Strain of stenotrophomonas HK11 and application thereof in removal of phosphorus in water body

Abstract

The invention discloses a strain of Stenotrophomonas sp. HK11. The preservation number of the Stenotrophomonas sp. HK11 in the Guangdong Microbial Culture Collection Center is GDMCC No. 66496, and the preservation number of the Stenotrophomonas sp. HK11 in the Guangdong Microbial Culture Collection Center is GDMCC No. 66496. The strain HK11 is applied to removal of inorganic phosphorus in a water body, and experimental results show that the strain HK11 has the capability of removing inorganic phosphorus in the water body and has a relatively good application prospect in treatment of phosphorus-polluted water bodies.

Description

Technical Field

[0001] The present invention belongs to the field of water eutrophication phosphorus pollution control, and specifically relates to a strain of Stenotrophomonas ( Stenotrophomonas sp.) HK11 and its application in bioremediation of water phosphorus removal. Background Art

[0002] Phosphorus is a key limiting factor that causes eutrophication in water bodies. Its excessive input leads to abnormal algal proliferation, dissolved oxygen depletion, and the collapse of aquatic ecosystems. Phosphorus in water bodies mainly exists in the form of orthophosphate, polyphosphate, and organic phosphorus. When the concentration exceeds 0.02 mg / L, it can induce algal blooms. The algal toxins and odorous substances released after the algae die and decompose not only threaten the safety of drinking water but also cause economic losses to the fishery. Traditional phosphorus removal technologies include chemical precipitation, electrocoagulation, and constructed wetlands, but these methods have disadvantages such as secondary pollution of chemical sludge, high energy consumption, or large land occupation. In contrast, biological phosphorus removal technology based on polyphosphate organisms (PAOs) has become a research hotspot because of its environmental friendliness, low cost, and ability to recover phosphorus resources.

[0003] Phosphorus accumulation bacteria achieves phosphorus enrichment and removal through the metabolic characteristics of "anaerobic phosphorus release - aerobic / anoxic excess phosphorus absorption". Candidatus Accumulibacter ) is extended to denitrifying phosphorus-accumulating bacteria (such as Dechloromonas ) and fermentative polyphosphate bacteria (such as Tetrasphaera ). In terms of microbial preparation development, patent CN202410085156.8 reports a device that combines electrocoagulation and biological phosphorus removal, but its core still relies on physical and chemical processes. The immobilized sludge technology achieves the simultaneous removal of nitrogen and phosphorus in eutrophic water bodies by embedding domesticated sludge in a sodium alginate carrier, and the TP removal rate can reach more than 80%. However, existing polyphosphate bacteria preparations are not effective in treating low-concentration phosphorus wastewater (TP<2mg / L), and there is an urgent need to develop new strains that are efficient in polyphosphate. Summary of the Invention

[0004] The present invention provides a Stenotrophomonas ( Stenotrophomonas sp . ) HK11, which was deposited in the Guangdong Provincial Microbial Culture Collection on June 11, 2025, with the deposit number GDMCC No. 66496, and the deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] Another object of the present invention is to apply Stenotrophomonas HK11 to remove phosphorus from eutrophic water bodies.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: 1. Silt and water samples were collected from a phosphate tailings accumulation area in Haikou, Yunnan. The mixed samples were enriched, separated, purified, and initially screened to obtain several phosphate-accumulating bacteria. The phosphate-accumulating bacteria seed liquid was inoculated into a culture medium containing synthetic wastewater. The culture was shaken at 28°C and 180 rpm. The phosphorus adsorption capacity of the strain was measured, and the strain with the highest phosphorus removal efficiency, HK11, was obtained. 2. A suspension of strain HK11 was spread on YG agar and incubated aerobically at 28°C for 48 hours. Morphological characteristics were then observed. HK11 colonies exhibited a regular, round, flattened morphology with a smooth surface and an opaque white color. Under an optical microscope, the bacteria appeared as straight rods lacking motility, ranging in length from 22.2 to 26.7 μm and in width from 10.9 to 12.1 μm. Further Gram staining confirmed that the strain was Gram-negative.

