A sphingobacterium strain and its application in river water treatment

Abstract

The present invention relates to a sphingobacterium strain and its application in river water management. In order to solve the problem that existing microbial strains cannot take into account the removal efficiency of ammonia nitrogen, COD and phosphorus at the same time, the present invention has developed a strain for river water management, which is a sphingobium Sphingobium sp.Q‑13 strain, which was deposited in the China Center for Type Culture Collection on May 27, 2022, with a deposit number of CCTCC No: M 2022746. The strain can remove more than 75% of COD, ammonia nitrogen and total phosphorus in eutrophic water bodies in rivers, can be used for river water management, restore the self-purification capacity of rivers, and has no pollution to the environment and low processing cost, which is of great significance to river water management.

Description

Technical Field

[0001] The present invention specifically relates to a sphingobacterium strain and application thereof in river water treatment. Background Art

[0002] Currently, the global water pollution situation is grim, with water functions gradually deteriorating and eutrophication a major concern. With the rapid development of the economy, industry, and agriculture, people are increasingly neglecting the protection of the water environment. Domestic sewage discharged from urban and rural areas contains large amounts of organic matter, nitrogen, phosphorus, and other pollutants. Over time, this leads to the proliferation of algae and other organisms in water bodies, resulting in eutrophication of lakes, rivers, and aquaculture water. Although rivers have a certain degree of self-purification function, this function is gradually impaired as eutrophication increases. Currently, there are various methods to control river water pollution, such as traditional physical and chemical methods. However, these more traditional methods are not only slow in degradation but also introduce new pollution, resulting in certain limitations. Biological remediation, on the other hand, is an effective long-term measure to eliminate endogenous pollution in river water. Aquatic plants and animals utilize nutrients, such as nitrogen, nitrite, and phosphorus, in river water during their metabolism. Biological remediation has been widely recognized by experts both domestically and internationally and holds great promise for the remediation of eutrophic river water bodies. In biological control, microbial biocontrol utilizes their physiological properties, such as mutualism and complementary interactions, to screen, amplify, and culture microorganisms to create corresponding microbial agents for release into river waters. This approach is both safe, harmless, and cost-effective, and has become a hot topic of research among experts in recent years. An increasing number of microbial strains have been developed for removing ammonia nitrogen, COD, or phosphorus from water bodies, achieving relatively good removal results. However, existing strains, when used individually, typically only offer good removal efficiency for one of these indicators, or some reports suggest that selected strains are simultaneously effective for both ammonia nitrogen and nitrite, but no strain has been found that can simultaneously achieve effective removal of ammonia nitrogen, COD, and phosphorus. To effectively remove ammonia nitrogen, COD, and phosphorus simultaneously, different strains must be combined to create a strain complex. However, due to the varying requirements of different strains for conditions such as water temperature, achieving effective treatment results for each strain in the complex is difficult, and the production cost of the complex is also high. Therefore, developing a strain that can simultaneously achieve effective removal of ammonia nitrogen, COD, and phosphorus could provide more options for river water management and restoration of river self-purification capacity, while also reducing wastewater treatment costs. Summary of the Invention

[0003] The purpose of the present invention is to provide a sphingobacterium strain that can simultaneously take into account the removal efficiency of ammonia nitrogen, COD and phosphorus in river water.

[0004] Another object of the present invention is to provide the use of the sphingobacterium strain in river water treatment.

[0005] Another object of the present invention is to provide a method for treating river water bodies using the sphingobacterium strain.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A sphingobacterium strain, wherein the sphingobacterium strain is a sphingobium sp. Q-13 strain, which was deposited in the China Center for Type Culture Collection on May 27, 2022, with a deposit number of CCTCC No: M 2022746, and the deposit address is Wuhan University, Wuhan, China.

[0008] Preferably, the sphingobacterium strain grows well at 25-30° C., and the sphingobacterium strain is identified as Sphingobium yanoikuyae strain.

[0009] Preferably, the sphingobacterium strain is non-motile and aerobic, and when cultured on a plate, the bacteria are yellow, round, and have a moist and smooth surface.

