Method for high-density shake flask fermentation of bradyrhizobium anhuoshanense and application of bradyrhizobium anhuoshanense

Abstract

The invention belongs to the technical field of peanut cultivation methods, and particularly relates to a method for high-density shake-flask fermentation of bradyrhizobium anhuoshanense and application of the bradyrhizobium anhuoshanense. The improved YGA culture medium is used for carrying out shake flask fermentation on the peanut bradyrhizobium strain WYCCWR 15459, the high-density peanut bradyrhizobium inoculant is prepared, the effective viable count of the inoculant is larger than 10 < 11 > cfu / mL, after fermentation is carried out for 168 h, the inoculant shows significant difference from a YMA culture medium, an MYMA culture medium, a TY culture medium, a YMAR culture medium, a ZJC culture medium and a YDS culture medium, and finally OD600 is 1.26 times that of the YMA culture medium. By optimizing the formula and fermentation conditions of the YGA culture medium, high-density shake flask fermentation of the bradyrhizobium anhuoshanense microbial inoculum is achieved, and the preparation method is efficient, environmentally friendly, simple in technological process and relatively low in cost and has production and application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of peanut cultivation methods, and particularly relates to a method for flask fermentation of slow-growing peanut rhizobia. Background Art

[0002] Peanut (Arachis hypogaea L.) originated in South America and is an important oilseed cash crop. Due to its relatively high oil content, it ranks third among the world's top five oilseed crops. Peanut is the oilseed crop with the highest yield potential per unit area and is planted throughout China, which is divided into 7 peanut production areas. Among them, the Northeast peanut production area, the Yellow River Basin peanut production area, the Yangtze River Basin peanut production area, and the Southern peanut production area are the main peanut production areas. Most of Henan Province is located in the Yellow River Basin peanut production area, and its peanut cultivation is further divided into four regions, namely the southern Henan production area, the eastern Henan production area, the central Henan production area, and the northern Henan production area. In the past two years, the peanut planting area in Henan Province has ranked first in the country, all above 22 million mu. Peanut is an important oilseed crop, and vigorously developing peanut production is of great significance in China's agriculture. However, in recent years, the abuse of chemical fertilizers and pesticides and unscientific fertilization methods have led to a significant decline in the yield and quality of leguminous plants. At the same time, problems such as soil compaction and acid-base imbalance have become increasingly prominent. Biological nitrogen fixation is the natural process of converting atmospheric nitrogen into ammonia, which can be achieved by free-living, associative, endophytic, and symbiotic rhizosphere bacteria, collectively referred to as rhizobia. They form a symbiotic relationship with leguminous plants, where the plant host provides nutrients to the rhizobia, and the rhizobia provide fixed atmospheric nitrogen to the host in the form of ammonia. Eventually, it can achieve the purposes of promoting crop growth, reducing crop diseases, and improving crop yield and quality.

[0003] Rhizobia are a type of rod-shaped bacteria that live in symbiosis with legumes. They form nodules and fix nitrogen in the air to provide plant nutrition. Normal cells move with flagella, are spore-free, and are Gram-negative. They are aerobic organisms. Rhizobia can utilize a variety of carbohydrates and produce a considerable amount of extracellular mucus. Currently, research on bradyrhizobium agents mainly focuses on soybean bradyrhizobium. Wu Ping et al. screened and optimized the culture conditions of soybean rhizobium AHM2B strain and found that soybean rhizobium AHM2B strain grew faster in BSE medium, followed by YMA and TY medium, and grew slowest in PA medium (Wu Ping, Li Zhengpeng, He Qingyuan, et al. Screening and optimization of culture conditions of soybean rhizobium AHM2B strain [J]. Soybean Science, 2014, 33(06): 953-956.). Wu Honghui and Zhou Junchu used soybean Bradyrhizobium japonicum USDA110 as the test bacteria and conducted a comparative test on five culture media, including YMA, TY, SM, PA and BSE. Both test strains grew fastest in BSE medium. They also conducted a culture medium optimization test on USDA110, which improved the growth efficiency of the strain. However, the concentration in the shake flask was still very low, and the shake flask preparation of Bradyrhizobium inoculant only reached 10 9 level, and cannot reach a very high density (Wu Honghui, Zhou Junchu. Optimization of rhizobium culture medium and comparative study of dosage forms [J]. Bulletin of Microbiology, 2004, (02): 14-19.). Patent 201510658106.5 provides a peanut rhizobium culture medium, which uses glycerol, mannitol, glucose, rhamnose, fructose, α-ketoglutaric acid and other substances to form a mixed carbon source, and optimizes the growth environment of rhizobia by precisely controlling the balance of carbon source, nitrogen source and mineral nutrients. The Japanese bradyrhizobium agent prepared by fermentation of the culture medium has a viable count of more than 10 billion per milliliter when stored at 20°C for 12 months. However, the culture medium contains a variety of chemical substances and nutrients, and the cost is high, which is not conducive to promotion and application. Fermentation culture medium plays an important role in the large-scale production and application of peanut bradyrhizobium. Therefore, by continuously optimizing the formula and fermentation conditions of the fermentation culture medium, it is expected to achieve efficient, environmentally friendly and low-cost production and application of peanut bradyrhizobium agent. Summary of the Invention

