Rhodopseudomonas palustris, culture medium, fermentation liquid and fermentation method

Abstract

The present application relates to the field of microbial fermentation, and specifically discloses a kind of Rhodopseudomonas palustris, culture medium, fermentation liquid and fermentation method. The culture medium of the Rhodopseudomonas palustris includes the following components in mass concentration: sodium acetate 0.5-2g / L, KH2PO4 0.5-2g / L, CaCl2 0.05-0.3g / L, NaHCO3 1-6g / L, MgCl2 0.2-2, NH4Cl 0.3-2g / L, NaCl 0.3-2g / L, sodium succinate 0.5-3g / L, yeast extract powder 0.2-1g / L and peptone 0.2-1g / L. The culture medium of the present application can be used for the fermentation culture of Rhodopseudomonas palustris, and the material cost of the culture medium is greatly reduced. The fermentation method of the present application can realize low-cost and high-density fermentation, which changes the production mode of extensive culture in the industry.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation, and more specifically, to Rhodopseudomonas palustris, a culture medium, a fermentation liquid and a fermentation method. Background Art

[0002] Photosynthetic bacteria Photosynthetic bacteria (Photosynthetic Bacteria) is the oldest and most widely existing bacterial group in nature. It has a primitive light energy synthesis system and is a general term for prokaryotes that can photosynthesize in anaerobic or facultative anaerobic environments without producing oxygen. Rhodopseudomonas palustris is the most widely used probiotic among photosynthetic bacteria. It is widely used in agriculture, fisheries, environmental protection, medicine and other fields. As a water purifier for aquaculture, Rhodopseudomonas palustris has long been widely used in aquaculture and can effectively reduce the chemical oxygen demand in shrimp aquaculture wastewater. Rhodopseudomonas palustris is rich in various bioactive proteins, pantothenic acid, folic acid and multiple vitamins. It is a safe green feed additive and one of the 12 live microorganisms allowed to be directly added to feed issued by the Ministry of Agriculture of my country. Rhodopseudomonas palustris can also accelerate the absorption and conversion of nutrients by plants, which is beneficial to improve the yield and quality of crops. At the same time, it can improve the distribution of soil flora and improve soil fertility. Feed products with Rhodopseudomonas palustris as additives are the product of the combination of modern biotechnology, animal microecology and animal nutrition. Researching and developing Rhodopseudomonas palustris microbial preparations will be of great significance in promoting the development of my country's aquaculture industry in an efficient, green, ecological and healthy direction, and its application prospects are also very broad. However, the research on the composition of Rhodopseudomonas palustris culture medium and various high-density culture conditions is still the main factor restricting the widespread application of this bacterium.

