CN102643770B - Colibacillus capable of generating succinic acid by anaerobic growth in synthetic medium pure and application thereof - Google Patents
Colibacillus capable of generating succinic acid by anaerobic growth in synthetic medium pure and application thereof Download PDFInfo
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- CN102643770B CN102643770B CN201210138292.6A CN201210138292A CN102643770B CN 102643770 B CN102643770 B CN 102643770B CN 201210138292 A CN201210138292 A CN 201210138292A CN 102643770 B CN102643770 B CN 102643770B
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 239000001384 succinic acid Substances 0.000 title claims abstract description 47
- 230000009604 anaerobic growth Effects 0.000 title claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 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 12
- 239000008103 glucose Substances 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 39
- 241000894006 Bacteria Species 0.000 claims description 26
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 24
- 230000000968 intestinal effect Effects 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 12
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 10
- 210000001072 colon Anatomy 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 8
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims 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 claims description 4
- 239000002253 acid Substances 0.000 abstract description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 abstract 1
- 239000001095 magnesium carbonate Substances 0.000 abstract 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 20
- 230000012010 growth Effects 0.000 description 19
- 238000000034 method Methods 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- 238000002703 mutagenesis Methods 0.000 description 11
- 231100000350 mutagenesis Toxicity 0.000 description 11
- 239000011521 glass Substances 0.000 description 7
- 239000012620 biological material Substances 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000588722 Escherichia Species 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000047 product Substances 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
- 241000233866 Fungi Species 0.000 description 2
- 230000003570 biosynthesizing effect Effects 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 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
- 238000011534 incubation Methods 0.000 description 2
- 230000000394 mitotic effect Effects 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
- 239000000243 solution Substances 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
- 230000009466 transformation Effects 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
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
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- 229920000229 biodegradable polyester Polymers 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- 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
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- 238000012827 research and development Methods 0.000 description 1
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- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a colibacillus capable of generating succinic acid by utilizing synthetic medium pure anaerobic growth. The colibacillus is classified and named as EscherichiacoliBER108 and has the preservation number of CCTCC (China Center for Type Culture Collection) NO.M2012068. The invention further discloses an application of the colibacillus to fermentation production of succinic acid. According to the invention, plasma is utilized to induce colibacillus, a synthetic medium flat screen is utilized to screen out a strain capable of growing rapidly under an anaerobic condition, and under the anaerobic condition, the strain grows by utilizing an inorganic nitrogen source and glucose and accumulates succinic acid; the strain is fermented in a shake flask for 72 hours by utilizing basic magnesium carbonate as a pH regulator; the OD600 of the strain reaches 7.6, the succinic acid yield reaches 11.2g/L, the yield of the succinic acid is increased by nearly three times compared with that of an original strain; and the original strain grows slowly and is low in acid yield under the pure anaerobic synthetic medium condition, so that the mutant strain BER108 has great social significance and economic value.
Description
Technical field
The invention belongs to industrial micro breeding and fermentation technical field thereof, relate to the intestinal bacteria that a strain utilizes the pure anaerobic growth succinic acid-producing of synthetic medium.
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 is 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 the focus that acid becomes Recent study.
Fermentation succinic acid-producing bacterial strain is one of biosynthetic key point of Succinic Acid, because 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 be that thalline obtains 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, so that the research of a 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 technical problem to be solved in the present invention is to provide a strain can utilize the intestinal bacteria of synthetic medium Fast Growth under pure anaerobic condition, makes it can accumulate 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 utilizes the intestinal bacteria of the pure anaerobically fermenting succinic acid-producing of synthetic medium, its Classification And Nomenclature be colon bacillus (
Escherichia coli) BER108, its preserving number is numbered: CCTCC NO:M 2012068.
Two, the colon bacillus of utilizing the pure anaerobically fermenting succinic acid-producing of synthetic medium of the present invention
Escherichia coliThe screening method of BER108 is with colibacillary starting strain
E.coliAFP111 utilizes the synthetic medium plate screening to obtain the bacterial strain that can under anaerobic grow after plasma body mutagenesis, through the anaerobism shake flask fermentation screening obtain can the high yield succinic acid-producing bacterial strain be aimed strain.Its concrete steps are as follows:
1) plasma mutagenesis: with the intestinal bacteria original strain
E.coliAFP111 activates in test tube, and 37 ℃, 200r/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 ~ 120W as radio frequency power, as gas flow, take 10 ~ 30s as irradiation time, bacterial strain is carried out plasma body mutagenesis with 10 ~ 30SLM.
