CN116254196A - Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method - Google Patents

Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method Download PDF

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CN116254196A
CN116254196A CN202211720555.4A CN202211720555A CN116254196A CN 116254196 A CN116254196 A CN 116254196A CN 202211720555 A CN202211720555 A CN 202211720555A CN 116254196 A CN116254196 A CN 116254196A
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fermentation
culture
value
culture medium
rhodopseudomonas palustris
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朱先峰
谢晓雨
张春发
敬科举
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Xiamen Canco Biotech Co ltd
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Abstract

The application relates to the field of microbial fermentation, and in particular discloses rhodopseudomonas palustris, a culture medium, a fermentation liquid and a fermentation method. The rhodopseudomonas palustris culture medium comprises the following components in mass concentration: sodium acetate 0.5-2g/L, KH 2 PO 4 0.5‑2g/L、CaCl 2 0.05‑0.3g/L、NaHCO 3 1‑6g/L、MgCl 2 0.2‑2、NH 4 Cl 0.3-2g/L, naCl 0.3-2g/L, sodium succinate 0.5-3g/L, yeast extract 0.2-1g/L and peptone 0.2-1g/L. The culture medium can be used for fermenting and culturing rhodopseudomonas palustris, the material cost of the culture medium is greatly reduced, the fermentation method can realize low-cost high-density fermentation, and the production mode of industrial extensive culture is changed.

Description

Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method
Technical Field
The application relates to the field of microbial fermentation, in particular to rhodopseudomonas palustris, a culture medium, a fermentation liquid and a fermentation method.
Background
Photosynthetic bacteria (Photosynthetic Bacteria) are the most widely existing bacterial group older in nature, have an original light energy synthesis system, and are a general term for prokaryotes capable of photosynthesis in anaerobic or facultative anaerobic environments without producing oxygen. Rhodopseudomonas palustris (Rhodopseudomonas palustris) is the most widely used probiotic in photosynthetic bacteria, is widely applied to the fields of agriculture, fishery, environmental protection, medicine and the like, is used as a culture water quality purifying agent, has been widely applied to aquaculture for a long time, and can effectively reduce the chemical oxygen demand in shrimp culture wastewater. Rhodopseudomonas palustris is rich in various bioactive proteins, pantothenic acid, folic acid and multiple vitamins, is a safe green feed additive, and is one of 12 living microorganisms allowed to be directly added into feeds issued by the agricultural department of China. Rhodopseudomonas palustris can also accelerate nutrient absorption and transformation of plants, is beneficial to improving the yield and quality of crops, and can improve the distribution of soil flora and improve the fertility of soil. The feed product with rhodopseudomonas palustris as additive is the product of combining modern biotechnology with animal microecology and animal nutrition. The research and development of rhodopseudomonas palustris microbial preparations has important significance for promoting the development of the breeding industry in China to the directions of high efficiency, green, ecology and health, and has very broad application prospect. However, the research on the culture medium components of rhodopseudomonas palustris and various high-density culture conditions is still the most important factor for restricting the wide application of the rhodopseudomonas palustris.
The culture medium is a main place for the growth and metabolism of microorganisms, and the fit of the culture medium is related to the quality and production efficiency of microbial products. In recent years, research on the growth conditions of rhodopseudomonas palustris has been conducted in the related art, and it has been found that factors such as pH, temperature, inoculum size and the like affect the growth of rhodopseudomonas palustris and are mutually affected. The related technology has carried out response surface optimization research on rhodopseudomonas palustris fermentation conditions, and based on a single factor, the optimal growth conditions of rhodopseudomonas palustris are determined by applying a Box-nhk center combination experiment and a response surface analysis method: the rhodopseudomonas palustris has the best growth situation under the conditions of pH value of 6.89, inoculation amount of 9.71%, culture temperature of 29.8 ℃ and illumination intensity of 2000 Lux. The prior related technology optimizes the formula and the culture condition of the rhodopseudomonas palustris fermentation medium, adopts a single-factor test design to respectively carry out