[0003] The culture medium is the main place for microbial growth and metabolism, and the compatibility of the culture medium is related to the quality and production efficiency of microbial products. In recent years, relevant technologies have been used to study the growth conditions of Rhodopseudomonas palustris, and it has been found that factors such as pH value, temperature, and inoculation amount affect the growth of Rhodopseudomonas palustris, and they affect each other. Relevant technologies have been used to optimize the fermentation conditions of Rhodopseudomonas palustris. On the basis of single factors, the Box-nhk central combination experiment and response surface analysis method were used to determine the optimal growth conditions of Rhodopseudomonas palustris: under the conditions of pH value of 6.89, inoculation amount of 9.71%, culture temperature of 29.8℃, and light intensity of 2000 Lux, the growth trend of Rhodopseudomonas palustris is the best. The relevant technology has been used to optimize the fermentation medium formula and culture conditions of Rhodopseudomonas palustris. The single-factor experimental design was used to compare the different levels of the four factors of inoculation amount, pH value, temperature, and light intensity of the Rhodopseudomonas palustris culture preserved in this laboratory, and their respective optimal experimental levels were determined. At the same time, the relevant technology also disclosed the use of orthogonal test L16 (45) design to optimize the fermentation medium formula, and the use of single-factor randomized test to optimize the culture conditions. The fermentation medium formula determined by the experiment was: 1.0 g glycerol, 1.0 g ammonium chloride, 0.3 g dipotassium hydrogen phosphate, 0.05 mL trace element solution, 0.15 mL vitamin solution, and 1000 mL distilled water; the optimal culture conditions were: initial pH value 7.0, culture temperature 30℃, inoculation amount 6%, liquid volume 50 mL / 250 mL conical flask, 40W incandescent lamp irradiated at a distance of 20 cm for static culture. Under these conditions, the number of Rhodopseudomonas palustris R1 can reach 5.6×108 / mL after 7 days of fermentation. The related technology also discloses a culture medium for aerobic fermentation of Rhodopseudomonas palustris, which includes the following components: 3.00-9.00 g / L of organic nitrogen source, 0.88-1.76 g / L of ammonium salt, 5.40-10.80 g / L of sodium succinate, 1.12-2.25 g / L of sodium glutamate, and 0.20-0.60 g / L of magnesium sulfate; using this culture medium and aerobic culture method, Rhodopseudomonas palustris can proliferate rapidly and has a high growth rate, and more Rhodopseudomonas palustris cells can be obtained in a short time, and the OD660nm after 48 hours of shake flask culture is 1.015.The related technology also provides another culture medium, which is composed of 0.75-1.25 g / L citric acid, 1.50-3.00 g / L beef extract, 0.42~0.62 g / L yeast extract, 1.50~2.00 g / L peptone, 0.40~0.60 g / L sodium acetate, 0.25~0.50 g / L magnesium sulfate, 5.00~8.00 mg / L ferrous sulfate, 0.1~0.3 mg / L VB6, 0.10~0.30 g / L calcium chloride, 0.10~0.25 g / L potassium dihydrogen phosphate, and 0.5~1.0 mL / L composite inorganic salt solution. This formula improves the quality of the palustrine Rhodopseudomonas inoculant, so that the effective live bacterial count of the inoculant is as high as 8.96*109 cells / mL, the color is positive, there is no stratification, the rate of foreign bacteria is low, and the quality is stable. The related technology also discloses a photosynthetic bacteria culture medium and a preparation method thereof, the components of which are sodium acetate trihydrate, ammonium sulfate, sodium bicarbonate, potassium dihydrogen phosphate, magnesium sulfate and water. When the photosynthetic bacteria are cultured using the culture medium, the OD660nm of the photosynthetic bacteria is 2.2.

[0004] However, the existing photosynthetic bacteria on the market, such as Rhodopseudomonas palustris, have a crude and backward upstream culture process, and are not produced in large-scale fermentation tanks. The general bacterial density is only 10 cfu / mL. 9 Due to the problem of cultivation process, most of them carry a considerable number of foreign bacteria and have poor quality. Moreover, the products can only be sold in liquid form. Due to cost reasons, they cannot be spray-dried and stored in powder form, and the product circulation cost also increases accordingly. Summary of the invention

[0005] In order to solve at least one of the above technical problems, the present application provides a Rhodopseudomonas palustris, a culture medium, a fermentation liquid and a fermentation method.

[0006] In a first aspect, the present application provides a Rhodopseudomonas palustris, using the following technical solution:

[0007] A Rhodopseudomonas palustris, whose deposit number is CCTCC M 20222088 CK-ZZH501.

[0008] The Rhodopseudomonas palustris of the present application is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M20222088 CK-ZZH501, the deposit date is December 28, 2022, and the deposit address is Wuhan University, Wuhan, China.

[0009] The Rhodopseudomonas palustris of the present application has high bacterial purity, short slant activation time, can reach a high bacterial density without illumination during the step-by-step enlargement of the tank culture from expansion culture to fermentation, and is not easily contaminated.

[0010] In a second aspect, the present application provides a seed culture medium of the above-mentioned Rhodopseudomonas palustris, which adopts the following technical solution:

[0011] A culture medium for Rhodopseudomonas palustris, comprising the following components in mass concentrations: 0.5-2 g / L sodium acetate, 0.5-2 g / L KH2PO4, 0.05-0.3 g / L CaCl2, 1-6 g / L NaHCO3, 0.2-2 g / L MgCl2, 0.3-2 g / L NH4Cl, 0.3-2 g / L NaCl, 0.5-3 g / L sodium succinate, 0.2-1 g / L yeast extract powder and 0.2-1 g / L peptone.

