Nitrogen-producing pseudomonas as well as culture method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a Pseudomonas azotoformans strain BH-11, a high-density fermentation culture method of the Pseudomonas azotoformans strain BH-11, an extracting solution prepared from the Pseudomonas azotoformans strain BH-11 and application of the Pseudomonas azotoformans strain BH-11 in agriculture. The 16S rRNA (ribosomal ribonucleic acid) gene sequence of the pseudomonas nitrogen-producing BH-11 is as shown in SEQ ID NO: 1, and the pseudomonas nitrogen-producing BH-11 is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC NO. 35761. According to the application, high-density and low-cost culture of the nitrogen-producing pseudomonas is realized, the thallus yield is increased by 43%, the fermentation cost is reduced by 10-15%, and the obtained extract has remarkable advantages in the aspects of promoting crop growth, increasing the yield and enhancing stress resistance and has a good application prospect.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a nitrogen-producing Pseudomonas aeruginosa, its culture method, and its application. Background Technology

[0002] *Pseudomonas azotoformans* is a Gram-negative bacterium belonging to the genus *Pseudomonas*, showing broad application potential in agriculture. Current technologies indicate that these strains can be used as biocontrol agents, effectively suppressing various agricultural pests and plant pathogens. However, existing technologies lack specific, quantifiable reports on the application of their bacterial extracts to crops, particularly regarding their specific effects on promoting crop growth, increasing yield, and alleviating salt stress.

[0003] Currently, the fermentation process for azotobacter is hampered by high costs and unsatisfactory cell density, directly limiting the development and market application of its microbial agents. High-density microbial fermentation is a complex systems engineering project involving the optimization of culture medium formulations, precise control of fermentation process parameters (such as pH, dissolved oxygen, and feeding strategies), and coordinated equipment configuration. Existing technologies have not yet provided a mature fermentation process for azotobacter, particularly one capable of achieving high-density cultivation at a controllable cost.

[0004] Therefore, there is an urgent need in this field to screen for a stable strain of nitrogen-producing Pseudomonas aeruginosa and establish a set of high-density, low-cost fermentation culture methods to enable the obtained bacterial extracts to promote crop growth, increase yield and enhance stress resistance, thereby promoting the practical application of such microbial preparations in agricultural production. Summary of the Invention

[0005] This application specifically discloses a strain of Pseudomonas azotoformans, BH-11, its high-density fermentation culture method, the extract prepared from it, and its application in agriculture.

[0006] In a first aspect, this application provides a nitrogen-producing Pseudomonas bacterium, the accession number of which is CGMCCNO.35761, and the name of which is Pseudomonas azotoformans BH-11.

[0007] Secondly, this application provides a method for culturing the aforementioned azotocinus, the method comprising fermenting the bacteria using a fermentation medium, wherein the fermentation medium comprises a carbon source, a nitrogen source, inorganic salts, and growth factors; wherein the carbon source is at least one of dextrin, glucose, and glycerol; the nitrogen source is at least one of soybean meal powder, ammonium sulfate, and peanut meal powder; the inorganic salt is at least two of dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate; and the growth factor is at least one of yeast extract, methionine, and para-aminobenzoic acid.

[0008] Optionally, the growth factor is yeast extract and methionine.

[0009] Optionally, the methionine concentration is 1 g / L, and the yeast extract concentration is 10 g / L.

[0010] Optionally, the nitrogen source is soybean meal powder and ammonium sulfate.

[0011] Optionally, the soybean meal powder concentration is 15 g / L, and the ammonium sulfate concentration is 10 g / L.

[0012] Optionally, the fermentation conditions include: pH 6.8-7.0, dissolved oxygen 30%-40%, rotation speed 200-300 rpm, temperature 34-37℃, aeration rate 1.0-2.7 vvm, and feeding rate 80-150 ml / h.

[0013] Optionally, the fermentation culture adopts a staged control strategy: the rotation speed, aeration rate, and pH are 200 rpm, 1.0-1.3 vvm, and 6.5 respectively during fermentation 0-5 h; the rotation speed, aeration rate, and pH are 250 rpm, 1.4-1.6 vvm, and 6.8 respectively after fermentation 5-8 h; the rotation speed, aeration rate, and pH are 300 rpm, 1.8-2.2 vvm, and 7.0 respectively after fermentation 8 h; and the aeration rate is further increased to 2.3-2.7 vvm after fermentation 14 h.