[0007] 3. The strain HK11 was sent to a sequencing company for strain identification. The bacterial 16S rDNA was amplified and sequenced using primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (AGAGTTTGATCMTGGCTCAG). The obtained sequence was compared with the sequence on NCBI by BLAST. It was found that the strain HK11 was Stenotrophomonas sp. MSW 101, and the similarity was as high as 99%, confirming that strain HK11 was Stenotrophomonas ( Stenotrophomonas sp.); 4. Inoculate strain HK11 into LB liquid medium for recovery culture. After shaking culture, allow the bacteria to enter the mid-logarithmic phase and obtain OD 600 =0.9±0.05 bacterial suspension; gradient experiments of temperature, pH, and phosphorus concentration were designed respectively. The bacterial suspension was inoculated into phosphorus-containing synthetic wastewater culture medium and dynamically cultured at 28°C. Samples were taken at regular intervals and the supernatant was collected by centrifugation. The total phosphorus concentration was determined according to the "Water Quality—Determination of Total Phosphorus—Ammonium Molybdate Spectrophotometric Method" (GB 11893-89), and the phosphorus removal efficiency of Stenotrophomonas HK11 was calculated.

[0008] The present invention has the following beneficial effects: (1) The strain HK11 of the present invention has activities such as catalase, catalase, and nitrate reduction, but lacks motility, which is consistent with its sessile growth characteristics in the sediment environment. In addition, HK11 is resistant to tetracycline and streptomycin (inhibition zone ≤ 5 mm), but is sensitive to most commonly used clinical antibiotics, and the hemolysis test is negative, indicating that its biosafety risk is controllable and has practical application potential; (2) Growth curve analysis showed that HK11 exhibited rapid adaptability, entering the logarithmic phase 1 hour after inoculation, growing rapidly, and reaching the stable phase 8 hours later. In terms of phosphorus removal kinetics, when the phosphorus concentration was 10 mg / L, HK11 reached a maximum phosphorus removal rate of 82.18% at 14 hours, which was highly consistent with its growth curve characteristics; (3) When the phosphorus concentration was 10 mg / L, strain HK11 could maintain its activity in the pH range of 6-11.5 and showed a phosphorus removal rate of 83.59% under strong alkaline conditions (pH 11.5); when the phosphorus concentration was 5 mg / L, the phosphorus removal rate of strain HK11 was higher than 97.15% at 15℃-30℃, and the efficiency dropped to 60.12% at 40℃; when the phosphorus concentration increased from 5 mg / L to 50 mg / L, the phosphorus removal rate of the strain dropped from 99.33% to 20.98%.

[0009] In conclusion, the strain of the present invention has the ability to remove inorganic phosphorus from water bodies and has good application prospects in the treatment of phosphorus-contaminated water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Figure 1 is a morphological diagram of strain HK11, where Figure a shows the colony morphology on LB solid medium; Figure b shows the morphology of the strain under a microscope; and Figure c shows the result of Gram staining. Figure 2 is the phylogenetic tree of strain HK11; Figure 3 This is a schematic diagram showing the effects of different carbon sources (left) and nitrogen sources (right) on the growth of Stenotrophomonas HK11; Figure 4 This is a schematic diagram of the growth of Stenotrophomonas HK11 (left) and phosphorus removal rate (right) under different treatment times; Figure 5 This is a schematic diagram of the effect of different pH water environments on the phosphorus removal rate of Stenotrophomonas HK11; Figure 6 is the phosphorus removal rate of Stenotrophomonas HK11 at different temperatures; Figure 7 This is a schematic diagram of the phosphorus removal rate of Stenotrophomonas HK11 at different initial phosphorus concentrations; Figure 8 It is the result of hemolysis test; Figure 9 This is a schematic diagram of the results of the actual application of Stenotrophomonas HK11 in aquaculture wastewater. DETAILED DESCRIPTION

[0011] The technical solutions of the present invention are further described in detail below with reference to specific embodiments and accompanying drawings. However, the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Those skilled in the art can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of the present invention to implement them.

