Citrobacter momusei and application thereof in dissolving and dissolving phosphorus

Abstract

The invention provides citrobacter mulesii and an application of the citrobacter mulesii in dissolving and dissolving phosphorus, the citrobacter mulesii is citrobacter mulesii LY2024080101 and is preserved in the China Center for Type Culture Collection, the preservation date is December 30, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20242931. Compared with the prior art, the strain capable of producing the acid phosphatase is obtained through screening, belongs to citrobacter mulesii, can be used for producing the acid phosphatase, can be used for dissolving phosphorus and is excellent in effect. The acid phosphatase produced by the strain obtained by screening has the characteristics of safety, environmental protection and high efficiency, and has wide application prospects in the fields of medical treatment, food and health.

Description

Technical Field

[0001] The present invention belongs to the technical field of Citrobacter murliniae, and particularly relates to a strain of Citrobacter murliniae and its application in phosphorus solubilization and phosphorus decomposition. Background Art

[0002] Acid Phosphatase (EC 3.1.3.2) is a type of hydrolase that can hydrolyze phosphomonoester bonds under acidic conditions. This enzyme is widely distributed in plant seeds, molds, the liver, and the prostate of the human body, and plays an important role in the metabolism of nucleotides, phosphoproteins, and phospholipids in organisms, bone formation, and the utilization of phosphorus. Currently, the main preparation method of this enzyme is to extract it from animal and plant cells or tissues. According to relevant literature, there are few reports at home and abroad on the research of preparing this enzyme using prokaryotic microbial fermentation technology. Although the technology for extracting and preparing acid phosphatase from animal and plant tissues or cells is mature and the material sources are extensive, the demand for raw materials is large and the enzyme production is low; the advantage of obtaining this enzyme by eukaryotic fermentation is high enzyme production, but there are also disadvantages such as high investment costs for related fermentation equipment, difficult control of fermentation process conditions, slow fermentation rate, and long cycle.

[0003] Due to its key enzymatic reaction (dephosphorylation), it has unique application prospects in agriculture and clinical practice. Based on the information obtained from some existing investigations, acid phosphatase can be applied in many more aspects, such as using this enzyme to improve agriculturally important plants, using it as a biomarker, using it as a therapeutic target for diseases, and building more useful biosensing devices. Summary of the Invention

[0004] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a strain of Citrobacter murliniae and its application in phosphorus solubilization and phosphorus decomposition.

[0005] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a strain of Citrobacter murliniae, which is Citrobacter murliniae LY2024080101, deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit number CCTCC NO: M 20242931.

[0007] In the second aspect, the present invention provides a composition, which contains the Citrobacter murliniae LY2024080101.

[0008] Third aspect, the present invention provides the application of the Citrobacter murliniae LY2024080101 as described above, or the composition as described above, in the production of acid phosphatase.

[0009] Fourth aspect, the present invention provides the application of the Citrobacter murliniae LY2024080101 as described above, or the composition as described above, in dissolving and decomposing phosphorus.

[0010] As a specific embodiment, the phosphorus in the dissolving and decomposing phosphorus includes inorganic phosphorus and / or organic phosphorus.

[0011] As a further scheme, the inorganic phosphorus includes calcium phosphate, and the organic phosphorus includes calcium phytate.

[0012] As a more specific scheme, the culture medium containing the calcium phytate uses water as a solvent and includes: glucose 10 g / L, calcium phytate 3 g / L, MgCl 2 5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, KCl 0.2 g / L, and (NH 4 ) 2 SO 4 0.1 g / L.

[0013] As a more specific scheme, the culture medium containing the calcium phosphate uses water as a solvent and includes: glucose 10 g / L, Ca 3 (PO 4 ) 2 5 g / L, MgCl 2 5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, KCl 0.2 g / L, and (NH 4 ) 2 SO 4 0.1 g / L.

[0014] Fifth aspect, the present invention provides the application of the Citrobacter murliniae LY2024080101 as described above, or the composition as described above, in decomposing phosphates in soil.

[0015] Sixth aspect, the present invention provides a method for decomposing phosphates in soil, the method comprising applying the Citrobacter murliniae LY2024080101 as described above, or the composition as described above, to the soil.

