Low-temperature-resistant burkholderia G38, burkholderia powder, preparation method of burkholderia powder and application of burkholderia powder

Abstract

The invention discloses low-temperature-resistant burkholderia G38, burkholderia G38 bacterial powder and a preparation method and application of the burkholderia G38 bacterial powder, and belongs to the technical field of microorganisms. The preservation number of the burkholderia sp. G38 is CCTCC (China Center For Type Culture Collection) NO: M 2026181. The invention also provides bacterial powder which contains the burkholderia G38, and the preparation method comprises the following steps: culturing the burkholderia G38 to obtain a G38 bacterial solution, and centrifuging the G38 bacterial solution to obtain bacterial sludge; and washing the bacterial sludge, adding skim milk powder, uniformly mixing, freeze-drying, and grinding into powder to obtain the bacterial powder. Experiments prove that the G38 bacterial powder can still keep efficient phosphate-solubilizing activity under low-temperature and low-rotating-speed culture conditions, and corresponding fermentation and preparation processes can be developed on the basis of the characteristic, so that the dependence of a traditional method on high-energy-consumption stirring is avoided, and a phosphate-solubilizing preparation which is more suitable for an actual soil environment and still effective under a low-temperature condition is produced.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a low-temperature resistant Burkholderia G38, its powder, preparation method, and application. Background Technology

[0002] Phosphorus is an essential nutrient element for crop growth, widely involved in key physiological processes such as photosynthesis, carbohydrate metabolism, energy transfer (ATP / ADP system), and cell signal transduction. However, phosphorus readily binds with cations such as calcium, iron, and aluminum in soil, forming insoluble phosphates that are difficult for plants to absorb directly. This strong binding effect significantly reduces its bioavailability. Although the total phosphorus content in global soils is typically between 400-1000 mg / kg, the available phosphorus that can be utilized by plants (mainly in the form of HPO4) is limited. 2- and H2PO4 - Phosphorus deficiency (in its most basic form) is present in extremely low concentrations, severely restricting the full potential of crop production. Crop phosphorus deficiency triggers a series of growth and developmental obstacles: sluggish root development, limited leaf expansion and premature senescence and shedding, and hindered accumulation of assimilates in stems and leaves, ultimately leading to decreased seed setting rate and significantly reduced yield, posing a direct threat to sustainable agricultural productivity. To address this challenge, agricultural production in the past few decades has relied heavily on and excessively applied chemical phosphate fertilizers. While this ensured food production in the short term, it also caused numerous ecological and environmental problems, including soil nutrient imbalance, heavy metal and byproduct pollution, soil compaction and acidification, eutrophication of water bodies, and decline in biodiversity, severely testing the path to sustainable agricultural development. Therefore, the development and promotion of environmentally friendly and efficient phosphorus resource utilization technologies are urgently needed. Among these, utilizing functional microorganisms capable of dissolving insoluble phosphates (phosphate-solubilizing microorganisms) to produce efficient and stable microbial agents or biofertilizers is considered one of the most promising solutions for fundamentally improving soil phosphorus bioavailability, reducing dependence on chemical phosphate fertilizers, and promoting the green transformation of agriculture.

[0003] Phosphate-solubilizing bacteria (PSBs) are a representative class of plant growth-promoting bacteria whose core function is to participate in and promote the soil-plant phosphorus cycle. In this cycle, phosphorus is easily adsorbed by soil particles or combines with metal ions to form insoluble phosphates such as iron-phosphorus (Fe-P), aluminum-phosphorus (Al-P), and calcium-phosphorus (Ca-P), significantly reducing its bioavailability and making it one of the main limiting nutrients for plant growth. Insufficient soil phosphorus supply significantly inhibits normal crop growth and development, specifically by hindering root morphogenesis and expansion, reducing leaf photosynthetic efficiency, weakening the plant's resistance to biotic stresses such as pests and diseases, and abiotic stresses such as drought and salinity, ultimately leading to decreased crop yield. The core value of phosphate-solubilizing bacteria lies in their ability to convert insoluble fixed phosphorus in the soil and phosphorus from plant and animal residues into a form available to plants. Simultaneously, they secrete metabolites such as auxins, organic acids, and siderophores, directly promoting crop growth and development, thus providing crucial support for food security and stable supply. These microorganisms have the dual ability to dissolve insoluble inorganic phosphorus and degrade organic phosphorus. When they are made into bio-fertilizers and applied to the soil, they can increase crop yields by promoting plant growth.

