CN103320367A - Screening and application of high-yield succinic acid escherichia coli by anaerobic utilization of synthetic culture medium - Google Patents
Screening and application of high-yield succinic acid escherichia coli by anaerobic utilization of synthetic culture medium Download PDFInfo
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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 101
- 239000001384 succinic acid Substances 0.000 title claims abstract description 48
- 239000001963 growth medium Substances 0.000 title claims abstract description 30
- 238000012216 screening Methods 0.000 title claims abstract description 22
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 16
- 239000002609 medium Substances 0.000 claims abstract description 51
- 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 27
- 239000008103 glucose Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000000413 hydrolysate Substances 0.000 claims abstract description 16
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 14
- 210000001072 colon Anatomy 0.000 claims abstract description 14
- 239000002253 acid Substances 0.000 claims abstract description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 48
- 241000894006 Bacteria Species 0.000 claims description 33
- 238000000855 fermentation Methods 0.000 claims description 27
- 230000004151 fermentation Effects 0.000 claims description 26
- 230000001580 bacterial effect Effects 0.000 claims description 24
- 229960003487 xylose Drugs 0.000 claims description 24
- 230000012010 growth Effects 0.000 claims description 22
- 230000000968 intestinal effect Effects 0.000 claims description 21
- 238000011218 seed culture Methods 0.000 claims description 18
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 claims description 17
- 238000002703 mutagenesis Methods 0.000 claims description 17
- 231100000350 mutagenesis Toxicity 0.000 claims description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 16
- 229910021591 Copper(I) chloride Inorganic materials 0.000 claims description 16
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 12
- 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 12
- 239000011521 glass Substances 0.000 claims 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 claims description 9
- 229930003756 Vitamin B7 Natural products 0.000 claims description 9
- 235000019156 vitamin B Nutrition 0.000 claims description 9
- 239000011720 vitamin B Substances 0.000 claims description 9
- 239000011735 vitamin B7 Substances 0.000 claims description 9
- 235000011912 vitamin B7 Nutrition 0.000 claims description 9
- 239000007789 gas Substances 0.000 claims description 8
- 235000000346 sugar Nutrition 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000000843 powder Substances 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 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 239000012531 culture fluid Substances 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 231100000219 mutagenic Toxicity 0.000 claims description 2
- 230000003505 mutagenic effect Effects 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 2
- 239000011780 sodium chloride Substances 0.000 claims 1
- 150000008163 sugars Chemical class 0.000 claims 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 abstract description 20
- 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 abstract description 11
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 abstract description 10
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 abstract description 10
- 230000001360 synchronised effect Effects 0.000 abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 4
- 229940023064 escherichia coli Drugs 0.000 abstract 2
- 108010009736 Protein Hydrolysates Proteins 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 239000000306 component Substances 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 241000588722 Escherichia Species 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000000394 mitotic effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 2
- 239000004631 polybutylene succinate Substances 0.000 description 2
- 229920002961 polybutylene succinate Polymers 0.000 description 2
- 239000000047 product Substances 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
- 241000196324 Embryophyta Species 0.000 description 1
- 241000605008 Spirillum Species 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- -1 poly butylene succinate Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000000192 social effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to escherichia coli capable of synchronously consuming main components of xylose and glucose in lignocellulose hydrolysate to synthesize succinic acid by using a cheap synthesis culture medium under pure anaerobic condition, a screening method and application thereof, wherein the escherichia coli is classified and named as escherichia coli Escherichiacoli) BA405, the accession number of which is: CCTCC NO: and M2013160. The invention adopts normal pressure low temperature plasma to mutate colon bacillus, and utilizes a synthetic medium plate to screen out a strain which can rapidly grow under an anaerobic condition, and the strain can synchronously consume main components of xylose and glucose in lignocellulose hydrolysate to grow and accumulate succinic acid under the anaerobic condition by utilizing an inorganic nitrogen source. The invention realizes the synchronous consumption of main components of the lignocellulose hydrolysate, namely xylose and glucose, by using the inorganic nitrogen source to grow and accumulate the succinic acid. The original starting strain grows very slowly and has low acid yield under the conditions of pure anaerobic and synthetic culture medium, so the mutant strain BA405 has great social significance and economic value.
