CN102936575A - Escherichia coli LL016 and application thereof - Google Patents
Escherichia coli LL016 and application thereof Download PDFInfo
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- CN102936575A CN102936575A CN2012105152181A CN201210515218A CN102936575A CN 102936575 A CN102936575 A CN 102936575A CN 2012105152181 A CN2012105152181 A CN 2012105152181A CN 201210515218 A CN201210515218 A CN 201210515218A CN 102936575 A CN102936575 A CN 102936575A
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- succinic acid
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- colon bacillus
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 17
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 81
- 239000001384 succinic acid Substances 0.000 claims abstract description 37
- 238000000855 fermentation Methods 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims description 36
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 24
- 210000001072 colon Anatomy 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 16
- 238000011218 seed culture Methods 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000012531 culture fluid Substances 0.000 claims description 2
- 230000012010 growth Effects 0.000 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 abstract description 12
- 239000008103 glucose Substances 0.000 abstract description 12
- 239000002253 acid Substances 0.000 abstract description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004321 preservation Methods 0.000 abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 229940023064 escherichia coli Drugs 0.000 abstract 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 24
- 230000001580 bacterial effect Effects 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 19
- 238000002703 mutagenesis Methods 0.000 description 13
- 231100000350 mutagenesis Toxicity 0.000 description 13
- 238000012216 screening Methods 0.000 description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 8
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 229930003756 Vitamin B7 Natural products 0.000 description 8
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 8
- 235000019156 vitamin B Nutrition 0.000 description 8
- 239000011720 vitamin B Substances 0.000 description 8
- 239000011735 vitamin B7 Substances 0.000 description 8
- 235000011912 vitamin B7 Nutrition 0.000 description 8
- 239000011521 glass Substances 0.000 description 7
- 230000000968 intestinal effect Effects 0.000 description 7
- 239000007789 gas Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- OGWLTJRQYVEDMR-UHFFFAOYSA-F tetramagnesium;tetracarbonate Chemical compound [Mg+2].[Mg+2].[Mg+2].[Mg+2].[O-]C([O-])=O.[O-]C([O-])=O.[O-]C([O-])=O.[O-]C([O-])=O OGWLTJRQYVEDMR-UHFFFAOYSA-F 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000000394 mitotic effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 230000003570 biosynthesizing effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005516 engineering process Methods 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
- 244000005700 microbiome Species 0.000 description 2
- 239000004631 polybutylene succinate Substances 0.000 description 2
- 229920002961 polybutylene succinate Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 241000606750 Actinobacillus Species 0.000 description 1
- 241000948980 Actinobacillus succinogenes Species 0.000 description 1
- 241000722954 Anaerobiospirillum succiniciproducens Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 244000134716 Hodgsonia macrocarpa Species 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229920000229 biodegradable polyester Polymers 0.000 description 1
- 239000004622 biodegradable polyester Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- -1 poly butylene succinate Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000004767 rumen Anatomy 0.000 description 1
- 230000000192 social effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses Escherichia coli (E.coli)Escherichiacoli) LL016 with the preservation number of CCTCC NO: m2012429. The invention also discloses application and a method of the Escherichia coli LL016 in pure anaerobic fermentation production of succinic acid by using a synthetic culture medium. The strain can grow by using an inorganic nitrogen source and glucose under an anaerobic condition, and can accumulate a large amount of succinic acid, the strain LL016 has a higher growth rate and a higher acid production amount, and has great social significance and economic value.
Description
Technical field
The present invention relates to strain colon bacillus LL016 and an application thereof, particularly a strain can utilize colon bacillus LL016 and the application thereof that the pure anaerobically fermenting of synthetic medium is produced Succinic Acid, belongs to industrial micro breeding and fermentation technical field thereof.