[0012] By adopting the above technical solution and optimizing the design of the culture medium components, the material cost of the culture medium is greatly reduced, and it can be suitable for large-scale fermentation. Specifically, the mass concentration of sodium acetate in the culture medium is 0.5-2g / L, for example, it can be 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 2g / L. Any value; the mass concentration of KH2PO4 is 0.5-2g / L, for example, it can be 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 2g / L. The mass concentration of CaCl2 is 0.05-0.3g / L, for example, 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 The mass concentration of NaHCO is 1-6g / L, for example, 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 6g / L; the mass concentration of MgCl2 is 0.2-2g / L, for example, it can be any value among 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 2g / L. The mass concentration of NH4Cl is 0.3-2g / L, for example, it can be any value among 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 2g / L; the mass concentration of NaCl is 0.3-2g / L, for example, 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 sodium succinate is 0.5-3g / L, for example, it can be 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. the mass concentration of yeast extract is 0.2-1g / L, for example, it can be any value among 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; the mass concentration of peptone is 0.2-1g / L, for example, it can be any value among 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. .

[0013] Preferably, the pH value of the culture medium is 6.8-7.5.

[0014] By adopting the above technical solution, the pH value of the culture medium is optimized and designed, and the pH value is specifically controlled to be 6.8-7.5, for example, it can be any value among 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5.

[0015] Preferably, each 50 L of the culture medium further comprises: 0.5-2 mL of trace element solution and 0.5-2 mL of vitamin solution.

[0016] By adopting the above technical solution, the components of the culture medium are optimized and designed, and 0.5-2mL of trace element solution and 0.5-2mL of vitamin solution are added for every 50L of culture medium. Specifically, the amount of trace element solution added is 0.5-2mL, for example, it can be any value among 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL and 2mL; the amount of vitamin solution added is 0.5-2mL, for example, it can be any value among 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1mL, 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL and 2mL.

[0017] By adopting the above technical solution, the culture effect can be further improved by controlling the pH value of the culture medium or adding trace element solution and vitamin solution.

[0018] In a third aspect, the present application provides a fermentation liquid, which adopts the following technical solution:

[0019] A fermentation liquid containing the above-mentioned Rhodopseudomonas palustris.

[0020] In a fourth aspect, the present application provides a fermentation method, which adopts the following technical solution:

[0021] A fermentation method for Rhodopseudomonas palustris according to claim 1, wherein the Rhodopseudomonas palustris is placed in the above-mentioned culture medium for anaerobic culture until the OD660nm value reaches a preset value.

[0022] Preferably, the fermentation method specifically comprises the following steps:

[0023] S1: taking the Rhodopseudomonas palustris and placing it in the culture medium for anaerobic culture until the OD660nm value reaches a preset value, thereby obtaining a seed solution;

[0024] S2: inoculating the seed liquid into the culture medium of a first preset volume for fermentation culture until the OD660nm value reaches the preset value, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32° C. during the fermentation culture process, to obtain a first bacterial liquid;

[0025] S3: inoculating the first bacterial liquid into the culture medium of a second preset volume for fermentation culture until the OD660nm value reaches the preset value, maintaining the pH value at 6.5-7.2 and controlling the temperature at 28-32° C. during the fermentation culture process to obtain a second bacterial liquid.

[0026] Preferably, the fermentation method specifically comprises the following steps:

[0027] S1: taking 0.5-1 mL of the Rhodopseudomonas palustris and placing it in the culture medium for anaerobic culture until the OD660nm value reaches 0.8-1 to obtain a seed solution;

[0028] S2: inoculating the seed liquid into 50 L of the culture medium at a volume inoculation rate of 5-10% 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° C. during the fermentation culture process, to obtain a first bacterial liquid;

[0029] S3: inoculating the first bacterial liquid into 500 L of the culture medium at a volume inoculation rate of 5-10% 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° C. during the fermentation culture process, to obtain a second bacterial liquid;

[0030] S4: inoculating the second bacterial liquid into 5000 L of the culture medium at a volume inoculation rate of 5-10% 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° C. during the fermentation culture process, to obtain a third bacterial liquid;

[0031] S5: inoculating the third bacterial liquid into 20000 L of the culture medium at a volume inoculation rate of 10-20% 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° C. during the fermentation culture process, to obtain a fourth bacterial liquid.

[0032] By adopting the above technical scheme, the above fermentation process suitable for large-scale expansion production is designed based on the optimized culture medium. From the preparation and preservation of pure strains of Rhodopseudomonas palustris at the source, the fermentation tank is gradually expanded to the final 20-ton tank production, which fully realizes modern large-scale production. Compared with the extensive and backward plastic barrel and transparent ton bag culture process, the indicators of large-scale fermentation tank batch production are controllable, avoiding the infection of miscellaneous bacteria and ensuring stable product quality. In addition, after the fermentation of the bacterial liquid in the fermentation tank is completed, it can be subsequently filled into 5-20L packaging bottles. According to the test, the number of bacteria after 3-7 days of illumination exceeds 3*10 10 cfu / mL, far exceeding the 10-50 billion / mL photosynthetic bacteria sold in the market. 30 billion / mL of bacterial liquid can also meet the feeding requirements of the spray drying tower after centrifugal concentration. In addition, it can be made into powder without much cost increase, thus enhancing the competitiveness of the product.

