CN115521945B - High polyphosphate and method for producing high polyphosphate by one-step fermentation - Google Patents
High polyphosphate and method for producing high polyphosphate by one-step fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 103
- 230000004151 fermentation Effects 0.000 title claims abstract description 100
- 229920000388 Polyphosphate Polymers 0.000 title claims abstract description 87
- 239000001205 polyphosphate Substances 0.000 title claims abstract description 87
- 235000011176 polyphosphates Nutrition 0.000 title claims abstract description 87
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 29
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 80
- 239000011574 phosphorus Substances 0.000 claims abstract description 80
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 80
- 239000001963 growth medium Substances 0.000 claims abstract description 45
- 230000001580 bacterial effect Effects 0.000 claims abstract description 39
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 35
- 238000011218 seed culture Methods 0.000 claims abstract description 32
- 238000004108 freeze drying Methods 0.000 claims abstract description 29
- 238000004380 ashing Methods 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 27
- 239000000047 product Substances 0.000 claims abstract description 24
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims abstract description 18
- 241000588919 Citrobacter freundii Species 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 15
- 238000000227 grinding Methods 0.000 claims abstract description 13
- 238000010000 carbonizing Methods 0.000 claims abstract description 4
- 238000001914 filtration Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000011780 sodium chloride Substances 0.000 claims description 13
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 11
- 229930027917 kanamycin Natural products 0.000 claims description 11
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 11
- 229960000318 kanamycin Drugs 0.000 claims description 11
- 229930182823 kanamycin A Natural products 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 11
- 239000012137 tryptone Substances 0.000 claims description 10
- 101100409044 Chlorobaculum tepidum (strain ATCC 49652 / DSM 12025 / NBRC 103806 / TLS) ppk1 gene Proteins 0.000 claims description 8
- 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 description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 238000003763 carbonization Methods 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 241001557688 Citrobacter freundii ATCC 8090 = MTCC 1658 Species 0.000 claims description 3
- 108020000161 polyphosphate kinase Proteins 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910000404 tripotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019798 tripotassium phosphate Nutrition 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 11
- 241001052560 Thallis Species 0.000 abstract description 10
- 230000000052 comparative effect Effects 0.000 description 17
- 239000012043 crude product Substances 0.000 description 15
- 239000010802 sludge Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 239000002609 medium Substances 0.000 description 12
- 235000010633 broth Nutrition 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 239000002245 particle Substances 0.000 description 9
- 238000009825 accumulation Methods 0.000 description 8
- 239000000306 component Substances 0.000 description 7
- 238000010304 firing Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 208000037062 Polyps Diseases 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000003837 high-temperature calcination Methods 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical group [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003575 carbonaceous material Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910021392 nanocarbon Inorganic materials 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 230000002572 peristaltic effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
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- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000122229 Acinetobacter johnsonii Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229920000037 Polyproline Polymers 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000192707 Synechococcus Species 0.000 description 1
- 241001123263 Zostera Species 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 238000004146 energy storage Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention discloses a high polyphosphate and a method for producing the high polyphosphate by one-step fermentation, belonging to the technical field of high polyphosphate production. The method of the invention comprises the following steps: selecting a monoclonal from LB plate with citrobacter freundii to seed culture medium to obtain seed culture solution, inoculating the seed culture solution to phosphorus polymerization culture medium for fermentation to obtain fermentation liquor; collecting fermentation liquor, centrifuging, filtering to obtain bacterial mud, freeze-drying bacterial mud, carbonizing, grinding, ashing, dissolving ashed products with lithium chloride, centrifuging, collecting supernatant, and freeze-drying to obtain the high polyphosphate. The preparation process flow provided by the invention does not need high-temperature or high-pressure equipment, is simple, reduces the production cost, has few production steps, shortens the fermentation period, and improves the phosphorus gathering speed and phosphorus gathering amount of thalli and the yield of high polyphosphate.
Description
Technical Field
The invention relates to the technical field of high polyphosphate production, in particular to high polyphosphate and a method for producing high polyphosphate by one-step fermentation.
Background
The Polyphosphate is a biological macromolecule with the most compact structure composed of orthophosphate residues and high-energy phosphorus-oxygen bonds, and is an energy storage high-molecular material which is widely existed in organism cells and is highly conserved in evolution. Although chemical synthesis is still the mainstream preparation method of the present high polyphosphate, namely, phosphoric acid and ammonia gas are melted and polymerized at 200-350 ℃ to obtain a high polyphosphate finished product (the chain length is less than 20 Pi), a method for synthesizing the high polyphosphate with higher polymerization degree (20 Pi < chain length is less than 700 Pi), higher unit negative charge density and higher functionality and characteristics is also available.