[0004] The present invention proposes a method for high-density shake flask fermentation of peanut bradyrhizobium and its application, and the high-density peanut rhizobium inoculum obtained by fermentation has the characteristics of high efficiency and environmental protection, simple process flow and low cost, providing technical support for the large-scale production and application of peanut bradyrhizobium inoculum.

[0005] The technical solution of the present invention is achieved in this way:

[0006] On the one hand, the present application provides an improved YGA medium, which comprises the following components: 7 - 15 g of glucose, 0.15 - 0.35 g of K2HPO4, 0.15 - 0.35 g of KH2PO4, 0.1 - 0.3 g of anhydrous MgSO4, 0.05 - 0.15 g of NaCl, 2 - 5 g of yeast powder, 1000 mL of water, and the pH is 6.8 - 7.2.

[0007] Preferably, the above - mentioned improved YGA medium specifically comprises the following components: 12 g of glucose, 0.25 g of K2HPO4, 0.25 g of KH2PO4, 0.1 g of anhydrous MgSO4, 0.1 g of NaCl, 4 g of yeast powder, 1000 mL of water, and the pH is 7.

[0008] Preparation of the seed liquid: The purified strain of Bradyrhizobium arachidis is inoculated into the YMA liquid medium, and cultured on a constant - temperature shaker at 28 °C for 5 d with a shaker speed of 180 rpm. The OD value of the well - cultured bacterial liquid is adjusted to 1.0 for standby. 600 value is 1.0 for standby.

[0009] On the second hand, the present application also provides a method for high - density shake - flask fermentation of Bradyrhizobium arachidis, which is realized based on the above - mentioned improved YGA medium.

[0010] Preferably, the steps of the above - mentioned method for high - density shake - flask fermentation of Bradyrhizobium arachidis are as follows: Inoculate the seed liquid of Bradyrhizobium arachidis into the improved YGA medium, and culture to obtain a high - density Bradyrhizobium arachidis inoculant, and the OD of the inoculant is 600 8.5 - 9.5.

[0011] Preferably, the inoculation amount of the above - mentioned seed liquid is 1 - 5%.

[0012] Preferably, the conditions for the above - mentioned culture are shake - flask culture at 25 - 32 °C and 150 - 200 r / min for 5 - 8 d.

[0013] Third invention: A high - density Bradyrhizobium arachidis inoculant prepared by the above - mentioned method for high - density shake - flask fermentation of Bradyrhizobium arachidis.

[0014] Preferably, the concentration of the above - mentioned inoculant is greater than 10 11 cfu / mL.

[0015] Fourth aspect: Application of the above - mentioned high - density Bradyrhizobium arachidis inoculant in peanut planting.

[0016] Preferably, the steps of the above - mentioned application are as follows: Mix the high - density Bradyrhizobium arachidis inoculant and peanut seeds evenly.

[0017] The present invention has the following beneficial effects:

[0018] In this application, an improved YGA medium is used for shake flask fermentation of Bradyrhizobium arachidis to prepare a high-density Bradyrhizobium arachidis inoculant, and the effective viable count of the inoculant is greater than 10 11 cfu / mL, showing significant differences from YMA medium, MYMA medium, TY medium, YMAR medium, ZJC medium, and YDS medium after 168 h of fermentation. Finally, the OD 600 is 1.26 times that of YMA medium, 1.1 times that of MYMA medium, 12.41 times that of TY medium, 4.42 times that of YMAR medium, 2.2 times that of ZJC medium, and 2.86 times that of YDS medium. The fermentation effect is significantly higher than that of the existing media. By optimizing the formula and fermentation conditions of the YGA medium, this application realizes high-density shake flask fermentation of Bradyrhizobium arachidis inoculant, and the preparation method is efficient, environmentally friendly, has a simple process flow, and relatively low cost, with the potential for production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is a comparison diagram of the optimized medium YGA and other media.