[0014] Optionally, the fermentation culture adopts a two-stage feeding strategy: feeding begins when the glucose content in the fermentation broth decreases to 5~10g / L, the feeding rate is 80~100ml / h before 9h, and the feeding rate is 100~150ml / h after 9h.

[0015] Thirdly, this application provides an extract, which is prepared by crushing and centrifuging azotocinus cells obtained by the culture method and then taking the supernatant.

[0016] Fourthly, this application provides a microbial inoculant containing the extract.

[0017] Optionally, the microbial agent may also contain amino acid liquid, brown algae oligosaccharides, humic acid, plant growth regulators and / or pesticides.

[0018] Fifthly, this application provides the application of the extract or microbial agent in promoting crop growth, increasing crop yield, and / or enhancing crop resistance to stress.

[0019] Optionally, the crop may be a chili pepper, tomato, Arabidopsis thaliana, spinach, grape, lettuce, or wheat.

[0020] In summary, this application includes at least one of the following beneficial technical effects: 1. High-density fermentation culture was carried out using BH-11 as the production strain. A two-stage feeding strategy and control of aeration and stirring during fermentation were employed. Methionine and para-aminobenzoic acid were added as growth factors. The final wet weight of BH-11 strain reached 300g, and the wet weight of the fermentation strain increased by 43%. 2. Using a cheaper nitrogen source instead of tryptone in the original culture medium reduces the cost per fermentation by 10% to 15%; 3. Applying BH-11 extract to crops can promote the growth of tomatoes, peppers, and Arabidopsis thaliana and increase crop yield. Adding 1 ng / L of BH extract increases the number of tomatoes and peppers and doubles the number of Arabidopsis thaliana flower buds. In the field, it can make grape berries plump and uniform from top to bottom. When used on spinach, it can reduce or eliminate leaf curling and dry leaf phenomena in the control group, and the spinach leaves are darker green. 4. BH-11 extract can alleviate salt or saline-alkali stress in tomato, lettuce and wheat crops. During the same period, the number of tomato flowers was higher than that of the control under salt stress, and the leaf width of lettuce was larger. In the field, under saline-alkali stress, wheat ear length and wheat tiller number were significantly increased, and wheat ear length could be increased by 21.6-26.7%. Attached Figure Description

[0021] Figure 1 The change in cell density during the fermentation process of fermentation culture method 1.

[0022] Figure 2 The change in cell density during the fermentation process of fermentation culture method 2.

[0023] Figure 3 The change in cell density during the fermentation process of fermentation culture method 3.

[0024] Figures 4A-4C The effects of BH-11 extract BH on crop growth and yield in an artificial climate chamber; among which, Figure 4A The crop is tomato. Figure 4B The crop is chili pepper. Figure 4C The crop is Arabidopsis thaliana.

[0025] Figures 5A-5B The effects of BH-11 extract BH on crop growth and yield in greenhouses and fields; among which, Figure 5A The crop is spinach. Figure 5B The crop is Shine Muscat grape.

[0026] Figures 6A-6B The BH-11 extract BH was used to mitigate the effects of soil salinity on crop growth and yield in an artificial climate chamber; among which, Figure 6A The crop is lettuce. Figure 6B The crop is tomato.

[0027] Figure 7 To investigate the effects of BH-11 extract BH on wheat growth and yield in the field. Detailed Implementation

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, the experimental methods in the following examples were performed according to standard procedures, and the materials and reagents used were all commercially available.

[0030] Example 1: Screening and identification of Pseudomonas azotoformans BH-11

[0031] Roots and stems of mangroves in Guangzhou were collected, and the strains were isolated and screened using a concentration gradient dilution method. Single colony streaking was repeated three times to obtain pure strains. Finally, a nitrogen-producing Pseudomonas aeruginosa that could stably grow under culture conditions was obtained and named BH-11.

[0032] Genomic DNA was extracted from BH-11 using a bacterial genomic DNA extraction kit. Using BH-11 DNA as a template, its 16S rRNA gene sequence was amplified by PCR. The amplified product was sequenced after purity testing by electrophoresis, and the sequencing result is shown in SEQ ID NO:1. The obtained 16S rRNA gene sequence SEQ ID NO:1 was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using BLAST. The sequence showed the highest similarity (99.86%) with Pseudomonas azotoformans strain: IAM 1603, confirming BH-11 as a nitrogen-producing Pseudomonas. BH-11 was deposited on August 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.35761.