[0033] 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; and stirring is continuously performed during the fermentation culture.

[0034] Preferably, the fermentation culture process in steps S3, S4 and S5 respectively maintains the pH value by intermittently adding an alkaline solution; when the alkaline solution is intermittently added, the solution is stirred and the stirring speed is adjusted, and the adjustment of the stirring speed specifically includes at least one operation in the following steps S3-S5:

[0035] In step S3, the stirring speed is 145-155 rpm in the initial period of fermentation culture, and when the dissolved oxygen value drops to zero, the stirring speed is controlled to 200-300 rpm;

[0036] In step S4, the stirring speed is 95-105 rpm in the initial period of fermentation culture, and when the dissolved oxygen value drops to zero, the stirring speed is controlled to 120-200 rpm;

[0037] In step S5, the stirring speed is 55-65 rpm in the initial period of fermentation culture, and when the dissolved oxygen value drops to zero, the stirring speed is controlled to 60-150 rpm.

[0038] By adopting the above technical scheme and optimizing the fermentation process, by intermittently adding alkaline solution (such as NaHCO3 and sodium acetate solution) to maintain the pH value and assisting in adjusting the stirring speed, the alkaline solution can be added according to the growth of the bacteria (OD660nm), specifically, low-speed continuous addition in the initial period of fermentation culture and high-speed low-frequency addition when the dissolved oxygen value drops to zero.

[0039] In summary, this application has the following beneficial effects:

[0040] 1. Since the present application adopts the above-mentioned optimized culture medium, the material cost of the culture medium is greatly reduced, and it is suitable for large-scale step-by-step expansion and fermentation of Rhodopseudomonas palustris.

[0041] 2. The above fermentation method is preferably used in this application to achieve low-cost and high-density fermentation in large fermentation tanks, changing the industry's extensive cultivation production model.

[0042] 3. In the fermentation method of the present application, the indicators of batch production are controllable, and the quality of the bacterial liquid product finally obtained is stable. According to the test, the number of bacteria after 3-7 days of illumination exceeds 3*10 10 cfu / mL, far exceeding the 10-50 billion / mL of photosynthetic bacteria sold on the market. The process is simple and controllable with low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Fermentation curve of a 20-ton tank of Rhodopseudomonas palustris culture liquid obtained in Example 11 of the present application. DETAILED DESCRIPTION

[0044] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0045] Ingredients

[0046] The Rhodopseudomonas palustris of the present application is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M20222088 CK-ZZH501, the deposit date is December 28, 2022, and the deposit address is Wuhan University, Wuhan, China.

[0047] All raw materials, reagents, etc. used in the examples of this application are common commercial products unless otherwise specified and can be purchased on the market. The experimental methods used are conventional methods unless otherwise specified. The percentage concentration of reagents in the examples is mass percentage unless otherwise specified. Example

[0048] Example 1

[0049] The components of the culture medium of this embodiment include (g / L): sodium acetate 0.5 g / L, KH2PO4 0.5 g / L, CaCl2 0.05 g / L, NaHCO3 1 g / L, MgCl2 0.2, NH4Cl 0.3 g / L, NaCl 0.3 g / L, sodium succinate 0.5 g / L, yeast extract powder 0.2 g / L and peptone 0.2 g / L, and the balance is water, and the pH value of the culture medium is 6.8.

[0050] Example 2

[0051] The components of the culture medium of this embodiment include (g / L): sodium acetate 2g / L, KH2PO4 2g / L, CaCl2 0.3g / L, NaHCO3 6g / L, MgCl22, NH4Cl 2g / L, NaCl 2g / L, sodium succinate 3g / L, yeast extract 1g / L and peptone 1g / L, and the balance is water, and the pH value of the culture medium is 7.5.