comparison tests on different levels of four factors of inoculum size, pH value, temperature and illumination intensity of the rhodopseudomonas palustris cultured in the laboratory, and determines the respective optimal test levels; meanwhile, the related technology also discloses that the design of the orthogonal test L16 (45) is adopted to optimize the fermentation medium formula, the single-factor random test is adopted to optimize the culture condition, and the fermentation medium formula determined by the test is as follows: glycerin 1.0 g, ammonium chloride 1.0 g, dipotassium hydrogen phosphate 0.3g, microelement solution 0.05 mL, vitamin solution 0.15 mL, distilled water 1000 mL; the optimal culture conditions are as follows: the initial pH value is 7.0, the culture temperature is 30 ℃, the inoculation amount is 6 percent, the liquid loading amount is a triangular flask of 50 mL/250 mL of the liquid culture medium, and the 40W incandescent lamp is irradiated and static cultured at a distance of 20 cm. Under the condition, 7d rhodopseudomonas palustris R1 bacteria are fermented and cultivated, and the number of the rhodopseudomonas palustris R1 bacteria can reach 5.6X108/mL. The related art also discloses a culture medium for aerobic fermentation of rhodopseudomonas palustris, which comprises the following components: 3.00 to 9.00 g/L of organic nitrogen source, 0.88 to 1.76 g/L of ammonium salt, 5.40 to 10.80 g/L of sodium succinate, 1.12 to 2.25 g/L of sodium glutamate and 0.20 to 0.60 g/L of magnesium sulfate; by using the culture medium and the aerobic culture method, rhodopseudomonas palustris can be rapidly proliferated, has higher growth rate, can obtain more rhodopseudomonas palustris thalli in a short time, and has OD660nm of 1.015 after 48h of shake flask culture. The related technology also provides another culture medium, which comprises 0.75-1.25 g/L of citric acid, 1.50-3.00 g/L of beef extract, 0.42-0.62 g/L of yeast extract, 1.50-2.00 g/L of peptone, 0.40-0.60 g/L of sodium acetate, 0.25-0.50 g/L of magnesium sulfate, 5.00-8.00 mg/L of ferrous sulfate, 0.1-0.3 mg/L of VB6, 0.10-0.30 g/L of calcium chloride, 0.10-0.25 g/L of monopotassium phosphate and 0.5-1.0 mL/L of compound inorganic salt solution, wherein the formula improves the quality of the rhodopseudomonas palustris bacterial agent, and the bacterial agent has the advantages of high effective viable count of 8.96×109 bacteria, positive color, no layering, low mixed bacteria rate and stability. The related art also discloses a photosynthetic bacteria culture medium and a preparation method thereof, wherein the photosynthetic bacteria culture medium comprises sodium acetate trihydrate, ammonium sulfate, sodium bicarbonate, potassium dihydrogen phosphate, magnesium sulfate and water, and the OD660nm of the photosynthetic bacteria is 2.2.
However, photosynthetic bacteria are available on the market-marshes are falseFor example, after the upstream culture process is widely put down, the production of a large-scale fermentation tank is not carried out, and the common bacterial density cfu/mL is only 10 9 The level is that the cultivation process problems mostly carry a considerable amount of mixed bacteria, the quality is poor, the product can only be sold in a liquid form, the product cannot be stored in a powder form due to the fact that the product cannot be subjected to spray drying due to cost, and the circulation cost of the product is increased.
Disclosure of Invention
In order to solve at least one of the technical problems, the application provides rhodopseudomonas palustris, a culture medium, a fermentation broth and a fermentation method.
In a first aspect, the present application provides rhodopseudomonas palustris, which adopts the following technical scheme:
rhodopseudomonas palustris with a preservation number of CCTCC M20222088 CK-ZZH501.
Rhodopseudomonas palustris of the application is preserved in China center for type culture collection (CCTCC M20222088 CK-ZH 501), the preservation date is No. 28 of 12 months in 2022, and the preservation address is Wuhan, university of Wuhan in China.
The rhodopseudomonas palustris has high strain purity and short bevel activation time, and can reach higher strain density without illumination from the step-by-step amplification tank culture process from the amplification to the fermentation, so that the rhodopseudomonas palustris is not easy to dye bacteria.
In a second aspect, the present application provides a seed culture medium of rhodopseudomonas palustris, which adopts the following technical scheme:
a culture medium of rhodopseudomonas palustris, which comprises the following components in mass concentration: sodium acetate 0.5-2g/L, KH 2 PO 4 0.5-2g/L、CaCl 2 0.05-0.3g/L、NaHCO 3 1-6g/L、MgCl 2 0.2-2、NH 4 Cl 0.3-2g/L, naCl 0.3-2g/L, sodium succinate 0.5-3g/L, yeast extract 0.2-1g/L and peptone 0.2-1g/L.