2) the dull and stereotyped primary dcreening operation of synthetic medium: the slide glass after the mutagenesis is placed the tool plug test tube of the physiological saline that 1mL is housed, concuss with the bacterium liquid wash-out on the slide glass, is diluted to different concentration and coats on the synthetic medium flat board, and 37 ℃ of anaerobism are cultivated 24h; Select growth larger, comparatively full bacterium colony.
3) the bacterial strain repeatedly turning point cultivation on synthetic medium that screens the dull and stereotyped multiple sieve of synthetic medium: with step 2), 37 ℃ of anaerobism are cultivated 24h, select growth larger, comparatively full bacterium colony.
4) anaerobism shake flask fermentation screening: with the inoculation that filters out in step 3) enlarged culturing in the seed culture medium, 37 ℃, 200r/min cultivates 48h, then ferments in fermention medium, and inoculum size is 2%(v/v), 37 ℃, 200r/min cultivates 72h; 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.
Three, utilize the application of intestinal bacteria in fermentation growth Succinic Acid of the pure anaerobic growth succinic acid-producing of synthetic medium: the dull and stereotyped colon bacillus BER108 that cultivates of solid synthetic medium is inoculated in the seed culture medium, carbonating is cultivated, and cultivates 48 h for 37 ℃ and obtains seed liquor; Seed liquor is inoculated in the fermention medium inoculum size 2%, carbonating fermentation culture 72h.
In above-mentioned screening method and fermentation process,
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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, agar 20g/L, glucose 10g/L.
Seed culture medium is: peptone 10g/L, yeast powder 5g/L, NaCl 5g/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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, magnesium basic carbonate 24 ~ 42g/L, glucose 30 ~ 40 g/L.
Beneficial effect of the present invention is:
The present invention uses plasma body mutagenesis intestinal bacteria, utilizes synthetic medium dull and stereotyped, and filter out under anaerobic and can utilize the synthetic medium Fast Growth, and the bacterial strain of high succinic acid-producing.This bacterial strain can be take inorganic nitrogen as nitrogenous source, and 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 product acid of synthetic medium and glucose growth, the 72h cell density has reached OD
600=7.6, Succinic Acid output is 11.2g/L, and original starting strain is extremely slow in this condition growth, 72h thalline OD
600Only be 0.34, so this bacterial strain have great social effect and economic worth.
Embodiment
Microorganism classification called after colon bacillus of the present invention (
Escherichia coli) BER108, depositary institution's full name is Chinese Typical Representative culture collection center, is called for short CCTCC, the address is: China. and Wuhan. Wuhan University.Preservation date is on March 8th, 2012, and deposit number is CCTCC NO:M 2012068.
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 claims.
Starting strain of the present invention
E.coliThere are two places in the source of the competence bacterial strain of AFP111:
（1）Biotechnol?Bioeng,?2001,?74:89～95。The applicant is at first by finding the above-mentioned document source of this biomaterial, and to have contacted the utterer be the David P. Clark professor of Univ Chicago USA, and its this biomaterial of gifting of mail requests, and freely obtained this biomaterial; And the applicant guaranteed to provide this biomaterial to the public in 20 years from the application's day;
(2) this biomaterial also discloses in the patent documentation of Chinese patent (application number 96198547.X, applying date 1996.10.31 authorize day on January 1st, 2003, Granted publication CN1097632C) and obtains the authorization.
Embodiment 1
The present embodiment explanation with colon bacillus (
Escherichia coli) screening method of BER108.
1, to carry out the method for the first step plasma body mutagenesis as follows for the intestinal bacteria original strain:
With intestinal bacteria
E.coliThe AFP111 original strain activates in the LB test tube, and 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 ~ 120W as radio frequency power, as gas flow, take 10 ~ 30s as irradiation time, bacterial strain is carried out plasma body mutagenesis with 10 ~ 30SLM; After the mutagenesis, the stroke-physiological saline solution of the mycoderm on the slide glass with 1 mL eluted, be coated on the synthetic medium flat board, in anaerobic box, cultivate.
In above-mentioned screening method, in the described plasma body mutagenesis method, preferred 100W is radio frequency power, and gas flow is 20SLM, and irradiation time is 15s.
2, screening step:
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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, agar 20g/L, glucose 10g/L.
(2) seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl 5g/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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, magnesium basic carbonate 24 ~ 42g/L, glucose 30 ~ 40 g/L.
The screening step:
A, 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 24h, pick out and select fast growth, comparatively full bacterium colony.
B, 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 BER49, BER93, and BER102 and BER108 have shown stronger growth velocity and growth stability.
