Use of lactococcus lactis psm16 in preparing bio-fertilizer

Abstract

The application discloses application of lactococcus lactis PSM16 in preparation of biofertilizer, and belongs to the technical field of microorganisms. The biofertilizer is any one or a mixed functional biofertilizer product of multiple of soil phosphorus solubilizing fertilizer, fertilizer for increasing effective phosphorus content in soil, seed germination promoting fertilizer, and fertilizer for improving soil pH and conductivity. The application finds that the lactococcus lactis PSM16 has great potential in soil improvement, so the lactococcus lactis PSM16 can be used as biofertilizer to convert phosphorus which is difficult for plants to absorb and utilize into a form that can be absorbed and utilized, improve crop phosphorus nutrition, improve soil environment, reduce use of chemical fertilizer, reduce environmental pollution, and promote sustainable development of agriculture. The application also discloses application of the lactococcus lactis PSM16 in production of acid phosphatase, and the lactococcus lactis PSM16 has very strong acid phosphatase activity and high production efficiency.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to the application of Lactococcus lactis PSM16 in the preparation of bio-fertilizer. Background Technology

[0002] Phosphorus is a crucial component of many enzymes in plants, playing a vital role in the transformation, transport, and storage of substances within the plant and thus in plant growth and development. Firstly, phosphorus promotes cell division and proliferation, significantly impacting plant growth and development. Sufficient phosphorus also promotes the synthesis and metabolism of substances within plants, leading to healthier plants, increased yields, and improved product quality. Secondly, phosphorus is an important component of soil organic matter, and its content and distribution in the soil affect soil fertility and crop growth. Soil phosphorus content is influenced by various factors, including soil moisture, temperature, and water erosion. Increased soil moisture promotes phosphorus accumulation, while rising temperatures and water erosion can reduce soil phosphorus content. Finally, from an environmental protection perspective, phosphorus also plays a positive role. Excessive phosphorus entering water bodies can lead to eutrophication, resulting in algal blooms and water quality deterioration. Available phosphorus in the soil can reduce phosphorus loss, thus protecting the environment. Available phosphorus can combine with elements such as calcium, iron, and aluminum in the soil to form insoluble salts, inhibiting phosphorus loss. Meanwhile, some soil bacteria can utilize available phosphorus to degrade organic matter, preventing excessive phosphorus from entering water bodies and causing environmental pollution. In conclusion, phosphorus in soil plays a vital role in promoting plant growth, improving product quality, enhancing soil fertility, and protecting the environment.

[0003] The phosphorus content in soil varies depending on factors such as soil type, geographical location, climate conditions, and land use. Generally, the total phosphorus content in Chinese soils ranges from 0.02% to 0.11%. The total phosphorus content in northern soils is typically higher than that in southern soils, with a gradual increasing trend from south to north. For example, the total phosphorus content in black soil in Northeast China is generally 0.06% to 0.15%, while the total phosphorus content in red soil and lateritic red soil in the south is lower, generally 0.01% to 0.03%. Therefore, finding a microorganism that can increase the available phosphorus content in soil is of great significance for areas in the south with relatively low phosphorus content. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an application of Lactococcus lactis PSM16 in the preparation of bio-fertilizer.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] The application of Lactococcus lactis PSM16 in the preparation of bio-fertilizer, wherein Lactococcus lactis PSM16 (Lactococcus sp.) was deposited at the China General Microbiological Culture Collection Center on July 21, 2021, with the accession number CGMCC NO.22933.

[0007] In the above applications, the bio-fertilizer is any one or more of the following functional bio-fertilizer products: soil phosphorus-solubilizing fertilizer, fertilizer that increases the available phosphorus content in the soil, fertilizer that promotes seed germination, and fertilizer that improves soil pH and electrical conductivity.

[0008] Furthermore, the method for preparing bio-fertilizer includes the following steps: inoculating and culturing the Lactococcus lactis PSM16, centrifuging to remove the supernatant, and obtaining bacterial powder or bacterial resuspension, thereby obtaining the bio-fertilizer.

[0009] Furthermore, the culture medium used for the culture is MRS medium.

[0010] Furthermore, the concentration of bacterial cells in the bacterial suspension is 1×10⁻⁶. 4 ~1×10 8 CFU / mL.

[0011] Based on a general inventive concept, the present invention also provides the application of Lactococcus lactis PSM16 in the production of acid phosphatase.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. This invention discovers the great potential of Lactococcus lactis PSM16 in soil improvement. Therefore, it can be used as a bio-fertilizer to convert phosphorus that is difficult for plants to absorb and utilize into an absorbable form, improve crop phosphorus nutrition, improve the soil environment, reduce the use of chemical fertilizers, reduce environmental pollution, and promote the sustainable development of agriculture.