[0052] Example 3

[0053] The components of the culture medium of this embodiment include (g / L): sodium acetate 1g / L, KH2PO4 1g / L, CaCl2 0.15g / L, NaHCO3 3g / L, MgCl2 1.5, NH4Cl 1.5g / L, NaCl 1.8g / L, sodium succinate 2g / L, yeast extract powder 0.8g / L and peptone 0.8g / L, and the balance is water, and the pH value of the culture medium is 6.9.

[0054] Example 4

[0055] The components of the culture medium of this embodiment include (g / L): sodium acetate 1.5 g / L, KH2PO4 1.8 g / L, CaCl2 0.2 g / L, NaHCO3 4 g / L, MgCl2 0.8, NH4Cl 1.2 g / L, NaCl 1.5 g / L, sodium succinate 1 g / L, yeast extract powder 0.5 g / L and peptone 0.6 g / L, and the balance is water, and the pH value of the culture medium is 7.1.

[0056] Example 5

[0057] The components of the culture medium of this embodiment include (g / L): sodium acetate 2g / L, KH2PO41.9g / L, CaCl20.22g / L, NaHCO34.5g / L, MgCl20.9, NH4Cl 1.6g / L, NaCl 1.3g / L, sodium succinate 2.2g / L, yeast extract powder 0.7g / L and peptone 0.9g / L, and the balance is water, and the pH value of the culture medium is 7.

[0058] Example 6

[0059] The culture medium of this example has a total of 50 L, and the components include (g / L): sodium acetate 1 g / L, KH2PO4 1 g / L, CaCl2 0.15 g / L, NaHCO3 3 g / L, MgCl2 1.5, NH4Cl 1.5 g / L, NaCl 1.8 g / L, sodium succinate 2 g / L, yeast extract powder 0.8 g / L, peptone 0.8 g / L, trace element solution 0.5 mL and vitamin solution 0.5 mL, the balance is water, and the pH value of the culture medium is 6.8.

[0060] Example 7

[0061] The culture medium of this example has a total of 50 L, and the components include (g / L): sodium acetate 1 g / L, KH2PO4 1 g / L, CaCl2 0.15 g / L, NaHCO3 3 g / L, MgCl2 1.5, NH4Cl 1.5 g / L, NaCl 1.8 g / L, sodium succinate 2 g / L, yeast extract powder 0.8 g / L, peptone 0.8 g / L, trace element solution 2 mL and vitamin solution 2 mL, the balance is water, and the pH value of the culture medium is 7.3.

[0062] Example 8

[0063] The culture medium of this example has a total of 50 L, and the components include (g / L): sodium acetate 1 g / L, KH2PO4 1 g / L, CaCl2 0.15 g / L, NaHCO3 3 g / L, MgCl2 1.5, NH4Cl 1.5 g / L, NaCl 1.8 g / L, sodium succinate 2 g / L, yeast extract 0.8 g / L, peptone 0.8 g / L, trace element solution 1.5 mL and vitamin solution 1.5 mL, the balance is water, and the pH value of the culture medium is 6.8.

[0064] Example 9

[0065] The culture medium of this example has a total of 50 L, and the components include (g / L): sodium acetate 1 g / L, KH2PO4 1 g / L, CaCl2 0.15 g / L, NaHCO3 3 g / L, MgCl2 1.5, NH4Cl 1.5 g / L, NaCl 1.8 g / L, sodium succinate 2 g / L, yeast extract powder 0.8 g / L, peptone 0.8 g / L, trace element solution 1.5 mL and vitamin solution 1 mL, the balance is water, and the pH value of the culture medium is 7.2.

[0066] Example 10

[0067] The culture medium of this example has a total of 50 L, and the components include (g / L): sodium acetate 1 g / L, KH2PO4 1 g / L, CaCl2 0.15 g / L, NaHCO3 3 g / L, MgCl2 1.5, NH4Cl 1.5 g / L, NaCl 1.8 g / L, sodium succinate 2 g / L, yeast extract 0.8 g / L, peptone 0.8 g / L, trace element solution 1.9 mL and vitamin solution 0.8 mL, the balance is water, and the pH value of the culture medium is 6.9.

[0068] Embodiment 11

[0069] The fermentation method of Rhodopseudomonas palustris of this embodiment specifically comprises the following steps:

[0070] (1) Seed culture medium preparation (g / L): sodium acetate 1, KH2PO4 1, CaCl2 0.15, NaHCO3 3, MgCl2 1.5, NH4Cl 1.5, NaCl 1.8, sodium succinate 2, yeast extract powder 0.8, peptone 0.8, trace element solution 1.5mL, vitamin solution 1.5mL, culture medium pH 6.8.