[0021] Figure 2 It is a comparison diagram of YGA, MYMA, and YMA media cultured for 5 days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following will clearly and completely describe the technical solutions of the present invention in combination with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0023] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0024] Example 1

[0025] A method for high-density shake flask fermentation of Bradyrhizobium arachidis is as follows:

[0026] 1. Preparation of strains

[0027] A Bradyrhizobium strain with good adaptability, Bradyrhizobium zhengyangense WYCCWR 15459, was selected from the Bradyrhizobium library we established (this strain is preserved in the Guangdong Provincial Microbial Culture Collection Center with the preservation number GDMCC No: 1.4969), and this strain was used to prepare the Bradyrhizobium inoculant for peanuts.

[0028] 2. Preparation of seed liquid

[0029] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate it on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD 600 value of the well-shaken bacterial liquid to 1.0 for standby.

[0030] 3. Preparation of medium and fermentation of the strain

[0031] Select YGA medium as the fermentation medium. The formula is 7 - 15 g of glucose, 0.15 - 0.35 g of K2HPO4, 0.15 - 0.35 g of KH2PO4, 0.1 - 0.3 g of anhydrous MgSO4, 0.05 - 0.15 g of NaCl, 2 - 5 g of yeast powder, 1000 mL of deionized water, and pH 6.8 - 7.2, which is more conducive to the cultivation of slower-growing rhizobia. After preparing the medium according to the above formula, sterilize it. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared YGA medium with an inoculation amount of 1%. Incubate it at 28 °C with a shaking speed of 180 revolutions per minute for 7 days until the bacterial cells grow to a high density, that is, the OD 600 is approximately 8.5 - 9.5.

[0032] Perform gradient dilution coating and counting on the high-density Bradyrhizobium inoculant for peanuts obtained by the above method. The results are shown in Table 1. The concentration of the inoculant reaches 10 11 , proving that this fermentation method is feasible.

[0033] Table 1 Gradient dilution coating and counting of high-density Bradyrhizobium inoculant for peanuts

[0034]

[0035] As can be seen from Table 1, YGA medium can enable high-density fermentation of Bradyrhizobium for peanuts, and the number of effective viable bacteria reaches 10 11 cfu / mL.

[0036] Comparative Example 1

[0037] A method for high-density shake-flask fermentation of Bradyrhizobium for peanuts is as follows:

[0038] 1. Preparation of strains

[0039] For the preparation of the microbial agent, a strain Bradyrhizobium zhengyangense WYCCWR 15459 with better adaptability was selected from the Bradyrhizobium arachidis strain library we established.

[0040] 2. Preparation of seed liquid

[0041] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate it on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD value of the well-shaken bacterial liquid to 1.0 for standby. 600 value to 1.0 for standby.

[0042] 3. Preparation of medium and fermentation of strains

[0043] Select YMA medium as the fermentation medium. This medium is the basic medium for Bradyrhizobium. The medium formula is as follows: mannitol 10 g, KH2PO4 0.25 g, K2HPO4 0.25 g, anhydrous magnesium sulfate 0.1 g, NaCl 0.1 g, yeast powder 3 g, agar powder 18 g, deionized water 1000 mL, pH 6.8 - 7.2. After preparing the medium according to the above formula, sterilize it. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared YMA medium with an inoculation amount of 1%. Incubate it at 28 degrees Celsius with a shaking speed of 180 revolutions per minute for 7 days, and the OD 600 is approximately 7.51.

[0044] Comparative Example 2

[0045] A method for high-density shake flask fermentation of Bradyrhizobium arachidis is as follows:

[0046] 1. Preparation of strains

[0047] For the preparation of the microbial agent, a strain Bradyrhizobium zhengyangense WYCCWR 15459 with better adaptability was selected from the Bradyrhizobium arachidis strain library we established.

[0048] 2. Preparation of seed liquid

[0049] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate it on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD 600 value to 1.0 for standby.