Bacteria that can produce high polyphosphate under aerobic conditions have been reported to date: the degree of polymerization of the polyphosphate and how the polyphosphate is separated from the bacterial liquid are not described in the above patents, although the engineering bacteria of the genus Zostera (CN 105368838A), acinetobacter johnsonii (CN 109022328A), synechococcus transgenosis (CN 106916775A) and Citrobacter freundii (CN 104531599B). CN 103687582B reports that the polyphosphate product mixture with a degree of polymerization of 20-300 can be obtained from yeast cells by extraction, but that the polyphosphate product cannot be separated from proteins or other endotoxin components in the yeast cells.
Recently, CN 112322664B discloses a method for producing polyphosphate with high polymerization degree by using citrobacter freundii ATCC 8090 as a host and introducing a polyphosphate kinase coding gene Ppk1 of the host, but the preparation process takes more than 35 h, high-density fermentation of 10-16 h is required to be carried out in a fermentation tank, bacterial sludge collected after fermentation is centrifuged, and bacterial liquid containing the polyphosphate product is obtained after putting the bacterial sludge into a sequencing batch bioreactor for culturing 16-19 h, and each gram of bacterial dry weight contains only 140mg of polyphosphate. In addition, CN 113073121A discloses a nano carbon material containing high-polymerization degree polyphosphate and a preparation method of the high-polymerization degree polyphosphate, but the nano carbon material needs to be subjected to aeration culture for 15-30 hours in a bioreactor, and then concentrated for 1-2 h by a PVDF flat membrane element in the bioreactor to obtain fermentation liquor, and the preparation process takes more than 30 hours.
High polyphosphate is representative of natural high energy products, but the bio-fermentation production process for preparing high polyphosphate economically, environmentally friendly, efficiently and simply has not yet been formed into a production scale.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a high polyphosphate and a method for producing the high polyphosphate by one-step fermentation. The preparation process flow provided by the invention does not need high-temperature or high-pressure equipment, is simple, reduces the production cost, has few production steps, shortens the fermentation period, and improves the phosphorus gathering speed and phosphorus gathering amount (the fermentation period is shortened) of the thalli and the high polyphosphate yield (the phosphorus content of the thalli).
The technical scheme of the invention is as follows:
the method for producing the high polyphosphate by one-step fermentation comprises the following steps:
(1) Selecting a monoclonal from LB plate with citrobacter freundii to seed culture medium to obtain seed culture solution, inoculating the seed culture solution to phosphorus polymerization culture medium for fermentation to obtain fermentation liquor;
(2) Collecting the fermentation liquor obtained in the step (1), centrifuging, filtering to obtain bacterial mud, freeze-drying, carbonizing, grinding, ashing, dissolving ashed products with lithium chloride, centrifuging at a speed of more than or equal to 12000rpm for 10-15 min, collecting supernatant, and freeze-drying at-75 to-80 ℃ for 5-6 h to obtain the high polyphosphate.
Further, the Citrobacter freundii is a phosphorus transfer gene Ppk1 Citrobacter freundii, and the phosphorus transfer gene Ppk1 Citrobacter freundii is a strain disclosed in patent CN 104531599B.
The method for producing the high polyphosphate by one-step fermentation comprises the following steps:
(1) Selecting a monoclonal strain from LB plates containing 20-25 mg/L kanamycin and growing a seed strain Citrobacter freundii to a seed culture medium to obtain a seed culture solution, inoculating the seed culture solution to a phosphorus polymerization culture medium for fermentation, and obtaining a fermentation solution;
(2) Collecting the fermentation liquor obtained in the step (1), centrifuging, filtering, freeze-drying, carbonizing, grinding, ashing, stirring, re-ashing, dissolving the ashed product by using lithium chloride, centrifuging at a speed of more than or equal to 12000rpm for 10-15 min, collecting supernatant, and freeze-drying at-80 to-75 ℃ for 5-6 h to obtain the high polyphosphate.
Further, in the step (1), the LB plate contains 20-25 mg/mL kanamycin; the seed culture medium comprises the following components in per liter: 5-6 g of tryptone, 0.35-10 g of sodium chloride, 18-20 g of yeast powder, 1-2 g of industrial glucose and 20-25 mg of kanamycin.