[0071] (2) Preparation of eggplant flask slant: Pipette 0.5-1 mL of bacterial solution from the glycerol culture tube into the prepared eggplant flask seed culture medium. Spread it evenly, plug it with cotton plug, and place it in an anaerobic box for static culture for 5-8 days to obtain the eggplant flask slant seeds.

[0072] (3) Preparation of anaerobic bottle seed liquid: The activated eggplant bottle seed slant colonies are eluted into sterilized and deoxygenated anaerobic bottle seed culture medium. The rotation speed is controlled at 150-200 rpm and the temperature is 28-32°C. After culturing for 2-5 days, the shake flask seed liquid is obtained.

[0073] (4) 50L tank seed culture medium (g / L): sodium acetate 0.5, KH2PO4 0.5, CaCl2 0.0.05, NaHCO3 1.5, MgCl2 0.25, NH4Cl 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, culture medium pH 6.8. Steps (1) to (4) correspond to step S1.

[0074] (5) 50 L tank seed culture process: Prepare a 50 L tank fermentation medium, weigh 25 g sodium acetate, 25 g KH2PO4, 2.5 g CaCl2, 75 g NaHCO3, 12.5 g MgCl2, 25 g NH4Cl, 25 g NaCl, 25 g sodium succinate, 12.5 g yeast extract powder, and 12.5 g peptone, dissolve and add to the 50 L fermentation tank, sterilize and use nitrogen to purge and replace the medium in the tank until the dissolved oxygen value DO is lower than 5%, then stop replacing, and then adjust the pH to 6.8-7.0 and the temperature to 30°C. When the anaerobic bottle seeds grow to a specific OD660nm value, inoculate the cultured anaerobic bottle seeds at a volume inoculation rate of 7% and inoculate them into the 50 L fermentation tank. Turn on the stirring at 250 rpm, do not ventilate, and start the fermentation culture. Use acetic acid and ammonia water to maintain the pH stable at around 6.8 in the early stage of fermentation. When the OD660nm value reaches 0.8-1.0, the fermentation culture is stopped and the next seed transfer operation is performed. Step (5) corresponds to step S2.

[0075] (6) 500 L tank fermentation process: First weigh 300 g sodium acetate, 300 g KH2PO4, 30 g CaCl2, 900 g NaHCO3, 150 g MgCl2, 300 g NH4Cl, 300 g NaCl, 300 g sodium succinate, 150 g yeast extract, 150 g peptone, 300 mL trace element solution, and 300 mL vitamin solution. After preliminary dissolution, add them into the 500 L fermentation tank, sterilize at 121°C for 30 min, and after cooling to 30°C, transplant the seeds cultured in the above 50 L tank into the 500 L tank at a volume inoculation rate of 7%. Then use a nitrogen generator to purge and replace the liquid in the 500 L tank until the dissolved oxygen value DO is lower than 5% and stop the replacement. Adjust the pH to 6.8-7.0 and the temperature to 30°C. Start stirring at 150 rpm without ventilation, and start fermentation. Use acetic acid and ammonia water to maintain the pH of the fermentation at about 6.8 in the early stage. After 12-15 hours of fermentation, when the dissolved oxygen drops to 0, gradually increase the stirring speed to 260 rpm, and intermittently feed NaHCO3 and sodium acetate solution according to the change of OD660nm value. When the OD660nm value reaches 0.8-1.0, stop fermentation and proceed to the next seed transfer operation. Step (6) corresponds to step S3.

[0076] (7) 5-ton tank fermentation process: weigh again 3000 g sodium acetate, 3000 g KH2PO4, 300 g CaCl2, 9000 g NaHCO3, 1500 g MgCl2, 3000 g NH4Cl, 3000 g NaCl, 3000 g sodium succinate, 1500 g yeast extract, 1500 g peptone, 3000 mL trace element solution, and 3000 mL vitamin solution. After preliminary dissolution, add them into the 5000 L fermentation tank, sterilize at 121°C for 40 min, and after cooling to 30°C, transplant the seeds cultured in the above 500 L tank into the 5-ton tank at a volume inoculation rate of 7%. Then use a nitrogen generator to purge and replace the liquid in the 5-ton tank until the dissolved oxygen value DO is lower than 5% and stop replacement. Adjust the pH to 6.8-7.0 and the temperature to 30°C. Start stirring at 100 rpm without ventilation, and start fermentation. Use acetic acid and ammonia water to maintain the pH of the fermentation at about 6.8 in the early stage. After 12-15 hours of fermentation, when the dissolved oxygen drops to 0, gradually increase the stirring speed to 160 rpm, and intermittently feed NaHCO3 and sodium acetate solution according to the change of OD660nm value. When the OD660nm value reaches 0.8-1.0, stop fermentation and proceed to the next seed transfer operation. Step (7) corresponds to step S4.