[0010] Preferably, the screening method comprises the following steps:

[0011] (1) Enrichment culture: Take a water sample from a river and culture it in simulated sewage with a COD concentration of 60-70 mg / L and an ammonia nitrogen concentration of 5-10 mg / L at 25-30°C and 150-200 r / min for 40-60 h to obtain an enrichment culture solution;

[0012] (2) Isolation and purification: The enriched culture fluid of step (1) is diluted in a gradient manner, and then spread on a beef extract peptone medium plate, cultured at 25-30°C for 40-60 hours, and a single colony is picked and transferred to a beef extract peptone slant to isolate multiple strains;

[0013] (3) Preliminary screening: The multiple strains isolated in step (2) were inoculated into simulated sewage with a COD concentration of 60-70 mg / L and an ammonia nitrogen concentration of 5-10 mg / L, and cultured at 25-30°C and 100-150 rpm for 2-3 days. After the culture was completed, the COD concentration and ammonia nitrogen concentration in the culture solution were detected. Strains that could achieve a COD removal rate of more than 65% and an ammonia nitrogen removal rate of more than 45% were selected as candidate strains;

[0014] (4) Secondary screening: The candidate strains of step (3) were inoculated into simulated sewage with a total phosphorus content of 0.25 to 0.35 mg / L, and cultured under shaking conditions of 25 to 30° C. and 100 to 150 r / min for 2 to 3 days. After the culture was completed, the total phosphorus content in the culture solution was detected, and the strain with the highest total phosphorus removal rate was selected, which was the sphingobacterium strain.

[0015] The present invention also provides application of the sphingobacterium strain or its fermentation liquid in river water treatment.

[0016] Preferably, the river water treatment includes using the sphingobacterium strain or its fermentation liquid to remove COD, ammonia nitrogen and total phosphorus in the river water.

[0017] The present invention also provides a method for treating river water bodies, which uses the sphingomyelinase bacteria strain or its fermentation liquid to remove COD, ammonia nitrogen and total phosphorus in the river water body.

[0018] Preferably, the Sphingobium sp. Q-13 strain is inoculated into the river water to be treated and cultured for 2 to 4 days.

[0019] More preferably, the concentration of the Sphingobium sp. Q-13 strain seed solution is adjusted to 1×10 8 ~9×10 8 CFU / mL, inoculated into river water at an inoculation rate of 1% to 5%, the temperature of the river water treatment is 25 to 30°C, and the pH value is 6.0 to 9.0.

[0020] Preferably, the COD concentration in the river water to be treated is 50-70 mg / L, the ammonia nitrogen concentration is 4-10 mg / L, and the total phosphorus content is 0.2-0.5 mg / L.

[0021] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0022] The sphingobacterium strain of the present invention has a good removal efficiency for COD, ammonia nitrogen and total phosphorus in river water bodies. The removal rate of COD, ammonia nitrogen and total phosphorus in eutrophic river water bodies can reach more than 75%. It can be used for river water body management and restore the self-purification capacity of the river. It has no cost to the environment and low treatment cost, which is of great significance to river water body management. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison of the removal rates of COD and ammonia nitrogen by different strains in Example 1;

[0024] Figure 2 This is a comparison of the total phosphorus removal rates of different strains in Example 1;

[0025] Figure 3 This is the phylogenetic tree of Sphingobium sp.Q-13 strain;

[0026] Figure 4 Comparison of the removal rates of ammonia nitrogen, COD, and total phosphorus in simulated sewage by the Sphingobium sp. Q-13 strain in Example 2;

[0027] Figure 5 This is a graph showing the change in ammonia nitrogen removal rate of the sphingobium sp. Q-13 strain in the eutrophic water of the river over time in Example 4;

[0028] Figure 6 This is a graph showing the change in total phosphorus removal rate of the sphingobium sp. Q-13 strain in eutrophic river water over time in Example 4;

[0029] Figure 7 This is a graph showing the change in COD removal rate of the sphingobium sp. Q-13 strain in the eutrophic water body of the river over time in Example 4. DETAILED DESCRIPTION

[0030] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0031] In the present invention, all the raw materials can be purchased commercially and / or prepared by known means. Unless otherwise specified, they all meet the requirements of standard chemical products.

[0032] In the present invention, unless otherwise specified, the "%" refers to mass percentage.

[0033] In the present invention, the seed solution of strain Q-13 was cultured using conventional methods, and the experimental methods in the following examples are all conventional methods unless otherwise specified.