Description
Technical field
The present invention relates to the intestinal bacteria of the pure anaerobic growth succinic acid-producing of utilized synthetic medium that a strain obtains by normal temperature low-voltage plasma mutagenic and breeding, with and application in fermentation industry, belong to industrial micro breeding and fermentation technical field thereof.Background technology
Succinic Acid claims again succsinic acid, is the intermediate product of tricarboxylic acid cycle, extensively is present in human body, animal, plant and the microorganism.It is as outstanding C
4The hardware and software platform compound can be used for organic chemicals and poly butylene succinate (PBS) the class Biodegradable materials such as synthetic BDO, tetrahydrofuran (THF), is thought one of biorefinery product of following 12 kinds of most worthies by USDOE.
Succinic Acid is the common intermediate product in some anaerobism and the amphimicrobe pathways metabolism, can obtain via the pathways metabolism of various bacteria.The Production by Microorganism Fermentation Succinic Acid has the renewable resources of utilization because of it, fixedly greenhouse gases CO
2Etc. advantage and more and more cause people's concern.The characteristics such as and intestinal bacteria are because to have a culture condition simple, and metabolism network is clearer and more definite, easily transforms, and is easy to operate become the study hotspot of biological process synthesizing succinic acid in recent years.At present, the research of Succinic Acid industrial fermentation bacterial strain mainly concentrates on succinic acid-producing anaerobism spirillum (Anaerobiospirillum succiniciproducens), succinic acid-producing actinobacillus (Actinobacillus succinogenes), Corynebacterium glutamicum (Corynebacterium glutamicum), and intestinal bacteria (Escherichia coli) etc.Wherein Escherichia coli is because genetic background is clear, and 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.
Intestinal bacteria are in the situation that oxygen lacks, and the main the way of production of Succinic Acid is glucose through Embden-Meyerhof-Parnaspathway through generating phosphoenolpyruvic acid, and and then the synthetic oxaloacetic acid of metabolism, oxysuccinic acid, fumaric acid, finally with the form accumulation of Succinic Acid.The people such as G.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.Yet in the two stage fermentation processes, need to consume the growth that a large amount of nutritive ingredients is used for thalline, cause the Succinic Acid total yield to descend, owing to the thalline grow aerobically dissolved oxygen is had relatively high expectations simultaneously, required higher energy consumption and the cost inputs such as stirring and cooling, therefore, a step anaerobically fermenting succinic acid-producing receives increasing concern.Yet under pure anaerobic condition, intestinal bacteria produce Succinic Acid needs the compound nitrogen sources such as yeast powder, peptone as medium component, with inorganic nitrogen-sourced (NH
4)
2HPO
4The synthetic medium that forms etc. is compared, and price is too expensive, for this reason, need to be by further screening producing bacterial strain, acquisition can utilize the production bacterial strain of synthetic medium growth synthesizing succinic acid.
The applicant's formerly patent Chinese patent application bacterial strain BA305(publication number CN102643775A, deposit number CCTCCNO:M2012102), this bacterial strain can be under anaerobic, utilize complex medium synchronous consumption ligno-cellulose hydrolysate main component xylose and glucose synthesizing succinic acid, but in synthetic medium, can only accumulate a small amount of Succinic Acid, therefore, the present invention is on the basis of this application, pass through strain improvement, make BA305 can under pure anaerobic condition, utilize cheap synthetic medium synchronous consumption ligno-cellulose hydrolysate main component xylose and glucose synthesizing succinic acid, in Succinic Acid manufacture from now on, have far-reaching significance.