By adopting the technical scheme, the components of the culture medium are optimally designed, the material cost of the culture medium is greatly reduced, and the method is applicable to mass seed expansion fermentation. Specifically, the mass concentration of sodium acetate in the culture medium0.5-2g/L, which may be, for example, any of 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L, and 2 g/L; KH (KH) 2 PO 4 The mass concentration of (C) is 0.5-2g/L, and may be, for example, any of 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L and 2 g/L; caCl (CaCl) 2 The mass concentration of (C) is 0.05-0.3g/L, and may be, for example, any of 0.05g/L, 0.06g/L, 0.07g/L, 0.08g/L, 0.09g/L, 1g/L, 0.1g/L, 0.11g/L, 0.12g/L, 0.13g/L, 0.14g/L, 0.15g/L, 0.16g/L, 0.17g/L, 0.18g/L, 0.19g/L, 0.2g/L, 0.21g/L, 0.22g/L, 0.23g/L, 0.24g/L, 0.25g/L, 0.26g/L, 0.27g/L, 0.28g/L, 0.29g/L and 0.3 g/L; naHCO (NaHCO) 3 The mass concentration of (C) is 1-6g/L, and may be, for example, any of 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, 5.5g/L and 6 g/L; mgCl 2 The mass concentration of (C) is 0.2-2g/L, and may be, for example, any of 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L and 2 g/L; NH (NH) 4 The Cl concentration by mass is 0.3-2g/L, and may be, for example, any of 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L and 2 g/L; the mass concentration of NaCl is 0.3-2g/L, and can be any value of 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L and 2 g/L; the sodium succinate has a mass concentration of 0.5 to 3g/L, and may be, for example, any of 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L, 2g/L, 2.1g/L, 2.2g/L, 2.3g/L, 2.4g/L, 2.5g/L, 2.6g/L, 2.7g/L, 2.8g/L, 2.9g/L and 3 g/L; the mass concentration of the yeast extract powder is 0.2-1g/L, and can be, for example, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L and 1g/LAny one of the values of (2); the concentration of peptone by mass is 0.2-1g/L, and may be, for example, any of 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L and 1g/L.
Preferably, the pH of the medium is between 6.8 and 7.5.
By adopting the technical scheme, the pH value of the culture medium is optimally designed, and the specific pH value is controlled to be 6.8-7.5, and can be any value of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 and 7.5.
Preferably, each 50L of the medium further comprises: 0.5-2mL of microelement solution and 0.5-2mL of vitamin solution.
By adopting the technical scheme, the components of the culture medium are optimally designed, and 0.5-2mL of microelement solution and 0.5-2mL of vitamin solution are added according to each 50L of culture medium. Specifically, the trace element solution may be added in an amount of 0.5 to 2mL, for example, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, 1.1mL, 1.2mL, 1.3 mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL and 2 mL; the amount of the vitamin solution to be added is 0.5 to 2mL, and may be, for example, any of 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, 1.1mL, 1.2mL, 1.3 mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL and 2mL.
By adopting the technical scheme, the culture effect can be further improved by controlling the pH value of the culture medium or adding the microelement solution and the vitamin solution.
In a third aspect, the present application provides a fermentation broth, which adopts the following technical scheme:
a fermentation broth containing the above Rhodopseudomonas palustris is provided. .
In a fourth aspect, the present application provides a fermentation method, which adopts the following technical scheme:
a fermentation method of rhodopseudomonas palustris as claimed in claim 1, wherein the rhodopseudomonas palustris is placed in the culture medium for anaerobic culture until the OD660nm value reaches a preset value.
Preferably, the fermentation method specifically comprises the following steps:
s1: placing the rhodopseudomonas palustris in the culture medium for anaerobic culture until the OD660nm value reaches a preset value to obtain seed liquid;
s2: inoculating the seed liquid into a first preset volume of the culture medium for fermentation culture until the OD660nm value reaches the preset value, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a first bacterial liquid;
s3: inoculating the first bacterial liquid into a second preset volume of the culture medium for fermentation culture until the OD660nm value reaches the preset value, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain the second bacterial liquid.
Preferably, the fermentation method specifically comprises the following steps:
s1: taking 0.5-1mL of rhodopseudomonas palustris to be placed in the culture medium for anaerobic culture until the OD660nm value reaches 0.8-1, and obtaining seed liquid;
s2: inoculating the seed liquid into 50L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a first bacterial liquid;
s3: inoculating the first bacterial liquid into 500L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a second bacterial liquid;
s4: inoculating the second bacterial liquid into 5000L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a third bacterial liquid;
s5: inoculating the third bacterial liquid into 20000L of culture medium according to the volume inoculation amount of 10-20% for fermentation culture until the OD660nm value reaches 0.8-1, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a fourth bacterial liquid.
By adopting the technical scheme, the method designs the suitable based on the optimized culture mediumThe fermentation process for mass seed expansion production comprises the steps of preparing and preserving pure rhodopseudomonas palustris seeds from a source, expanding seeds step by a fermentation tank to the production of the last 20 ton tank, completely realizing the modern mass production, and compared with the cultivation process of a plastic barrel and a transparent ton bag after rough placement, the fermentation process has the advantages that the index of mass production of the large-scale fermentation tank is controllable, the infection of mixed bacteria is avoided, and the product quality is stable; in addition, after the fermentation of the bacterial liquid in the fermentation tank is finished, the subsequent packaging bottle with the volume of 5-20L can be filled, and the bacterial quantity of the bacterial liquid subjected to illumination for 3-7d exceeds 3 x 10 through test 10 The cfu/mL and the photosynthetic bacteria of 10-50 hundred million/mL which are sold in far-reaching super market can meet the feeding requirement of a spray drying tower after being centrifugally concentrated, and in addition, the product can be prepared into powder under the condition of low cost rise, so that the competitiveness of the product is enhanced.