Background technology
Succinic Acid has another name called succsinic acid, is a kind of common natural organic acids, extensively is present in human body, animal, plant and the microorganism.Succinic Acid occupies very important status as one of the intermediate product of TCA circulation and terminal reduzate of anaerobic metabolism in bio-metabolic process.Result of study in recent years shows that Succinic Acid can also be as bulk chemical and poly butylene succinate (PBS) class biodegradable polyesters such as C4 platform compou nd synthesis BDO, tetrahydrofuran (THF), gamma-butyrolactones.In recent years, day by day serious along with the day by day exhausted and environmental problem of fossil resource adopts the Biological preparation Succinic Acid to get most of the attention, and USDOE is classified Succinic Acid as one of biorefinery product of 12 kinds of most worthies in future.
With respect to chemical synthesis, the method that biosynthesizing prepares Succinic Acid receives increasing concern.The biosynthesizing Succinic Acid, to utilize bacterium, the various microorganisms such as fungi, take glucose or other various hydrolyzed solutions as carbon source, through producing succinic acid by microbial fermentation, with respect to the method for chemosynthesis, the large advantage of one is that starting material are cheap biomass resource, utilizes the biological process synthesizing succinic acid to become the focus of Recent study.
Fermentation strain is one of biosynthetic key point of Succinic Acid, most bacteriums and fungi can both produce Succinic Acid, but only have the part bacterial strain can produce the Succinic Acid of high density, Succinic Acid industrial production bacterium comprises some propionate production bacterium, typical stomach and intestine bacterium and rumen bacteria.At present, the research of Succinic Acid industrial fermentation bacterial strain mainly concentrate on succinic acid-producing anaerobism spirillum (
Anaerobiospirillum succiniciproducens), the succinic acid-producing actinobacillus (
Actinobacillus succinogenes), Corynebacterium glutamicum (
Corynebacterium glutam icum) and intestinal bacteria (
Escherichia coli) etc.Wherein
Escherichia coliBecause genetic background is clear, genetic manipulation is simple, and fast growth, easy-regulating and used medium are comparatively cheap, becomes in recent years the study hotspot of Biological preparation Succinic Acid.
The fermentation and acid ability that improves Succinic Acid has a lot of methods, and especially on the means of fermentation control, a lot of researchers all conduct in-depth research.G. the people such as N. Vemuri report utilizes the method for intestinal bacteria two stage fermentation succinic acid-producings, and principle is to make thalline obtain Rapid Accumulation in the aerobic stage, then transfers anaerobism to and makes thalline produce a large amount of Succinic Acid.Because limitation and the complicacy of two stage fermentations, a nearest step anaerobically fermenting succinic acid-producing receives increasing concern.Yet under the pure anaerobic condition, intestinal bacteria utilize the research of synthetic medium growth and accumulation Succinic Acid but to rarely have report.As seen, by improved strain, make it can under pure anaerobic condition, utilize synthetic medium Fast Growth and a large amount of accumulation to produce acid, in Succinic Acid growth industry from now on, have very important effect.
Summary of the invention
The object of the present invention is to provide a strain colon bacillus, can under anaerobic utilize the synthetic medium growth, and can accumulate Succinic Acid.
Another object of the present invention provides the application of above-mentioned colon bacillus in utilizing the pure anaerobically fermenting production of synthetic medium Succinic Acid.
The 3rd purpose of the present invention provides the method that above-mentioned colon bacillus utilizes the pure anaerobically fermenting of synthetic medium to produce Succinic Acid.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
One, a strain colon bacillus (
Escherichia coli) LL016, its preserving number is CCTCC NO:M 2012429.
Colon bacillus of the present invention (
Escherichia coli) LL016 be with colibacillary starting strain colon bacillus (
Escherichia coli) BA016(CCTCC NO:M 2012350) after plasma body mutagenesis, utilize the synthetic medium plate screening to obtain the bacterial strain that can under anaerobic grow, through the anaerobism shake flask fermentation screening obtain can high succinic acid-producing bacterial strain be aimed strain.