[0014] 2. This invention discovers that Lactococcus lactis PSM16 can solubilize phosphorus, increase the proportion of available phosphorus in the soil, and also promote seed germination.

[0015] 3. This invention has found that Lactococcus lactis PSM16 can improve soil pH and electrical conductivity, which plays an important role in improving soil fertility.

[0016] 4. This invention discovers that Lactococcus lactis PSM16 has very strong acid phosphatase production activity and can be used to produce acid phosphatase with high production efficiency. 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 description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are 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 It is the enzyme activity of Lactococcus lactis PSM16.

[0019] Figure 2 The effects of PSM16 on soil phosphorus content, including (a) available phosphorus content in PSM16-containing soils without and with added phosphate fertilizer, (b) total phosphorus content in PSM16-containing soils without and with added phosphate fertilizer, and (c) the ratio of available phosphorus to total phosphorus in PSM16-containing soils without and with added phosphate fertilizer. Data represent mean ± SD, N = 3.

[0020] Figure 3 The effect of PSM16 on the germination rate of Arabidopsis thaliana was investigated. The results are presented as mean ± SD, N = 3. *P < 0.05, **P < 0.01, ***P < 0.001, determined by t-test.

[0021] Figure 4 The study investigated the effect of recombinant bacteria on the available phosphorus content in soil. (a) showed the S-ACP of each strain after E. coli inoculation; (b) showed the available phosphorus content in soil after adding strains of E. coli inoculation; (c) showed the S-ACP of each strain after Bacillus inoculation; and (d) showed the available phosphorus content in soil after adding strains of Bacillus inoculation. Results were expressed as mean ± SD, N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, and determined by t-test.

[0022] Figure 5 The effects of adding KT2440 and PSM16 on Arabidopsis thaliana germination were investigated. (a) Germination rate of Arabidopsis thaliana after adding KT2440 and PSM16, with results as mean ± SD, N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, determined by t-test; (b) Arabidopsis thaliana germinating after adding KT2440 and PSM16.

[0023] Figure 6 The effect of adding recombinant strains on Arabidopsis thaliana germination is shown in (a)-(d), where (a)-(d) represent the germination rates of Arabidopsis thaliana after adding recombinant bacterial groups 834, 917, 925, and 974, respectively. The results are expressed as mean ± SD, N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, and were determined by t-test.

[0024] Figure 7 It refers to Arabidopsis thaliana germination after the addition of recombinant strains.

[0025] Figure 8 The effects of PSM16 on conductivity and pH were investigated, with (a) the effect of PSM16 on conductivity and (b) the effect of PSM16 on pH. The results are average ± SD, N = 3, *P < 0.05, **P < 0.01, ***P < 0.001, and were determined by t-test. Detailed Implementation

[0026] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0029] The *Lactococcus* sp. psm16 described in the following examples, classified as *Lactococcus lactis* subsp. *lactis*, was deposited on July 21, 2021, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 22933. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, 100101, China. For the source and isolation method of this strain, please refer to Chinese Patent Document CN113652372A.

[0030] Example:

[0031] 1. Bacterial culture

[0032] Using a disposable inoculation loop, gently dip a small amount of PSM16 culture stored at -80°C into an MRS solid plate (prepared by adding 20.8g MRS medium powder and 6g agar to 400mL of single-distilled water). Then, place the plate in a 37°C incubator and incubate it upside down overnight.

[0033] 2. Determination of enzyme activity of API-ZYM strain

[0034] The API-ZYM kit was used. First, single colonies were picked from the plates in step 1 and cultured in MRS liquid medium at 37°C for 18 h using a thermostat. Next, the samples were centrifuged at 9000 rpm for 10 min to remove the supernatant. Then, the bacterial cells were resuspended in 2 mL of sterile physiological saline, and the concentration was adjusted to approximately 1 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL. Then, place the reagent strip in a dish, add 65 μL of bacterial suspension, cover, and incubate at 37°C. After 4 hours, add one drop of ZYMB solution to each dish. After 5 minutes, observe and record the color change to assess the enzyme production capacity of strain PSM16.

[0035] Experimental results show that PSM16 exhibits strong activity against acid phosphatase. Figure 1 This provides strong evidence for further investigation into the effects of Lactococcus lactis PSM16 on phosphorus in soil.

