacterial strain screening method, it is thus achieved that a strain in the fermentation medium can be efficient
Utilize glycerine growth and the bacterial strain of metabolism.When with glycerine for primary carbon source, under anaerobic condition, bacterial strain can utilize glycerine to grow
And synthesizing reduced form metabolite: after fermenting 48 hours in complex medium under anaerobic condition, glycerine consumption is up to 60 g/
L, total reductic acid cumulative amount reaches 97 g/L.Demonstrate growth and the metabolism performance that can be changed thalline by ARTP mutagenesis means,
Reduce intracellular reducing power level, recover the energy for growth of cell, simultaneously on this basis, the beneficially accumulation of reduced form product
And the generation of electric energy.
Accompanying drawing explanation
Fig. 1 is starting strain destruction curve during ARTP mutagenesis.
Biomaterial of the present invention, its Classification And Nomenclature is Actinobacillus succinogenes (Actinobacillus
Succinogenes) JF1315, on March 31st, 2016 be preserved in China typical culture collection center (be called for short CCTCC,
Address: China. Wuhan. Wuhan University), its deposit number is: CCTCC NO:M 2016160.
Detailed description of the invention
According to following example, the present invention can be better understood from.Concrete material proportion described in case study on implementation,
Process conditions and result thereof are merely to illustrate the present invention, and should be also without limitation on basis 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 screens the starting strain Actinobacillus succinogenes (Actinobacillus that JF1315 bacterial strain uses
Succinogenes) during NJ113 bacterial classification has been disclosed in the patent of same inventor's earlier application, the patent No.
ZL200610085415.9, preserves numbered CGMCC No.1716.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 bacterial strain is by producing butanedioic acid unwrapping wire
Bacillus (Actinobacillus succinogenes) NJ113 is through ARTP mutagenesis screening.
Concrete mutagenesis screening step is as follows:
ARTP mutagenesis: starting strain Actinobacillus succinogenes NJ113 is inoculated in the anaerobism serum bottle equipped with seed culture medium
Middle cultivate 6-12 hour with obtain exponential phase bacterial strain.Seed culture fluid is suitably diluted to OD660After=0.5-1.5
Coat to meet and carry out mutagenesis on cold micro slide.Mutagenic condition is chosen for: using helium as carrier gas, throughput 10SLM, power supply merit
Rate 80-120W, mutation time 0-300s, and measure fatal rate drafting mutagenesis destruction curve.On the basis of destruction curve, choose cause
The time of dead rate relatively big (more than 90%) is mutation time, under the same terms, bacterial strain is carried out mutagenic treatment.
Fig. 1 is starting strain destruction curve during ARTP mutagenesis.Under identical mutagenesis intensity, investigate mutation time to bacterial strain
The impact of growth.It will be seen from figure 1 that mutation time is the longest, Strain survival rate is the lowest, and mutagenesis fatal rate is the highest.
After mutagenesis, single bacterium colony separates: after being coated with mutagenesis, the micro slide of seed culture fluid is put in SPSS,
Coat after mixing in solid plate, cultivate for 35-37 DEG C and separate single bacterium colony after 8-15 hour.By list colony inoculation in containing seed
In the anaerobism serum bottle of culture medium, cultivate for 35-37 DEG C and obtain seed culture fluid after 8-15 hour.
O-nitrobenzene-beta-d-galactopyranoside glycosides (ONPG) screening: seed culture fluid is diluted to OD660=0.5-2.0, takes 50-
After 200 j diluent mix with 5-15 microlitre ONPG, under 405nm, after reacting 2 hours at 35-37 DEG C, detect absorbance
Value, and bacterial strain higher for absorbance is preserved.
Fluorescent dye NPN screens: after the above-mentioned bacterial strain screened carries out Anaerobic culturel acquisition seed liquor, enter seed liquor
Row is diluted to OD660=0.5-2.0, takes after 1-3 mL dilution mixes with 10-30 microlitre NPN, reacts 2-10 and divide at 35-37 DEG C
Detecting the size of fluorescent value after clock, the relatively low bacterial strain of picking fluorescence angle value preserves, and the bacterial strain that being the present invention provides produces amber
Amber acid Actinobacillus JF1315.
The formula of above-mentioned solid plate culture medium and seed culture medium is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/
L, dusty yeast 10 g/L, 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 of the Actinobacillus succinogenes JF1315 that above-described embodiment 1 obtains, the most such as
Under:
After mutagenesis, bacterial strain there is no with original bacteria Actinobacillussuccinogenes NJ113 colonial morphology and growth performance
Notable difference: bacterial strain Gram-negative, flat-plate bacterial colony is rounded, and neat in edge is smooth, can metabolizable glucose, wood under anaerobic condition
Sugar synthesis organic acid, wherein main acid consist of succinic acid, acetic acid, lactic acid and formic acid.Bacterial strain is glutamic acid deficient strain,
Synthetic media need to add glutamic acid during growth.