[0050] 3. Preparation of culture medium and fermentation of strains

[0051] Select MYMA medium as the fermentation medium, which is mainly used for the cultivation of Bradyrhizobium. The medium formula is as follows: yeast powder 1 g, mannitol 10 g, sodium glutamate 0.5 g, K2HPO4 0.5 g, anhydrous MgSO4 0.1 g, CaCl2 0.04 g, FeCl3 0.04 g, NaCl 0.05 g, agar powder 18 g, deionized water 1000 mL, pH 6.8 - 7.2. After preparing the medium according to the above formula, perform sterilization treatment. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared MYMA medium, with an inoculation amount of 1%. Incubate at 28 °C with a shaking speed of 180 revolutions per minute for 7 days, and the OD 600 is approximately 8.24.

[0052] Comparative Example 3

[0053] A method for high-density shake-flask fermentation of Bradyrhizobium arachidis, the steps are as follows:

[0054] 1. Preparation of strains

[0055] From the Bradyrhizobium arachidis library we established, a strain Bradyrhizobium zhengyangense WYCCWR 15459 with better adaptability was selected for the preparation of the bacterial agent.

[0056] 2. Preparation of seed liquid

[0057] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD 600 value to 1.0 for standby.

[0058] 3. Preparation of culture medium and fermentation of strains

[0059] Select TY medium as the fermentation medium, which is mainly used for the cultivation of fast-growing rhizobia. The medium formula is as follows: yeast powder 3 g, anhydrous calcium chloride 0.53 g, tryptone 5 g, deionized water 1000 mL, pH 6.8 - 7.2. After preparing the medium according to the above formula, perform sterilization treatment. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared TY medium, with an inoculation amount of 1%. Incubate at 28 °C with a shaking speed of 180 revolutions per minute for 7 days, and the OD 600 is approximately 0.73.

[0060] Comparative Example 4

[0061] A method for high-density shake flask fermentation of Bradyrhizobium sp. in peanuts, the steps are as follows:

[0062] 1. Preparation of the strain

[0063] From the Bradyrhizobium sp. library we established, a strain Bradyrhizobium zhengyangense WYCCWR 15459 with better adaptability was selected for the preparation of the bacterial agent.

[0064] 2. Preparation of the seed liquid

[0065] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate it on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD of the well-shaken bacterial liquid 600 value to 1.0 for standby.

[0066] 3. Preparation of the medium and fermentation of the strain

[0067] Select YMAR medium as the fermentation medium. This medium is an optimized medium based on YMA and is used for the cultivation of Bradyrhizobium sp. in chickpeas. The medium formula is as follows: mannitol 12 g, KH2PO4 0.25 g, K2HPO4 0.25 g, anhydrous magnesium sulfate 0.1 g, NaCl 0.1 g, yeast powder 8.75 g, agar powder 18 g, deionized water 1000 mL, pH 6.8 - 7.2. After preparing the medium according to the above formula, sterilize it. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared YMAR medium with an inoculation amount of 1%. Incubate it at 28 degrees Celsius with a rotation speed of 180 revolutions per minute for 7 days. OD 600 is approximately 2.05.

[0068] Control Example 5

[0069] A method for high-density shake flask fermentation of Bradyrhizobium sp. in peanuts, the steps are as follows:

[0070] 1. Preparation of the strain

[0071] From the Bradyrhizobium sp. library we established, a strain Bradyrhizobium zhengyangense WYCCWR 15459 with better adaptability was selected for the preparation of the bacterial agent.

[0072] 2. Preparation of the seed liquid

[0073] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate it on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD of the well-shaken bacterial liquid600 Reserved with a value of 1.0.

[0074] 3. Preparation of culture medium and fermentation of strains

[0075] Select ZJC medium as the fermentation medium. This medium is optimized based on BSE medium and is suitable for the growth of Bradyrhizobium japonicum. The medium formula is as follows: 15 g of glucose, 4 g of yeast powder, 0.5 g of K2HPO4, 0.2 g of MgSO4, 0.1 g of NaCl, 0.05 g of CaCl2, 4 mL of Rh solution, 1000 mL of deionized water, pH 6.8 - 7.2. After preparing the medium according to the above formula, perform sterilization treatment. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared ZJC medium, with an inoculation amount of 1%. Incubate at 28 degrees Celsius with a shaking speed of 180 revolutions per minute for 7 days, OD 600 is approximately 4.12.

[0076] Comparative Example 6

[0077] A method for high-density shake-flask fermentation of Bradyrhizobium arachidis, the steps are as follows:

[0078] 1. Preparation of strains

[0079] A strain of Bradyrhizobium arachidis was selected from the Bradyrhizobium arachidis library we established, and a strain with better adaptability, Bradyrhizobium zhengyangense WYCCWR 15459, was used for the preparation of the microbial agent.