Further, in the step (1), the specific preparation method of the seed culture solution comprises the following steps: and (3) selecting a monoclonal, inoculating the monoclonal to a 500mL baffle shake flask filled with 100-150 mL of seed culture medium, and culturing for 12-13 h at a shaking table temperature of 35-37 ℃ and a rotating speed of 180-200 rpm overnight.
Further, in the step (1), the specific process of the fermentation is as follows: inoculating the seed culture solution into 2.5L of phosphorus polymerization culture medium with an inoculum size of 6-8% so that the initial OD600 is 0.25-0.3, and fermenting.
Further, in the step (1), the fermentation conditions are as follows: the fermentation temperature is 35-37 ℃, the initial rotation speed is 200-300 r/min, the dissolved oxygen concentration is 30%, and the air inlet ratio is 1:1, the pH value is 7.0, and the fermentation time is 4-12 hours.
Further, in the step (1), the phosphorus polymerization culture medium comprises the following components per liter:
1.5-2 g of sucrose and 0.18-2 g of ammonium chloride; 0.1-0.7 g of tryptone; 0.1-0.2 g of yeast powder; 0.5-1.776 g of tripotassium phosphate; 0.05-0.35 g of sodium chloride; 0.236-1.652 g of magnesium sulfate heptahydrate; the phosphorus content of the phosphorus polymerization culture medium is 60-210 mg/L.
Further, in the step (2), the speed of centrifugation is 6000-8000 r/min, and the time is 5-10 min.
Further, in the step (2), the specific process of freeze-drying is as follows: freezing the bacterial mud at the temperature of-75 to-80 ℃ for 5-6 hours, and then freeze-drying the bacterial mud in a vacuum freeze dryer for 1-3 days.
Further, in the step (2), the carbonization is to fire the freeze-dried bacterial sludge for 1 hour at the temperature of more than or equal to 700 ℃; the specific process of ashing is that the ground product is ashed for 1h at the temperature of more than or equal to 700 ℃, and then ashed again for 0.5h at the temperature of more than or equal to 700 ℃ after stirring.
A high polyphosphate prepared by the above method. The high polyphosphate is a crude product of the high polyphosphate, the chain length distribution is 40-60 Pi, and the pure product of the high polyphosphate can be obtained through an alcohol precipitation method.
The beneficial technical effects of the invention are as follows:
(1) The invention improves the seed culture medium and the phosphorus polymerization culture medium components and the proportion thereof required by production, improves the concentration of strains, realizes the synchronous (one-step fermentation) production of the growth of the strains and the production of the phosphorus polymerization, and obtains the one-step fermentation production of the high polyphosphate, thereby avoiding the complicated process of the prior production process that the growth of the strains is performed by pre-fermentation to regenerate the fermentation (or the pre-culture and the re-fermentation in a bioreactor), simplifying the process flow, improving the phosphorus polymerization speed and the phosphorus polymerization amount of the thalli, shortening the fermentation period to 8-11h, saving the production cost while saving the time and improving the fermentation efficiency. Meanwhile, the strain cultured by adopting the culture medium has higher phosphorus concentration than the strain activated in CN 113073121A.
(2) The invention optimizes the process conditions of firing and collecting the high polyphosphate, and improves the recovery rate of the product by approximately 20 percent; by carbonization and multiple ashing processes, proteins or other endotoxin components within the yeast cells can be burned off leaving only phosphate.
(3) The method for producing the high-polymer phosphate by one-step fermentation does not need a high-temperature high-pressure device, establishes reasonable fermentation and purification process routes and corresponding process parameters, can be used for process flows of shake flask fermentation, batch fermentation, constant-speed fed-batch fermentation, variable-speed constant-concentration fed-batch fermentation and the like, is beneficial to the amplification and large-scale production of the process, and has great significance for basic scientific research and market development.
Drawings
FIG. 1 is a data graph of shake flask fermentation according to example 1 of the present invention.
FIG. 2 is a data graph of batch fermentation according to example 2 of the present invention.
FIG. 3 is a data graph of constant rate feed for example 3 of the present invention.
FIG. 4 is a product diagram of a batch fermentation according to example 2 of the present invention.
