A type of *Pseudomonas putrefactiveis* P683 and its application in saline-alkali soil improvement.

Abstract

This invention discloses a *Pseudomonas putrefactiveis* P683 strain and its application in saline-alkali soil improvement, relating to the field of microbial technology. The key technical points are as follows: the *Pseudomonas putrefactiveis* P683 strain is specifically *Pseudomonas putrefactiveis* P683, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27636, and deposited on June 15, 2023; the 16S rRNA gene sequence of *Pseudomonas putrefactiveis* P683 is shown in Seq ID No. 1. This invention provides a novel acid-producing and alkali-reducing *Pseudomonas putrefactiveis* P683 strain and its fermentation broth. Strain P683 can produce various organic acids, lowering the environmental pH value, enabling the strain to grow in highly alkaline environments. The fermentation broth of strain P683 can effectively reduce the pH value of soda-alkali soil, alleviating alkaline stress. Strain P683 and its fermentation broth not only effectively improve saline-alkali soil and increase crop emergence rate, but also possess safety, environmental friendliness, and feasibility for industrial production, providing a new and effective solution for saline-alkali soil improvement.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a *Pseudomonas putrefactiveus* P683 and its application in the improvement of saline-alkali soil. Background Technology

[0002] Saline-alkali soil is a general term for both saline and alkaline soils. Based on the severity of salinization, it is generally classified as mild, moderate, and severe. Saline-alkali soil is a widely distributed obstacle soil worldwide. According to incomplete statistics, the global area of ​​saline-alkali land is 954.38 million hectares, accounting for approximately 10% of the global land area. In my country, the area of ​​saline-alkali soil reaches 36 million hectares, accounting for 4.9% of the exploitable land area. Saline-alkali land is widely distributed in my country, with over 30% located in the Northeast, Northwest, and coastal areas. Saline-alkali soil severely affects plant root growth, reduces arable land utilization, and negatively impacts the agricultural economy. Furthermore, salt in saline-alkali land can migrate with water, potentially leading to the salinization of groundwater and surface water, affecting water resource quality and posing a potential threat to drinking water and irrigation water.

[0003] Soil is the foundation of agricultural production, and saline-alkali soil is one of my country's important reserve arable land resources. In recent years, my country's total grain demand has continued to grow, and the improvement and management of saline-alkali land is of great practical significance for regional ecological restoration and reconstruction, local economic stability and development, and national food security. Currently, saline-alkali soil improvement includes physical improvement, chemical improvement, and biological improvement. Among them, biological improvement has received widespread attention due to its effectiveness and sustainability. Saline-alkali stressed soils often exist in high pH environments, and lowering soil pH is key to the reclamation of saline-alkali soils. By screening and applying acid-producing microorganisms, lowering the pH of saline-alkali soils has become an effective and sustainable biological means of improving saline-alkali soils.

[0004] Therefore, the present invention aims to provide a *Pseudomonas putrefactiveis* P683 and its application in the improvement of saline-alkali soil. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems by providing *Pseudomonas putrefactiveis* P683 and its application in saline-alkali soil improvement, aiming to address the issues in saline-alkali soil improvement. *Pseudomonas putrefactiveis* strain P683 can produce various organic acids, lowering the environmental pH value and enabling it to grow in highly alkaline environments.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a *Pseudomonas putrefactiveis* P683 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27636 and accession date of June 15, 2023; the 16S rRNA gene sequence of the *Pseudomonas putrefactiveis* P683 is shown in Seq ID No. 1.

[0007] Another object of the present invention is to provide a fermentation broth of the above-mentioned *Pseudomonas putrefactiveis* P683, wherein the fermentation broth is a mixture of bacterial cells and substances secreted into the culture medium produced by the *Pseudomonas putrefactiveis* P683 strain during the cultivation process.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned *Pseudomonas putrefactiveis* P683 fermentation broth, wherein *Pseudomonas putrefactiveis* strain P683 is inoculated into NB medium and cultured with shaking at 28°C and 220 rpm until saturation.

[0009] Another object of the present invention is to provide a method for lowering the environmental pH value using *Pseudomonas putrefactiveis* P683 or its fermentation broth, wherein *Pseudomonas putrefactiveis* P683 or / and its fermentation broth are used to produce a variety of organic acids, thereby lowering the environmental pH value and enabling the strain to grow in a highly alkaline environment.