[0004] Currently, commercial phosphate-solubilizing bacterial agents generally suffer from three main shortcomings. First, they have poor environmental adaptability, with activity significantly decreasing at low temperatures (<15℃), limiting their application in early spring, late autumn, and cold regions. Second, their quality evaluation systems are incomplete, heavily relying on the "viable cell count" indicator. However, "high biomass" does not equate to "high functional activity," making it impossible to accurately predict and guarantee the actual phosphate-solubilizing effect of the product. Third, their formulations and production processes have defects: liquid formulations are inconvenient to store and transport and have a short shelf life; more importantly, existing preparation technologies typically rely on high-speed, high-energy-consuming fermentation processes to activate the bacterial strains, which not only increases production costs but also makes it difficult for the resulting agents to adapt to the low dissolved oxygen and minimal disturbance environments of soil. Summary of the Invention

[0005] The purpose of this invention is to provide a low-temperature resistant Burkholderia G38, its powder, its preparation method, and its application, in order to solve the problems existing in the prior art. The G38 powder can still maintain high phosphorus-solubilizing activity under low temperature and low speed cultivation conditions, and can be developed into corresponding phosphorus-solubilizing preparations.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a low-temperature resistant Burkholderia (… Burkholderia pyrrociniaBurkholderia G38 was deposited on January 20, 2026 at the China Center for Type Culture Collection (CCTCCNO: M 2026181) at Wuhan University, Wuhan, China.

[0007] The present invention also provides the application of the aforementioned Burkholderia G38 in the preparation of bacterial powder.

[0008] The present invention also provides a bacterial powder containing Burkholderia G38.

[0009] The present invention also provides a method for preparing the bacterial powder, comprising the following steps: Burkholderia G38 was cultured to obtain G38 bacterial solution, and the G38 bacterial solution was centrifuged to obtain bacterial sludge; The bacterial sludge is washed and then mixed with skim milk powder. After being freeze-dried and ground into powder, the bacterial powder is obtained.

[0010] Optionally, the culture conditions are 120 rpm and 30℃ for 42 h; the centrifugation conditions are 3500 rpm for 10 min. The volume ratio of the skim milk powder to the bacterial sludge is 1:1.

[0011] Optionally, the concentration of the skim milk powder is 10%-20% by weight / volume.

[0012] Preferably, the concentration of the skim milk powder is 10% by weight / volume.

[0013] The present invention also provides the application of Burkholderia G38 or the bacterial powder thereon in phosphorus solubilization under low temperature and / or low speed conditions, wherein the low temperature includes 10°C-20°C and the low speed includes 0 rpm-50 rpm.

[0014] This invention also provides the application of Burkholderia G38 or the bacterial powder described above in the preparation of phosphate-solubilizing agents, wherein the phosphate-solubilizing agents are used for phosphate solubilization under low temperature and / or low rotation speed conditions; The low temperature includes 10℃-20℃, and the low speed includes 0 rpm-50 rpm.

[0015] The present invention also provides a method for phosphorus dissolution under low temperature and / or low rotation speed conditions, comprising the step of adding the Burkholderia G38 or the bacterial powder.

[0016] The present invention discloses the following technical effects: To address the numerous limitations of existing technologies, this invention aims to provide a comprehensive solution. The primary objective is to provide a specific strain of Burkholderia (…). Burkholderia pyrrociniaThis study develops strain G38 and its corresponding solid-state bacterial powder preparation method to overcome the shortcomings of liquid formulations and significantly improve the storage stability and portability of the bacterial agent. The key technological breakthrough lies in utilizing the characteristic of strain G38 to maintain highly efficient phosphorus-solubilizing activity under low-temperature and low-speed cultivation conditions. This allows for the development of corresponding fermentation and formulation processes, thereby eliminating the reliance on energy-intensive stirring in traditional methods and producing phosphorus-solubilizing formulations that are more adapted to actual soil environments and remain effective even at low temperatures. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram showing the colony morphology of strain G38 on a solid plate. Figure 2 Phylogenetic tree of strain G38 Figure 3 Flowchart of the G38 bacterial powder preparation process; Figure 4 This is a picture of the finished product of G38 mycelium powder; Figure 5 A bar chart comparing the release of soluble phosphorus from G38 bacterial powder and G38 bacterial liquid at different temperatures; Figure 6 The results show the determination of soluble phosphorus content and pH of G38 bacterial powder at different rotation speeds; the bar chart represents soluble phosphorus content, and the line graph represents pH. Figure 7 The results show the soluble phosphorus release and pH of G38 bacterial powder under the same rotation speed conditions, using different insoluble phosphorus sources as culture media; the bar chart represents the soluble phosphorus content, and the line graph represents the pH. Figure 8 Line graph showing the change in soluble phosphorus release from G38 bacterial powder over 7 days at 50 rpm. Figure 9 The results show the soluble phosphorus release and pH of G38 bacterial powder prepared with different concentrations of freeze-drying protectant at 50 rpm; the bar chart represents the soluble phosphorus content, and the line graph represents the pH. Figure 10 The viable cell survival rate of G38 bacterial powder prepared with different concentrations of freeze-drying protectant. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] In the following examples, the culture medium (liquid medium) used for bacterial culture and isolation comprises the following components: glucose 10 g / L, tricalcium phosphate 5 g / L, sodium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ammonium sulfate 0.5 g / L, potassium chloride 0.3 g / L, manganese sulfate 0.03 g / L, and ferrous sulfate 0.03 g / L. The solid medium is the liquid medium with 20% agar powder added by mass.