[0033] Example 2: Cultivation method of BH-11

[0034] Step 1, Mother Culture: The BH-11 strain sample was inoculated onto a solid culture medium and cultured at 34℃ for 20 h to obtain an activated strain. The solid culture medium formula was: 5 g yeast extract, 20 g tryptone, 20 g sodium chloride, 2 g agar, 1000 ml water, adjusted to pH 6.8, and sterilized.

[0035] Step 2, Original Seed Culture: The strain obtained in step 1 was inoculated into liquid culture medium and cultured for 18 h at a temperature of 34℃ and a rotation speed of 120 rpm. The liquid culture medium formula was: 5 g yeast extract, 20 g tryptone, 20 g sodium chloride, 1000 ml water, adjusted to pH 6.8, and sterilized.

[0036] Step 3a, Fermentation Cultivation Method 1: The bacterial culture obtained in step 2 was inoculated into a 30 L fermenter containing fermentation medium at a 2% inoculation rate. The pH was maintained at 6.8 with 10% sodium hydroxide, dissolved oxygen at 30%, the fermentation speed was 150 rpm, the temperature was set at 34℃, the aeration rate was maintained at 1.0 vvm, and the feed rate was 80 ml / h. The feeding of carbon and nitrogen sources was stopped after 16 h, and fermentation ended after 20 h. Samples were taken every 2 h to measure the cell density of the fermentation broth. The changes in cell density are shown below. Figure 1 As shown.

[0037] Step 3b, Fermentation Cultivation Method 2: Two-stage feeding strategy: Inoculate 2% of the culture medium into a 30 L fermenter, maintain pH at 6.8 with 10% sodium hydroxide solution, control dissolved oxygen at 30%, spin speed at 150 rpm, temperature at 34℃, and aeration rate at 1.1 vvm. Feed rate is 80 ml / h for the first 9 hours, and 100 ml / h thereafter. Stop feeding carbon and nitrogen sources at 16 hours, and end fermentation at 20 hours. Samples are taken every 2 hours to measure cell density. Changes in cell density are shown below. Figure 2 As shown.

[0038] Step 3c, Fermentation Cultivation Method 3: Fermentation strategy with gradually increasing aeration and rotation speed: Inoculate 2% of the culture into a 30 L fermenter. Maintain pH at 6.8 using 10% sodium hydroxide solution, control dissolved oxygen at 30%, and set the temperature to 34℃. Feed rate was 80 ml / h for the first 9 hours, then 100 ml / h after 9 hours. For fermentation 0-5 hours, maintain constant stirring speed, aeration rate, and pH at 200 rpm, 1.0 vvm, and 6.5, respectively. From 5-8 hours, maintain constant stirring speed, aeration rate, and pH at 250 rpm, 1.4 vvm, and 6.8, respectively. After 8 hours, increase stirring speed and aeration rate to 300 rpm and 1.8 vvm, and adjust pH to 7.0. After 14 hours, increase aeration rate to 2.3 vvm. Stop feeding carbon and nitrogen sources at 16 hours, and end fermentation at 20 hours. Samples were taken every 2 hours to measure cell density in the fermentation broth. Changes in cell density are shown below. Figure 3 As shown.

[0039] Method for detecting cell density in BH-11 fermentation broth: Every 2 hours, take 10 ml of fermentation broth and centrifuge at 10000 r / min for 5 min. Collect the cell precipitate, wash twice with physiological saline, resuspend in physiological saline and dilute appropriately so that the OD value of the diluted suspension is between 0.2 and 0.8. Measure the absorbance at 600 nm using a spectrophotometer. The product of the absorbance value and the dilution factor is the cell density (OD600) of the fermentation broth.

[0040] Fermentation medium formulation: 600g carbon source, 400g nitrogen source, 100g inorganic salts, 70g growth factors; carbon source is dextrin and glycerol (added in a 2:1 ratio), nitrogen source is soybean meal and ammonium sulfate, inorganic salts are dipotassium hydrogen phosphate, magnesium sulfate, and sodium chloride (added in a 1:1:1 ratio), growth factors are yeast extract and methionine, 18L water, sterilized at 121℃. Fed medium formulation: 100g / L carbon source, 70g / L nitrogen source.

[0041] The optimization results of nitrogen source and growth factors are shown in Tables 1 and 2, respectively. Table 1 shows that the highest cell density was achieved with 15 g / L soybean meal powder and 10 g / L ammonium sulfate as nitrogen sources. Table 2 shows that the highest cell density was achieved when 1 g / L methionine and 10 g / L yeast extract were added simultaneously.