[0010] Another object of the present invention is to provide a method for improving soda saline-alkali soil using *Pseudomonas putida* P683 or its fermentation broth, wherein *Pseudomonas putida* P683 or / and the fermentation broth of *Pseudomonas putida* P683 are used to effectively reduce the pH value of soda saline-alkali soil and alleviate alkaline stress.

[0011] Another object of the present invention is to provide a method for improving the germination rate of corn in saline-alkali soil by using *Pseudomonas putida* P683 or its fermentation broth, wherein *Pseudomonas putida* P683 or / and the fermentation broth of *Pseudomonas putida* P683 are used to coat corn seeds, thereby significantly improving the germination rate of corn in saline-alkali soil.

[0012] Another object of the present invention is to provide a method for improving saline-alkali soil and increasing crop emergence rate in saline-alkali soil using *Pseudomonas putida* P683 or its fermentation broth, wherein the *Pseudomonas putida* P683 or / and the fermentation broth of *Pseudomonas putida* P683 are used in the improvement of saline-alkali soil and the increase of crop emergence rate in saline-alkali soil.

[0013] Compared with existing technologies, the beneficial effects of this solution are as follows: This invention provides a *Pseudomonas putida* P683 strain and its application in saline-alkali soil improvement, specifically addressing the problem of saline-alkali soil improvement. The *Pseudomonas putida* P683 strain can produce various organic acids, effectively lowering the environmental pH value, enabling it to grow in highly alkaline environments, which is of great significance for the agricultural utilization of saline-alkali land. First, the fermentation broth of *Pseudomonas putida* P683 can significantly reduce the pH value of soda saline-alkali soil from alkaline to near neutral, which is crucial for alleviating soil alkalinity stress, improving soil fertility, and enhancing crop growth conditions. This biological method of saline-alkali soil improvement has advantages such as low cost, high effectiveness, and environmental friendliness compared to traditional physical and chemical improvement methods. Furthermore, coating corn seeds with the fermentation broth can significantly improve the germination rate of seeds in saline-alkali soil, which directly promotes increased crop yield and agricultural economic benefits.

[0014] Secondly, the application of *Pseudomonas putrefactiveis* P683 not only enhances the agricultural value of saline-alkali soil but also offers safety and environmental friendliness. This strain is harmless to humans and animals and does not cause environmental pollution, meeting the requirements of modern green agriculture and environmental protection. Furthermore, due to its simple cultivation conditions and ease of preservation, this strain is suitable for industrial production, helping to reduce costs and improve efficiency, making the technical solution of this invention more readily adopted and applied. These characteristics give this invention broad application prospects and significant ecological and economic benefits in the field of saline-alkali soil improvement.

[0015] In summary, this invention provides a novel *Pseudomonas putida* P683 and its fermentation broth, which can not only effectively improve saline-alkali soil and increase crop emergence rate, but also has safety, environmental friendliness, and feasibility for industrial production, providing a new and effective solution for saline-alkali soil improvement. Attached Figure Description

[0016] Figure 1 This is a litmus plate test image of the acid-producing characteristics of Pseudomonas P683 in Example 2 of the present invention;

[0017] Figure 2 This is a growth curve and pH monitoring graph of Pseudomonas P683 during fermentation in Example 2 of the present invention;

[0018] Figure 3 This is the HPLC chromatogram of organic acid substances in the concentrated supernatant of Pseudomonas P683 fermentation in Example 3 of the present invention.

[0019] Figure 4 This is a graph showing the growth and pH dynamics of Pseudomonas P683 in a highly alkaline culture medium in Example 4 of this invention.

[0020] Figure 5This is a soil pH value detection diagram after treating saline-alkali soil with Pseudomonas P683 fermentation broth in Example 5 of the present invention.

[0021] Figure 6 This is a graph showing the corn germination rate after corn seeds were coated with Pseudomonas P683 fermentation broth in Example 6 of this invention. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. Furthermore, the experimental methods in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc., used in the following embodiments can be obtained commercially to replicate the experiments of this application unless otherwise specified.

[0024] The present invention will now be described in detail with reference to embodiments.

[0025] Example 1: A type of *Pseudomonas putrefactiveis* P683

[0026] A species of *Pseudomonas taetrolens*, P683, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27636, deposited on June 15, 2023, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), postal code 100101. *Pseudomonas taetrolens* P683 is an aerobic, Gram-negative bacterium that fluoresces on King's B medium.