[0025] Example 1: Isolation, Identification and Preservation of Strains 1. Separation Soil samples were collected from soils in Yueshan Village, Liangzihu Town, and Baoerdong Village, Xinhua Town, Shennongjia County, where Gaoshi No. 15 bio-organic fertilizer was applied. A sterilized conical flask containing glass beads was used. 90 mL of sterile water was added, along with 10 g of the collected soil sample. The mixture was shaken at 25°C and 180 rpm for 30 minutes to ensure thorough mixing. 1 mL of the supernatant was gradually diluted into test tubes containing 9 mL of sterile water. The experiment was repeated three times to prepare 10 samples. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Soil suspensions were prepared. 100 μL of soil suspensions with different dilution gradients were evenly spread on the surface of inorganic phosphorus medium and incubated at 30°C for 48–72 hours. Typical colonies producing clear phosphate-solubilizing zones were screened, and the cultures were purified by streak plating on the same medium using a four-zone method. After five rounds of streak plating, pure strain G38 was obtained.

[0026] 2. Colony morphology characteristics Colonies of strain G38 are white, opaque, round, with regular edges, smooth and moist surface, and no halo. Figure 1 ).

[0027] 3. Molecular identification of strains Using bacterial genomic DNA as a template, 16S rRNA was amplified using 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) primers. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 25 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25 cycles; and a final extension at 72℃ for 5 min. After amplification, the PCR products were subjected to 1% agarose gel electrophoresis to check their integrity. Finally, the PCR products were sent to the sequencing department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The effective length of the 16S rDNA gene of the amplified strain was approximately 1430 bp, and the 16S rDNA sequence is shown in SEQ ID NO.3. The 16S rDNA sequence was submitted to GenBank, and homology sequence alignment was performed using Blast software. A phylogenetic tree was constructed based on the homology of the 16S rDNA sequence. Figure 2 ), and strain G38 was identified as Burkholderia pyrrocinia .

[0028] SEQ ID NO.3:

[0029] Based on the phylogenetic analysis of the 16S rDNA gene and physiological and biochemical characteristics, strain G38 was identified as Burkholderia burkermansia (…). Burkholderia pyrrocinia The strain was named G38. It was deposited on January 20, 2026, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026181, located at Wuhan University, China.

[0030] Example 2: Preparation of G38 bacterial powder See flowchart Figure 3 A single colony of purified G38 was picked from the solid culture medium and inoculated into LB liquid culture medium. The culture was placed on a shaker and incubated at 120 rpm and 30°C for 42 h. The bacterial culture was then collected to obtain the G38 bacterial culture.

[0031] Centrifuge the G38 bacterial culture at 3500 rpm for 10 min, remove the supernatant, and retain the bacterial sludge. Wash the sludge twice with physiological saline to remove residual culture medium and metabolic products, avoiding impurities from affecting subsequent drying and storage. Add an equal volume of skim milk powder (as a freeze-drying protectant, which helps improve bacterial survival rate and ensures uniform mixing with the sludge to protect cell structure from damage during drying) to the obtained sludge and mix thoroughly. Aliquot the sludge into 50 mL centrifuge tubes, pre-freeze to -20°C, and then vacuum dry for 48 hours. Grind the freeze-dried sludge into powder to obtain G38 bacterial powder. Figure 4 Store in a dry, sealed, and dark place.

