CN105462904A - Method for industrially and fast producing flavobacterium mizutaii with high yield - Google Patents
Method for industrially and fast producing flavobacterium mizutaii with high yield Download PDFInfo
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- CN105462904A CN105462904A CN201610038230.6A CN201610038230A CN105462904A CN 105462904 A CN105462904 A CN 105462904A CN 201610038230 A CN201610038230 A CN 201610038230A CN 105462904 A CN105462904 A CN 105462904A
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Abstract
The invention discloses a method for industrially and fast producing flavobacterium mizutaii with the high yield. The method comprises the specific steps that a culture solution with the sugar content of 2% to 6% is inoculated with a flavobacterium mizutaii strain, the flavobacterium mizutaii strain is cultured at the temperature of 28 DEG C to 37 DEG C till the number of cells ranges from 20-60 hundred million per milliliter, and a seed culture solution is obtained; then a fermentation cylinder containing a culture solution is inoculated with the seed culture solution, culture is carried out till the number of cells is 40-80 hundred million per milliliter, and a fermentation cylinder seed culture solution is obtained; then a fermentation cylinder containing a culture solution is inoculated with the fermentation cylinder seed culture solution, fermentation is carried out till the number of cells is 100-200 hundred million per milliliter, and fermentation is finished. The total number of the cells of a flavobacterium mizutaii cylinder-out solution fermented through the method reaches up to 100-200 hundred million per milliliter, and the method can be used for industrially producing flavobacterium mizutaii.
Description
Technical field
The invention belongs to field of microbial fermentation, be specifically related to a kind of method that industrialization high yield produces Flavobacterium fast.
Background technology
Microorganism can be sole carbon source and the energy with organic pollutant or carry out Co metabolism and degradable organic pollutant with other organic substances.The microorganism remediation technology utilizing microbiological deterioration to develop is a kind of recovery technique common during Farmland Soil Pollution is repaired, and this bioremediation technology is applied in agricultural chemicals or oil-polluted soils.In China, agricultural chemicals efficient degrading bacteria triage techniques, microorganism renovation agent technology of preparing and pesticide residue microbiological deterioration Field information technology are constructed; And being separated to can using the microorganism of polycyclic aromatic hydrocarbons (PAHs) as sole carbon source, as Rhodopseudomonas, Flavobacterium etc.Particularly Flavobacterium (Flavobacteriummizutaii) is a kind of excellent improvement agricultural land soil and environment remediation microbial preparation, but the fermentation process of routine is only limitted to laboratory stage, cell quantity cannot be obtained in the industrial production many, active good Flavobacterium.
Therefore, be badly in need of a kind of method that industrialization high yield produces Flavobacterium fast, the method is simple, and fermentation period is short, can obtain the Flavobacterium that total cellular score reaches 100-200 hundred million/ml within a short period of time.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of industrialization high yield to produce the method for Flavobacterium fast, processing method is simple, can obtain out tank liquid total cellular score within a short period of time large, the bacterium liquid that living cell rate is high.
For achieving the above object, the invention provides following technical scheme:
Industrialization high yield produces a method for Flavobacterium fast, comprises the steps:
(1) spawn culture: by Flavobacterium (Flavobacteriummizutaii) inoculation in the nutrient solution of sugar degree 2 ~ 6%, being then cultured to cell count under temperature is 28 ~ 37 DEG C of conditions is 20 ~ 6,000,000,000/ml, obtains seed culture fluid;
(2) fermentor tank seed culture: it is in the fermentor tank of 2 ~ 6% that step (1) gained seed culture fluid is seeded to nutrient solution sugar degree, is 6 ~ 14m by air flow
3/ h passes into sterile air, and under temperature is 28 ~ 37 DEG C of conditions, be cultured to cell count is 40 ~ 8,000,000,000/ml, obtains fermentor tank seed culture fluid;
(3) step (2) gained fermentor tank seed culture fluid being seeded to nutrient solution sugar degree is in the fermentor tank of 4 ~ 10%, is first 16 ~ 28m by air flow
3/ h passes into sterile air, and being 28 ~ 37 DEG C of condition bottom fermentations after 6 ~ 8 hours in temperature, is 28 ~ 50m by air flow
3/ h passes into sterile air, and continuing fermentation is 100-200 hundred million/ml to cell count, obtains Flavobacterium bacterium liquid.
Preferably, described step (1) for Flavobacterium is seeded to by 8 ~ 12% inoculum sizes in the nutrient solution of sugar degree 3 ~ 6%, then temperature be 30 ~ 35 DEG C, to be cultured to cell count under being 7-7.5 condition be 20 ~ 8,000,000,000/ml to pH, obtains seed culture fluid; Preferred, described pH is 7.0.