[0027] The 16S rRNA gene sequence of Pseudomonas P683 strain is shown in Seq ID No.1.

[0028] In this embodiment, a fermentation broth of *Pseudomonas putrefactiveis* P683 is also provided: the fermentation broth is a mixture of bacterial cells and substances secreted into the culture medium produced by the *Pseudomonas putrefactiveis* P683 strain during the cultivation process;

[0029] The fermentation broth is prepared as follows:

[0030] Pseudomonas P683 strain was inoculated into NB medium and cultured at 28°C and 220 rpm with shaking until saturation, which is the fermentation broth of strain P683.

[0031] The Pseudomonas P683 or / and Pseudomonas P683 fermentation broth provided in this embodiment can be used to produce a variety of organic acids, reduce the pH value of the environment, and enable the strain to grow in a highly alkaline environment; effectively reduce the pH value of soda saline-alkali soil and alleviate alkaline stress; and coat corn seeds to significantly improve the germination rate of corn in saline-alkali soil.

[0032] The following is the method for preparing the culture medium used in the examples:

[0033] Nutrient Agar (NA) medium: 3g beef extract, 5g peptone, 2.5g glucose, 12g agar, pH 7.0, distilled water to a final volume of 1000mL.

[0034] Nutrient Broth (NB) medium: 3g beef extract, 5g peptone, 2.5g glucose, pH 7.0, diluted with distilled water to 1000mL.

[0035] King's B(KB) solid medium: peptone 3 20g, MgSO4·7H2O 0.4g, glycerol 10mL, K2HPO4 1.5g, agar 12g, distilled water to a final volume of 1000mL.

[0036] King's B(KB) liquid medium: peptone 3 20g, MgSO4·7H2O 0.4g, glycerol 10mL, K2HPO4 1.5g, distilled water to a final volume of 1000mL.

[0037] Example 2: Detection of Acid-Producing Characteristics of Pseudomonas putrefactiveis P683

[0038] The acid production of strain P683 was detected using litmus solution. Litmus solution, as an acid-base indicator, turns red under acidic conditions and blue under alkaline conditions. Plates were prepared by adding 0.1% litmus solution to NA solid medium. Strain P683 was cultured overnight in KB liquid medium with shaking until saturation. 10 μL of the bacterial culture was spotted onto the center of the plate, dried, and then incubated at 28°C. The color change around the colonies was observed. Simultaneously, strain P683 was streaked onto the plate, and the color change around the colonies was also observed. The results showed that a red halo appeared around the colonies of strain P683, as shown in the attached image. Figure 1 As shown, strain P683 produces and secretes acidic substances during cultivation, making the surrounding culture medium acidic, and litmus solution turns red under acidic conditions.

[0039] To further investigate the changes in acid production of strain P683 during its growth, strain P683 was inoculated into Erlenmeyer flasks containing 50 mL of NB medium, with an initial inoculation concentration of OD0.05. 600 =0.05, and incubated in a shaker at 28℃ and 220rpm. The absorbance (OD) of the fermentation broth at 600nm was measured every 4 hours. 600 The results showed that strain P683 entered the stationary phase after 12 hours of culture, and the pH value decreased from neutral to below 4 and remained stable, as shown in the attached figure. Figure 2 As shown, strain P683 does indeed produce acidic substances during fermentation, which lowers the pH value of the environment.

[0040] Example 3: Identification of organic acids produced by *Pseudomonas putrefactiveis* P683

[0041] To identify the acidic substances produced and secreted by strain P683 during fermentation, the fermentation supernatant of strain P683 was lyophilized and concentrated before being analyzed by high-performance liquid chromatography (HPLC). Specifically, strain P683 was inoculated into 50 mL of NB medium with an initial inoculum size of OD0.05. 600 =0.01, and cultured in a shaker at 28℃ and 220rpm for 24h. The fermentation supernatant was collected by centrifugation, filtered through a 0.22μm filter membrane, and 24mL of the supernatant was subjected to vacuum freeze-drying. The freeze-dried powder was reconstituted in 1mL of sterile water and filtered through a 0.22μm filter membrane to obtain the concentrated fermentation supernatant (24-fold concentrated). The content of 11 organic acids in the concentrated fermentation supernatant was quantitatively detected using a high-performance liquid chromatography (HPLC) system (Thermo Fisher U3000). The results showed that strain P683 produced various organic acids during fermentation, as shown in the attached figure. Figure 3 As shown in Table 1, strain P683 produced the most gluconic acid, with a concentration of 1.16 mg / mL before concentration. In addition, strain P683 also produced a lot of lactic acid and acetic acid, with concentrations of 0.60 mg / mL and 0.32 mg / mL before concentration, respectively.