[0036] 3. Determination of available phosphorus and total phosphorus content in soil treated with PSM16

[0037] A single colony was precisely selected from the plate from step 1 and inoculated into 2 mL of MRS liquid medium. Then, it was incubated at 37°C and 900 rpm for 12 hours. Control group 1 consisted of 1.9 mL of MRSbroth medium added to 15 g of vermiculite, followed by 20 mL of single-distilled water. Control group 2 consisted of control group 1 plus 2.2 mg of phosphate fertilizer. Experimental group 1 consisted of 1.9 mL of bacterial cells added to 15 g of vermiculite, followed by 20 mL of single-distilled water. Experimental group 2 consisted of experimental group 1 plus 2.2 mg of phosphate fertilizer. After mixing, the samples were incubated at 37°C for 24 hours. Subsequently, the samples were evenly distributed on a drying tray to form a 2-3 cm thin layer, and were crushed and turned as needed. After crushing with a wooden stick to remove impurities, the mixture was pulverized and thoroughly mixed. Subsequently, the soil was sieved through a 1mm soil sieve, mixed again, and the resulting soil treated with PSM16 was placed in a ground glass bottle for later use.

[0038] There is a direct and close correlation between acid phosphatase activity and available phosphorus content. To accurately determine the available phosphorus content in soil, this invention employs a sodium bicarbonate extraction-molybdenum antimony spectrophotometric method to detect the PSM16-treated soil. Figure 2 As shown in figure a, the content of available phosphorus in the PSM16 treatment group was significantly increased compared with the control group (CON).

[0039] The present invention further utilizes the alkali fusion-molybdenum antimony anti-spectrophotometric method to detect the soil treated with PSM16. Figure 2 b shows the total phosphorus content in the soil. The total phosphorus content in the PSM16 treatment group decreased slightly but was not significantly different. Figure 2 c shows the ratio of available phosphorus to total phosphorus, which is an indicator of phosphorus use efficiency in the soil. The increase in this ratio in the PSM16 treatment group indicates that when phosphate fertilizer is added, a larger proportion of total phosphorus is available in a plant-available form, suggesting that PSM16 can improve the bioavailability of phosphorus in the soil.

[0040] It is evident that PSM16 treatment of soil, as an effective method, not only significantly increases the content of available phosphorus but also ensures that phosphorus exists in large quantities in a form easily absorbed by plants. These data provide strong support for optimizing fertilizer use in agricultural practices, ensuring crops receive sufficient phosphorus for optimal growth, while helping to reduce the need for excessive fertilization, thereby avoiding potential negative environmental impacts.

[0041] 4. Arabidopsis thaliana cultivation

[0042] A single colony was precisely selected from the plate from step 1 and inoculated into 2 mL of MRS liquid medium. Then, it was incubated at 37°C with constant temperature mixing at 900 rpm for 12 hours to obtain the bacterial suspension.

[0043] Before planting Arabidopsis thaliana, vermiculite needs to be sterilized, then mixed with bacterial solution and incubated at 37°C for 24 hours. After cooling, the vermiculite is placed in square boxes on a red tray. Seeds are then spread evenly on weighing paper and planted on the vermiculite using a pipette tip, 5 seeds per box, each labeled to prevent omissions. This ensures the seeds grow in a clean environment rich in bacterial solution, improving experimental accuracy. Five seeds are planted in each small box. A total of 36 small square boxes were planted, arranged in groups of three, for a total of 12 groups: phosphorus-free nutrient solution, phosphorus-free nutrient solution + P1, phosphorus-free nutrient solution + P2, phosphorus-free nutrient solution + PSM16, phosphorus-free nutrient solution + PSM16 + P1, phosphorus-free nutrient solution + PSM16 + P2, phosphorus-containing nutrient solution, phosphorus-containing nutrient solution + P1, phosphorus-containing nutrient solution + P2, phosphorus-containing nutrient solution + PSM16, phosphorus-containing nutrient solution + PSM16 + P1, and phosphorus-containing nutrient solution + PSM16 + P2. P1 indicates the addition of low-phosphorus fertilizer (75 mg / kg), and P2 indicates the addition of high-phosphorus fertilizer (300 mg / kg). The phosphorus fertilizer was superphosphate (ω(P2O5) = 12%).