[0016] Beneficial effects: Compared with the prior art, a strain of acid phosphatase-producing bacteria, belonging to Citrobacter murliniae, is screened and obtained in the present invention. The strain can produce acid phosphatase and can be used for dissolving phosphorus, with excellent effects. The acid phosphatase produced by the strain screened in the present invention has the characteristics of safety, environmental protection and high efficiency, and has broad application prospects in the fields of medicine, food and health. Description of the Drawings

[0017] Figure 1 It is the prediction result of non-coding RNA, where Type: non-coding RNA type; Copy num: copy number; Avg.length (bp): average sequence length; Total length (bp): total sequence length; Percent of Genome (%): percentage of the total length of the non-coding RNA sequence in the total length of the genome sequence.

[0018] Figure 2 It is the circular map of the genome of Citrobacter murliniae LY2024080101.

[0019] Figure 3 It is the clustering result map of the gene family analysis of Citrobacter murliniae LY2024080101.

[0020] Figure 4 It is the Gram staining map of Citrobacter murliniae LY2024080101.

[0021] Figure 5 It is the effect diagram after culturing Citrobacter murliniae LY2024080101 in the calcium phytate (organic phosphorus) solid medium for 5 days. Among them, the left test tube is the experimental group, and the right test tube is the control group.

[0022] Figure 6 It is the effect diagram after culturing Citrobacter murliniae LY2024080101 in the disodium phenyl phosphate (inorganic phosphorus) solid medium for 5 days. Among them, the left test tube is the experimental group, and the right test tube is the control group.

[0023] Figure 7 It is the effect diagram of Citrobacter murliniae LY2024080101 used for decomposing phosphorus in the contaminated soil. Among them, the left test tube is the soil group added with the bacterial agent, and the right test tube is the untreated soil group. Detailed Embodiments

[0024] The technical solutions of the present invention will be further described in detail below with reference to the drawings and embodiments.

[0025] The culture medium formula used in the embodiment is as follows:

[0026] LB liquid medium: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L, sterilized at 121 °C for 20 min. LB solid medium is prepared by adding 15 g of agar to the liquid medium.

[0027] The calcium phosphate medium uses water as a solvent and includes: glucose 10 g / L, Ca 3 (PO 4 ) 2 5 g / L, MgCl 2 5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, KCl 0.2 g / L, and (NH 4 ) 2 SO 4 0.1 g / L.

[0028] The calcium phytate medium uses water as a solvent and includes: glucose 10 g / L, calcium phytate 3 g / L, MgCl 2 5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, KCl 0.2 g / L, and (NH 4 ) 2 SO 4 0.1 g / L.

[0029] Example 1:

[0030] 1. Screening and preservation of strains.

[0031] (1) Soil was collected from Laoshan National Park in Nanjing, Jiangsu Province and stored at low temperature in a sterile bag.

[0032] (2) 1 g of soil was placed into 100 ml of inorganic phosphorus screening liquid medium (peptone 10.0 g / l; sodium chloride 5.0 g / l; yeast extract powder 5.0 g / l; glucose 1.0 g / l; disodium phenyl phosphate 2.0 g / l), and shaken at 37 °C in an incubator for 24 hours.

[0033] (3) 1 mL of the culture suspension was inoculated into 100 mL of freshly prepared inorganic phosphorus screening liquid medium and shaken at 37 °C in an incubator for 24 hours.

[0034] (4) After repeating (3) once, the culture suspension was serially diluted 10 4 and 10 5 times. 200 μL was taken and spread on the organic phosphorus screening medium plate, and incubated at 28 °C in an incubator for 24 - 48 hours.

[0035] (5) Select the strain with a larger transparent circle (i.e., phosphate-solubilizing circle), named LY202409191, and calculate the phosphate-solubilizing index (diameter of phosphate-solubilizing circle / diameter of colony). And purify the strain on the organic phosphorus screening plate, with monoclonal purification at least 3 times. The purified strain is mixed with 50% glycerol and fresh bacterial liquid at a ratio of 1:1 and stored in a -40°C ultra-low temperature refrigerator.

[0036] 2. Strain identification

[0037] Extract the total DNA of strain LY2024080101 using a rapid bacterial genomic DNA extraction kit, and detect the concentration and purity of the extracted DNA with Qubit2.0. Using this DNA as a template and 16SrRNA27F / 1429R as primers, perform polymerase chain reaction (PCR). PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, 30 cycles; extension at 72°C for 10 min. After detecting the quality of the PCR amplification product by 1.5% agarose gel electrophoresis, the amplified fragment is sequenced by the Sanger method.