[0042] Table 1 A: Soybean meal powder (g / L) B: Ammonium sulfate (g / L) C: Peanut cake powder (g / L) Y:OD600 1 15 10 30 2.075 2 30 0 15 2.055 3 15 10 0 2.185 4 30 10 15 2.155 5 15 5 15 2.12 6 0 5 30 2.12 7 15 5 15 2.12 8 15 5 15 2.12 9 0 10 15 1.855 10 0 5 0 2.005 11 15 0 0 2.12 12 15 5 15 1.995 13 0 0 15 2.04 14 30 5 0 2.3 15 15 0 30 1.95 16 15 5 15 2 17 30 5 30 2.1

[0043] Table 2 A: Methionine (g / L) B: Yeast extract (g / L) C: Para-aminobenzoic acid (g / L) Y:OD600 1 1 10 0 2.8 2 1 0 0 2.45 3 2 5 5 2.52 4 1 5 2.5 2.54 5 1 10 5 2.65 6 1 5 2.5 2.54 7 1 5 2.5 2.54 8 0 10 2.5 2.355 9 2 0 2.5 2.05 10 1 5 2.5 2.54 11 1 5 2.5 2.54 12 2 5 0 2.45 13 0 5 5 2.4 14 1 0 5 2.295 15 0 5 0 2 16 2 10 2.5 2.39 17 0 0 2.5 1.96

[0044] Example 3: Application of BH-11 extract BH in agriculture After fermentation, the bacterial broth was centrifuged at 8000×g for 5 min to obtain bacterial cells. The obtained bacterial cells were resuspended in water and ultrasonically disrupted at 100W for 5 h. After disruption, the cells were centrifuged at 6000×g at 4℃ for 5 min to separate solids and liquids. 1 ml of the supernatant was collected and concentrated in a vacuum centrifuge until the solid weight no longer changed. The concentrate was then diluted with water to the required concentration of BH-11 extract BH.

[0045] 3.1 Application of BH-11 extract (BH) in promoting crop growth and yield

[0046] In an artificial climate chamber, tomatoes, Arabidopsis thaliana, and peppers were thoroughly mixed with field soil and vermiculite in a 1:1 volume ratio. The mixture was then poured into planting bowls, each containing 150 ml of the mixture. Seedlings were then transplanted into the planting bowls. The temperature was 17°C, humidity 45% RH, and light intensity 30,000 Lux (supplementary lighting 12h / d). Every seven days after transplanting, extract BH or a microbial inoculant containing extract BH (combined with 2 mg / L alginate oligosaccharides (AOS), plant growth regulators (PGRs), and / or humic acid (HA)) was added. The control group (CK) used only water throughout the process. Results are as follows... Figures 4A-4C As shown, the BH11 extract BH of the present invention, when applied to crops, promotes the growth of tomatoes, Arabidopsis thaliana, and peppers and increases crop yield. Figure 4A The fruits harvested from four tomato plants showed that the average number of fruits per plant increased by about 59% compared with the control group in the BH (10ng / L) group. The BH+AOS group and the BH+HA+PGRs group also showed a significant increase in the number of fruits compared with the BH group. Figure 4B The study showed the effects of different concentrations of BH on the growth of chili seedlings, with a concentration of 10 ng / L effectively promoting the growth of chili seedlings and leaves. Figure 4C The number of Arabidopsis thaliana flower buds in the BH (10 ng / L) group was significantly increased by several times compared with the CK group, and the plant height was also significantly increased.

[0047] Spinach and Shine Muscat grapes were used in greenhouses and fields. After planting, the crops were foliar sprayed once during the seedling stage with 1 ng / L extract BH, and during the growing season, they were foliar sprayed with 10 ng / L extract BH or a mixture of extract BH and 2 mg / L fucoidan (AOS) and humic acid (HA), for a total of 3 treatments. The control group (CK) used water throughout the treatment. The results are as follows: Figures 5A-5B As shown, this method promotes the yield of spinach and grapes in field soil. Figure 5A It is evident that the use of extract BH on spinach can reduce or eliminate leaf curling and dryness in the CK group, and the spinach leaves are darker green. Figure 5B It is evident that, compared to the CK group, the grape berries in the BH+HA+AOS group are fuller and more uniform from top to bottom.