The genetic stability test of the Actinobacillus succinogenes JF1315 of embodiment 1, strain passage fermenting experiment is as follows
Shown in.
Strains A ctinobacillus succinogenes JF1315 after mutagenesis enters under the conditions of low pH (5.0-6.0)
Row continuous passage is cultivated, the growth of period sampling measuring difference generation cell cell at low ph conditions constantly and product acid activity, knot
The most as shown in table 1 below.When cell continuous passage was cultivated to 10 generations, and growth and product acid activity are uninfluenced in acid condition.
The growth of passage cell and product acid activity under table 1. low ph condition
Embodiment 3
This example demonstrates that in the present invention after mutagenesis that strains A ctinobacillus succinogenes JF1315 compares to set out
The superiority of bacterial strain.
Inoculate after Actinobacillus succinogenes NJ113 and JF1315 bacterial strain are passed through solid plate medium culture by the present invention
Cultivate to seed culture medium and obtain seed liquor；Then seed liquor is inoculated in low pH fermentation medium and carries out anaerobic fermentation.
Described method may comprise steps of:
(1) Actinobacillus succinogenes NJ113 and JF1315 bacterial strain are forwarded to anaerobism serum after solid plate culture medium activates
Bottle, transfers in seed culture medium after cultivating 12-14 hour under anaerobic condition by 37 DEG C, at 37 DEG C, trains under conditions of 200 revs/min
Support and obtain seed liquor in 6-8 hour；
(2) above-mentioned seed liquor is inoculated in the blood containing low pH fermentation medium (pH5.8) according to the inoculum concentration of 6-10 % (v/v)
In clear bottle, carry out anaerobic fermentation 48 in 37 DEG C and as a child sampled, measure cell concentration and organic acid content.
The formula of above-mentioned solid plate culture medium and seed culture medium is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/
L, dusty yeast 10 g/L, 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 is: glucose 10 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, second
Acid sodium 1.36 g/L, 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
According to table 2, either original strain NJ113 or mutagenic strain JF1315, all can survive in the range of pH=4.8-6.8
And fermentation production of organic acid, and reach optimal growth and metabolism performance when pH=6.8.
Embodiment 4 this example demonstrates that the method that in the present invention, bacterial strain anaerobic fermentation produces organic acid.
Actinobacillus succinogenes bacterial strain is seeded to seed culture by after solid plate medium culture by the present invention
Base is cultivated and obtains seed liquor；Then seed liquor is inoculated in fermentation medium and carries out anaerobic fermentation.Described method can be wrapped
Include following steps:
(1) Actinobacillus succinogenes bacterial strain is forwarded to anaerobism serum bottle after solid plate culture medium activates, 37 DEG C, anaerobism bar
Transfer in seed culture medium after cultivating 12-14 hour under part, at 37 DEG C, cultivate under conditions of 200 revs/min and obtain for 6-8 hour
Seed liquor；
(2) above-mentioned seed liquor is inoculated in the electrochemical appliance containing fermentation medium according to the inoculum concentration of 6-10 % (v/v),
Anaerobic fermentation is carried out in 37 DEG C.
(3) sterile sampling is carried out the most at set intervals, to measuring carbon source after sample centrifugal treating and having
Machine acid concentration.
The formula of above-mentioned solid plate culture medium and seed culture medium is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/
L, dusty yeast 10 g/L, 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 and Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 is carried out anaerobism
The method of fermentation production of organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 anaerobic fermentation method is as follows:
By Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113 in cryopreservation tube by embodiment 4 institute
The method of stating activates, after activation and seed culture 12 hours, after secondary seed is cultivated 10 hours, after obtaining seed liquor
It is inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), is passed through carbon dioxide 2 minutes to protect simultaneously
Card anaerobic environment.After centrifugal for fermentation broth sample, retain supernatant after fermenting 48 hours, detect organic acid by high performance liquid chromatography
Content.
Described fermentation medium consists of: glycerine 10 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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 table 3 anaerobic fermentation 48h
Embodiment 6
This example demonstrates that and Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 is carried out anaerobism
The method of fermentation production of organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 in cryopreservation tube by embodiment 4
Described method activates, after activation and seed culture 12 hours, after secondary seed is cultivated 10 hours, will obtain seed liquor
After be inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), be passed through carbon dioxide 2-3 minute simultaneously
To ensure anaerobic environment.After centrifugal for fermentation broth sample, retain supernatant after fermenting 48 hours, had by high performance liquid chromatography detection
Machine acid content.
Described fermentation medium consists of: glycerine 10 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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 table 4 anaerobic fermentation 48h
Embodiment 7
This example demonstrates that and Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 is carried out anaerobism
The method of fermentation production of organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 in cryopreservation tube by embodiment 4
Described method activates, after activation and seed culture 12 hours, after secondary seed is cultivated 10 hours, will obtain seed liquor
After be inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), be passed through carbon dioxide to ensure simultaneously
Anaerobic environment.After centrifugal for fermentation broth sample, retain supernatant after fermenting 48 hours, contained by high performance liquid chromatography detection organic acid
Amount.