[0038] The sequence is compared and analyzed in the 16SrRNA database of NCBI to determine that LY2024080101 is Citrobacter murliniae.

[0039] The 16S rDNA of Citrobacter murliniae LY2024080101 strain is shown in SEQ ID NO.1.

[0040] Example 2 Sequencing of the whole genome sequence of the strain

[0041] (1) Genomic DNA extraction: Extract high-quality genomic DNA from soil samples, and detect the integrity of the extracted DNA by agarose gel electrophoresis

[0042] (2) Sequencing data statistics and quality control: Adopt the whole genome shotgun (WGS) sequencing strategy to construct libraries with different insert fragments. Using the second-generation sequencing technology (Next-Generation Sequencing, NGS), based on the Illumina NovaSeq sequencing platform, perform paired-end (PE) sequencing on the libraries.

[0043] (3) Sequencing data filtering: The sequencing data contains some reads with adapters and low quality, which will interfere with subsequent information analysis. To ensure the quality of subsequent information analysis, it is necessary to further filter the data downloaded from the sequencer. The fastp software is used to filter the original downloaded data (rawdata) to generate high-quality sequences (high quality data). Adapter contamination removal; length filtering: If the length of any one of the paired-end reads ≤ 50 bp, then remove the paired-end reads; reads quality filtering: If the average quality value of any one of the paired-end reads < 20, then remove the paired-end reads; ambiguous base N filtering: If the number of N bases in the paired-end reads ≥ 5, then remove the paired-end reads.

[0044] (4) Genome assembly and evaluation: The SPAdes software is used to perform de novo assembly on the second-generation data. Scaffolds with a length greater than 500 bp and an average depth greater than 10 are selected as the assembly results to construct scaffolds and contigs. The Pilon software is used for single-base correction to obtain the assembly results. Before performing formal genome assembly, the basic characteristics of the genome (such as genome size) can be estimated using the reads information of the short insert fragment library (library insert fragment less than 1,500 bp) obtained by sequencing. That is, a sequence of K bases in length is iteratively selected from a continuous sequence. If the length of the read is L and the length of the K-mer is K, then for each read, L - K + 1 K-mers can be obtained. The K-mer depth distribution curve belongs to a standard Poisson distribution curve, and the shape of this curve is affected by the following factors: sequencing error rate, genome heterozygosity, and repetitive sequences.

[0045] (5) Non-coding RNA prediction: The tRNAscan-SE is used to predict tRNA genes in the whole genome, and Barrnap is used to predict rRNA genes. The prediction of the remaining non-coding RNAs is mainly obtained by comparison with the Rfam database (https: / / rfam.org / ). The non-coding RNA prediction results are shown in Figure 1 .

[0046] (6) Coding gene annotation: The protein sequences of the predicted genes are respectively aligned with the NR, GO, eggNOG, KEGG, and Swiss-Prot databases by blastp to obtain the annotation information of the predicted genes.

[0047] (7) Genome complete map: The Circos software (v0.64) was used to draw the genome circle map, comprehensively showing the genome characteristics, including the distribution of genes on the sense and antisense strands, the COG functional classification of genes, GC content, genomic islands, and homologous genes, etc., providing a more comprehensive and intuitive understanding of the genomic characteristics of the strain.

[0048] The genome circle map of Citrobacter murliniae LY2024080101 is shown in Figure 2 .

[0049] (8) Gene family analysis: The orthofinder software was used to perform gene family analysis on the sample protein sequences with the parameter diamond (-e 0.001) to find potential homologous genes. The clustering results are as shown in Figure 3 (shown as a venn diagram when the number of samples is less than 5 and as a petal diagram when greater than 5):

[0050] Example 3 Gram staining of the acid phosphatase-producing strain.

[0051] Overview: This experiment requires ammonium oxalate crystal violet staining solution, Lugol's iodine solution, safranin staining solution, and 95% ethanol. It is mainly used to determine whether the bacteria are Gram-negative or Gram-positive.

