CN110982750A - High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method - Google Patents
High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method Download PDFInfo
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- CN110982750A CN110982750A CN201911371787.1A CN201911371787A CN110982750A CN 110982750 A CN110982750 A CN 110982750A CN 201911371787 A CN201911371787 A CN 201911371787A CN 110982750 A CN110982750 A CN 110982750A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
Abstract
The invention belongs to the technical field of microorganisms, and relates to a rhodopseudomonas palustris high-density fermentation method and application thereof. The rhodopseudomonas palustris strain is derived from China industrial microorganism strain preservation management center, and has the preservation number: CICC 23812. The high-density fermentation method of rhodopseudomonas palustris sequentially adopts shake flasks to activate seeds, the activated shake flask seeds are inoculated into a primary fermentation tank to perform propagation to prepare primary seeds, the primary seeds are transferred into a secondary fermentation tank to perform fermentation production, fed-batch materials are adopted in the fermentation process to improve the strain density of fermentation liquor, and the strain density can reach at least 500 hundred million CFU/ml after 5 days of fermentation. The invention solves the problems of illumination requirement, long period, high rate of mixed bacteria, difficult control of product quality and the like in the prior art. Has the advantages of short fermentation period, stable product quality, low production cost and the like. The rhodopseudomonas palustris fermentation liquor obtained by the method can be further processed into a microbial feed additive, a microbial fertilizer and a microbial water purifying agent.
Description
Technical Field
The invention relates to a rhodopseudomonas palustris high-density fermentation method and application thereof, belonging to the technical field of microorganisms.
Background
Rhodopseudomonas palustris (Rhodopseudomonas palustris) is a photosynthetic bacterium which is widely researched and applied, belongs to Rhodopseudomonas palustris of the family Exoseriaceae, has various metabolic pathways such as nitrogen fixation, hydrogen absorption, sulfide utilization and the like, and can secrete various beneficial substances such as B vitamins, coenzyme Q, carotenoid and the like.
Currently, the artificial culture of rhodopseudomonas palustris is concentrated on light fermentation culture, namely, rhodopseudomonas palustris seeds are inoculated into a liquid culture medium in a fully transparent plastic barrel or a film bag and are subjected to anaerobic fermentation for 4-5 days, and finally rhodopseudomonas palustris fermentation liquor is obtained. However, the method can not ensure that the product is not polluted by mixed bacteria, the production is influenced under the conditions of cloudy days or low temperature, the density of the thalli of the fermentation liquor is not higher than 50 hundred million/mL, and the quality of the final product can not be guaranteed.
Disclosure of Invention
The invention aims to solve the defects of the problems and provides a rhodopseudomonas palustris high-density fermentation method and application thereof.
The invention is realized by adopting the following technical scheme:
the invention cultures activated strain through liquid seed culture medium, transfers to the first-stage fermentation tank to culture and obtain first-stage liquid, transfers to the fermentation tank to obtain high-density fermentation liquor through parameter control and fed-batch culture.
The technical scheme adopted by the invention is as follows:
a rhodopseudomonas palustris high-density fermentation method comprises the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
Preferably, the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, adjusting pH to 5.0-8.0, and sterilizing at 121 deg.C for 30 min.
Preferably, the sterile liquid fermentation medium in step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
Preferably, the fed-batch fermentation medium in step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
Preferably, the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
Preferably, the pH of the liquid seed culture medium or the liquid fermentation medium is 7.0 to 8.0.
Preferably, in the step a, the rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculation amount of 1 per thousand, and cultured for 3 days on a constant temperature shaking bed with the temperature of 28 ℃ and the humidity of 60% at the rotation speed of 110-.
Preferably, in the step b, the activated rhodopseudomonas palustris liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after the culture is cultured for 2 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1: 0.5.
Preferably, in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1:0.5, a metabolizable carbon source and a metabolizable nitrogen source are provided in a feeding and supplementing manner during the fermentation, and the rhodopseudomonas palustris fermentation liquid is obtained, and the density of bacteria is up to 560 hundred million CFU/ml.
The application of the high-density rhodopseudomonas palustris as a feed additive belongs to the field of feed additives of poultry, livestock, aquatic animals and special animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
Compared with the prior art, the invention has the remarkable advantages that:
(1) the method can avoid contamination of bacteria, ensure smooth production, and ensure product quality.
(2) The method can avoid the dependence of equipment and process on illumination condition, and can be used for production in the absence of light.
(3) The method can obtain the bacterial density of more than 500 hundred million CFU/ml, and is the highest level in China at present.
(3) The method has low cost and low investment, and is suitable for mass production.
(4) The invention not only provides an industrialized high-density fermentation method for rhodopseudomonas palustris, but also enables the strain to have wide application prospect in the fields of livestock breeding, aquaculture, planting industry, wastewater treatment, river treatment and the like, and has higher social benefit.
Drawings
FIG. 1 is a diagram of activated liquid of Rhodopseudomonas palustris provided by the present invention.
FIG. 2 is a primary liquid diagram of Rhodopseudomonas palustris provided by the present invention.
FIG. 3 is a diagram of a high-density fermentation broth of Rhodopseudomonas palustris provided by the present invention.
Detailed Description
The following describes in detail a specific embodiment of the present invention with reference to the drawings.
See fig. 1-3.