[0048] 3.2 Application of BH-11 extract (BH) in alleviating the effects of soil salinity stress on crop growth and yield

[0049] In an artificial climate chamber, tomatoes and lettuce were used. Field soil and vermiculite were thoroughly mixed in a 1:1 volume ratio and then filled into planting bowls, each containing 150 ml of the mixture. Seedlings were then transplanted into planting pots containing 100 mM sodium chloride. The cultivation temperature and light were: 17°C, 45% RH, and 30,000 Lux light intensity (supplementary lighting 12 h / d). Every seven days after transplanting, a concentration of 10 ng / L extract BH or 10 ng / L extract BH + 2 mg / L amino acid solution (AA) was added. Results are as follows... Figures 6A-6B As shown, BH11 extract can also alleviate salt stress in tomato and lettuce crops, such as Figure 6A It is evident that the number of tomato flowers during the same period was higher than that of the control under salt stress. Figure 6B As can be seen, the lettuce leaves are significantly larger than the control group.

[0050] Wheat was used in the field, with saline-alkali soil selected. After planting, during the seedling stage, the leaves were sprayed once with 10 ng / L extract BH or a mixture of 10 ng / L insecticide (INSC) and 2 mg / L amino acid solution (AA). During the growing season, the leaves were sprayed with 50 ng / L extract BH or a mixture of 10 ng / L insecticide (INSC) and 2 mg / L amino acid solution (AA). A total of 3 treatments were performed. The control group (CK) used water throughout the treatment. Results are as follows: Figure 7 As shown, under salt-alkali stress, the extract BH significantly increased wheat spike length and wheat tiller number, with the spike length increasing by 21.6–26.7%.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0052] sequence list BH11 16s SEQ ID NO:1

Claims

1. A nitrogen-producing Pseudomonas bacterium, characterized in that, The azotoform bacteria described has the accession number CGMCC NO.35761 and is named Pseudomonas azotoformans BH-11.

2. A method for culturing *Pseudomonas aeruginosa* according to claim 1, characterized in that, The method includes fermentation culture using a fermentation medium, wherein the fermentation medium contains a carbon source, a nitrogen source, inorganic salts, and growth factors; wherein the carbon source is at least one of dextrin, glucose, and glycerol; the nitrogen source is at least one of soybean meal powder, ammonium sulfate, and peanut meal powder; the inorganic salt is at least two of dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate; and the growth factor is at least one of yeast extract, methionine, and para-aminobenzoic acid; preferably, the growth factor is yeast extract and methionine, and the nitrogen source is soybean meal powder and ammonium sulfate.

3. The cultivation method according to claim 2, characterized in that, The fermentation conditions include: pH 6.8-7.0, dissolved oxygen 30%-40%, rotation speed 200-300 rpm, temperature 34-37℃, aeration rate 1.0-2.7 vvm, and feeding rate 80-150 ml / h.

4. The cultivation method according to claim 3, characterized in that, The fermentation culture adopts a two-stage feeding strategy: feeding begins when the glucose content in the fermentation broth decreases to 5-10 g / L, with a feeding rate of 80-100 ml / h before 9 hours and a feeding rate of 100-150 ml / h after 9 hours.

5. The cultivation method according to claim 4, characterized in that, The fermentation culture also adopts a staged control strategy: the rotation speed, aeration rate and pH are 200 rpm, 1.0~1.3 vvm and 6.5 respectively for 0~5 h of fermentation; the rotation speed, aeration rate and pH are 250 rpm, 1.4~1.6 vvm and 6.8 respectively for 5~8 h of fermentation; the rotation speed, aeration rate and pH are 300 rpm, 1.8~2.2 vvm and 7.0 respectively for 8 h of fermentation; and the aeration rate is further increased to 2.3~2.7 vvm after 14 h of fermentation.

6. An extract, characterized in that, The extract is prepared by crushing and centrifuging azotobacterial cells obtained by any one of the culture methods described in claims 2 to 5, and then taking the supernatant.

7. A microbial inoculant, characterized in that, The microbial agent contains the extract described in claim 6.

8. The microbial agent according to claim 7, characterized in that, The microbial agent also contains amino acid liquid, brown algae oligosaccharides, humic acid, plant growth regulators and / or insecticides.

9. The application of the extract according to claim 6 or the microbial agent according to any one of claims 7-8 in promoting crop growth, increasing crop yield and / or enhancing crop stress resistance.

10. The application according to claim 9, characterized in that, The crops mentioned are chili peppers, tomatoes, Arabidopsis thaliana, spinach, grapes, lettuce, or wheat.

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