Preferably, in step S2 or S3, after inoculation and before fermentation culture, the dissolved oxygen value of the culture medium is controlled to be lower than 5% and the pH value is adjusted to 6.8-7.0; stirring is continuously carried out in the fermentation culture process.
Preferably, the fermentation culture process in steps S3, S4 and S5 is maintained at pH by intermittent addition of alkaline solution, respectively; when the alkaline solution is intermittently added, stirring the solution and adjusting the stirring speed, wherein the adjustment of the stirring speed specifically comprises at least one operation of the following steps S3-S5:
in the step S3, when the stirring speed in the initial period of fermentation culture is 145-155rpm and the dissolved oxygen value is reduced to zero, controlling the stirring speed to be 200-300rpm;
in the step S4, when the stirring speed in the initial period of fermentation culture is 95-105rpm and the dissolved oxygen value is reduced to zero, controlling the stirring speed to be 120-200rpm;
in the step S5, the stirring speed in the initial period of fermentation culture is 55-65rpm, and when the dissolved oxygen value is reduced to zero, the stirring speed is controlled to be 60-150rpm.
By adopting the technical scheme, the fermentation process is optimized, and the alkaline solution (such as NaHCO is intermittently added 3 And sodium acetate solution) to maintain pH value and assist in adjusting stirring speed, namely, alkaline solution is added according to the growth condition (OD 660 nm) of the thallus, and the preparation method comprises the following steps ofThe body can be fed-batch at a low speed and continuously in the initial period of fermentation culture and fed-batch at a high speed and at a low frequency when the dissolved oxygen value is reduced to zero.
In summary, the present application has the following beneficial effects:
1. due to the adoption of the optimized culture medium, the material cost of the culture medium is greatly reduced, and the method is suitable for large-batch step-by-step seed expansion fermentation of rhodopseudomonas palustris.
2. The fermentation method is preferably adopted in the application, so that low-cost high-density fermentation is realized in a large-scale fermentation tank, and the production mode of industrial extensive culture is changed.
3. In the fermentation method, the index of mass production is controllable, the quality of the finally obtained bacterial liquid product is stable, and the bacterial quantity of the bacterial liquid product subjected to illumination for 3-7d exceeds 3 x 10 through experiments 10 cfu/mL far exceeds 10-50 hundred million/mL of photosynthetic bacteria sold in the market, and the technology is simple and controllable and the cost is low.
Drawings
FIG. 1A fermentation graph of a 20 ton tank of rhodopseudomonas palustris broth obtained in example 11 of the present application.
Detailed Description
The present application is described in further detail below with reference to the drawings and examples.
Description of raw materials
Rhodopseudomonas palustris of the application is preserved in China center for type culture collection (CCTCC M20222088 CK-ZH 501), the preservation date is No. 28 of 12 months in 2022, and the preservation address is Wuhan, university of Wuhan in China.
All materials, reagents, etc. used in the examples of the present application are commercially available products, unless otherwise specified. The experimental methods used are all conventional methods unless specified. The percentage concentration of the reagents in the examples is mass percent, unless specified otherwise.
Examples
Example 1
The components of the medium of this example included (g/L): sodium acetate 0.5g/L, KH 2 PO 4 0.5g/L、CaCl 2 0.05g/L、NaHCO 3 1g/L、MgCl 2 0.2、NH 4 Cl 0.3g/L, naCl g/L, sodium succinate 0.5g/L, yeast extract 0.2g/L and peptone 0.2g/L, and the balance water, the pH of the medium was 6.8.
Example 2
The components of the medium of this example included (g/L): sodium acetate 2g/L, KH 2 PO 4 2g/L、CaCl 2 0.3g/L、NaHCO 3 6g/L、MgCl 2 2、NH 4 Cl 2g/L, naCl g/L, sodium succinate 3g/L, yeast extract 1g/L, peptone 1g/L, and water in balance, wherein the pH value of the culture medium is 7.5.
Example 3
The components of the medium of this example included (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl 1.8.8 g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, and water in balance, the pH of the culture medium was 6.9.
Example 4
The components of the medium of this example included (g/L): sodium acetate 1.5g/L, KH 2 PO 4 1.8g/L、CaCl 2 0.2g/L、NaHCO 3 4g/L、MgCl 2 0.8、NH 4 Cl 1.2g/L, naCl 1.5.5 g/L, sodium succinate 1g/L, yeast extract 0.5g/L, peptone 0.6g/L, and water in balance, the pH of the culture medium was 7.1.
Example 5
The components of the medium of this example included (g/L): sodium acetate 2g/L, KH 2 PO 4 1.9g/L、CaCl 2 0.22g/L、NaHCO 3 4.5g/L、MgCl 2 0.9、NH 4 Cl 1.6g/L, naCl 1.3.3 g/L, sodium succinate 2.2g/L, yeast extract 0.7g/L and peptone 0.9g/L, and the balance water, the pH of the medium was 7.