[0052] Steps: Smear, dry, fix with a gentle flame of an alcohol lamp, stain with ammonium oxalate crystal violet, wash with distilled water, mordant with Lugol's iodine solution, wash with distilled water, decolorize with ethanol, wash with distilled water, counterstain with safranin, wash with distilled water, and examine under an oil immersion microscope. Gram-positive bacteria have a cell wall mainly composed of peptidoglycan, which can firmly retain the complex of crystal violet and iodine, making the bacterial cells remain blue. As can be seen from + , the acid phosphatase-producing strain is a Gram-positive bacterium. Figure 4

[0053] Example 4 Acid phosphatase activity at different inorganic phosphate concentrations

[0054] 1. Take 6 test tubes and number them sequentially from 0 to 5, with the blank being tube 0. According to Table 1, add 0.4 nM phenol standard application solution, 0.2 M acetate buffer at pH 5.6, 1 M sodium carbonate solution, and Folin-Ciocalteu reagent to each test tube in sequence. Shake well and incubate at 35 °C for more than 10 minutes (first place a beaker filled with water at 35 °C in a water bath, then put the test tubes into the beaker for incubation to prevent the test tubes from slipping into the water). Using tube 0 as the blank, read the absorbance A680 of each tube at a wavelength of 680 nm on a visible light spectrophotometer. Plot a standard curve with A680 as the abscissa and the volume of the phenol standard application solution in milliliters as the ordinate.

[0055] C = 1.8228x

[0056] ​C: Volume of Folin - phenol reagent (ml)

[0057] X: Absorbance value

[0058] 2. Determination of enzyme activity

[0059] Take 8 test tubes, numbered 1, 2, 3, 4, 5, 6, 7, 8. Add 0.5 ml of 5 nM disodium phenyl phosphate solution to each of the 8 test tubes, preheat at 35 °C for 2 minutes, and then add 0.5 ml of enzyme solution preheated at 35 °C (enzyme solution of Citrobacter murliniae LY2024080101 cultured in 100 ml of LB medium) to the 8 test tubes (the phosphate contents in the medium are 0, 0.1 g, 0.2 g, 0.5 g, 0.8 g, 1 g, 2 g, 5 g respectively). Immediately start timing, shake well, and after precisely reacting at 35 °C for 10 minutes (the time when the enzyme solution is added is the starting time, and the time when the sodium carbonate solution is added is the ending time), immediately add 5 ml of 1 M sodium carbonate solution to each of the 8 test tubes, then add 0.5 ml of dilute Folin - phenol solution to each, mix well. After the 8 test tubes are shaken well, incubate and develop color at 35 °C for about 10 minutes or more, and measure the light absorption value A680 at 680 nm of the 8 test tubes with a visible light spectrophotometer.

[0060] Table 1

[0061]

[0062] The enzyme activities of acid phosphatase in LY2024080101 under different inorganic phosphate concentrations are shown in the following table:

[0063]

[0064] Example 5 Determination of the phosphorus activation ability of acid phosphatase - producing strains

[0065] (1) Drawing of the standard curve: Accurately pipette 0, 1, 2, 4, 6, 8, 10 mL of 5 mg / L phosphorus standard solution into 50 - mL volumetric flasks respectively, add 5 mL of molybdenum antimony anti - reagent, and make up the volume to 50 mL with water. Shake well, and after developing color, measure the absorbance value of the solution at 880 nm with a spectrophotometer.

[0066] (2) Preparation of the bacterial suspension: Use an inoculation loop to pick 1 loop of the strain preserved in Example 1 and inoculate it into a test tube containing LB medium, and culture it overnight at 28 °C with constant shaking. Transfer it to a triangular flask containing LB medium according to an inoculation amount of 2%, and culture it at 28 °C with constant shaking until the OD600 of the bacterial suspension is approximately 1.

[0067] (3) Cultivation: Pipette 1 mL of the bacterial suspension into a 1.5 - mL centrifuge tube, wash it 3 times with sterile water, and resuspend it in sterile water.

[0068] (4) Pipette 0.2 mL of the washed bacterial suspension (inoculation amount: 1%), and transfer it to a liquid medium containing 20 mL (for measuring the ability to dissolve organic phosphorus: the organic phosphorus screening liquid medium is the same as in Example 1; for measuring the ability to dissolve inorganic phosphorus: disodium phenyl phosphate medium (glucose: 10 g / L, disodium phenyl phosphate: 5 g / L, MgCl 2 : 5 g / L, MgSO 4 ·7H 2 O: 0.25 g / L, KCl: 0.2 g / L, (NH 4 ) 2 SO 4 : 0.1 g / L)), and culture it at a constant temperature of 28°C with shaking for 7 days. Set up 3 parallels and sample every 24 hours; at the same time, pipette 0.2 mL of sterile water to replace the bacterial suspension as a negative control.