[0044] Place the trays containing Arabidopsis thaliana seeds in a 22℃ greenhouse and cover them with plastic wrap. After about two days, the plastic wrap can be removed. The planting day is counted as day 0, and so on, recording the germination rate of the Arabidopsis thaliana seeds. During the growth process, the corresponding nutrient solutions can be applied. The phosphorus-free nutrient solution group is applied with phosphorus-free nutrient solution, and the phosphorus-containing nutrient solution group is applied with phosphorus-containing nutrient solution.

[0045] Figure 3 The germination rates of Arabidopsis thaliana in phosphorus-containing and phosphorus-free nutrient solutions after the addition of PSM16 are shown as mean ± SD. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001, determined by t-test. Figure 3 It is evident that, compared to phosphorus-free nutrient solutions without PSM16, the treatment group with added PSM16 likely provided more abundant available phosphorus, meeting the phosphorus requirements of Arabidopsis thaliana during germination and thus significantly promoting its germination rate. This result is because PSM16 can act as a phosphate-solubilizing bacterium to convert unavailable phosphorus in the soil into available phosphorus, improving phosphorus utilization. Even under phosphorus-free nutrient solution conditions, PSM16 can still effectively convert and release phosphorus, providing sufficient phosphorus source for Arabidopsis thaliana germination. In other words, PSM16 can convert unavailable phosphorus in the soil into available phosphorus, thereby significantly improving the germination rate of plants. This discovery not only reveals the key role of PSM16 in phosphorus conversion but also provides strong theoretical support and practical guidance for optimizing soil phosphorus utilization and promoting healthy plant growth. By utilizing PSM16, phosphorus resources in the soil can be utilized more efficiently, reducing phosphorus loss, lowering environmental pollution, and simultaneously improving crop growth vigor and yield. This research result has an important role in promoting sustainable agricultural development and achieving green agriculture.

[0046] 5. Screening for phosphatase-related genes

[0047] First, a single colony of Bacillus subtilis 168 was picked and inoculated into an appropriate amount of LB liquid medium, and cultured aerobically overnight at 37°C and 900 rpm. The next day, 50 μL of the overnight bacterial culture was transferred to 1.5 mL of LB medium and cultured at 37°C until the OD value reached approximately 1.5, which took about 3.5-4 hours. Subsequently, 80 μL of 20% xylose solution was added to bring the final concentration to 1%, and the temperature was adjusted to 30°C for another 2 hours. Afterward, the bacterial cells were collected by centrifugation, the supernatant was removed, and the bacterial cells were resuspended in 200 μL of antibiotic-free LB liquid medium, along with 10-20 μL of plasmid. The mixture was incubated at 30°C for 1 hour, and then 100 μL was spread onto a plate containing 30 μL / mL kanamycin and incubated overnight at 37°C.

[0048] To further investigate the mechanism of PSM16 in phosphorus conversion, this invention conducted a genome-wide analysis, screened for genes related to phosphatase, and transformed these genes into *E. coli* and *Bacillus*, testing their enzyme activity and determining the available phosphorus content. The results showed that GM000834, GM000917, GM000925, and GM000974 are associated with the phosphorus-solubilizing function of PSM16. Subsequently, this invention used heat transfer technology to transform these phosphatase-active genes into *E. coli*. Experimental results showed that the acid phosphatase (S-ACP) activity of strains transformed with the GM000834, GM000917, GM000925, and GM000974 genes was significantly enhanced (e.g., ...). Figure 4 (As shown in a). This finding indicates that the expression of these genes in *E. coli* significantly enhances their acid phosphatase activity. Subsequently, this invention further investigated the effects of these genes on the available phosphorus content in soil. Experimental data showed that, compared with the control group, the available phosphorus content in the treated soil was significantly increased (e.g., as shown in a). Figure 4 (As shown in b). This result fully demonstrates the positive role of these genes in increasing the available phosphorus content in the soil. These genes were then transferred into Bacillus subtilis (…). Figure 4 c) It was found that S-ACP also increased, and the content of available phosphorus also increased. Figure 4 d). Thus, this invention screened out four genes that can increase the content of available phosphorus in soil, and based on this, subsequent experiments were conducted.

[0049] The experimental results showed that, compared with the non-transgenic control strain, the strains transformed with these four genes significantly exhibited acid phosphatase activity. This means that these genes were successfully expressed in the transgenic strains and conferred the function of acid phosphatase on the strains.