[0077] (8) 20-ton tank fermentation process: Finally, weigh 15 kg of sodium acetate, 15 kg of KH2PO4, 1500 g of CaCl2, 45 kg of NaHCO3, 7.5 kg of MgCl2, 15 kg of NH4Cl, 15 kg of NaCl, 15 kg of sodium succinate, 7.5 kg of yeast extract, 7.5 kg of peptone, 15 L of trace element solution, and 15 L of vitamin solution. After preliminary dissolution, add them into the 20-ton fermentation tank and sterilize at 121°C for 40 min. After cooling to 30°C, transplant the seeds cultured in the above 5-ton tank into the 20-ton tank at a volume inoculation rate of 14%. Then use a nitrogen generator to purge and replace the liquid in the 20-ton tank until the dissolved oxygen value DO is lower than 5%, stop replacement, adjust pH 6.8-7.0, and temperature 30°C. Turn on stirring at 60 rpm, do not ventilate, and start fermentation. Acid and alkali feeds are used to maintain the pH stable at around 6.8 in the early stage of fermentation with acetic acid and ammonia water. After 12-15 hours of fermentation, when the dissolved oxygen drops to 0, gradually increase the stirring speed by 100 rpm, and intermittently feed NaHCO3 and sodium acetate solution according to the change of OD660nm value. When the OD660nm value reaches 0.8-1.0, stop fermentation and proceed to the next step of tanking and 5-20L barrel filling. Step (8) corresponds to step S5.

[0078] It should be noted that CaCl2 and MgCl2 should be dissolved separately and added to the fermenter as the last material before sterilization to avoid changes in the inorganic salt composition of the culture medium due to precipitation. Intermittent feeding of NaHCO3 and sodium acetate solution can only be carried out according to the growth of bacteria (OD660nm), including any form of feeding operation of this material such as low-speed continuous feeding and high-speed low-frequency feeding. During the deoxygenation process of the fermenter culture medium after sterilization, the use of inert gases other than the nitrogen generator (nitrogen) including carbon dioxide can achieve the requirement of replacing the oxygen in the tank to a low content (<5%).

[0079] Example 12

[0080] The fermentation method of Rhodopseudomonas palustris in this embodiment is basically the same as that in embodiment 11, except that: in step (2), the culture is statically cultured in the anaerobic box for 5 days; in step (3), the culture is carried out for 2 days; in step (5), the cultured anaerobic bottle seeds are inoculated at a volume of 5% and inoculated into 50 L fermentation tank; in step (6), the seeds cultured in the above 50L tank are transplanted into the 500L tank at a volume inoculation rate of 5%, the stirring speed when stirring is turned on is 145rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 200rpm; in step (7), the seeds cultured in the above 500L tank are transplanted into the 5-ton tank at a volume inoculation rate of 5%, the stirring speed when stirring is turned on is 95rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 120rpm; in step (8), the seeds cultured in the above 5-ton tank are transplanted into the 20-ton tank at a volume inoculation rate of 10%, the stirring speed when stirring is turned on is 55rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 60rpm.

[0081] Among them, in steps (6)-(8), NaHCO3 and sodium acetate solutions are intermittently fed according to the change of OD660nm value. When stirring is turned on, the stirring speed is controlled to perform low-speed continuous flow addition. When the dissolved oxygen drops to 0, the stirring speed is adjusted to perform high-speed low-frequency flow addition.

[0082] Embodiment 13

[0083] The fermentation method of Rhodopseudomonas palustris in this embodiment is basically the same as that in embodiment 11, except that: in step (2), the culture is statically cultured in the anaerobic box for 8 days; in step (3), the culture is carried out for 5 days; in step (5), the cultured anaerobic bottle seeds are inoculated at a volume of 10% to 50 L fermentation tank; in step (6), the seeds cultured in the above 50L tank are transplanted into the 500L tank at a volume inoculation rate of 10%, the stirring speed when stirring is turned on is 155rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 300rpm; in step (7), the seeds cultured in the above 500L tank are transplanted into the 5-ton tank at a volume inoculation rate of 10%, the stirring speed when stirring is turned on is 105rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 200rpm; in step (8), the seeds cultured in the above 5-ton tank are transplanted into the 20-ton tank at a volume inoculation rate of 20%, the stirring speed when stirring is turned on is 65rpm, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 150rpm.