Preferably, in step (2), described seed culture fluid is by volume for 1:40 is seeded in seed fermentation tank.
In the present invention, described nutrient solution contains nitrogenous source, phosphorus source, magnesium sulfate and calcium carbonate, and each constituent mass per-cent is as follows: nitrogenous source 5-8%, phosphorus source 0.1-0.2%, magnesium sulfate 0.1-0.2%, calcium carbonate 0.1-0.5%.
Preferably, described nitrogenous source is beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, ammoniacal liquor, urea, saltpetre, SODIUMNITRATE, ammonium nitrate, ammonium phosphate, one or more in ammonium hydrogen phosphate or Secondary ammonium phosphate; Described phosphorus source is phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.
Preferred, the preparation method of described beef soup is as follows: beef steam water is boiled 30min, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
Most preferred, the feed postition of described nutrient solution is nitrogenous source, carbon source, phosphorus source, magnesium sulfate, and calcium carbonate adds with seed fermentation tank nutrient solution.
Beneficial effect of the present invention is: the method for a kind of industrialization quick fermentation production Flavobacterium disclosed by the invention, and fermentation period is short, and cost is low, and the Flavobacterium total cellular score going out to fill with reaches 100-200 hundred million/ml, is obviously better than existing fermentation process.
Embodiment
To be described in detail the preferred embodiments of the present invention below.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
In the present invention, Flavobacterium (Flavobacteriummizutaii) derives from INST OF AGRICULTURAL RESOURCES, does is open network address: http://www.accc.org.cn/search/accc/show.asp? jzbc=03071; Deposit number is ACCC03071.
Embodiment 1
Industrialization high yield produces a method for Flavobacterium fast, comprises the steps:
(1) spawn culture: Flavobacterium (Flavobacteriummizutaii) is seeded in the nutrient solution of sugar degree 6% by 8% inoculum size, then temperature be 35 DEG C, to be cultured to cell count under being 7.0 conditions be 4,000,000,000/ml to initial pH, obtains seed culture fluid;
(2) fermentor tank seed culture: be in the nutrient solution of 2% for 1:40 is seeded to sugar degree by volume by step (1) gained seed culture fluid is 8m by air flow
3/ h passes into sterile air, and under temperature is 35 DEG C of conditions, be cultured to cell count is 5,000,000,000/ml, obtains fermentor tank seed culture fluid;
(3) being in the fermentor tank of 5% nutrient solution by step (2) gained fermentor tank seed culture fluid inoculation sugar degree, is first 24m by air flow
3/ h passes into sterile air, and being 35 DEG C of condition bottom fermentations after 7 hours in temperature, is 32m by air flow
3/ h passes into sterile air, continues fermentation and then ferments complete to cell count at 100 ~ 20,000,000,000/ml, go out tank.
Detect tank liquid after going out to fill with, be coated with flat board and determine viable count; Result shows, and using method of the present invention fermentation Flavobacterium to go out tank number is 12,000,000,000/ml.
In the present embodiment, Flavobacterium (Flavobacteriummizutaii) is provided by INST OF AGRICULTURAL RESOURCES, and is preserved by our company.Above-mentioned nutrient solution also includes nitrogenous source 5%, phosphorus source 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.1%.Nitrogenous source is selected from beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, urea, saltpetre, SODIUMNITRATE, at least one in ammonium nitrate; Phosphorus source is selected from phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.Wherein beef chopping steam water is boiled 30min by the preparation method of beef soup as follows, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
Embodiment 2
Industrialization high yield produces a method for Flavobacterium fast, comprises the steps:
(1) spawn culture: Flavobacterium (Flavobacteriummizutaii) is seeded in the nutrient solution of sugar degree 3% by 10% inoculum size, then temperature be 30 DEG C, to be cultured to cell count under being 7.0 conditions be 4,500,000,000/ml to initial pH, obtains seed culture fluid;
(2) fermentor tank seed culture: be in the nutrient solution of 4% for 1:40 is seeded to sugar degree by volume by step (1) gained seed culture fluid is 10m by air flow
3/ h passes into sterile air, and under temperature is 35 DEG C of conditions, be cultured to cell count is 6,000,000,000/ml, obtains fermentor tank seed culture fluid;
(3) being in the fermentor tank of 6% nutrient solution by step (2) gained fermentor tank seed culture fluid inoculation sugar degree, is first 26m by air flow
3/ h passes into sterile air, and being 35 DEG C of condition bottom fermentations after 8 hours in temperature, is 40m by air flow
3/ h passes into sterile air, continues fermentation to cell count and then ferments complete at 100 ~ 20,000,000,000/ml.Go out after tank goes out to fill with and detect tank liquid, be coated with flat board and determine viable count; Result shows, and using method of the present invention fermentation Flavobacterium to go out tank number is 15,000,000,000/ml.