Example 6
The medium of this example totals 50L and the components include (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, trace element solution 0.5mL, vitamin solution 0.5mL, water in balance, and pH value of the culture medium is 6.8.
Example 7
The medium of this example totals 50L and the components include (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, trace element solution 2mL, vitamin solution 2mL, water in balance, and pH value of the culture medium is 7.3.
Example 8
The medium of this example totals 50L and the components include (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, trace element solution 1.5mL, vitamin solution 1.5mL, water in balance, and pH value of the culture medium is 6.8.
Example 9
The medium of this example totals 50L and the components include (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, trace element solution 1.5mL, vitamin solution 1mL, water in balance, and pH value of the culture medium is 7.2.
Example 10
The medium of this example totals 50L and the components include (g/L): sodium acetate 1g/L, KH 2 PO 4 1g/L、CaCl 2 0.15g/L、NaHCO 3 3g/L、MgCl 2 1.5、NH 4 Cl 1.5g/L, naCl g/L, sodium succinate 2g/L, yeast extract 0.8g/L, peptone 0.8g/L, trace element solution 1.9mL, vitamin solution 0.8mL, water in balance, and pH value of the culture medium is 6.9.
Example 11
The fermentation method of rhodopseudomonas palustris of the embodiment specifically comprises the following steps:
(1) Seed culture preparation (g/L): sodium acetate 1, KH 2 PO 4 1，CaCl 2 0.15，NaHCO 3 3，MgCl 2 1.5，NH 4 Cl 1.5, naCl 1.8, sodium succinate 2, yeast extract 0.8, peptone 0.8, trace element solution 1.5mL, vitamin solution 1.5mL, and the pH value of the culture medium is 6.8.
(2) Preparing an eggplant bottle inclined plane: sucking 0.5-1mL of bacterial liquid from a glycerol strain tube into a prepared eggplant-shaped bottle seed culture medium, uniformly coating, then plugging a cotton plug, and placing into an anaerobic box for static culture for 5-8d to obtain the eggplant bottle slant seed.
(3) Anaerobic bottle seed liquid preparation: the activated seed slant colony of the eggplant bottle is eluted into a sterilized and deoxidized anaerobic bottle seed culture medium, the rotating speed is controlled at 150-200rpm, the temperature is 28-32 ℃, and the shake bottle seed liquid is obtained after 2-5d culture.
(4) 50L tank seed Medium (g/L): sodium acetate 0.5 KH 2 PO 4 0.5，CaCl 2 0.0.05，NaHCO 3 1.5，MgCl 2 0.25，NH 4 Cl 0.5, naCl 0.5, sodium succinate 0.5, yeast extract 0.25, peptone 0.25, trace element solution 1.5mL, vitamin solution 1.5mL, and pH value of the culture medium is 6.8. Steps (1) - (4) correspond to step S1.
(5) 50L pot seed culture process: preparing 50L jar fermentation medium, weighing 25g sodium acetate, KH 2 PO 4 25 g，CaCl 2 2.5 g，NaHCO 3 75 g，MgCl 2 12.5 g，NH 4 Cl 25g, naCl 25g, sodium succinate 25g, yeast extract 12.5 g and peptone 12.5 g are added into a 50L fermentation tank after dissolution, nitrogen is used for blowing and replacing the culture medium in the tank after sterilization until the dissolved oxygen value DO is lower than 5%, replacement is stopped, and the pH is adjusted to 6.8-7.0, and the temperature is 30 ℃. And (3) inoculating the cultured anaerobic bottle seeds to a 50L fermentation tank according to the inoculation amount of 7% by volume when the anaerobic bottle seeds grow to a specific OD660nm value. Stirring was started at 250rpm and aeration was not performedAnd starting fermentation culture, and maintaining the pH of the acid-base feed at about 6.8 by using acetic acid and ammonia water. Stopping fermenting and culturing when the OD660nm value reaches 0.8-1.0, and performing the next seed transferring operation. Step (5) corresponds to step S2.
(6) 500L tank fermentation process: firstly, weighing 300 g of sodium acetate and KH 2 PO 4 300 g，CaCl 2 30 g，NaHCO 3 900 g，MgCl 2 150 g，NH 4 300 g of Cl, 300 g of NaCl, 300 g of sodium succinate, 300 g of yeast extract powder, 150 g of peptone, 150 g of microelement solution, 300 mL of vitamin solution and 300 mL of vitamin solution. Adding into 500L fermenter after preliminary dissolution, sterilizing at 121deg.C for 30min, cooling to 30deg.C, inoculating the above 50L seed into 500L tank according to 7% volume inoculum size, purging and replacing the 500L tank with nitrogen generator until dissolved oxygen DO is lower than 5%, and adjusting pH to 6.8-7.0 and temperature to 30deg.C. Stirring at 150rpm was started, aeration was not performed, fermentation was started, and pH of the acid-base feed was maintained at about 6.8 with acetic acid and aqueous ammonia at the early stage of fermentation. Fermenting to 12-15 h, gradually increasing stirring rotation speed to 260rpm when dissolved oxygen is reduced to 0, and performing NaHCO according to OD660nm value change condition 3 And intermittent feeding of sodium acetate solution. When the OD660nm value reaches 0.8-1.0, stopping fermentation and carrying out the next seed transferring operation. Step (6) corresponds to step S3.
