Serratia marcescens HK16 and application of serratia marcescens HK16 in phosphorus removal of water body

Abstract

The invention discloses serratia sp. HK16, and the preservation number of the serratia sp. HK16 in Guangdong Microbial Culture Collection Center is GDMCC No.66497. The strain is applied to removal of inorganic phosphorus in water, and experimental results show that the strain HK16 has the capability of removing inorganic phosphorus in water and has a better application prospect in treatment of phosphorus-polluted water.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental microorganisms and specifically relates to a Serratia marcescens ( Serratia sp.) HK16 and its application in phosphorus removal in water. Background Art

[0002] Eutrophication refers to the phenomenon in which excessive nutrients such as nitrogen and phosphorus enter water bodies due to human activities, leading to the rapid growth of algae and other plankton, which in turn causes a decrease in dissolved oxygen, deterioration of water quality, and an imbalance in the ecosystem. The main sources of eutrophication include substandard discharge of industrial and domestic wastewater, as well as fertilizer and pesticide residues carried by agricultural runoff. Although natural water bodies can maintain ecological balance through self-purification, the human input of pollutants far exceeds their purification capacity, resulting in extreme eutrophication phenomena such as algal blooms and red tides in the short term.

[0003] At present, a variety of technologies including physical, chemical and biological methods are mainly used to treat eutrophic water bodies. Among them, physical methods remove nutrients from water bodies through physical means such as filtration and sedimentation; chemical methods use chemical substances such as coagulants and algaecides to control algae growth; biological methods focus on using microorganisms and aquatic plants to absorb nutrients, and improve the water environment through ecological restoration technologies such as microbial re-regulation technology. Among them, although chemical methods are quick to take effect, they are prone to secondary pollution; physical methods are difficult to promote due to large equipment investment and high operating costs. In contrast, biological phosphorus removal technology based on polyphosphate bacteria (PAOs) has become a research hotspot due to its advantages such as environmental friendliness and low cost. Polyphosphate bacteria play an important role in the synthesis and degradation of polyphosphates, and their metabolic characteristics depend on the conditions of an anaerobic-aerobic alternating environment. In addition, research progress on polyphosphate bacteria shows that they have great potential in treating eutrophic water bodies. The phosphorus content in water bodies can be effectively reduced through biological phosphorus removal technology. Traditional polyphosphate bacteria (such as Candidatus Accumulibacter ) rely on the metabolic pathway of “anaerobic phosphorus release-aerobic phosphorus uptake” to achieve phosphorus enrichment, while denitrifying phosphorus accumulation bacteria (such as Dechloromonas ) and fermentative polyphosphate bacteria (such as Tetrasphaera ) further expanded the scope of application of biological phosphorus removal. However, existing technologies still have significant limitations: (1) insufficient removal efficiency for low-concentration phosphorus (TP < 5 mg / L); (2) limited ability to degrade organic phosphorus (such as pesticide residues); and (3) poor environmental adaptability of strains, with activity dropping sharply under low temperatures or alkaline conditions. Summary of the Invention

[0004] The present invention provides a Serratia ( Serratiasp.) HK16, which was deposited in Guangdong Provincial Microbial Culture Collection on June 11, 2025, with the deposit number GDMCC No. 66497. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0005] Another object of the present invention is to use Serratia HK16 to remove phosphorus from rich 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 Yunnan. The mixed samples were enriched, separated, purified, and initially screened to obtain several phosphate-accumulating bacteria. The seed liquid of the phosphate-accumulating bacteria was inoculated into a culture medium containing synthetic wastewater. The culture was shaken at 28°C and 180 rpm. The phosphorus removal capacity of the phosphate-accumulating bacteria was measured, and strain HK16 with high phosphorus removal ability was obtained. 2. A bacterial suspension of strain HK16 was spread on YG agar medium and incubated aerobically at 28°C for 48 h. Morphological observation revealed that HK16 colonies were relatively regular, round, flat, smooth, and opaque white. Under an optical microscope, the bacteria were spherical, nonmotile, 12-14 μm in size, and Gram-negative. 3. The strain HK16 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 HK16 was Serratiamarcescens FD24 is the closest relative, with a similarity of up to 99%. Combined with its morphological characteristics, the strain HK16 was identified as Serratia marcescens ( Serratia sp.); 4. Inoculate the strain HK16 into LB liquid culture medium and culture it with shaking. The bacteria enter the logarithmic growth phase and obtain a bacterial suspension. Design gradient tests of temperature, pH, and phosphorus concentration. Inoculate the bacterial suspension into a phosphorus-containing synthetic wastewater culture medium and culture it dynamically at 28°C. Take samples at regular intervals and centrifuge the supernatant to obtain the total phosphorus concentration according to the "Water Quality - Determination of Total Phosphorus - Ammonium Molybdate Spectrophotometry" (GB11893-89). Calculate the phosphorus removal rate of Serratia marcescens HK16.