The technical solutions of the present invention are further described below, but not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The invention relates to a rhodopseudomonas palustris high-density fermentation method, which comprises the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
Preferably, the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, adjusting pH to 5.0-8.0, and sterilizing at 121 deg.C for 30 min.
Preferably, the sterile liquid fermentation medium in step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
Preferably, the fed-batch fermentation medium in step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
Preferably, the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
Preferably, the pH of the liquid seed culture medium or the liquid fermentation medium is 7.0 to 8.0.
Preferably, in the step a, the rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculation amount of 1 per thousand, and cultured for 3 days on a constant temperature shaking bed with the temperature of 28 ℃ and the humidity of 60% at the rotation speed of 110-.
Preferably, in the step b, the activated rhodopseudomonas palustris liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after the culture is cultured for 2 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1: 0.5.
Preferably, in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1:0.5, a metabolizable carbon source and a metabolizable nitrogen source are provided in a feeding and supplementing manner during the fermentation, and the rhodopseudomonas palustris fermentation liquid is obtained, and the density of bacteria is up to 560 hundred million CFU/ml.
The application of the high-density rhodopseudomonas palustris as a feed additive belongs to the field of feed additives of poultry, livestock, aquatic animals and special animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
Example 1 preparation of activating solution
1) Preparing a liquid seed culture medium. The liquid seed culture medium of claim, wherein the liquid seed culture medium comprises the following components, sodium acetate 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, pH 7.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
2) Activating the liquid. Adding 1% of the production seeds into the culture medium, and culturing in a constant temperature shaking table at 28 deg.C and humidity of 60%, and rotating speed of 120 rpm. After 3 days of culture, activated seed solution was obtained as shown in FIG. 1.
Example 2 preparation of a first grade liquor
1) The liquid seed culture medium of claim, wherein the liquid seed culture medium comprises the following components, sodium acetate 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, pH 7.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
2) Inoculating the activated seed solution into a primary fermentation tank filled with a liquid seed culture medium, and carrying out constant temperature treatment at 28 ℃, stirring at 120rpm, and ventilation volume of 1: culturing for 3 days under 0.5 condition to obtain the primary liquid of Rhodopseudomonas palustris, as shown in FIG. 2.
Example 3 high Density fermentation
1) The liquid fermentation medium of claim is prepared by weighing each component, sodium acetate 30g/L, yeast powder 10g/L, ammonium sulfate 10g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 1.0g/L, ferrous sulfate heptahydrate 0.4g/L, adjusting pH to 7.0-8.0, and sterilizing at 121 deg.C for 30 min.
2) Accurately weighing the components, 300g/L sodium acetate, 100g/L yeast powder and 100g/L ammonium sulfate according to the mixture ratio of the components in the liquid feed culture medium, adjusting the pH value to 5.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
3) Inoculating all the first-stage liquid into a fermentation tank filled with a liquid fermentation culture medium, and carrying out constant temperature of 28 ℃, stirring at 120rpm and ventilation volume of 1: culturing for 5 days under the condition of 0.5, and feeding the supplemented culture medium timely according to the dissolved oxygen condition in the fermentation process to finally obtain the high-density fermentation liquor of the rhodopseudomonas palustris.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (10)
1. A rhodopseudomonas palustris high-density fermentation method is characterized by comprising the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
2. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, and adjusting pH to 5.0-8.0, sterilizing at 121 deg.C for 30 min.
3. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the sterile liquid fermentation medium in the step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
4. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the feed fermentation medium in the step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
5. The rhodopseudomonas palustris high-density fermentation method according to one of the claims 2 or 3, wherein the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
6. The Rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the pH of the liquid seed culture medium or liquid fermentation medium is 7.0-8.0.
7. The method for high-density fermentation of Rhodopseudomonas palustris as claimed in claim 1, wherein in step a, Rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculum size of 1 ‰, and cultured on a constant temperature shaking bed with a temperature of 28 ℃ and a humidity of 60% for 3 days at a rotation speed of 110-.
8. The rhodopseudomonas palustris high-density fermentation method and the application thereof as claimed in claim 1, wherein in the step b, the rhodopseudomonas palustris activated liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after 2 days of culture under the conditions of the temperature of 28 ℃, the stirring speed of 100rpm and the ventilation amount of 1: 0.5.
9. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions of 28 ℃ of temperature, 100rpm of stirring speed and 1:0.5 of ventilation, and a metabolizable carbon source and a metabolizable nitrogen source are provided by feeding and supplementing in the fermentation process to obtain the rhodopseudomonas palustris fermentation liquid with the bacterial density of 560 hundred million CFU/ml.
10. The use of rhodopseudomonas palustris of claim 1 as a feed additive in the field of poultry, livestock, aquatic animals, specialty animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
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| CN112625956A (en) * | 2020-12-25 | 2021-04-09 | 江苏苏港和顺生物科技有限公司 | Method for culturing photosynthetic bacteria by utilizing membrane concentrated biogas slurry |
| CN113151012A (en) * | 2021-05-11 | 2021-07-23 | 河北大河生物科技有限公司 | High-density fermentation method of antrodia camphorata |
| CN114891682A (en) * | 2022-05-18 | 2022-08-12 | 北京蓝晶微生物科技有限公司 | Rapid culture method and culture medium for rhodopseudomonas palustris |
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Application publication date: 20200410 |