(7) A fermentation process of a 5 ton tank: 3000 g of sodium acetate and KH are weighed again 2 PO 4 3000 g，CaCl 2 300 g，NaHCO 3 9000 g，MgCl 2 1500 g，NH 4 Cl 3000 g,NaCl 3000 g, sodium succinate 3000 g, yeast extract 1500 g, peptone 1500 g, trace element solution 3000 mL, and vitamin solution 3000 mL. Adding into a 5000L fermentation tank after preliminary dissolution, sterilizing at 121deg.C for 40min, cooling to 30deg.C, inoculating the cultured seeds of the 500L tank into a 5 ton tank according to 7% of inoculation amount, purging and replacing the liquid in the 5 ton tank by using a nitrogen generator until dissolved oxygen DO is lower than 5%, stopping replacement, adjusting pH to 6.8-7.0, and maintaining the temperature at 30deg.C. Stirring at 100rpm is started, aeration is not carried out, fermentation is started, and the pH of the acid-base feed is maintained to be stable at about 6.8 in the earlier stage of fermentation by using acetic acid and ammonia water. Fermenting to 12-15 h, dissolving oxygenWhen the stirring speed is reduced to 0, the stirring speed is gradually increased by 160rpm, and NaHCO is carried out according to the change condition of OD660nm value 3 And intermittent feeding of sodium acetate solution. When the OD660nm value reaches 0.8-1.0, stopping fermentation and carrying out the next seed transferring operation. Step (7) corresponds to step S4.
(8) A 20 ton tank fermentation process: finally weighing 15 kg of sodium acetate and KH 2 PO 4 15 kg，CaCl 2 1500 g，NaHCO 3 45 kg，MgCl 2 7.5 kg，NH 4 Cl 15 kg, naCl 15 kg, sodium succinate 15 kg, yeast extract 7.5 kg, peptone 7.5 kg, trace element solution 15L, vitamin solution 15L. Adding into a 20 ton fermentation tank after preliminary dissolution, sterilizing at 121 ℃ for 40min, cooling to 30 ℃, transferring the cultured seeds in the 5 ton tank into the 20 ton tank according to 14% of inoculation amount by volume, then using a nitrogen making machine to purge and replace the liquid in the 20 ton tank until the dissolved oxygen value DO is lower than 5%, stopping replacement, adjusting the pH value to 6.8-7.0, and controlling the temperature to 30 ℃. Stirring at 60rpm is started, aeration is not carried out, fermentation is started, and the pH of the acid-base feed is maintained to be stable at about 6.8 in the earlier stage of fermentation by using acetic acid and ammonia water. Fermenting to 12-15 h, gradually increasing stirring rotation speed to 100rpm when dissolved oxygen is reduced to 0, and performing NaHCO according to OD660nm value change 3 And intermittent feeding of sodium acetate solution. When the OD660nm value reaches 0.8-1.0, stopping fermentation, and performing the next tank discharge and 5-20L barrel filling operation. Step (8) corresponds to step S5.
Note that CaCl 2 And MgCl 2 The materials which are taken as the last throwing materials before sterilization are added into a fermentation tank after the materials are singly and preliminarily dissolved so as to avoid the change of inorganic salt components of the culture medium caused by precipitation. NaHCO (NaHCO) 3 And the intermittent feeding of sodium acetate solution can only be carried out according to the growth condition (OD 660 nm) of the thalli, and comprises any form of feeding operation of the materials such as low-speed continuous feeding, high-speed low-frequency feeding and the like. In the deoxidizing process of the fermentation tank culture medium after sterilization, inert gases except a nitrogen generator (nitrogen) including carbon dioxide and the like can meet the requirement of replacing oxygen in the tank to low content (less than 5 percent).
Example 12
The fermentation method of rhodopseudomonas palustris of this example is basically the same as that of example 11, except that: in the step (2), standing culture is carried out for 5d in an anaerobic box; in the step (3), culturing for 2d; in the step (5), the cultured anaerobic bottle seeds are inoculated into a 50L fermentation tank according to the inoculation amount of 5 percent by volume; in the step (6), the seeds which are cultured in the 50L tank are transplanted into the 500L tank according to the inoculation amount of 5 percent by volume, the stirring speed is 145rpm when stirring is started, and the stirring speed is 200rpm when dissolved oxygen is reduced to 0; in the step (7), the seeds which are cultured in the 500L tank are transplanted into a 5 ton tank according to the inoculation amount of 5 percent by volume, the stirring speed is 95rpm when stirring is started, and the stirring speed is gradually increased to 120rpm when dissolved oxygen is reduced to 0; in the step (8), the seeds which are cultured in the 5-ton tank are transplanted into the 20-ton tank according to the inoculation amount of 10 percent by volume, the stirring speed is 55rpm when stirring is started, and the stirring speed is gradually increased to 60rpm when dissolved oxygen is reduced to 0.