C, shake flask fermentation screening
With bacterial strain BER49, BER93, enlarged culturing in BER102 and the BER108 access seed culture medium, carbonating 2min, 37 ℃, 200r/min cultivates 48h.Then be inoculated in the Medium of shaking flask fermentation 100mL anaerobism serum bottle liquid amount 30mL, inoculum size 2%(v/v), carbonating 2min, 37 ℃, 200r/min cultivates 72h.Detect each bacterial strain cell density and Succinic Acid output as shown in table 1:
Under pure anaerobic condition, the starting strain speed of growth is extremely slow, and the accumulation of Succinic Acid also very low, 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, the strain growth speed of BER108 is very fast, and product acid is higher.
Embodiment 2
The present embodiment explanation colon bacillus (
Escherichia coli) mitotic stability of BER108.
On the synthetic medium flat board, many turning points of mutant strain BER108 to be cultivated, and will be obtained the bacterial strain checking of fermenting respectively, experimental result is as shown in table 2:
From experimental result as can be known, through 8 continuous passages, the Succinic Acid output of mutant strain BER108 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 3
The present embodiment explanation colon bacillus (
Escherichia coli) technique of BER108 fermentation production of succinic acid.
The described culture medium prescription of the 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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, agar 20g/L, glucose 10g/L.
Seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl 5g/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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, magnesium basic carbonate 24 ~ 42g/L, glucose 30 ~ 40g/L.
With colon bacillus
Escherichia coliBER108 is seeded to plate culture medium, cultivates 37 ℃ of culture temperature, incubation time 24h in anaerobic box.The BER108 that flat board is cultivated is inoculated in the seed culture medium 100mL serum bottle liquid amount 30mL, carbonating 2min, 37 ℃ of culture temperature, 200r/min, incubation time 48h; Seed is inoculated in the fermention medium inoculum size 2%(v/v), 100mL serum bottle liquid amount 30mL, carbonating 2min, the output that detects Succinic Acid behind the fermentation culture 72h is 11.2g/L, the Succinic Acid transformation efficiency is 75%.
Claims (3)
1. a strain utilizes the intestinal bacteria of the pure anaerobic growth succinic acid-producing of synthetic medium, its Classification And Nomenclature be colon bacillus (
Escherichia coli)BER108, its preserving number is numbered: CCTCC NO:M 2012068.
2. the application of intestinal bacteria in fermentation production of succinic acid that utilizes the pure anaerobic growth succinic acid-producing of synthetic medium claimed in claim 1.
3. application according to claim 2 is characterized in that concrete steps are: the dull and stereotyped colon bacillus BER108 that cultivates of solid synthetic medium is inoculated in the seed culture medium, and carbonating is cultivated, and cultivates 48 h for 37 ℃ and obtains seed liquor; Seed liquor is inoculated in the fermention medium inoculum size 2%, carbonating fermentation culture 72h;
Wherein, the dull and stereotyped prescription of described solid synthetic 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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, agar 20g/L, glucose 10g/L;
Described seed culture based formulas is: peptone 10g/L, yeast powder 5g/L, NaCl 5g/L;
Described fermentative medium formula 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(SO4)
31.77 mgL
-1, Na
2MoO
42H
2O 0.5 mgL
-1, ironic citrate 16.1 mgL
-1, magnesium basic carbonate 24 ~ 42g/L, glucose 30 ~ 40 g/L.
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| CN102864113B (en) * | 2012-10-16 | 2014-01-22 | 南京工业大学 | Strain capable of producing succinic acid, method for producing succinic acid and application thereof |
| CN102936575B (en) * | 2012-12-05 | 2014-05-07 | 南京工业大学 | Escherichia coli LL016 and application thereof |
| CN103451126B (en) * | 2013-07-04 | 2015-07-01 | 南京工业大学 | High-concentration ammonium ion tolerant escherichia coli capable of producing succinic acid and application thereof |
| CN103320367B (en) * | 2013-07-10 | 2014-12-31 | 南京工业大学 | Screening method and application of Escherichia coli for preparing high-yield succinic acids by utilizing synthetic media under anaerobic conditions |
| CN105586283A (en) * | 2014-10-20 | 2016-05-18 | 中国石化扬子石油化工有限公司 | Escherichia coli YPC-SA-1 and application thereof |
| CN104877941B (en) * | 2015-05-27 | 2018-06-08 | 南京工业大学 | The Escherichia coli of one plant of ammonium ion tolerance type succinic acid-producing and its application |
| CN106337064B (en) * | 2016-04-07 | 2019-11-26 | 南京工业大学 | A kind of production method of L MALIC ACID |
| CN110527630B (en) * | 2019-05-24 | 2021-04-20 | 浙江工业大学 | Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof |
| CN112239738B (en) * | 2020-10-29 | 2022-11-25 | 江南大学 | Escherichia coli capable of producing succinic acid and application thereof |
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