Its concrete steps are as follows:
(1) plasma mutagenesis: the intestinal bacteria original strain is activated in test tube, 37 ℃, 200 r/min, incubated overnight.The bacterium liquid that obtains is diluted to OD with stroke-physiological saline solution
600=1.0, drip on aseptic slide glass, dry up with sterile wind; Take helium as discharge gas, take 80 ~ 120 W as radio frequency power, as gas flow, take 10 ~ 30 s as irradiation time, bacterial strain is carried out plasma body mutagenesis with 10 ~ 30 SLM;
(2) the dull and stereotyped primary dcreening operation of synthetic medium: the tool plug test tube that the slide glass after the mutagenesis is placed the physiological saline that 1mL is housed, concuss, with the bacterium liquid wash-out on the slide glass, be diluted to different concentration and coat on the synthetic medium flat board, 37 ℃ of anaerobism are cultivated 24 h.It is larger to select growth, comparatively full bacterium colony;
(3) the dull and stereotyped multiple sieve of synthetic medium: with the bacterial strain repeatedly turning point cultivation on synthetic medium that screens in the step (2), 37 ℃ of anaerobism are cultivated 24 h, and it is larger to select growth, comparatively full bacterium colony;
(4) anaerobism shake flask fermentation screening: with the inoculation that filters out in the step (3) enlarged culturing in the seed culture medium, 37 ℃, 200 r/min, cultivate 48 h, then ferment in fermention medium, inoculum size is 2%(v/v), 30 ℃, 170 r/min cultivate 72 h; Investigate in the bacterium colony that filters out in the step 3) and filter out fast growth, produce the high bacterial strain of acid amount.
In above-mentioned screening method, in the plasma body mutagenesis method described in the step (1), selecting 100 W is radio frequency power, and 20 SLM are gas flow, and 15 s are irradiation time.
In above-mentioned screening method,
Seed culture medium is: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermention medium is: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, magnesium basic carbonate 16 g/L, glucose 20 g/L.
The solid plate substratum is: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, agar 20 g/L, glucose 10 g/L.
Two, above-mentioned colon bacillus (
Escherichia coli) application of LL016 in utilizing the pure anaerobically fermenting production of synthetic medium Succinic Acid.
Three, above-mentioned colon bacillus (
Escherichia coli) LL016 utilizes the pure anaerobically fermenting of synthetic medium to produce the method for Succinic Acid, comprises following steps:
(1) the dull and stereotyped cultivation: colon bacillus LL016 is seeded in anaerobism cultivation on the plate culture medium, and culture temperature is 37 ℃, and incubation time is 24 h;
(2) seed culture: the colon bacillus LL016 that flat board is cultivated is inoculated in the seed culture medium, carbonating 2 min, and culture temperature is 37 ℃, 200 r/min, incubation time are 48 h;
(3) fermentation production of succinic acid: seed culture fluid is inoculated in the fermention medium, and the inoculum size volume ratio is 2%, carbonating 2 min, and culture temperature is 30 ℃, 170 r/min, incubation time are 72 h.
Wherein, plate culture medium: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, agar 20 g/L, glucose 10 g/L.
Seed culture medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermention medium: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, magnesium basic carbonate 16 g/L, glucose 20 g/L.
Beneficial effect of the present invention is: using plasma mutagenesis recombination bacillus coli of the present invention, utilize synthetic medium dull and stereotyped, filter out and under anaerobic can utilize the synthetic medium growth, and the colon bacillus LL016 of high succinic acid-producing, this bacterial strain can be take inorganic nitrogen as nitrogenous source, glucose is under anaerobic Fast Growth of carbon source, and accumulates in a large number Succinic Acid; In the anaerobism shaking flask, can utilize the growth of synthetic medium and glucose and produce acid, 72 h cell densities have reached OD
600=5.6, Succinic Acid output is 11.64 g/L, and acid can't be grown and produce to original starting strain under this condition, so this bacterial strain has great social effect and economic worth.
Description of drawings
Fig. 1 is colibacillary plasma body mutagenesis survival rate curve.