In the figure: A. is the fungus mud after freeze-drying; B. is carbonized particles; C. to obtain a crude polyp product.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
The citrobacter freundii used in the embodiment of the invention is a phosphorus transfer gene Ppk1 citrobacter freundii, the phosphorus transfer gene Ppk1 citrobacter freundii is a strain disclosed in patent CN 104531599B, the citrobacter freundii ATCC 8090 is taken as a host, and a polyphosphate kinase numbering gene Ppk1 of the host itself is introduced.
Example 1
A polyphosphate salt produced by one-step fermentation, the one-step fermentation process comprising the steps of:
(1) Preparation of seed solution from LB plates containing 25 mg/L Kanamycin grown with the seed strain Citrobacter freundii, were picked up and placed in 500mL baffle shake flasks containing 100 mL seed medium, and incubated at 37℃overnight at 200 rpm on a shaker for 12h (seed medium composition: tryptone 5g, sodium chloride 10g, yeast powder 20 g, industrial glucose 1g, kanamycin 25mg per liter).
(2) And (3) phosphorus polymerization fermentation production: inoculating the seed culture solution obtained in the step (1) into 2.5L of phosphorus polymerization culture medium with an inoculum size of 8% so that the initial OD600 is about 0.25, and performing shake flask one-step fermentation (the phosphorus polymerization culture medium comprises 1.5g of sucrose, 0.18/g of ammonium chloride, 0.1/g of tryptone, 0.1/g of yeast powder, 0.3/g of dipotassium phosphate trihydrate, 0.05/g of sodium chloride and 0.236/g of magnesium sulfate heptahydrate).
The fermentation conditions are as follows: 37. at a temperature of 300 rpm at initial speed, pH 7.0.
The maximum phosphorus accumulating amount of 38 mg/L can be obtained after the culture is measured for 8 hours, and the phosphorus accumulating amount of unit thalli reaches 149 mg P/g dry weight. Wherein the maximum phosphorus accumulation amount is measured in the fermentation broth, the phosphorus accumulation amount of unit thallus is measured by a potassium persulfate digestion method, and the result is shown in figure 1, and OD600 is the absorbance value of the bacterial culture broth at 600 nm; p/dw is the phosphorus content in the dry weight of the bacteria; polyp represents the amount of phosphorus; p remove is the concentration of phosphorus in the supernatant after centrifugation of the broth.
(3) Collecting high polyphosphate: and (3) collecting and centrifuging the fermentation liquor in the step (2), and sequentially carrying out freeze-drying, carbonization, grinding, ashing, re-ashing, dissolution, centrifugation and freeze-drying on bacterial mud obtained after centrifugation to obtain a high polyphosphate crude product.
The specific operation is as follows: a. collecting fermentation liquor, and centrifuging at 8000 rpm for 5min at 4 ℃ to obtain bacterial sludge; b. freezing bacterial mud 5h at-80 ℃ and then freeze-drying in a vacuum freeze dryer for 1 day; c. firing the freeze-dried bacterial sludge at 700 ℃ to carbonize 1h; d. grinding the black carbonized particles and then ashing 1h at 700 ℃; e. stirring the ashed product, and ashing again at 700 ℃ for 0.5h until no black particles exist; f. dissolving the ashed product with 250 mM lithium chloride; g.12000 Centrifuging at rpm for 10 min; h. collecting the supernatant and freeze-drying at-80 ℃ for 5 hours to obtain the crude product of the high polyphosphate. The data of yield, molecular weight, chain length and the like of the obtained high polyphosphate after the high-temperature calcination crude extraction are shown in table 1.
Table 1: the experimental data of the high polyphosphate detection obtained by high-temperature calcination and crude extraction are as follows:
the high polyphosphate yield was calculated as 2.5L broth; the yield of the high polyphosphate is calculated by dry weight of the degerming body of the crude high polyphosphate; the average relative molecular weight of the polyphosphate is calculated by taking glucan as a standard sample; the average chain length is calculated using type45 as a standard.
Example 2
A polyphosphate salt produced by one-step fermentation, the one-step fermentation process comprising the steps of:
(1) Preparing seed liquid: from LB plates containing 25 mg/L Kanamycin, which had grown with the seed strain Citrobacter freundii CPP, single clones were picked up in 500mL baffle shake flasks containing 100 mL seed medium, shake flasks were incubated at 37℃overnight at 200 rpm for 12h (the seed medium composition was tryptone 5g, sodium chloride 0.35 g, yeast powder 20 g, industrial glucose 1g, kanamycin 25mg per liter).