[0069] (5) Let it stand at room temperature for 15 minutes, pour the fermentation broth into a centrifuge tube, centrifuge at 1500 rpm for 3 minutes, and collect the supernatant A; centrifuge a part of the supernatant A again at 10000 rpm for 10 minutes to collect the supernatant B.

[0070] (6) Measure the available phosphorus: Pipette an appropriate amount of supernatant B and place it in a 50 mL volumetric flask. Add 8 mL of molybdenum antimony anti-color reagent and make up to 50 mL with deionized water. Shake well. After color development, measure the absorbance of the solution at 880 nm using a spectrophotometer.

[0071] (7) Phosphorus solubilization amount (mg / L) = ρ × V2 / V1

[0072] Where: ρ—the mass concentration of phosphorus in the test solution (mg / L);

[0073] V1—the volume of supernatant B pipetted (mL);

[0074] V2—the volume of the colored solution (mL).

[0075] (8) After Citrobacter murliniae LY2024080101 was cultured on the calcium phytate (organic phosphorus) solid medium for 5 days, the phosphorus solubilization amount was 236.72 mg / L. The effect is as Figure 5 shown.

[0076] (9) After Citrobacter murliniae LY2024080101 was cultured on the disodium phenyl phosphate (inorganic phosphorus) solid medium for 5 days, the phosphorus solubilization amount was 206.32 mg / L. The effect is as Figure 6 shown.

[0077] Example 6 Application of Acid Phosphatase-Producing Strains in Contaminated Soil

[0078] (1) Preparation of bacterial agent: The monoclonal colony of Citrobacter murliniae LY2024080101 on the purification plate was inoculated into 5 mL of LB liquid medium and cultured overnight at 37 °C with shaking at 160 rpm. It was transferred to 100 mL of LB medium according to an inoculation amount of 2% and cultured at 28 °C with shaking at 160 rpm until the logarithmic growth phase. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded and the cells were collected. The cell pellet was resuspended in sterile water and centrifuged at 8000 rpm for 10 min, and this step was repeated 2 - 3 times. The cells were finally resuspended in sterile water to obtain a bacterial agent suspension with OD600 = 1.0.

[0079] (2) 1 kg of soil contaminated with phosphate was selected, and 100 ml of the bacterial agent was added. Samples were taken after 3 days. 5 g of the soil was immersed in sterile water, and after thorough mixing, it was centrifuged at 8000 rpm for 5 min. Then the supernatant was taken, and the method for detecting organic phosphorus and inorganic phosphorus was the same as that in Example 4.

[0080] (3) The phosphate content in the supernatant of the soil with the added bacterial agent decreased significantly, and the effect is as Figure 7 shown.

[0081] The embodiments of the present invention have been described in detail above in conjunction with specific embodiments. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention.

Claims

1. A strain of Citrobacter moorei, characterized in that The Citrobacter murliniae is Citrobacter murliniae LY2024080101, which is deposited in the China Center for Type Culture Collection on December 30, 2024, and the deposit number is CCTCC NO: M 20242931.

2. A composition, characterized in that The composition comprises the Citrobacter rodentium LY2024080101 described in claim 1.

3. Use of the Citrobacter muehii LY2024080101 according to claim 1 or the composition according to claim 2 in producing acid phosphatase.

4. Use of the Citrobacter muehii LY2024080101 described in claim 1 or the composition described in claim 2 in phosphate solubilization.

5. The use according to claim 4, characterized in that: The phosphorus in the phosphorus dissolving and solution includes inorganic phosphorus and / or organic phosphorus.

6. The use according to claim 5, characterized in that: The inorganic phosphorus includes calcium phosphate, and the organic phosphorus includes calcium phytate.

7. Use of the Citrobacter moorei LY2024080101 according to claim 1 or the composition according to claim 2 for decomposing phosphate in soil.

8. A method for decomposing phosphate in soil, characterized in that: The method comprises applying the Citrobacter rodentium LY2024080101 of claim 1 or the composition of claim 2 to the soil.

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