[0007] The present invention has the following beneficial effects: (1) The strain HK16 of the present invention exhibited multiple enzyme activities, including catalase, catalase, amylolytic enzyme, and nitrate reduction, but lacked motility. The strain HK16 was resistant to tetracycline and streptomycin (inhibition zone ≤ 5 mm), but was sensitive to most commonly used clinical antibiotics and had a negative hemolysis test, indicating that its biosafety risk is controllable and has practical application potential. (2) Growth curve analysis revealed that HK16 has excellent rapid adaptability. It entered the logarithmic growth phase only 1 hour after inoculation and grew rapidly until it reached a stable state after 16 hours. In terms of phosphorus removal kinetics, HK16 reached a maximum phosphorus removal rate of 60.88% at the 22nd hour, which is highly consistent with its growth curve characteristics. (3) Strain HK16 exhibited a wide pH adaptability range (6-11.5). Under extremely alkaline conditions (pH = 11), its phosphorus removal rate reached 84.34%, demonstrating its excellent alkali resistance. The phosphorus removal rate of this strain remained above 92.96% in the range of 15-30°C. When the temperature rose to 40°C, the phosphorus removal efficiency dropped to 62.47%. In the phosphorus concentration range of 5-50 mg / L, the phosphorus removal rate of this strain dropped sharply from a near-perfect 99.4% to 12.96%. This change pattern indicates that the phosphorus transport system works efficiently under low concentration conditions, while at high concentrations, the phosphorus removal efficiency decreases due to saturation of the transport protein. In conclusion, the strain HK16 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

[0008] Figure 1 Figure 1 is a morphological diagram of strain HK16, where Figure a shows the colony morphology on LB solid medium; Figure b shows the result of Gram staining; and Figure c shows the morphology of the strain under a microscope. Figure 2 is the phylogenetic tree of strain HK16; Figure 3 This is a schematic diagram of the effects of different carbon sources (left) and nitrogen sources (right) on the growth of Serratia HK16; Figure 4 The diagram shows the growth curve of Serratia HK16 (left) and phosphorus removal rate (right) at different treatment times; Figure 5 This is a schematic diagram of the effect of different pH water environments on the phosphorus removal rate of Serratia HK16; Figure 6 is the phosphorus removal rate of Serratia HK16 at different temperatures; Figure 7 This is a schematic diagram of the phosphorus removal rate of Serratia HK16 under different initial phosphorus concentrations; Figure 8It is the result of hemolysis test; Figure 9 This is a schematic diagram of the results of the practical application of Serratia HK16 in agricultural wastewater. DETAILED DESCRIPTION

[0009] 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.

[0010] Example 1: Isolation, screening and identification of Serratia HK16 1. Sludge and water samples were collected from the phosphate tailings accumulation area of ​​Yunnan Phosphate Chemical Group Haikou Phosphate Industry Co., Ltd., and stored in dry ice boxes before being 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 ~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 colony morphology (diameter, color, and edge characteristics) and purified by three-zone streak.

[0011] 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, HK16, was obtained; 5. Identification of strain HK16 ① Morphological characteristics of strains: Figure 1 As shown, the colonies of strain HK16 on YG plates are relatively regular, round, flat, and smooth in surface. The colony diameter is 2 mm and the colony color is opaque white. Under an optical microscope, the bacteria are spherical, non-motile, and 12-14 μm in size. Gram staining is negative. ②Physiological and biochemical characteristics of Stenotrophomonas HK16 According to the methods in the Manual of Identification of Common Bacteria, the physiological and biochemical characteristics of strain HK16 were tested. The results are shown in Table 1. The catalase test, catalase test, nitrate reduction test, starch hydrolysis test, and gelatin test of strain HK16 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 HK16

[0012] (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 the sequences with high similarity were downloaded. The phylogenetic tree of polyphosphate bacteria was established using MEGA 7.0 analysis software ( Figure 2 ), by comparison, strain HK16 and SerratiamarcescensFD24 (OR551376.1) is the closest relative, and the similarity is as high as 99%, confirming that strain HK16 is Serratia marcescens ( Serratia sp.).

[0013] Example 2: Preparation of Serratia HK16 bacterial suspension For strains that were stored using the double-sided preservation method (YG slant at 4°C for short-term storage, 50% glycerol suspension at -80°C for freezing), a sterile spatula was used to peel off the slant bacterial lawn (stored at 4°C for no more than 30 days), and then inoculated into 5 mL of LB liquid medium for recovery culture. The culture was shaken at 28°C ± 0.5°C and 150g for 12 h 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.

[0014] Example 3: Growth and phosphorus removal rate of Serratia HK16 under different conditions 1. Utilization of carbon and nitrogen sources by Serratia HK16 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; HK16 bacterial suspension was added to 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.

[0015] 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. HK16 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 ; Effects of different carbon and nitrogen sources on the growth of strain HK16 are shown in Figure 3 The carbon source that is beneficial to the growth of strain HK16 is sucrose; the nitrogen source is ammonium chloride.

[0016] 2. Growth and phosphorus removal rate of HK16 under different treatment times The HK16 bacterial suspension was inoculated at 6% into a culture medium containing phosphorus-containing synthetic wastewater (same as Example 1, 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 was measured using a UV spectrophotometer. 600 value, continue until OD 600 When the value decreased or the phosphorus content tended to be stable, the culture was centrifuged at 12,000 rpm for 5 minutes, and the total phosphorus content of the culture supernatant was measured.