[0032] Example 3: G38 Fertilizer Powder Test (1) Effect of temperature on phosphorus solubility in G38 bacterial powder G38 bacterial powder was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of culture medium at an inoculation ratio of 0.1 g. At the same time, the control group was inoculated into the culture medium with G38 bacterial suspension at an inoculation ratio of 1% (V' / V). The flasks were cultured at different temperatures (10℃, 15℃, 20℃, 25℃) and a rotation speed of 50 rpm. After seven days of culture, samples were taken to determine the amount of phosphorus dissolved.

[0033] Figure 5The study showed the soluble phosphorus release of G38 bacterial powder and liquid at different culture temperatures. Results indicated that even at a low temperature of 10°C, the phosphorus solubility of the powder remained at a high level (approximately 106 mg / L), reaching over 50% of the phosphorus solubility at the optimal temperature of 25°C (approximately 193 mg / L), demonstrating that G38 bacterial powder can effectively activate its phosphorus-soothing function even at low temperatures. Regardless of the temperature, the phosphorus solubility of the bacterial powder prepared by this invention was several times that of the liquid formulation with the same bacterial volume (approximately 3.5 times at room temperature and up to 5.5 times at low temperatures). The preparation method of this invention ensures that the final product (bacterial powder) exhibits an overwhelming performance advantage over traditional liquid bacterial agents at any test temperature.

[0034] (2) Effect of rotation speed on phosphorus solubility of G38 bacterial powder G38 bacterial powder was inoculated at a ratio of 0.1g into 250mL Erlenmeyer flasks containing 100mL of culture medium and cultured at different speeds (0, 50, 90, 130, 170, and 210 rpm). After seven days of culture, samples were taken to determine the amount of phosphorus dissolved and the pH.

[0035] The results are as follows Figure 6 As shown, the phosphorus solubility in the 0 rpm group (approximately 167 mg / L) was 18 times that of the 210 rpm group (approximately 9 mg / L), and the phosphorus solubility in the 50 rpm group (approximately 190 mg / L) was 21 times that of the 210 rpm group (approximately 9 mg / L). This demonstrates that the G38 bacterial powder obtained by this invention can effectively exert its phosphorus-solidating effect under low or 0 rpm conditions, while excessively high rpm will severely inhibit its functional expression. This overcomes the dependence of traditional bacterial agents on high-energy-consuming stirring and improves environmental adaptability.

[0036] (3) Phosphorus solubilization effect of G38 bacterial powder on different poorly soluble phosphorus sources G38 bacterial powder was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of culture medium at a ratio of 0.1 g. The flasks were then cultured with different phosphorus sources (calcium phosphate, iron phosphate, or aluminum phosphate) at a shaking speed of 50 rpm. Samples were taken after seven days of culture to determine the dissolved phosphorus and pH.

[0037] The results are as follows Figure 7 As shown, the solubility of this bacterial powder varies greatly for different phosphorus sources: its solubility for calcium phosphate (approximately 193 mg / L) is much stronger than that for aluminum phosphate (approximately 20 mg / L) and iron phosphate (approximately 14.9 mg / L), indicating that it is more suitable for soil environments where calcium-bound phosphorus is the main component.

[0038] (4) Changes in phosphorus solubility of G38 bacterial powder at 50 rpm G38 bacterial powder was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of culture medium at an inoculation ratio of 0.1 g. The flask was incubated at 50 rpm. Samples were taken at 0, 24, 48, 72, 96, 120, 144, and 168 hours after incubation. The soluble phosphorus content in the supernatant was measured after centrifugation.

[0039] like Figure 8 As shown, the phosphate-solubilizing effect of the G38 bacterial powder begins immediately after inoculation, and the amount of phosphate dissolved increases steadily over time. At 72 hours, the phosphate dissolved reaches approximately 135 mg / L, accounting for more than 65% of the total release, demonstrating rapid initiation. By the end of the 168-hour culture period, the phosphate dissolved tends to stabilize, ultimately reaching approximately 195 mg / L. This dynamic curve confirms the potential of the bacterial powder of this invention to have rapid efficacy and sustained phosphate dissolution in practical applications.