[0012] Example 1: Isolation, screening and identification of Stenotrophomonas HK11 1. Sludge and water samples were collected from the phosphate tailings accumulation area of ​​Yunnan Phosphate Chemical Group Haikou Phosphate Industry Co., Ltd., stored in dry ice boxes and brought back to the laboratory; 2. Weigh 10g of the collected and preserved sample and add it to 90mL of sterile saline containing sterile glass beads. After shaking at 150rpm for half an hour, let it stand to form a suspension. Take 1mL of the supernatant and dilute it 10 times (10 -1 to 10 -8 ), take 10 -8 200 μL of the diluted suspension was inoculated into YG solid medium (yeast extract 1 g, glucose 1 g, KH2PO4 0.25 g, K2HPO4 0.3 g, MgSO4·7H2O 0.2 g, agar 20 g, water 1000 mL), and cultured at 28±0.5℃ for 4-5 days. Single colonies were picked based on their morphological differences (diameter, color, and edge characteristics) and purified by three-zone streak. 3. After the purified strains were shaken, 2 μL was respectively applied to the phosphorus-limited medium (take 25 mL of 10× glucose-MOPS medium, add it to 200 mL of agar medium (50°C) containing 0.0087 g of K2HPO4, pour it into a plate, cool it and set it aside), and the phosphorus-excessive medium (take 25 mL of 10× MOPS medium, add it to 200 mL of agar medium (50°C) containing 0.173 g of K2HPO4, pour it into a plate, cool it and set it aside), and cultured in a constant temperature incubator at 28°C for 48 h. After preliminary screening by BCIP colorimetry, the strains that showed blue (phosphatase activity positive) in both systems were identified as polyphosphate bacteria. Fifteen polyphosphate bacteria strains were obtained and stored on YG slants at 4°C for a short period of time. 4. Use a sterile spatula to peel off the polyphosphate bacteria lawn on the slope (stored at 4℃, ≤30d), inoculate it into 5mL LB liquid medium, and culture it at 28℃±0.5℃ and 150g shaking for 12h until the bacteria enter the mid-logarithmic phase and the bacterial solution concentration OD 600=0.9±0.05; the seed liquid of polyphosphate bacteria was inoculated into phosphorus-containing synthetic wastewater culture medium (0.68 g sodium acetate, 0.1 g peptone, 0.01 g yeast powder, 0.05 g sodium chloride, 0.03 g dipotassium hydrogen phosphate, 0.075 g sodium carbonate, 0.075 g magnesium sulfate, 0.025 g calcium chloride, 1 L distilled water, pH 7.0, sterilized at 121°C for 20 min), and cultured in a shaking incubator at 28°C and 150 rpm. The phosphorus adsorption capacity of the strain was measured, and the strain with the highest phosphorus removal efficiency, HK11, was obtained; 5. Identification of strain HK11 ① Morphological characteristics of strains: Figure 1 As shown, the colonies of strain HK11 on YG plates are relatively regular, round, flat, and smooth in surface. The colony diameter is 4 mm and the colony color is opaque white. Under an optical microscope, the bacteria are straight rod-shaped, with a length ranging from 22.2 to 26.7 μm and a width ranging from 10.9 to 12.1 μm. Gram staining is negative. ②Physiological and biochemical characteristics of Stenotrophomonas HK11 According to the methods in the Manual of Identification of Common Bacteria, the physiological and biochemical tests of strain HK11 were performed. The results are shown in Table 1. The catalase test, catalase test, nitrate reduction test, starch hydrolysis test, and gelatin test of strain HK11 were all positive, while the Gram staining test, oxidase test, methyl red test, VP test, and motility test were all negative. Table 1: Physiological and biochemical indexes of strain HK11

[0013] (Note: “+” represents positive, “-” represents negative); ③ Molecular identification: Using bacterial liquid as a template, primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (AGAGTTTGATCMTGGCTCAG) were used to amplify the 16S rRNA gene by polymerase chain reaction (PCR). The process was as follows: pre-denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s, followed by extension at 72°C for 5 min. After the amplification was completed, it was confirmed that the target fragment had been successfully amplified and then sent to a sequencing company for sequencing to obtain and screen the 16S rRNA gene sequence of polyphosphate bacteria. The 16S rRNA gene sequences of polyphosphate bacteria were compared in the NCBI database and sequences with high similarity were downloaded. The phylogenetic tree of polyphosphate bacteria was established using MEGA 7.0 analysis software ( Figure 2 ), strain HK11 was compared with Stenotrophomonassp. MSW 101 (KX094420.1) showed the closest relationship with a similarity of 99%, based on which the strain HK11 was identified as Stenotrophomonas ( Stenotrophomonas sp.).

[0014] Example 2: Preparation of Stenotrophomonas HK11 Bacterial Suspension For strains that were stored on two sides (YG slant at 4°C for short-term storage and 50% glycerol suspension at -80°C for freezing), a sterile spatula was used to peel off the slant lawn (stored at 4°C for ≤30 days) and inoculated into 5 mL LB liquid medium for recovery culture. The culture was shaken at 28°C ± 0.5°C and 150g for 12 hours to allow the bacteria to enter the mid-logarithmic phase. The bacterial solution concentration OD 600 =0.9±0.05, and the bacterial suspension was obtained.