Biomaterial Classification And Nomenclature of the present invention be colon bacillus (
Escherichia coli) LL016, preservation date is on October 25th, 2012, depositary institution's full name is Chinese Typical Representative culture collection center, and referred to as CCTCC, depositary institution address: China. Wuhan. Wuhan University; Deposit number: CCTCC NO:M 2012429.
Starting strain colon bacillus used in the present invention (
Escherichia coli) source of BA016: carried out patented procedure preservation (deposit number: CCTCC NO:M 2012350) at the Chinese Typical Representative culture collection center that Patent Office of the People's Republic of China or international monopoly tissue are admitted, and submitted Chinese patent application (cut-off the application's the applying date is also unexposed) to, the number of patent application that relates to this bacterial strain is 201210392834.2, and patent application day is on October 16th, 2012.
The starting strain colon bacillus (
Escherichia coli) BA016, its preservation date is on 09 14th, 2012, and depositary institution's full name is Chinese Typical Representative culture collection center, and referred to as CCTCC, the address is: China. Wuhan. Wuhan University, deposit number: CCTCC NO:M 2012350.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described further, according to following examples, can better understand the present invention.Concrete material proportion described in the case study on implementation, processing condition and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in the claims.
Embodiment 1
The method that the present embodiment explanation is carried out the mutagenesis of the first step plasma body with the intestinal bacteria original strain.
The method that the intestinal bacteria original strain carries out the mutagenesis of the first step plasma body is as follows:
Intestinal bacteria BA016 original strain is activated in the LB test tube, 37 ℃, 200 r/min incubated overnight; Get the cell dilution of fresh culture to cell concn OD
600=1.0, drip on aseptic slide glass, dry up with sterile wind; Take helium as discharge gas, take 80 ~ 120 W as radio frequency power, as gas flow, take 10 ~ 30 s as irradiation time, bacterial strain is carried out plasma body mutagenesis with 10 ~ 30 SLM; After the mutagenesis, the stroke-physiological saline solution of the mycoderm on the slide glass with 1mL eluted, be coated on the synthetic medium flat board, in anaerobic box, cultivate.Experimental result as shown in Figure 1, wherein, X-coordinate is irradiation time, ordinate zou is survival rate.
Embodiment 2
The good colibacillary method of present embodiment explanation screening.
Wherein, employed culture medium prescription is as follows:
(1) the solid synthetic medium is dull and stereotyped: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, agar 20 g/L, glucose 10 g/L.
(2) seed culture medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
(3) Medium of shaking flask fermentation: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, magnesium basic carbonate 16 g/L, glucose 20 g/L.
The screening step is as follows:
(1) the dull and stereotyped primary dcreening operation of synthetic medium
Slide glass after the mutagenesis is placed the tool plug test tube that 1mL physiological saline is housed, and concuss elutes the thalline on the slide glass fully, being diluted to different concentration coats on the synthetic medium flat board, 37 ℃ of anaerobism are cultivated 24 h, pick out fast growth, comparatively full bacterium colony.
(2) the dull and stereotyped multiple sieve of synthetic medium
Bacterial strain repeatedly turning point cultivation on flat board with screening has finally obtained bacterial strain LL010, LL012, LL014 and LL016 and has shown stronger growth velocity and growth stability.
(3) shake flask fermentation screening
(4) with enlarged culturing in bacterial strain LL010, LL012, LL014 and the LL016 access seed culture medium, carbonating 2 min, 37 ℃, 200 r/min cultivate 48 h.Then be inoculated in the fermention medium 100 mL anaerobism serum bottle liquid amounts, 30 mL, inoculum size 2%(v/v), carbonating 2 min, 30 ℃, 170 r/min cultivate 72 h.It is as shown in table 1 to detect each bacterial strain cell density and Succinic Acid output:
The growth of table 1 mutant strain and starting strain and product acid are relatively
| Bacterial strain | OD 600 | Succinic Acid output (g/L) | Output of pyruvic acid (g/L) |
| BA016 | 0.23 | 0.00 | 0.00 |
| LL010 | 3.2 | 5.55 | 2.01 |
| LL012 | 3.4 | 6.45 | 3.21 |
| LL014 | 3.8 | 7.02 | 3.24 |
| LL016 | 5.6 | 11.64 | 5.67 |
Under pure anaerobic condition, starting strain can't utilize the synthetic medium growth and produce acid, but through obtaining under pure anaerobic condition, utilizing the bacterial strain of synthetic medium Fast Growth after the ARTP mutagenesis.Through the shake flask fermentation screening, bacterial strain LL016 growth velocity is very fast, and product acid is higher.