(2) And (3) phosphorus polymerization fermentation production: inoculating the seed culture solution obtained in the step (1) into 2.5L of phosphorus polymerization culture medium with an inoculum size of 8% so that the initial OD600 is about 0.25, and performing batch one-step fermentation (the phosphorus polymerization culture medium comprises 1.5g of sucrose, 0.18g of ammonium chloride, 0.1g of tryptone, 0.1g of yeast powder, 0.596 g of dipotassium phosphate trihydrate, 0.05 g of sodium chloride and 0.236 g of magnesium sulfate heptahydrate per liter).
The fermentation conditions are as follows: the initial rotation speed of the phosphorus polymerization culture medium is 300 rpm at 37 ℃, the pH value is 7.0, and the air inlet ratio is 1:1.
The maximum phosphorus concentration of about 4. 4 h and about 30 mg P/L are measured, and the high polyphosphate content of unit bacteria reaches 175 mg P/g-dry weight. The results are shown in FIG. 2, where Glc is the sucrose concentration; OD600 is absorbance of the bacterial culture at 600 nm; p/dw is the phosphorus content in the dry weight of the bacteria; polyp represents the amount of phosphorus and Pi represents the concentration of phosphorus in the supernatant after centrifugation of the fermentation broth.
(3) Collecting high polyphosphate: and (3) collecting and centrifuging the fermentation liquor in the step (2), and sequentially carrying out freeze-drying, carbonization, grinding, ashing, re-ashing, dissolution, centrifugation and freeze-drying on bacterial mud obtained after centrifugation to obtain a high polyphosphate crude product.
The specific operation is as follows: a. collecting fermentation liquor, and centrifuging at 8000 rpm for 5min at 4 ℃ to obtain bacterial sludge; b. freezing bacterial mud 5h at-80 ℃ and then freeze-drying in a vacuum freeze dryer for 1-3 days; c. firing the freeze-dried bacterial sludge at 700 ℃ to carbonize 1h; d. grinding the black carbonized particles and then ashing 1h at 700 ℃; e. stirring the ashed product, and ashing again at 700 ℃ for 0.5h until no black particles exist; f. dissolving the ashed product with 250 mM lithium chloride; g.12000 Centrifuging at rpm for 10 min; h. collecting the supernatant and freeze-drying at-80 ℃ for 5 hours to obtain the crude product of the high polyphosphate.
Example 3
A polyphosphate salt produced by one-step fermentation, the one-step fermentation process comprising the steps of:
(1) Preparation of seed solution from a LB plate containing 25 mg/L Kanamycin grown with the seed strain Citrobacter freundii CPP, single clones were picked up in 500mL baffle shake flasks containing 100 mL seed culture medium, and incubated at 37℃overnight at 200 rpm for 12h (seed culture medium composition: tryptone 5g, sodium chloride 0.35 g, yeast powder 20 g, industrial glucose 1g, kanamycin 25mg per liter).
(2) And (3) performing phosphorus polymerization fermentation production, namely inoculating the seed culture solution obtained in the step (1) into 2.5L of phosphorus polymerization culture medium with an inoculum size of 8 percent, so that the initial OD600 is about 0.25, and performing constant-speed feed one-step fermentation (the phosphorus polymerization culture medium comprises sucrose 2g, ammonium chloride 1.26 g, tryptone 0.7g, yeast powder 0.2g, dipotassium phosphate trihydrate 1.776 g, sodium chloride 0.35 g and magnesium sulfate heptahydrate 1.652 g per liter). The fermentation conditions are as follows: 37. at a temperature of 300 rpm, pH 7.0, DO 30%, and an air inlet ratio of 1:1, sucrose was supplemented at a rate of 1 g/(L h).
After supplementing the carbon source, CPP can convert almost all phosphorus in the supernatant into polyp, and the unit cell yield in a fermentation tank can reach 245 mg P/L, and the fermentation period is 11 h. The results are shown in FIG. 3, where Glc is the sucrose concentration; OD600 is absorbance of the bacterial culture at 600 nm; p/dw is the phosphorus content in the dry weight of the bacteria; polyp represents the amount of phosphorus and Pi represents the concentration of phosphorus in the supernatant.
(3) Collecting high polyphosphate, collecting and centrifuging the fermentation liquor in the step (2), and sequentially carrying out freeze-drying, carbonization, grinding, ashing, re-ashing, dissolving, centrifuging and freeze-drying on bacterial mud obtained after centrifugation to obtain a high polyphosphate crude product.