[0015] Example 3: Growth of Stenotrophomonas HK11 and Phosphorus Removal Rate under Different Conditions 1. Utilization of carbon and nitrogen sources by Stenotrophomonas HK11 1.1 Carbon source utilization test Carbon sources were added to the basal culture medium ((NH4)2SO4 2.0 g, MgSO4•7H2O 0.2 g, NaH2PO4•H2O 0.5 g, K2HPO4 0.5 g, CaCl2•2H2O 0.1 g, and distilled water 1000 mL); the mass volume concentration of sugar alcohol carbon sources (ethanol, glycerol, glucose, sucrose) was 0.5%, and the mass volume concentration of other carbon sources (sodium acetate, sodium propionate, sodium thiosulfate, sodium bicarbonate, L-malic acid, L-glutamic acid) was 0.2%. The pH was adjusted to 7.0. The HK11 bacterial suspension was inoculated into the above culture medium at a 6% inoculum volume and cultured at 28°C for 22 h. The experiment was repeated three times and the absorbance of the bacterial suspension was measured at 600 nm.

[0016] 1.2 Nitrogen source utilization test Nitrogen sources (peptone, yeast extract, ammonium chloride, ammonium sulfate, ammonium nitrate, diammonium dihydrogen phosphate, ammonium bicarbonate, sodium nitrate) were added to a basal culture medium (KH2PO4 1.36 g, CaCl2•2H2O 0.5 g, Na2HPO4 2.13 g, glucose 10 g, MgSO4•7H2O 0.2 g, FeSO4•7H2O 0.05 g, distilled water 1000 mL) to a nitrogen source mass volume concentration of 0.1%, and the pH was adjusted to 7.0. HK11 bacterial suspension was inoculated into the above culture medium at a 6% inoculum volume and cultured at 28°C for 22 h. The experiment was repeated three times and the absorbance of the bacterial suspension was measured at 600 nm. 600 .

[0017] See the results Figure 3 Different carbon and nitrogen sources had a significant effect on the growth of strain HK11. Glucose as a carbon source was particularly beneficial to the growth of strain HK11, while the culture medium with peptone as a nitrogen source was more conducive to the growth of strain HK11.

[0018] 2. Growth and phosphorus removal rate of HK11 under different treatment times The phosphorus-containing synthetic wastewater culture medium in Example 1 was used, except that the phosphorus concentration was 10 mg / L (i.e., the amount of potassium hydrogen phosphate added was 0.0556 g). The culture was shaken at 28°C and 200 rpm. Samples were taken every 2 hours, and the OD of the bacterial solution was measured using an ultraviolet spectrophotometer at a wavelength of 600 nm. 600 value, continue until OD 600 When the value decreased or the phosphorus content stabilized, the culture was centrifuged at 12,000 rpm for 5 minutes, and the total phosphorus content in the supernatant was measured.

[0019] The results are as follows Figure 4 As can be seen from the figure, strain HK11 has a strong adaptability to the new environment and grows faster. It enters the logarithmic growth period from the 1st hour and reaches the stable period at the 8th hour. The phosphorus adsorption ability of strain HK11 changes with time. It can be observed that the less phosphorus content in the culture medium, the better the phosphorus adsorption effect of the strain. The results show that the highest phosphorus adsorption efficiency of HK11 reaches 82.18% at the 14th hour.

[0020] 3. Phosphorus removal rate of strain HK11 under different pH conditions The phosphorus-containing synthetic wastewater culture medium in Example 1 was used, except that the phosphorus concentration was 10 mg / L (i.e., the amount of potassium hydrogen phosphate added was 0.0556 g), and the pH of the culture medium was adjusted to 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 11.5 (precisely controlled using 1 mol / L HCl / NaOH). The HK11 bacterial suspension was inoculated into 10 mL of the above culture medium at a 6% inoculum size, and the culture was shaken at 28°C and 200 rpm for 22 h. After the culture solution was centrifuged at 12000 rpm for 5 min, the phosphorus content in the liquid was detected, and the phosphorus removal rate of HK11 was calculated.

[0021] from Figure 5 It can be seen that HK11 has better phosphorus adsorption effect in alkaline environment than in acidic environment. Under the condition of pH value of 11.5, the phosphorus removal rate of HK11 reached the highest value, i.e. 83.59%.