[0042] Table 1. Quantitative results of organic acid detection by HPLC

[0043]

[0044] Note: "ND" indicates not detected.

[0045] Example 4: Growth and pH monitoring of *Pseudomonas putrefactiveis* P683 in highly alkaline medium.

[0046] To test whether strain P683 can grow in an alkaline environment and effectively lower the alkaline pH, strain P683 was inoculated into 50 mL of NB medium (pH = 10), with an initial inoculum size of OD. 600 =0.05, placed in a shaker at 28℃ and 220rpm for incubation, and OD was measured every 12 hours. 600 The results showed that strain P683, in alkaline NB medium at pH=10, was in a lag phase with virtually no growth for the first 12 hours; from 12 to 24 hours, it entered the logarithmic growth phase and began to grow rapidly; it reached the stationary phase at 48 hours. The pH value rapidly decreased to below 4 from 12 to 36 hours and stabilized after 36 hours, as shown in the attached figure. Figure 4 As shown above, the results indicate that strain P683 can grow in a highly alkaline environment and lower the alkaline pH value by producing acid, revealing its potential for acid production and alkali reduction.

[0047] Example 5: Effect of *Pseudomonas putrefactiveis* P683 on pH value of soda saline-alkali soil

[0048] To investigate the application of the acid-producing and alkali-reducing characteristics of strain P683 in the improvement of saline-alkali soil, the fermentation broth (OD) of strain P683 was tested. 600 =0.231 (pH=3.92) and soda saline-alkali soil from Northeast China (pH=10.22) were mixed at a 1:1 ratio and allowed to stand at room temperature for 1 day. The pH value of the treated soil was then measured according to the National Environmental Protection Standard of the People's Republic of China (HJ 962-2018). The results showed that on day 0 of treatment, the soil pH value of the P683 fermentation broth treatment group decreased to 7.96, significantly lower than the pH value of the H2O treatment group (pH=10.14). After standing at room temperature for 1 day, the soil pH value of the P683 fermentation broth treatment group further decreased to 6.75, becoming neutral, as shown in the attached figure. Figure 5 As shown, the fermentation broth of strain P683 has the potential to improve saline-alkali soil, especially in reducing the pH value of saline-alkali soil.

[0049] Example 6: Effect of Pseudomonas putida P683 coating treatment on corn seeds and its germination rate in soda saline-alkali soil.

[0050] This embodiment uses maize as a model crop to investigate the effect of coating maize seeds with the fermentation broth of strain P683 on their germination rate in saline-alkali soil. A portion of colonies of strain P683 were inoculated into 5 mL of NB medium and cultured at 28℃ and 220 rpm for 12 h to obtain the fermentation broth (OD). 600=0.877 (pH=3.49). Corn seeds were coated using the fermentation broth of strain P683, with sterile water as a blank control. The specific coating method was as follows: A film-forming agent with a final concentration of 8% was added to the P683 bacterial solution and sterile water and mixed thoroughly to obtain the coating agent; an appropriate number of whole, plump corn seeds were selected and weighed; the prepared coating agent was mixed with the seeds at a mass ratio of 1:30, stirred thoroughly, and air-dried to obtain the coated corn seeds. The coated corn seeds were planted in pots filled with soda-saline soil (pH=10.22), 16 seeds / pot, with 3 pots planted per treatment as replicates. They were placed in a greenhouse for cultivation, and the germination rate was observed and recorded. The germination rate of corn under both treatments was statistically analyzed on day 18 after planting. The results showed that compared with the H2O coating treatment, the germination rate of corn seeds coated with the fermentation broth of strain P683 was significantly improved (see attached figure). Figure 6 As shown.

[0051] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for improving soda-saline-alkali soil using *Pseudomonas putida* P683 or its fermentation broth, characterized in that: The fermentation broth of Pseudomonas P683 or / and Pseudomonas P683 is used to effectively reduce the pH value of soda saline-alkali soil and alleviate alkaline stress.

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