Summary of the invention
One of the technical problem to be solved in the present invention is to cultivate a strain can utilize cheap synthetic medium synchronous consumption ligno-cellulose hydrolysate main component xylose and glucose synthesizing succinic acid under pure anaerobic condition intestinal bacteria, and this colibacillary screening method is provided simultaneously.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
One, the present invention has cultivated a strain can utilize cheap synthetic medium synchronous consumption ligno-cellulose hydrolysate main component xylose and glucose synthesizing succinic acid under pure anaerobic condition intestinal bacteria, its Classification And Nomenclature is colon bacillus (Escherichia coli) BA405, and its preserving number registration number is: CCTCCNO:M2013160.
Two, the invention provides the described screening method that under pure anaerobic condition, utilizes intestinal bacteria (Escherichia coli) BA405 of cheap synthetic medium synchronous consumption ligno-cellulose hydrolysate main component xylose and glucose synthesizing succinic acid.
Screening method of the present invention adopts colibacillary starting strain BA305 after plasma body mutagenesis, screening obtains under anaerobic can utilizing synthetic medium, the bacterial strain of synchronous consumption xylose and glucose mixing sugar growth, again through the screening of anaerobism shake flask fermentation obtain can high succinic acid-producing aimed strain.
Wherein, the described intestinal bacteria bacterium that sets out is BA305, deposit number CCTCCNO:M2012102.
Screen more specifically the thalline step as follows:
1, plasma mutagenesis: the intestinal bacteria starting strain is activated in test tube, 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 10～20s 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 6h, then ferments in fermention medium, and inoculum size is 2%(v/v), 37 ℃, 200r/min cultivates 48h; 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), selection 100W is radio frequency power, and 20SLM is gas flow, and 15s is irradiation time.
The present invention further provides the application of above-mentioned intestinal bacteria in fermentation production of succinic acid.
Particularly, utilize the step of above-mentioned Escherichia coli fermentation production Succinic Acid as follows:
(1) the dull and stereotyped cultivation: colon bacillus (Escherichia coli) BA405 is seeded in anaerobism cultivation on the plate culture medium, and culture temperature is 37 ℃, and incubation time is 24h;
(2) seed culture: colon bacillus (Escherichia coli) BA405 that flat board is cultivated is inoculated in the seed culture medium, 500ml triangular flask liquid amount 100ml, and culture temperature is 37 ℃, 200r/min, incubation time are 6h;
(3) fermentation production of succinic acid: seed culture fluid is inoculated in the fermention medium, and 100ml anaerobism shaking flask liquid amount is 30ml, and magnesium basic carbonate is as pH adjusting agent, carbonating 2min; Culture temperature is 37 ℃, and 200r/min, incubation time are 48-96h.
In the method for above-mentioned fermentation production of succinic acid,
The prescription of seed culture medium comprises: peptone 10g/L, yeast powder 5g/L, NaCl5g/L.
The dull and stereotyped prescription of solid synthetic medium is: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, agar 15g/L, glucose 5g/L, wood sugar 5g/L, pH7.
Application of the present invention is in step 3 fermentation culture, take the wood sugar of 1:1 and glucose or corn hydrolyzed solution as carbon source.
Particularly, fermentative medium formula is: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, magnesium basic carbonate 16g/L, glucose 10g/L, wood sugar 10g/L;
Or citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, magnesium basic carbonate 16g/L, wood sugar 15g/L, glucose 15g/L;
Or citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, magnesium basic carbonate 16g/L contains the Corncob hydrolysate of 30g/L reducing sugar.
Beneficial effect of the present invention is:
The present invention filters out under anaerobic and can utilize the synthetic medium Fast Growth with atmospheric low-temperature plasma mutagenesis intestinal bacteria, and the bacterial strain of high succinic acid-producing.This bacterial strain can be take inorganic nitrogen as nitrogenous source, synchronous consumption glucose and xylose Fast Growth under anaerobic, and accumulate a large amount of Succinic Acid; In the 1.5L anaerobic fermentation tank, utilize synthetic medium, acid is produced in the Corncob hydrolysate growth, and the 96h cell density has reached OD
600=3.07, Succinic Acid output is 25g/L, and original starting strain is extremely slow in this condition growth, so this bacterial strain has great social effect and economic worth.