[0022] 4. Phosphorus removal rate of Stenotrophomonas HK11 at different temperatures This example uses a phosphorus-containing synthetic wastewater culture medium with a phosphorus concentration of 5 mg / L (i.e., the addition amount of potassium hydrogen phosphate is 0.0278 g). The difference is that the culture medium pH is 7. The HK11 bacterial suspension is inoculated into 10 mL of the culture medium at a 6% inoculum size. The culture is cultured with constant temperature and shaking (200 rpm) at 15°C, 25°C, 30°C, 35°C, and 40°C for 22 hours. The culture medium is centrifuged at 12,000 rpm for 5 minutes, the phosphorus content in the liquid phase is detected, and the HK11 phosphorus removal efficiency is calculated.

[0023] Figure 6 The results showed that low temperature did not affect its phosphorus removal efficiency. The phosphorus removal rate of strain HK11 was higher than 97.15% at 15℃~30℃, but as the temperature increased, the phosphorus removal efficiency decreased. The phosphorus removal effect was the worst at 40℃, with a phosphorus removal rate of 60.12%. Under high temperature conditions, although some bacteria may survive, their metabolic activity decreased and the phosphorus removal efficiency would also decrease.

[0024] 5. Phosphorus removal rate of HK11 at different initial phosphorus concentrations The culture medium used in this example is the same as the phosphorus-containing synthetic wastewater culture medium in Example 1, except that the phosphorus concentrations are 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L (i.e., the amount of potassium hydrogen phosphate added is 0.0278 g, 0.0556 g, 0.1111 g, 0.1667 g, 0.2222 g, and 0.2778 g). The HK11 bacterial suspension is inoculated into 10 mL of the culture medium at a 6% inoculum size. After shaking culture at 28°C and 200 rpm for 22 h, the culture solution is centrifuged at 12,000 rpm for 5 min, the phosphorus content in the liquid is detected, and the phosphorus removal rate of HK11 is calculated.

[0025] See the results Figure 7 When the phosphorus concentration increased from 5 mg / L to 50 mg / L, the phosphorus removal efficiency decreased from 99.33% to 20.98%; the results showed that the abundance of environmentally available phosphorus had a significant effect on the phosphorus solubilization effect of HK11, and optimizing culture conditions such as temperature and pH value could further improve the phosphorus removal efficiency.

[0026] Example 4: Safety evaluation of Stenotrophomonas HK11 ① Antibiotic susceptibility testing of strain HK11 was performed using the paper strip method described in the Manual of Common Bacterial Systematic Identification. The bacterial mix was densely spread on a growth medium plate using streaking or plate spreading. At the same time, a paper strip containing antibiotics was placed on the plate with sterile tweezers. The plate was incubated at 30°C for 24-48 hours, and then the size of the inhibition zone was observed. Table 2: Antibiotic sensitivity test of strain HK11

[0027] Note: 0-5mm: no effect; 6-15mm: weak effect; >15mm: strong inhibitory effect.

[0028] ②Hemolysis test was performed using blood (sheep blood) agar plates (Changde Beekman Biotechnology Co., Ltd.), and antimicrobial susceptibility test was performed using antimicrobial susceptibility disks (Hangzhou Microbiological Reagent Co., Ltd., Hangzhou, China) according to the European Committee on Antimicrobial Susceptibility Testing disk diffusion method.

[0029] The results are shown in Table 2. Strain HK11 showed resistance to tetracycline and streptomycin (inhibition zone ≤ 5 mm), but was sensitive to antibiotics such as chloramphenicol and kanamycin (inhibition zone ≥ 10 mm). Hemolysis experiments showed that the strain had no hemolytic activity (see Figure 8 ), in compliance with biosafety requirements.

[0030] Example 5: Practical application of strain HK11 in aquaculture wastewater The collected aquaculture wastewater (phosphorus content is 3.052 mg / L) was packaged, the experimental group was inoculated with 6% HK11 bacterial suspension, and the control group was inoculated with 6% LB liquid culture medium. Three biological replicates were performed for each group. The culture was shaken at 28°C and 150 r / min for 36 hours. Samples were taken every 12 hours, and the culture solution was centrifuged at 12000 rpm for 5 minutes. The phosphorus content in the liquid was detected, and the phosphorus removal rate of HK11 in practical application was calculated.

[0031] like Figure 9 As shown in the figure, the phosphorus removal rate of strain HK11 increased with time, reaching 76.57% at 36 hours. As for the control group, the phosphorus removal rate was only 3.31% at 36 hours, which further illustrates the practical application value of HK11 in treating aquaculture wastewater.

Claims

1. A strain of Stenotrophomonas ( Stenotrophomonas sp.) HK11, whose deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 66496.

2. Use of the Stenotrophomonas HK11 according to claim 1 in removing phosphorus from water.

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