The specific operation is as follows: a. collecting fermentation liquor, and centrifuging at 8000 rpm for 5min at 4 ℃ to obtain bacterial sludge; b. freezing bacterial mud 5h at-80 ℃ and then freeze-drying in a vacuum freeze dryer for 2 days; c. firing the freeze-dried bacterial sludge at 700 ℃ to carbonize 1h; d. grinding the black carbonized particles and then ashing 1h at 700 ℃; e. stirring the ashed product, and ashing again at 700 ℃ for 0.5h until no black particles exist; f. dissolving the ashed product with 250 mM lithium chloride; g.12000 Centrifuging at rpm for 10 min; h. collecting the supernatant and freeze-drying at-80 ℃ for 6 hours to obtain the crude product of the high polyphosphate.
Comparative example 1
A high polyphosphate salt was produced in the same manner as in example 1, except that a phosphorus polymerization medium was used. The components of the phosphorus polymerization medium used in comparative example 1 were: glucose 0.5 g, peptone 0.3 g, yeast powder 0.1g, anhydrous sodium acetate 0.3 g, sodium chloride 0.3 g, magnesium sulfate heptahydrate 0.1g, dipotassium phosphate trihydrate 0.75 g, ammonium chloride 0.5 g).
Comparative example 2
A high polyphosphate salt was produced in the same manner as in example 1, except that the seed medium was used. The seed culture medium used in comparative example 2 is a high-density fermentation broth comprising the following components in percentage by mass: 3% of yeast extract, 3% of peptone, 2% of sodium chloride, inorganic salt ions and regulating the pH value to be neutral.
Comparative example 3
A production method of the high polyphosphate comprises the following steps of
(1) A monoclonal colony was picked from the LB plate used in example 3, inoculated into a 500mL baffle shake flask containing 100 mL seed medium, and cultured overnight at 200 rpm at 37℃on a shaker 16 h. ( The seed culture medium is as follows: 3% of yeast extract, 3% of peptone, 2% of sodium chloride, inorganic salt ions and regulating the pH value to be neutral. )
(2) Pre-fermentation: the resulting fermentation broth was centrifuged to obtain a bacterial slurry, which was then poured into 2.5L of a phosphorus-containing medium to an initial OD600 of 0.25 and subjected to pre-fermentation at room temperature of 6. 6 h.
(3) And (3) producing fermentation, namely starting to circulate water in and out (inserting peristaltic pump pipes into the phosphorus-containing culture medium), setting the flow rate of the peristaltic pump to be 60 rpm, and then adjusting the flow rate in time according to the culture condition. And (3) inserting a flat membrane, starting a water pumping device, circularly feeding and discharging water, and culturing 24 h to obtain fermentation liquor.
(4) Collecting high polyphosphate: and (3) collecting and centrifuging the fermentation liquor in the step (3), and sequentially carrying out freeze-drying, carbonization, grinding, ashing, re-ashing, dissolution, centrifugation and freeze-drying on bacterial mud obtained after centrifugation to obtain a high polyphosphate crude product.
The specific operation is as follows: a. collecting fermentation liquor, and centrifuging at 8000 rpm for 5min at 4 ℃ to obtain bacterial sludge; b. freezing bacterial mud 5h at-80 ℃ and then freeze-drying in a vacuum freeze dryer for 2 days; c. firing the freeze-dried bacterial sludge at 700 ℃ to carbonize 1h; d. grinding the black carbonized particles and then ashing 1h at 700 ℃; e. stirring the ashed product, and ashing again at 700 ℃ for 0.5h until no black particles exist; f. dissolving the ashed product with 250 mM lithium chloride; g.12000 Centrifuging at rpm for 10 min; h. collecting the supernatant and freeze-drying at-80 ℃ for 6 hours to obtain the crude product of the high polyphosphate.
Comparative example 4
The fermentation process of the high polyphosphate is the same as in example 1, except that the process for collecting the high polyphosphate is as follows:
(1) High-temperature calcination: the same weight of the freeze-dried bacterial sludge obtained by the same operation as in example 1 was subjected to high temperature firing at 700℃in a tube furnace for 2h, and naturally cooled to room temperature to obtain carbonized bacterial sludge.
(2) Grinding: the carbonized bacterial sludge was manually ground with a mortar pestle.