Description of drawings
Fig. 1 is colibacillary plasma body mutagenesis survival rate curve;
Fig. 2 is that mutant strain BA405 utilizes one step of synthetic medium mixing sugar anaerobically fermenting 96h experimental result graphic representation;
Fig. 3 is that mutant strain BA405 utilizes one step of synthetic medium Corncob hydrolysate anaerobically fermenting 96h experimental result graphic representation.
Embodiment
Microorganism classification called after colon bacillus of the present invention (Escherichia coli) BA405, be preserved in Chinese Typical Representative culture collection center on April 25th, 2013 and (be called for short CCTCC, address: China. Wuhan. Wuhan University), deposit number is CCTCCNO:M2013160.
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.
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 starting strain carries out the mutagenesis of the first step plasma body is as follows:
Original strain intestinal bacteria BA305 is activated in the LB test tube, 37 ℃, the 200r/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 1ml eluted, be coated on the synthetic medium flat board, in anaerobic box, cultivate.
Embodiment 2
The good colibacillary method of this example explanation screening.
Wherein, employed culture medium prescription is as follows:
(1) plate culture medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, agar 15g/L, glucose 5g/L, wood sugar 5g/L, pH7.
(2) seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl5g/L.
(3) Medium of shaking flask fermentation: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, magnesium basic carbonate 16g/L, glucose 10g/L, wood sugar 10g/L.
The screening step:
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 24h, pick out and select 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 BA403, BA405, and B411 and BA423 have shown stronger growth velocity and growth stability.
3, shake flask fermentation screening
4, with bacterial strain BA403, BA405, enlarged culturing in B411 and the BA423 access seed culture medium, 37 ℃, 200r/min cultivates 48h.Then be inoculated in the fermention medium 100ml anaerobism serum bottle liquid amount 30ml, inoculum size 2%(v/v), carbonating 2min, 37 ℃, 200r/min cultivates 48h.Detect each bacterial strain cell density and Succinic Acid output as shown in table 1:
The growth of table 1 mutant strain and starting strain and productivity ratio are
Under pure anaerobic condition, the starting strain speed of growth is extremely slow, and the accumulation of Succinic Acid is also very low, but obtains utilizing under pure anaerobic condition the bacterial strain of synthetic medium Fast Growth after the mutagenesis of normal temperature low-voltage plasma.Through the shake flask fermentation screening, the strain growth speed of BA405 is very fast, and product acid is higher.
The mitotic stability of the present embodiment explanation mutant strain BA405
On the synthetic medium flat board, many turning points of mutant strain BA405 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 BA405 mitotic stability is analyzed
From experimental result as can be known, through 8 continuous passages, the Succinic Acid output of mutant strain BA405 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
The technique of the present embodiment explanation colon bacillus EscherichiacoliBA405 fermentation production of succinic acid
The described culture medium prescription of the present embodiment:
Plate culture medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, agar 15g/L, glucose 5g/L, wood sugar 5g/L, pH7.
Seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl5g/L.
Fermention medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, magnesium basic carbonate 16g/L, glucose 10g/L, wood sugar 10g/L.
Concrete grammar: colon bacillus Escherichia coliBA405 is applied to plate culture medium, in anaerobic box, cultivates, 37 ℃ of culture temperature, incubation time 24h.The BA405 that flat board is cultivated is inoculated in the seed culture medium 500ml triangular flask liquid amount 100ml, 37 ℃ of culture temperature, 200r/min, incubation time 6h; 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 48h is 7.88g/L, the Succinic Acid transformation efficiency is 89%.
The present embodiment explanation colon bacillus Escherichia coliBA405 utilizes wood sugar and glucose ratio to produce the technique of Succinic Acid for carbon source through fermentation for the simulation sugar of 1:1
The described culture medium prescription of the present embodiment:
Plate culture medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, agar 15g/L, glucose 5g/L, wood sugar 5g/L, pH7.
Seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl5g/L.
Fermention medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, magnesium basic carbonate 16g/L, wood sugar 15g/L, glucose 15g/L.
Concrete grammar: colon bacillus Escherichia coliBA405 is applied to plate culture medium, in anaerobic box, cultivates, 37 ℃ of culture temperature, incubation time 24h.The BA405 that flat board is cultivated is inoculated in the seed culture medium 500ml triangular flask liquid amount 100ml, 37 ℃ of culture temperature, 200r/min, incubation time 6h; Seed is inoculated in the fermention medium inoculum size 10%(v/v), 3L fermentor tank liquid amount 1.5L, CO
2Flow velocity is per minute 0.2L, and 200 rpms, 37 ° of C cultivate 96h.
Fermentation results as shown in Figure 2, the output that detects Succinic Acid behind the fermentation culture 96h is 23.6g/L, the Succinic Acid transformation efficiency is 84.7%, fermenting process has realized utilizing synthetic medium synchronous consumption glucose sugar and wood sugar.
The present embodiment explanation colon bacillus Escherichia coliBA405 utilizes the technique of Corncob hydrolysate fermentation production of succinic acid.
The described culture medium prescription of the present embodiment:
Plate culture medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, vitamins B
140mgL
-1, vitamin H 4mg.L
-1, agar 15g/L, wood sugar 10g/L, pH7.
Seed culture medium: peptone 10g/L, yeast powder 5g/L, NaCl5g/L.
Fermention medium: citric acid 3gL
-1, Na
2HPO
412H
2O4gL
-1, KH
2PO
48gL
-1, (NH
4)
2HPO
48gL
-1, NH
4Cl0.2gL
-1, (NH
4)
2SO
40.75gL
-1, MgSO
47H
2O1gL
-1, CaCl
22H
2O10.0mgL
-1, ZnSO
47H
2O0.5mgL
-1, CuCl
22H
2O0.25mgL
-1, MnSO
4H
2O2.5mgL
-1, CoCl
26H
2O1.75mgL
-1, H
3BO
30.12mgL
-1, Al
2(SO4)
31.77mgL
-1, Na
2MoO
42H
2O0.5mgL
-1, ironic citrate 16.1mgL
-1, magnesium basic carbonate 16g/L contains the Corncob hydrolysate of 30g/L reducing sugar.The main component of Corncob hydrolysate is as shown in table 3.
The main component of table 3 Corncob hydrolysate
Concrete grammar: colon bacillus Escherichia coliBA405 is applied to plate culture medium, in anaerobic box, cultivates, 37 ℃ of culture temperature, incubation time 24h.The BA405 that flat board is cultivated is inoculated in the seed culture medium 500ml triangular flask liquid amount 100ml, 37 ℃ of culture temperature, 200r/min, incubation time 6h; Seed is inoculated in the fermention medium inoculum size 10%(v/v), 3L fermentor tank liquid amount 1.5L, CO
2Flow velocity is per minute 0.2L, and 200 rpms, 37 ° of C cultivate 96h.
Fermentation results as shown in Figure 3, mutant strain BA405 utilize the Corncob hydrolysate anaerobically fermenting with utilize the mixing sugar anaerobically fermenting not have larger difference, acetic acid is unique by product.The output that detects Succinic Acid behind the cultivation 96h is 25g/L, and the Succinic Acid transformation efficiency is 83.3%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a strain utilizes the intestinal bacteria of synthetic medium synthesizing succinic acid, its Classification And Nomenclature be colon bacillus (
Escherichia coli) BA405, its preserving number registration number is: CCTCC NO:M 2013160.
2. the screening method of coli strain claimed in claim 1, it is characterized in that: the intestinal bacteria starting strain is after plasma body mutagenesis, utilize the pure anaerobic condition of synthetic medium to sieve to get the bacterial strain of Fast Growth, again through the serum bottle anaerobically fermenting sieve high succinic acid-producing the intestinal bacteria aimed strain (
Escherichia coli) BA405.