[0080] 2. Preparation of seed liquid

[0081] Pick the purified test strain and inoculate it into a 5 mL test tube containing YMA liquid medium. Incubate on a constant temperature shaker at 28 °C for 5 days with a shaker speed of 180 rpm. Adjust the OD of the shaken bacterial liquid 600 to a value of 1.0 for standby.

[0082] 3. Preparation of culture medium and fermentation of strains

[0083] Select YDS medium as the fermentation medium. This medium is optimized based on YMA medium and is suitable for Bradyrhizobium japonicum. The medium formula is as follows: 9.45 g of mannitol, 0.5 g of yeast powder, 0.38 g of K2HPO4, 0.2 g of MgSO4, 0.1 g of NaCl, 1000 mL of deionized water, pH 6.8 - 7.2. After preparing the medium according to the above formula, perform sterilization treatment. Then, inoculate the seed liquid of strain WYCCWR 15459 into the prepared YDS medium, with an inoculation amount of 1%. Incubate at 28 degrees Celsius with a shaking speed of 180 revolutions per minute for 7 days, OD600 It is approximately 3.17.

[0084] The results are as Figure 1 shown. The optimized medium YGA showed significant differences from the YMA medium, MYMA medium, TY medium, YMAR medium, ZJC medium, and YDS medium of Comparative Examples 1-6 after 168 h. Moreover, the final OD of the fermentation inoculum in the optimized YGA medium 600 was 1.26 times that of the YMA medium before optimization, 1.1 times that of the MYMA medium, 12.41 times that of the TY medium, 4.42 times that of the YMAR medium, 2.2 times that of the ZJC medium, and 2.86 times that of the YDS medium. The effect was significantly higher than that of the YMA medium, MYMA medium, TY medium, YMAR medium, ZJC medium, and YDS medium of Comparative Examples 1-6.

[0085] Figure 2 Comparing the YGA, MYMA, and YMA media cultured for 5 days, it can be seen from Figure 2 this that the turbidity of the strain after fermenting in the YGA medium for 5 days was significantly higher than that in the MYMA and YMA media. Combining Figure 1 this indicates that the effect of the YGA medium was significantly higher than that of the MYMA and YMA media.

[0086] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An improved YGA medium, characterized in that, It comprises the following components: 7 - 15 g of glucose, 0.15 - 0.35 g of K2HPO4, 0.15 - 0.35 g of KH2PO4, 0.1 - 0.3 g of anhydrous MgSO4, 0.05 - 0.15 g of NaCl, 2 - 5 g of yeast powder, 1000 mL of water, and the pH is 6.8 - 7.

2.

2. The improved YGA medium according to claim 1, characterized in that, Specifically, it comprises the following components: 12 g of glucose, 0.25 g of K2HPO4, 0.25 g of KH2PO4, 0.1 g of anhydrous MgSO4, 0.1 g of NaCl, 4 g of yeast powder, 1000 mL of water, and the pH is 7.

3. A method for fermenting Bradyrhizobium arachidis in a shake flask with high density, characterized in that: The method is implemented based on the improved YGA medium described in claim 1 or 2.

4. The method for fermenting Bradyrhizobium arachidis in a high-density shake flask according to claim 3, wherein The steps are as follows: inoculate the seed liquid of Bradyrhizobium arachidis into the improved YGA medium and culture to obtain a high - density Bradyrhizobium arachidis inoculant.

5. The method for fermenting Bradyrhizobium arachidis with high density in shake flasks according to claim 4, characterized in that: The inoculation amount of the seed liquid is 1 - 5%.

6. The method for fermenting Bradyrhizobium arachidis in a high-density shake flask according to claim 5, wherein: The conditions for the culture are shake - flask culture at 25 - 32 °C and 150 - 200 r / min for 5 - 8 d.

7. A high - density Bradyrhizobium arachidis inoculant prepared by the method for high - density shake - flask fermentation of Bradyrhizobium arachidis according to any one of claims 3 - 6.

8. The high-density Bradyrhizobium arachidis inoculant according to claim 7, characterized in that: The concentration of the bacterial agent is greater than 10 11 cfu / mL.

9. The application of the high - density Bradyrhizobium arachidis inoculant described in claim 8 in peanut planting.

10. The application according to claim 9, wherein The steps are as follows: mix the high - density Bradyrhizobium arachidis inoculant and peanut seeds evenly.

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