(3) Dissolving: the solution obtained in the step (2) was dissolved in 250 mM lithium chloride, and then centrifuged at 12000rpm for 10 minutes, and the supernatant was collected to obtain an aqueous solution of high polyphosphate.
(4) Preparing a crude product: and (3) freezing the high polyphosphate water solution in the step (3) in a refrigerator at the temperature of minus 80 ℃ for 2h, and then placing the high polyphosphate water solution in a vacuum freeze dryer for dehydration and drying for 1 day to obtain a high polyphosphate crude product 93.513 mg.
Test example:
and (3) measuring the phosphorus content in the supernatant obtained by centrifuging the fermented thalli by using an ammonium molybdate spectrophotometry, and indirectly obtaining the phosphorus content consumed by the actual thalli. The intracellular polyP content of the cells was measured by potassium persulfate digestion to obtain the maximum phosphorus accumulation amounts of the fermentation broths obtained in examples 1 to 3 and comparative examples 1 to 3, and the results are shown in FIGS. 1 to 3, respectively.
As is clear from FIG. 1, in example 1, the maximum phosphorus concentration was 38 mg/L and the phosphorus concentration per cell was 149 mg P/g-dry weight after 8 hours fermentation. As is clear from FIG. 2, example 2 reached a maximum phosphorus concentration of about 30 mg P/L and a high polyphosphate content per cell of 175 mg P/g-dry weight after about 4. 4 h. As is clear from FIG. 3, in example 3, the CPP of the strain Citrobacter freundii after supplementing the carbon source can convert almost all of the phosphorus in the supernatant into polyp, and the yield per unit cell in the fermenter can reach 245 mg P/L, and the fermentation period is 11 h.
As is clear from examination of examples and comparative example 1, the phosphorus accumulation rate of the phosphorus accumulation medium used in the fermentation in step (2) of example 1 was about 38 mg/L and only 20 mg/L, which is approximately doubled when the phosphorus accumulation medium was cultured for fermentation in the fermentation culture of 8h, compared with the phosphorus accumulation medium used in step (2) of comparative example 1. The improved phosphorus polymerization culture medium has greatly improved yield of high polyphosphate. As is evident from the comparison of example 1 and example 2, the seed culture medium used in step (1) of example 1 was cultured under the same conditions and time as the high-density fermentation broth used in the liquid activation of example 2, the OD600 of the seed strain was 12 in the shake flask, and the OD600 of the seed strain was only 5 to 6. To achieve the initial OD600 value required for fermentation, the amount of high density broth required is twice that of the seed medium. Comparison of example 1 with comparative example 2 shows that the improved seed medium facilitates the growth of seed strains.
Comparative example 3 and comparative example 3 it is clear that the seed culture medium used in step (1) of example 3 allows the OD600 of the seed strain to reach 12 in shake flasks and the OD600 of the latter is only 5-6 when compared to the high density broth used for liquid activation of comparative example 3 under the same culture conditions and time. The pre-fermentation step of 4-6 h can be omitted, and the fermentation period is shortened. Realizes synchronous fermentation growth of strains and phosphorus accumulation production. The phosphorus-containing culture medium used in the fermentation in the step (2) of example 3 was at most 11h per gram of cells per dry weight of phosphorus-containing culture medium as compared with the phosphorus-containing culture medium in the step (2) of comparative example 3, and the phosphorus-containing culture medium had a phosphorus concentration of 245 mg P/g dry weight and a fermentation time of about 30h per gram of cells per dry weight of phosphorus-containing culture medium of 140mg per gram of phosphorus-containing culture medium. Not only shortens the fermentation period by half, but also improves the content of high polyphosphate in each gram of dry weight of thalli by about 75 percent.
Comparative example 4 examined the effect of the conditions of the process for collecting the high polyphosphate, and comparison of example 1 with comparative example 4 shows that the firing time and the ashing degree have a great effect on the yield of the crude high polyphosphate product. The process of the invention adopts the method that the carbonized bacterial sludge is ashed at 700 ℃ for 1h, and then ashed at 700 ℃ for 0.5h after stirring, thus the crude product of the high polyphosphate of 110.2 mg can be obtained, and the yield of the prepared crude product of the high polyphosphate is improved.
The amounts of phosphorus accumulating mg P/g-dry weight per unit cell obtained by the preparation methods described in examples 1-3 and comparative examples 1-3 are shown in the following Table.
Table 2: high polyphosphate content in cells
As shown in the table, the reasonable fermentation and purification process route and the corresponding process parameters are established, and the phosphorus accumulating amount of the thalli is greatly improved.