3. screening method according to claim 2 is characterized in that: the described intestinal bacteria bacterium that sets out is BA305, deposit number CCTCC NO:M2012102.
4. screening method according to claim 2, it is characterized in that: concrete screening step is as follows:
1) plasma mutagenesis: the intestinal bacteria starting 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; 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 1 ml is housed, concuss, with the bacterium liquid wash-out on the slide glass, being diluted to different concentration coats on the synthetic medium flat board, 37 ℃ of anaerobism are cultivated 24 h, 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 24 h, 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 ℃, 200 r/min, cultivate 6 h, then ferment in fermention medium, inoculum size is 2%(v/v), 37 ℃, 200 r/min cultivate 48 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.
5. screening method according to claim 4, it is characterized in that described step 1) ionic medium mutagenic condition is: 100 W are as radio frequency power, and 10 SLM are as gas flow, and 15 s are as irradiation time.
6. the application of coli strain claimed in claim 1 in fermentation production of succinic acid.
7. application according to claim 6 is characterized in that described concrete applying step is as follows:
(1) the dull and stereotyped cultivation: with colon bacillus (
Escherichia coli) BA405 is seeded on the plate culture medium anaerobism and cultivates, culture temperature is 37 ℃, incubation time is 24 h;
(2) seed culture: the colon bacillus that flat board is cultivated (
Escherichia coli) BA405 is inoculated in the seed culture medium, 500 ml triangular flask liquid amounts, 100 ml, culture temperature is 37 ℃, 200 r/min, incubation time are 6 h;
(3) fermentation production of succinic acid: seed culture fluid is inoculated in the fermention medium, and 100 ml anaerobism shaking flask liquid amounts are 30 ml, and magnesium basic carbonate is as pH adjusting agent, carbonating 2 min; Culture temperature is 37 ℃, and 200 r/min, incubation time are 48-96h.
8. application according to claim 7 is characterized in that: the prescription of described seed culture medium comprises: peptone 10 g/L, yeast powder 5 g/L, NaCl 5 g/L;
The dull and stereotyped prescription of 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, vitamins B
140 mgL
-1, vitamin H 4 mg.L
-1, agar 15 g/L, glucose 5 g/L, wood sugar 5 g/L, pH 7.
9. according to claim 7 or 8 described application, it is characterized in that: in described step 3 fermentation culture, take the wood sugar of 1:1 and glucose or corn hydrolyzed solution as carbon source.
10. according to claim 7 or 8 described application, it is characterized in that: 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, vitamins B
140 mgL
-1, vitamin H 4 mg.L
-1, magnesium basic carbonate 16 g/L, glucose 10 g/L, wood sugar 10g/L;
Or 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 16 g/L, wood sugar 15 g/L, glucose 15 g/L;
Or 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 16 g/L contain the Corncob hydrolysate of 30 g/L reducing sugars.
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| CN105820991A (en) * | 2016-04-01 | 2016-08-03 | 中国科学院上海高等研究院 | Genetically engineered bacterium of Escherichia coli |
| CN110241024A (en) * | 2019-06-13 | 2019-09-17 | 西北农林科技大学 | The screening technique of one type succinate production bacterium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630290A (en) * | 2013-11-12 | 2015-05-20 | 中国石油化工股份有限公司 | Method for utilizing flocculation to prolong cycle of bacterial fermentation production of succinic acid |
| CN105820991A (en) * | 2016-04-01 | 2016-08-03 | 中国科学院上海高等研究院 | Genetically engineered bacterium of Escherichia coli |
| CN105820991B (en) * | 2016-04-01 | 2019-11-19 | 中国科学院上海高等研究院 | A kind of Recombinant organism |
| CN110241024A (en) * | 2019-06-13 | 2019-09-17 | 西北农林科技大学 | The screening technique of one type succinate production bacterium |
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