Wherein in the steps (6) - (8), naHCO is carried out according to the change condition of OD660nm value 3 And intermittent feeding of sodium acetate solution, wherein the stirring speed is controlled to perform low-speed continuous feeding when stirring is started, and the stirring speed is adjusted to perform high-speed low-frequency feeding when dissolved oxygen is reduced to 0.
Example 13
The fermentation method of rhodopseudomonas palustris of this example is basically the same as that of example 11, except that: in the step (2), standing culture is carried out for 8d in an anaerobic box; in the step (3), culturing for 5d; in the step (5), the cultured anaerobic bottle seeds are inoculated into a 50L fermentation tank according to the inoculation amount of 10 percent by volume; in the step (6), the seeds which are cultured in the 50L tank are transplanted into the 500L tank according to the inoculation amount of 10 percent by volume, the stirring speed is 155rpm when stirring is started, and when dissolved oxygen is reduced to 0, the stirring speed is gradually increased to 300rpm; in the step (7), the seeds which are cultured in the 500L tank are transplanted into a 5 ton tank according to the inoculation amount of 10 percent by volume, the stirring speed is 105rpm when stirring is started, and the stirring speed is gradually increased to 200rpm when dissolved oxygen is reduced to 0; in the step (8), the seeds which are cultured in the 5-ton tank are transplanted into the 20-ton tank according to the inoculation amount of 20% by volume, the stirring speed is 65rpm when stirring is started, and the stirring speed is gradually increased to 150rpm when dissolved oxygen is reduced to 0.
Wherein in the steps (6) - (8), naHCO is carried out according to the change condition of OD660nm value 3 And intermittent feeding of sodium acetate solution, wherein the stirring speed is controlled to perform low-speed continuous feeding when stirring is started, and the stirring speed is adjusted to perform high-speed low-frequency feeding when dissolved oxygen is reduced to 0.
Example 14
The fermentation method of rhodopseudomonas palustris of this example is basically the same as that of example 11, except that: in the step (2), standing culture is carried out for 5-8d in an anaerobic box; in the step (3), culturing for 4d; in the step (5), the cultured anaerobic bottle seeds are inoculated into a 50L fermentation tank according to the inoculation amount of 5-10% by volume; in the step (6), the seeds which are cultured in the 50L tank are transplanted into the 500L tank according to the inoculation amount of 8 percent by volume, the stirring speed is 150rpm when stirring is started, and the stirring speed is gradually increased to 250rpm when dissolved oxygen is reduced to 0; in the step (7), the seeds which are cultured in the 500L tank are transplanted into a 5 ton tank according to the inoculum size of 5 to 10 percent by volume, the stirring speed is 100rpm when stirring is started, and the stirring speed is gradually increased to 180rpm when dissolved oxygen is reduced to 0; in the step (8), the seeds which are cultured in the 5 ton tank are transplanted into the 20 ton tank according to the inoculation amount of 10-20 percent by volume, the stirring speed is 60rpm when stirring is started, and the stirring speed is gradually increased to 120rpm when dissolved oxygen is reduced to 0.
Wherein in the steps (6) - (8), naHCO is carried out according to the change condition of OD660nm value 3 And intermittent feeding of sodium acetate solution, wherein the stirring speed is controlled to perform low-speed continuous feeding when stirring is started, and the stirring speed is adjusted to perform high-speed low-frequency feeding when dissolved oxygen is reduced to 0.
Performance test
1. As an example, the bacterial liquids obtained in examples 11 to 14 were used as samples of rhodopseudomonas palustris after tank discharge, and the bacterial count was measured to be 8.5×10 in order 9 cfu/mL；
2. The bacterial liquids obtained in examples 11 to 14 (rhodopseudomonas palustris 20 ton tank fermentation liquid) were taken as samples after putting rhodopseudomonas palustris in tank, and the samples after putting rhodopseudomonas palustris in tank were respectively subjected to light irradiation for 3 to 7 days, and the bacterial numbers obtained in examples 11 to 14 were detected to be greater than 3 x 10 10 cfu/mL。
3. Referring to FIG. 1, FIG. 1 shows the fermentation curve of the rhodopseudomonas palustris 20 ton tank broth corresponding to example 11.