According to the invention, the fermentation process in the preparation of the high polyphosphate is completed in one step by optimizing the seed culture medium and the phosphorus polymerization culture medium, and the recovery of the high polyphosphate crude product in the thalli is improved, namely the yield of the high polyphosphate crude product is improved by optimizing the collecting process of the high polyphosphate.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. A method for producing high polyphosphate by one-step fermentation, which is characterized by comprising the following steps:
(1) Selecting a monoclonal from LB plate with citrobacter freundii to seed culture medium to obtain seed culture solution, inoculating the seed culture solution to phosphorus polymerization culture medium for fermentation to obtain fermentation liquor;
the citrobacter freundii is a phosphorus transfer gene Ppk1 citrobacter freundii, takes citrobacter freundii ATCC 8090 as a host, and is introduced with a polyphosphate kinase numbering gene Ppk1 of the host;
the seed culture medium comprises the following components in per liter: 5-6 g of tryptone, 0.35-10 g of sodium chloride, 18-20 g of yeast powder, 1-2 g of industrial glucose and 20-25 mg of kanamycin; (2) Collecting the fermentation liquor obtained in the step (1), centrifuging, filtering to obtain bacterial mud, freeze-drying, carbonizing, grinding, ashing, dissolving ashed products with lithium chloride, centrifuging at a speed of more than or equal to 12000rpm for 10-15 min, collecting supernatant, and freeze-drying at-75 to-80 ℃ for 5-6 h to obtain high polyphosphate;
in the step (1), the specific fermentation process is as follows: inoculating the seed culture solution into a phosphorus polymerization culture medium with an inoculum size of 6-8% so that the initial OD600 is 0.25-0.3, and fermenting;
in the step (1), the components of the phosphorus polymerization culture medium are as follows per liter: 1.5-2 g of sucrose and 0.18-2 g of ammonium chloride; 0.1-0.7 g of tryptone; 0.1-0.2 g of yeast powder; 0.5-1.776 g of tripotassium phosphate; 0.05-0.35 g of sodium chloride; 0.236-1.652 g of magnesium sulfate heptahydrate; the phosphorus content of the phosphorus polymerization culture medium is 60-210 mg/L;
in the step (2), the specific process of freeze-drying is as follows: freezing the bacterial mud at-80 to-75 ℃ for 5-6 hours, and then freeze-drying the bacterial mud in a vacuum freeze dryer for 1-3 days;
in the step (2), the carbonization is to fire the freeze-dried bacterial mud for 1h at 700 ℃; the ashing is carried out by ashing the ground product at 700 deg.C for 1h, stirring, and re-ashing at 700 deg.C for 0.5h.
2. The method of claim 1, wherein in step (1), the LB plate contains 20-25 mg/mL kanamycin.
3. The method according to claim 1, wherein in the step (1), the seed culture solution is prepared by the following steps: and (3) selecting the monoclonal, inoculating the monoclonal into 100-150 mL of seed culture medium, and culturing the monoclonal at 35-37 ℃ and 180-200 rpm overnight for 12-13 hours.
4. The method according to claim 1, wherein in step (1), the fermentation is performed by: inoculating the seed culture solution into 2.5L of phosphorus polymerization culture medium with an inoculum size of 6-8% so that the initial OD600 is 0.25-0.3, and fermenting.
5. The method of claim 4, wherein in step (1), the fermentation conditions are: the fermentation temperature is 35-37 ℃, the initial rotation speed is 200-300 r/min, the dissolved oxygen concentration is 30%, and the air inlet ratio is 1:1, the pH value is 7.0, and the fermentation time is 4-12 hours.
6. The method according to claim 1, wherein in the step (2), the centrifugation is performed at a speed of 6000 to 8000r/min for 5 to 10 min.
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| CN104531599A (en) * | 2015-01-07 | 2015-04-22 | 南京大学 | Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof |
| WO2020109371A1 (en) * | 2018-11-28 | 2020-06-04 | Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen | Polyphosphate-rich yeast extracts and method for producing same |
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| WO2020109371A1 (en) * | 2018-11-28 | 2020-06-04 | Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen | Polyphosphate-rich yeast extracts and method for producing same |
| CN112322664A (en) * | 2020-11-02 | 2021-02-05 | 江苏南创化学与生命健康研究院有限公司 | Method for producing polyphosphate with high polymerization degree |
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