In the present embodiment, Flavobacterium (Flavobacteriummizutaii) is provided by INST OF AGRICULTURAL RESOURCES, and is preserved by our company.Above-mentioned nutrient solution also includes nitrogenous source 6%, phosphorus source 0.15%, magnesium sulfate 0.1%, calcium carbonate 0.2%.Nitrogenous source is selected from beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, urea, saltpetre, SODIUMNITRATE, at least one in ammonium nitrate; Phosphorus source is selected from phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.Wherein beef chopping steam water is boiled 30min by the preparation method of beef soup as follows, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
Embodiment 3
Industrialization high yield produces a method for Flavobacterium fast, comprises the steps:
(1) spawn culture: Flavobacterium (Flavobacteriummizutaii) is seeded in the nutrient solution of sugar degree 4% by 10% inoculum size, then temperature be 37 DEG C, to be cultured to cell count under being 7.0 conditions be 6,000,000,000/ml to initial pH, obtains seed culture fluid;
(2) fermentor tank seed culture: be in the nutrient solution of 5% for 1:40 is seeded to sugar degree by volume by step (1) gained seed culture fluid is 14m by air flow
3/ h passes into sterile air, and under temperature is 37 DEG C of conditions, be cultured to cell count is 8,000,000,000/ml, obtains fermentor tank seed culture fluid;
(3) being in the fermentor tank of 8% nutrient solution by step (2) gained fermentor tank seed culture fluid inoculation sugar degree, is first 28m by air flow
3/ h passes into sterile air, and being 35 DEG C of condition bottom fermentations after 7 hours in temperature, is 50m by air flow
3/ h passes into sterile air, continues fermentation to cell count and then ferments complete at 100 ~ 20,000,000,000/ml.Go out after tank goes out to fill with and detect tank liquid, be coated with flat board and determine viable count; Result shows, and using method of the present invention fermentation Flavobacterium to go out tank number is 20,000,000,000/ml.
In the present embodiment, Flavobacterium (Flavobacteriummizutaii) is provided by INST OF AGRICULTURAL RESOURCES, and is preserved by our company.Above-mentioned nutrient solution also includes nitrogenous source 8%, phosphorus source 0.2%, magnesium sulfate 0.2%, calcium carbonate 0.5%.Nitrogenous source is selected from beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, urea, saltpetre, SODIUMNITRATE, at least one in ammonium nitrate; Phosphorus source is selected from phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.Wherein beef chopping steam water is boiled 30min by the preparation method of beef soup as follows, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
Embodiment 4
Industrialization high yield produces a method for Flavobacterium fast, comprises the steps:
(1) spawn culture: Flavobacterium (Flavobacteriummizutaii) is seeded in the nutrient solution of sugar degree 2% by 12% inoculum size, then temperature be 28 DEG C, to be cultured to cell count under being 7.5 conditions be 2,000,000,000/ml to initial pH, obtains seed culture fluid;
(2) fermentor tank seed culture: be in the nutrient solution of 3% for 1:40 is seeded to sugar degree by volume by step (1) gained seed culture fluid is 6m by air flow
3/ h passes into sterile air, and under temperature is 28 DEG C of conditions, be cultured to cell count is 4,000,000,000/ml, obtains fermentor tank seed culture fluid
(3) being in the fermentor tank of 4% nutrient solution by step (2) gained fermentor tank seed culture fluid inoculation sugar degree, is first 16m by air flow
3/ h passes into sterile air, and being 28 DEG C of condition bottom fermentations after 8 hours in temperature, is 28m by air flow
3/ h passes into sterile air, continues fermentation to cell count and then ferments complete at 100 ~ 20,000,000,000/ml.Go out after tank goes out to fill with and detect tank liquid, be coated with flat board and determine viable count; Result shows, and using method of the present invention fermentation Flavobacterium to go out tank number is 10,000,000,000/ml.