[0050] 6. Seed germination

[0051] The model bacterium *Pseudomonas putida* KT2440 can dissolve insoluble soil phosphates by secreting gluconic acid. Soil was treated with a bacterial suspension of PSM16 and the phosphate-solubilizing model bacterium KT2440, and *Arabidopsis thaliana* was planted. The germination rate of *Arabidopsis thaliana* was then observed and statistically analyzed (e.g., germination rate of *Arabidopsis thaliana*). Figure 5 (as shown in a). By comparing the germination rate data of different treatment groups, this invention clearly shows that PSM16, like the phosphate-solubilizing model bacterium KT2440, has a positive effect on the growth potential of Arabidopsis thaliana. Figure 5 Image b shows the last germinated Arabidopsis thaliana. It can be seen that there are obvious differences between the two experimental groups and the control group. Both the experimental groups and the control group have advantages in terms of the number of Arabidopsis thaliana germinated and the growth.

[0052] Subsequently, strains infused with the genes “GM1000834”, “GM1000917”, “GM1000925”, and “GM1000974” were introduced into the soil for Arabidopsis thaliana cultivation experiments. Throughout the experiments, the invention strictly adhered to scientific experimental principles, ensuring consistency in variables such as soil conditions, cultivation methods, and the quality and quantity of Arabidopsis thaliana seeds to obtain accurate and reliable experimental results.

[0053] like Figure 6 As shown in the diagram, treatment with the experimental bacteria promotes faster germination and better growth in Arabidopsis thaliana. This effect of increasing germination rate remains unchanged after the addition of phosphorus nutrient solution, and it still contributes to better growth in Arabidopsis thaliana. Figure 7 This is a picture of the final germination of Arabidopsis thaliana.

[0054] In summary, through this experiment, the present invention has provided insights into the specific effects of these genetically modified strains on Arabidopsis thaliana cultivation, offering strong support for the development of agricultural production technologies and the improvement of crop yields.

[0055] 7. Determination of pH and electrical conductivity of soil mixed with PSM16 bacterial solution.

[0056] The present invention subsequently determined the pH and electrical conductivity of soil mixed with PSM16 bacterial solution. Most plants do indeed grow best in near-neutral soils, with the optimal soil pH typically between 5.2 and 6.8, within which nutrients are more readily absorbed and utilized by plants. Therefore, treated soil with a pH within this range will be more conducive to plant growth. As for soil electrical conductivity, this indicator reflects the content of exchangeable salt ions in the soil and also has a significant impact on plant growth. The suitable range of electrical conductivity varies depending on the crop species and soil conditions. Generally, the soil electrical conductivity range conducive to plant growth and development is between 350 and 1000 μS / cm. When the electrical conductivity is too high, exceeding 1200 μS / cm, the salt concentration in the soil is too high, which may lead to salinization damage to plants. Conversely, if the electrical conductivity value is too low, it indicates insufficient exchangeable salt ion content in the soil, which may lead to a lack of necessary nutrients for plants. Treated soil, if its electrical conductivity can be reduced to a range suitable for plant growth, will be more conducive to plant development and growth. In summary, maintaining soil pH between 5.2 and 6.8 through appropriate treatment, while ensuring soil electrical conductivity within the range of 350–1000 μS / cm, can provide a more suitable growth environment for plants. Therefore, soil treated with PSM16, if meeting these standards, will help improve plant growth and yield.

[0057] Depend on Figure 8As shown in the figure, the experimental results show that the electrical conductivity of the mixed soil after PSM16 treatment is about 775 μS / cm and the pH value is about 6.5, indicating that the soil treated with PSM16 can improve plant growth and yield.

[0058] In summary, PSM16 can be used as a bio-fertilizer to convert phosphorus, which is difficult for plants to absorb and utilize, into an absorbable form, improve crop phosphorus nutrition, improve the soil environment, reduce the use of chemical fertilizers, reduce environmental pollution, and promote sustainable agricultural development.

Claims

1. The application of Lactococcus lactis PSM16 in the preparation of bio-fertilizer, characterized in that, The bio-fertilizer is a functional bio-fertilizer product that is a mixture of soil phosphorus-solubilizing fertilizer, fertilizer that increases the available phosphorus content in the soil, fertilizer that promotes seed germination, and fertilizer that improves soil pH and electrical conductivity. The method for preparing bio-fertilizer includes the following steps: inoculating and culturing *Lactococcus lactis* PSM16, centrifuging to remove the supernatant, and obtaining bacterial powder or bacterial suspension, wherein the bacterial suspension contains bacterial cells at a concentration of 1×10⁻⁶. 4 ~ 1×10 8 The bio-fertilizer is obtained by measuring CFU / mL.

2. The application of Lactococcus lactis PSM16 according to claim 1 in the preparation of bio-fertilizer, characterized in that, The culture medium used was MRS medium.

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