By combining the embodiments 1-14, it can be seen that the optimized culture medium is designed specifically based on the preserved rhodopseudomonas palustris, the fermentation process suitable for mass seed expansion production is designed based on the optimized culture medium, the preservation is prepared from pure seeds of the rhodopseudomonas palustris from the source, the fermentation tank is expanded step by step to the final 20 ton tank production, the modern mass production is completely realized, compared with the roughly-placed plastic barrel and transparent ton bag culture process, the index of mass production of the large-scale fermentation tank is controllable, the infection of mixed bacteria is avoided, and the product quality is stable; in addition, after the fermentation of the bacterial liquid in the fermentation tank is finished, 5-20L packaging bottles can be filled in later. The number of the bacteria subjected to illumination for 3-7d exceeds 3 x 10 10 The cfu/mL and the photosynthetic bacteria of 10-50 hundred million/mL which are sold in far-reaching super market can meet the feeding requirement of a spray drying tower after being centrifugally concentrated, and in addition, the product can be prepared into powder under the condition of low cost rise, so that the competitiveness of the product is enhanced.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims (10)

1. The rhodopseudomonas palustris is characterized by being preserved in China Center for Type Culture Collection (CCTCC) M20222088 CK-ZZH501.
2. A culture medium of rhodopseudomonas palustris as claimed in claim 1, characterized in that it comprises the following components in mass concentrations: sodium acetate 0.5-2g/L, KH 2 PO 4 0.5-2g/L、CaCl 2 0.05-0.3g/L、NaHCO 3 1-6g/L、MgCl 2 0.2-2、NH 4 Cl 0.3-2g0.3-2g/L of/L, naCl, 0.5-3g/L of sodium succinate, 0.2-1g/L of yeast extract powder and 0.2-1g/L of peptone.
3. The rhodopseudomonas palustris culture medium according to claim 2, wherein the pH of the culture medium is 6.8-7.5.
4. A rhodopseudomonas palustris culture medium according to claim 2, wherein each 50L of the culture medium further comprises: 0.5-2mL of microelement solution and 0.5-2mL of vitamin solution.
5. A fermentation broth comprising rhodopseudomonas palustris of claim 1.
6. A fermentation method of rhodopseudomonas palustris as claimed in claim 1, wherein rhodopseudomonas palustris is placed in the culture medium as claimed in any one of claims 2 to 5 for anaerobic culture until the OD660nm reaches a preset value.
7. The fermentation process of rhodopseudomonas palustris according to claim 6, characterized in that it comprises in particular the following steps:
s1: placing the rhodopseudomonas palustris in the culture medium for anaerobic culture until the OD660nm value reaches a preset value to obtain seed liquid;
s2: inoculating the seed liquid into a first preset volume of the culture medium for fermentation culture until the OD660nm value reaches the preset value, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a first bacterial liquid;
s3: inoculating the first bacterial liquid into a second preset volume of the culture medium for fermentation culture until the OD660nm value reaches the preset value, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain the second bacterial liquid.
8. The fermentation process of rhodopseudomonas palustris according to claim 7, characterized in that it comprises in particular the following steps:
s1: taking 0.5-1mL of rhodopseudomonas palustris to be placed in the culture medium for anaerobic culture until the OD660nm value reaches 0.8-1, and obtaining seed liquid;
s2: inoculating the seed liquid into 50L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a first bacterial liquid;
s3: inoculating the first bacterial liquid into 500L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a second bacterial liquid;
s4: inoculating the second bacterial liquid into 5000L of the culture medium according to the inoculation amount of 5-10% by volume for fermentation culture until the OD660nm value reaches 0.8-1, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a third bacterial liquid;
s5: inoculating the third bacterial liquid into 20000L of culture medium according to the volume inoculation amount of 10-20% for fermentation culture until the OD660nm value reaches 0.8-1, and maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32 ℃ in the fermentation culture process to obtain a fourth bacterial liquid.
9. The method for fermenting rhodopseudomonas palustris according to claim 8, wherein in the step S2 or S3, after inoculation and before fermentation culture, the dissolved oxygen value of the culture medium is controlled to be lower than 5% and the pH value is adjusted to 6.8-7.0; stirring is continuously carried out in the fermentation culture process.
10. Fermentation process of rhodopseudomonas palustris according to claim 8 or 9, characterized in that the fermentation culture in steps S3, S4 and S5 is maintained at pH by intermittent addition of alkaline solution, respectively; when the alkaline solution is intermittently added, stirring the solution and adjusting the stirring speed, wherein the adjustment of the stirring speed specifically comprises at least one operation of the following steps S3-S5:
in the step S3, when the stirring speed in the initial period of fermentation culture is 145-155rpm and the dissolved oxygen value is reduced to zero, controlling the stirring speed to be 200-300rpm;
in the step S4, when the stirring speed in the initial period of fermentation culture is 95-105rpm and the dissolved oxygen value is reduced to zero, controlling the stirring speed to be 120-200rpm;
in the step S5, the stirring speed in the initial period of fermentation culture is 55-65rpm, and when the dissolved oxygen value is reduced to zero, the stirring speed is controlled to be 60-150rpm.
CN202211720555.4A 2022-12-30 2022-12-30 Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method Pending CN116254196A (en)

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