Embodiment 3
The mitotic stability of present embodiment explanation mutant strain LL016
On the synthetic medium flat board, many turning points of mutant strain LL016 to be cultivated, and will be obtained the bacterial strain checking of fermenting respectively, experimental result is as shown in table 2:
Table 2 mutant strain LL016 mitotic stability is analyzed
From experimental result as can be known, through 8 continuous passages, the Succinic Acid output of mutant strain LL016 and Succinic Acid transformation efficiency are all comparatively stable, have good mitotic stability, can be used as the production bacterial classification of further research and development.
Embodiment 4
Present embodiment explanation colon bacillus (
Escherichia coli) method of LL016 fermentation production of succinic acid
The described culture medium prescription of present embodiment:
Plate culture medium: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, agar 20 g/L, glucose 10 g/L.
Seed culture medium: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L.
Fermention medium: citric acid 3 gL
-1, Na
2HPO
412H
2O 4 gL
-1, KH
2PO
48 gL
-1, (NH
4)
2HPO
48 gL
-1, NH
4Cl 0.2 gL
-1, (NH
4)
2SO
40.75 gL
-1, MgSO
47H
2O 1 gL
-1, CaCl
22H
2O 10.0 mgL
-1, ZnSO
47H
2O 0.5 mgL
-1, CuCl
22H
2O 0.25 mgL
-1, MnSO
4H
2O 2.5 mgL
-1, CoCl
26H
2O 1.75 mgL
-1, H
3BO
30.12 mgL
-1, Al
2(SO
4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, vitamins B
120 mgL
-1, vitamin H 2 mgL
-1, magnesium basic carbonate 16 g/L, glucose 20 g/L.
With colon bacillus
Escherichia coliLL016 is seeded to plate culture medium, cultivates 37 ℃ of culture temperature, incubation time 24 h in anaerobic box.The LL016 that flat board is cultivated is inoculated in the seed culture medium 100 mL serum bottle liquid amounts, 30 mL, carbonating 2 min, 37 ℃ of culture temperature, 200 r/min, incubation time 48 h.Seed is inoculated in the fermention medium inoculum size 2%(v/v), 100 mL serum bottle liquid amounts, 30 mL, carbonating 2 min, culture temperature is 30 ℃, 170 r/min, the output that detects Succinic Acid behind fermentation culture 72 h is 11.64 g/L, OD
600Be 5.6.
Claims (3)
- One strain colon bacillus ( Escherichia coli) LL016, its preserving number is CCTCC NO:M 2012429.
- 2. the application of colon bacillus LL016 claimed in claim 1 in utilizing the pure anaerobically fermenting production of synthetic medium Succinic Acid.
- 3. colon bacillus LL016 claimed in claim 1 utilizes the method that the pure anaerobically fermenting of synthetic medium is produced Succinic Acid, comprises following steps:(1) the dull and stereotyped cultivation: colon bacillus LL016 is seeded in anaerobism cultivation on the plate culture medium, and culture temperature is 37 ℃, and incubation time is 24 h;(2) seed culture: the colon bacillus LL016 that flat board is cultivated is inoculated in the seed culture medium, carbonating 2 min, and culture temperature is 37 ℃, 200 r/min, incubation time are 48 h;(3) fermentation production of succinic acid: seed culture fluid is inoculated in the fermention medium, and the inoculum size volume ratio is 2%, carbonating 2 min, and culture temperature is 30 ℃, 170 r/min, incubation time are 72 h.
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