In the present embodiment, Flavobacterium (Flavobacteriummizutaii) is provided by INST OF AGRICULTURAL RESOURCES, and is preserved by our company.Above-mentioned nutrient solution also includes nitrogenous source 5%, phosphorus source 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.1%.Nitrogenous source is selected from beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, urea, saltpetre, SODIUMNITRATE, at least one in ammonium nitrate; Phosphorus source is selected from phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.Wherein beef chopping steam water is boiled 30min by the preparation method of beef soup as follows, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
In above-described embodiment, the feed postition of described nutrient solution is nitrogenous source, carbon source, magnesium sulfate, and calcium carbonate is better with seed fermentation tank nutrient solution effect.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (8)
1. industrialization high yield produces a method for Flavobacterium fast, it is characterized in that, comprises the steps:
(1) spawn culture: by Flavobacterium (Flavobacteriummizutaii) inoculation in the nutrient solution of sugar degree 2 ~ 6%, being then cultured to cell count under temperature is 28 ~ 37 DEG C of conditions is 20 ~ 6,000,000,000/ml, obtains seed culture fluid;
(2) fermentor tank seed culture: it is in the fermentor tank of 2 ~ 6% that step (1) gained seed culture fluid is seeded to nutrient solution sugar degree, is 6 ~ 14m by air flow
3/ h passes into sterile air, and under temperature is 28 ~ 37 DEG C of conditions, be cultured to cell count is 40 ~ 8,000,000,000/ml, obtains fermentor tank seed culture fluid;
(3) step (2) gained fermentor tank seed culture fluid being seeded to nutrient solution sugar degree is in the fermentor tank of 4 ~ 8%, is first 16 ~ 28m by air flow
3/ h passes into sterile air, and being 28 ~ 37 DEG C of condition bottom fermentations after 6 ~ 8 hours in temperature, is 28 ~ 50m by air flow
3/ h passes into sterile air, and continuing fermentation is 100-200 hundred million/ml to cell count, obtains Flavobacterium bacterium liquid.
2. industrialization high yield produces the method for Flavobacterium fast according to claim 1, it is characterized in that: described step (1) is for be seeded to Flavobacterium by 8 ~ 12% inoculum sizes in the nutrient solution of sugar degree 3 ~ 6%, then temperature be 30 ~ 35 DEG C, to be cultured to cell count under being 7-7.5 condition be 20 ~ 8,000,000,000/ml to pH, obtains seed culture fluid.
3. industrialization high yield produces the method for Flavobacterium fast according to claim 1, it is characterized in that: in step (2), and described seed culture fluid is by volume for 1:40 is seeded in seed fermentation tank.
4. industrialization high yield produces the method for Flavobacterium fast according to claim 1, it is characterized in that: described pH is 7.0.
5. industrialization high yield produces the method for Flavobacterium fast according to claim 1, it is characterized in that: described nutrient solution contains nitrogenous source, phosphorus source, magnesium sulfate and calcium carbonate, each constituent mass per-cent is as follows: nitrogenous source 5-8%, phosphorus source 0.1-0.2%, magnesium sulfate 0.1-0.2%, calcium carbonate 0.1-0.5%.
6. industrialization high yield produces the method for Flavobacterium fast according to claim 5, it is characterized in that: described nitrogenous source is beef soup, peptone, corn juice, ammonium sulfate, bicarbonate of ammonia, ammoniacal liquor, urea, saltpetre, SODIUMNITRATE, ammonium nitrate, ammonium phosphate, one or more in ammonium hydrogen phosphate or Secondary ammonium phosphate; Described phosphorus source is phosphoric acid, ammonium phosphate, ammonium hydrogen phosphate, Secondary ammonium phosphate, potassium primary phosphate, one or more in dipotassium hydrogen phosphate.
7. industrialization high yield produces the method for Flavobacterium fast according to claim 6, it is characterized in that: the preparation method of described beef soup is as follows: beef steam water is boiled 30min, then filters to obtain beef soup; Described corn liquid is prepared by following methods: Dried Corn Steep Liquor Powder steam water boiled, and filters to obtain corn slurries.
8. industrialization high yield produces the method for Flavobacterium fast according to claim 5, it is characterized in that: the feed postition of described nutrient solution is nitrogenous source, carbon source, phosphorus source, magnesium sulfate, and calcium carbonate adds with seed fermentation tank nutrient solution.
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Citations (3)
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| CN103374525A (en) * | 2012-04-29 | 2013-10-30 | 中国石油化工股份有限公司 | Wastewater treating microbial agent and preparation method thereof |
| WO2014210372A1 (en) * | 2013-06-26 | 2014-12-31 | Symbiota, Inc. | Seed-origin endophyte populations, compositions, and methods of use |
| CN105176852A (en) * | 2015-09-14 | 2015-12-23 | 重庆富农现代生物科技有限公司 | Method for producing trichosporon cutaneum through industrialized rapid fermentation |
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2016
- 2016-01-20 CN CN201610038230.6A patent/CN105462904A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374525A (en) * | 2012-04-29 | 2013-10-30 | 中国石油化工股份有限公司 | Wastewater treating microbial agent and preparation method thereof |
| WO2014210372A1 (en) * | 2013-06-26 | 2014-12-31 | Symbiota, Inc. | Seed-origin endophyte populations, compositions, and methods of use |
| CN105176852A (en) * | 2015-09-14 | 2015-12-23 | 重庆富农现代生物科技有限公司 | Method for producing trichosporon cutaneum through industrialized rapid fermentation |
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