Described fermentation medium consists of: glycerine 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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 table 5 anaerobic fermentation 48h
Embodiment 8
This example demonstrates that and Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 is carried out anaerobism
The method of fermentation production of organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 in cryopreservation tube by embodiment 4
Described method activates, after activation and seed culture 12 hours, after secondary seed is cultivated 10 hours, will obtain seed liquor
After be inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), be passed through carbon dioxide to ensure simultaneously
Anaerobic environment.After centrifugal for fermentation broth sample, retain supernatant after fermenting 48 hours, contained by high performance liquid chromatography detection organic acid
Amount.
Described fermentation medium consists of: glycerine 30 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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 table 6 anaerobic fermentation 48h
Embodiment 9
This example demonstrates that and Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 is carried out anaerobism
The method of fermentation production of organic acid.
Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 anaerobic fermentation method is as follows:
By Actinobacillus succinogenes (Actinobacillus succinogenes) JF1315 in cryopreservation tube by embodiment 4
Described method activates, after activation and seed culture 12 hours, after secondary seed is cultivated 10 hours, will obtain seed liquor
After be inoculated in the anaerobism serum bottle containing fermentation medium (fermentation pH control 6.8), be passed through carbon dioxide to ensure simultaneously
Anaerobic environment.After centrifugal for fermentation broth sample, retain supernatant after fermenting 48 hours, contained by high performance liquid chromatography detection organic acid
Amount.
Described fermentation medium consists of: glycerine 60 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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 table 7 anaerobic fermentation 48h
Claims (8)
1. the bacterial strain of a strain strengthening glycerol metabolism, it is characterised in that the named Actinobacillus succinogenes of described strain classification
(Actinobacillussuccinogenes) JF1315, is preserved in China typical culture collection on March 31st, 2016
Center, its deposit number is: CCTCC NO:M 2016160.
2. the application in producing organic acid of the bacterial strain described in claim 1.
Bacterial strain application in producing organic acid the most according to claim 2, it is characterised in that the fermented and cultured of described bacterial strain
PH scope is 4.8-6.8.
4. according to bacterial strain described in Claims 2 or 3 in the application produced in organic acid, it is characterised in that the fermentation of described bacterial strain
Cultivating pH is 6.8.
Bacterial strain application in producing organic acid the most according to claim 2, it is characterised in that the fermented and cultured of described bacterial strain
During, with glycerine or glucose for fermented and cultured carbon source.
Bacterial strain application in producing organic acid the most according to claim 2, it is characterised in that the fermentation training of described bacterial strain
During Yanging, with high concentration glycerine as carbon source, described glycerol concentration is 10 g/L ~ 60 g/L.
Bacterial strain application in producing organic acid the most according to claim 2, it is characterised in that comprise the steps:
(1) Actinobacillus succinogenes bacterial strain is forwarded to anaerobism serum bottle after solid plate culture medium activates, 37 DEG C, anaerobism bar
Transfer in seed culture medium after cultivating 12-14 hour under part, at 37 DEG C, cultivate under conditions of 200 revs/min and obtain for 6-8 hour
Seed liquor；
(2) step (1) described seed liquor is inoculated in the electrochemistry containing fermentation medium according to the inoculum concentration of 6-10 % (v/v)
In device, carry out anaerobic fermentation in 37 DEG C；
(3) sterile sampling is carried out the most at set intervals, to measuring carbon source and organic acid after sample centrifugal treating
Concentration.
Bacterial strain application in producing organic acid the most according to claim 7, it is characterised in that
The formula of described solid plate culture medium and seed culture medium is: glucose 20 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, ferment
Female powder 10 g/L, 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.
Described fermentative medium formula is: glycerine 10 ~ 60 g/L, Dried Corn Steep Liquor Powder 7.5 g/L, dusty yeast 10 g/L, 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。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701637A (en) * | 2017-02-13 | 2017-05-24 | 广西科学院 | Bacterial strain used for producing succinic acid through fermentation of biodiesel by-product crude glycerol and production method of bacterial strain |
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| CN112111418A (en) * | 2019-06-20 | 2020-12-22 | 华东师范大学 | High-yield strain of succinyl polysaccharide and preparation and application of succinyl polysaccharide |
| CN112111418B (en) * | 2019-06-20 | 2022-09-20 | 华东师范大学 | High-yield strain of succinyl polysaccharide and preparation and application of succinyl polysaccharide |
| CN113980868A (en) * | 2021-12-02 | 2022-01-28 | 广西科学院 | Actinobacillus succinogenes capable of tolerating pentamethyl furfural and breeding method and application thereof |
| CN113980868B (en) * | 2021-12-02 | 2023-02-03 | 广西科学院 | Actinobacillus succinogenes capable of tolerating pentamethyl furfural and breeding method and application thereof |
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