[0084] Among them, in steps (6)-(8), NaHCO3 and sodium acetate solutions are intermittently fed according to the change of OD660nm value. When stirring is turned on, the stirring speed is controlled to perform low-speed continuous flow addition. When the dissolved oxygen drops to 0, the stirring speed is adjusted to perform high-speed low-frequency flow addition.

[0085] Embodiment 14

[0086] The fermentation method of Rhodopseudomonas palustris in this embodiment is basically the same as that in embodiment 11, except that: in step (2), the culture is statically cultured in the anaerobic box for 5-8 days; in step (3), the culture is carried out for 4 days; in step (5), the cultured anaerobic bottle seeds are inoculated at a volume of 5%-10% and inoculated into 50 L fermentation tank; in step (6), the seeds cultured in the above 50L tank are transplanted into the 500L tank at an inoculum rate of 8% by volume, the stirring speed is 150 rpm when stirring is turned on, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 250 rpm; in step (7), the seeds cultured in the above 500L tank are transplanted into the 5-ton tank at an inoculum rate of 5%-10% by volume, the stirring speed is 100 rpm when stirring is turned on, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 180 rpm; in step (8), the seeds cultured in the above 5-ton tank are transplanted into the 20-ton tank at an inoculum rate of 10%-20% by volume, the stirring speed is 60 rpm when stirring is turned on, and when the dissolved oxygen drops to 0, the stirring speed is gradually increased to 120 rpm.

[0087] Among them, in steps (6)-(8), NaHCO3 and sodium acetate solutions are intermittently fed according to the change of OD660nm value. When stirring is turned on, the stirring speed is controlled to perform low-speed continuous flow addition. When the dissolved oxygen drops to 0, the stirring speed is adjusted to perform high-speed low-frequency flow addition.

[0088] Performance testing

[0089] 1. As an example, the bacterial solution obtained in Examples 11-14 was used as a sample of Rhodopseudomonas palustris after tanking, and the bacterial counts obtained were 8.5*10 9 cfu / mL;

[0090] 2. The bacterial liquid obtained in Examples 11-14 (20-ton tank fermentation liquid of Rhodopseudomonas palustris) was used as a sample of Rhodopseudomonas palustris after tanking, and the samples of Rhodopseudomonas palustris after tanking were respectively exposed to light for 3-7 days, and the bacterial counts of Examples 11-14 were detected to be greater than 3*10 10 cfu / mL.

[0091] 3. See Figure 1 , Figure 1 This is the fermentation curve of the 20-ton tank culture liquid of Rhodopseudomonas palustris corresponding to Example 11.

[0092] In combination with Examples 1-14, it can be seen that the present application specifically designs an optimized culture medium based on the preserved palustris Rhodopseudomonas, and designs the above-mentioned fermentation process suitable for large-scale expansion production based on the optimized culture medium, from the preparation and preservation of pure strains of palustris Rhodopseudomonas at the source, to the gradual expansion of the fermentation tank to the final 20-ton tank production, modern large-scale production is fully realized. Compared with the extensive and backward plastic barrel and transparent ton bag culture process, the indicators of large-scale fermentation tanks for mass production are controllable, which avoids infection by foreign bacteria and ensures stable product quality. In addition, after the fermentation of the bacterial liquid in the fermentation tank is completed, it can be subsequently filled into 5-20L packaging bottles. According to the test, the number of bacteria after 3-7 days of illumination exceeded 3*10 10 cfu / mL, far exceeding the 10-50 billion / mL photosynthetic bacteria sold in the market. 30 billion / mL of bacterial liquid can also meet the feeding requirements of the spray drying tower after centrifugal concentration. In addition, it can be made into powder without much cost increase, thus enhancing the competitiveness of the product.

[0093] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A Rhodopseudomonas palustris, characterized in that The Rhodopseudomonas palustris is deposited in China Center for Type Culture Collection with a deposit number of CCTCC M 20222088.

2. A fermentation broth containing the Rhodopseudomonas palustris according to claim 1.

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