[0040] (5) Effect of different concentrations of freeze-drying protectant on phosphorus solubility in G38 bacterial powder To optimize the freeze-drying protectant formulation, 5%, 10%, 15%, and 20% (w / v) skim milk powder were added as protectants during the preparation of the bacterial sludge, and bacterial powder was prepared using the same process. Subsequently, G38 bacterial powder was inoculated into 250mL Erlenmeyer flasks containing 100mL of culture medium at an inoculation ratio of 0.1g, and the phosphorus solubility and pH were measured.

[0041] The results showed that ( Figure 9 While a 20% concentration yields the highest phosphorus solubility (220 mg / L), a 10% concentration (190 mg / L) achieves a similar level of activity and is far superior to a 5% concentration (107 mg / L). Given that a 10% concentration significantly reduces raw material costs while maintaining excellent phosphorus solubility, it has been determined as the optimal concentration for adding the protective agent in production.

[0042] (6) Effect of different concentrations of freeze-drying protectant on the survival rate of live bacteria in G38 bacterial powder In preparing the bacterial sludge, 5%, 10%, 15%, and 20% (w / v) skim milk powder were added as a preservative, respectively, and the bacterial powder was prepared using the same process. The viable count of the obtained samples before freeze-drying was calculated using the plate count method. After freeze-drying, the samples were reconstituted to the original volume before freeze-drying to determine the viable count and calculate the survival rate.

[0043] Experimental results show that ( Figure 10Skim milk powder has a significant protective effect on bacterial cells during freeze-drying, with the survival rate increasing with increasing concentration. Although the survival rate is highest at a concentration of 20% (approximately 83%), considering the protective effect, production cost, and the process applicability of subsequent formulations, a 10% skim milk powder concentration was determined to be the optimal choice for this invention. At this concentration, the bacterial cell survival rate reaches a relatively high level (approximately 71%), effectively ensuring a sufficient functional bacterial load in the bacterial powder. More importantly, combined with... Figure 8 The results show that the phosphorus-solubilizing capacity of the bacterial powder prepared with this concentration of preservative is not significantly different from that prepared with a higher concentration, fully meeting the application requirements. At the same time, this choice avoids the activity loss caused by insufficient protection with low concentrations (5%), as well as the uneconomical raw material costs and potential clumping issues during rehydration caused by excessively high concentrations. Therefore, using 10% skim milk powder as the freeze-drying preservative achieves the optimal balance between bacterial powder activity, preparation cost, and product processability.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cold-resistant Burkholderia ( Burkholderia pyrrocinia G38, characterized in that, The Burkholderia G38 strain was deposited on January 20, 2026, at the China Center for Type Culture Collection (CCTCCNO: M 2026181), located at Wuhan University, Wuhan, China.

2. The application of Burkholderia G38 as described in claim 1 in the preparation of mycelial powder.

3. A bacterial powder, characterized in that, The bacterial powder contains Burkholderia G38 as described in claim 1.

4. The method for preparing the bacterial powder as described in claim 3, characterized in that, Includes the following steps: Burkholderia G38 was cultured to obtain G38 bacterial solution, and the G38 bacterial solution was centrifuged to obtain bacterial sludge; The bacterial sludge is washed and then mixed with skim milk powder. After being freeze-dried and ground into powder, the bacterial powder is obtained.

5. The preparation method according to claim 4, characterized in that, The culture conditions were 120 rpm and 30℃ for 42 h; the centrifugation conditions were 3500 rpm for 10 min. The volume ratio of the skim milk powder to the bacterial sludge is 1:

1.

6. The preparation method according to claim 5, characterized in that, The concentration of the skim milk powder is 10%-20% by weight / volume.

7. The preparation method according to claim 6, characterized in that, The concentration of the skim milk powder is 10% by weight / volume.

8. The application of Burkholderia G38 as described in claim 1 or the bacterial powder as described in claim 3 in phosphorus solubilization under low temperature and / or low rotation speed conditions, characterized in that, The low temperature includes 10℃-20℃, and the low speed includes 0 rpm-50 rpm.

9. The application of Burkholderia G38 as described in claim 1 or the bacterial powder as described in claim 3 in the preparation of phosphate-solubilizing agents, characterized in that, The phosphorus-solubilizing agent is used for phosphorus dissolution under low temperature and / or low rotation speed conditions; The low temperature includes 10℃-20℃, and the low speed includes 0 rpm-50 rpm.

10. A method for dissolving phosphorus under low temperature and / or low rotation speed conditions, characterized in that, The step includes adding Burkholderia G38 as described in claim 1 or the bacterial powder as described in claim 3.

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