[0017] The results are as follows Figure 4 As can be seen from the figure, strain HK16 has a strong adaptability to the new environment and grows faster. It enters the logarithmic growth phase from the 1st hour and reaches the stable period at the 16th hour. The phosphorus adsorption capacity of strain HK16 is highly consistent with its growth curve characteristics. Strain HK16 reaches the highest phosphorus adsorption efficiency of 60.88% at the 22nd hour.

[0018] 3. Phosphorus removal rate of strain HK16 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 HK16 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 HK16 was calculated.

[0019] See the results Figure 5 It can be seen that HK16 has better phosphorus adsorption effect in alkaline environment than in acidic environment. The phosphorus removal rate of HK16 is the highest at a solution pH of 11, which is 84.34%.

[0020] 4. Phosphorus removal rate of Serratia HK16 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 dipotassium hydrogen phosphate is 0.0278 g). The difference is that the culture medium pH is 7. The HK16 bacterial suspension is inoculated into 10 mL of the culture medium at a 6% inoculum size. The culture is cultured with constant temperature shaking (200 rpm) at 15°C, 25°C, 30°C, 35°C, and 40°C for 22 hours. The culture solution is centrifuged at 12,000 rpm for 5 minutes, the phosphorus content in the liquid phase is detected, and the HK16 phosphorus removal rate is calculated. Figure 6 The results showed that the phosphorus removal efficiency was high in the range of 15℃~30℃, with a phosphorus removal rate higher than 92.96%. Above 30℃, the phosphorus removal efficiency decreased significantly. The phosphorus removal effect was the worst at 40℃, with a phosphorus removal rate of only 62.47%. Under high temperature conditions, although some bacteria may survive, their metabolic activity decreased and the phosphorus removal efficiency would also decrease.

[0021] 5. Growth and phosphorus removal rate of HK16 under different initial phosphorus concentrations The culture medium used in this example was the same as the phosphorus-containing synthetic wastewater culture medium in Example 1, except that the phosphorus concentrations were 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 was 0.0278 g, 0.0556 g, 0.1111 g, 0.1667 g, 0.2222 g, and 0.2778 g). The HK16 bacterial suspension was inoculated into 10 mL of the phosphorus-containing synthetic wastewater culture medium at a 6% inoculum, and then cultured at 28°C with constant shaking (200 rpm) for 22 hours. After the incubation period, the culture was centrifuged at 12,000 rpm for 5 minutes, the phosphorus concentration in the supernatant was measured, and the phosphorus removal efficiency of HK16 was calculated. See the results Figure 7 When the phosphorus concentration increased from 5 mg / L to 50 mg / L, the phosphorus removal efficiency of HK16 decreased from 99.40% to 12.96%, and the phosphorus removal rate decreased with the increase of phosphorus concentration.

[0022] Example 4: Safety Evaluation of Serratia HK16 ① Antibiotic susceptibility testing of strain HK16 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. ②Hemolysis tests were performed using blood (sheep blood) agar plates (Changde Beekman Biotechnology Co., Ltd.). Antimicrobial susceptibility testing was performed using antimicrobial susceptibility discs (Hangzhou Microbiological Reagent Co., Ltd., Hangzhou, China) according to the European Committee on Antimicrobial Susceptibility Testing (ECAST) disk diffusion method.

[0023] The results are shown in Table 2. Strain HK16 showed resistance to tetracycline and streptomycin (inhibition zone ≤ 5 mm), but was sensitive to antibiotics such as chloramphenicol and kanamycin (inhibition zone ≥ 10 mm). The hemolysis test showed that the strain had no hemolytic activity (see Figure 8 ), in compliance with biosafety requirements.

[0024] Table 2: Antibiotic sensitivity test of strain HK16

[0025] Note: 0-5mm: no effect; 6-15mm: weak effect; >15mm: strong inhibitory effect.

[0026] Practical application of strain HK16 in aquaculture wastewater After collecting aquaculture wastewater (phosphorus concentration of 3.052 mg / L), the experimental group was inoculated with a 6% suspension of HK16, while the control group was inoculated with a 6% suspension of LB liquid medium. Three biological replicates were set up for each group. Cultures were shaken at 28°C and 150 rpm for 36 hours. Samples were taken every 12 hours, and the culture medium was centrifuged at 12,000 rpm for 5 minutes. The phosphorus content in the liquid was then measured, and the phosphorus removal efficiency of HK16 in practical applications was calculated.

[0027] like Figure 9 As shown in the figure, the phosphorus removal rate of strain HK16 increased with time. The phosphorus removal rate reached 63.55% at 36 hours, while the phosphorus removal rate of the control group was only 3.48% at 36 hours, further demonstrating the practical application value of HK16 in treating aquaculture wastewater.

Claims

1. A species of Serratia marcescens ( Serratia sp.) HK16, whose deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No. 66497.

2. Use of the Serratia HK16 according to claim 1 in phosphorus removal in water bodies.

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