[0034] In the present invention, the ammonia nitrogen detection method refers to the national standard HJ535-2009 Water Quality Ammonia Nitrogen Determination Nessler Reagent Spectrophotometry; the COD detection method refers to the national standard HJ 828-2017 Water Quality Chemical Oxygen Demand Determination Dichromate Method. The total phosphorus detection method refers to the potassium persulfate digestion ammonium molybdate spectrophotometry method (GB11893-89).

[0035] In the following examples and comparative examples, the simulated sewage containing COD and ammonia nitrogen concentrations of 70 mg / L and 7.0 mg / L, respectively, was prepared by dissolving glucose and NH4Cl in water to a glucose concentration of 70 mg / L and an NH4Cl concentration of 21 mg / L, and adjusting the pH to 7.0 to obtain the simulated sewage for the experiment.

[0036] In the following examples and comparative examples, the preparation method of simulated sewage with a total phosphorus content of 0.3 mg / L is as follows: weigh 70 mg of glucose and 21 mg of NH4Cl, add 300 μL of 1 mg / mL total phosphorus mother solution, make the volume to 1000 mL, and adjust the pH value to 7.0.

[0037] Example 1

[0038] Isolation and screening of strains capable of simultaneously removing COD, ammonia nitrogen and total phosphorus

[0039] (1) Enrichment culture: 20 mL of river water sample was collected from the Shengze River in Suzhou and inoculated into a 500 mL Erlenmeyer flask containing 200 mL of simulated sewage with COD and ammonia nitrogen concentrations of 70 mg / L and 7.0 mg / L, respectively. The sample was placed at 28°C and 180 rpm for 48 h to obtain the enrichment culture solution.

[0040] (2) Isolation and purification: The enriched culture fluid was diluted with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 There were 5 concentration gradients in total. 100 μL of samples from each concentration gradient were taken using a pipette and spread on a beef extract peptone medium plate. The plate was placed in a constant temperature incubator and cultured at 28°C for 48 hours. Single colonies were picked from the plate and transferred to a beef extract peptone slant. Six strains were isolated and screened.

[0041] (3) Preliminary screening: The six different strains were transferred to the slant of beef extract peptone culture medium, cultured at 28°C for 24 h, and the bacteria were prepared with 0.85% saline to a concentration of 1×10 8 CFU / mL of bacterial suspension, aspirate 1 ml of the bacterial suspension and transfer it to a conical flask containing 100 mL of simulated sewage with COD and ammonia nitrogen concentrations of 75 mg / L and 7.5 mg / L respectively, and culture it in a shaking incubator at 28°C and 180 r / min for 48 hours. After the culture is completed, let it stand for 20 minutes, measure the COD concentration and ammonia nitrogen concentration of the supernatant, and use the simulated sewage without bacterial suspension as a control to calculate the removal rates of COD and ammonia nitrogen by different strains. Strains that can make the COD removal rate reach more than 65% and the ammonia nitrogen removal rate reach more than 45% are selected as candidate strains. Figure 1The results showed that the COD removal rates of strains 1, 5 and 6 could reach over 65%, and the ammonia nitrogen removal rates could reach over 45%. Strain 1, 5 and 6 were selected as candidate strains.

[0042] (4) Secondary screening: strains 1, 5, and 6 were transferred to a slant of beef extract peptone culture medium and cultured at 28°C for 24 h. The bacteria were prepared with 0.85% saline to a concentration of 1 × 10 8 CFU / mL of bacterial suspension, aspirate 1 ml of the bacterial suspension and transfer it to a conical flask containing 100 mL of simulated sewage containing 0.3 mg / L of total phosphorus, and culture it in a shaking incubator at 28°C and 180 r / min for 48 hours. After the culture is completed, let it stand for 20 minutes, and determine the total phosphorus content of the supernatant. Use the phosphorus-containing simulated sewage without bacterial suspension as a control, calculate the total phosphorus removal rate of different strains, and select the strain with the best total phosphorus removal effect. Figure 2 The results showed that the total phosphorus removal rate of strain 5 could reach 86.48%. Strain 5 was named Q-13.

[0043] Identification of Q-13 strain:

[0044] (1) Cultivation characteristics of strain Q-13: This strain is non-motile and aerobic. When cultured on plates, the bacteria are yellow, round, and have a smooth and moist surface.

[0045] (2) 16S rRNA sequence analysis: The strain Q-13 was sent to Shanghai Sangon Biotechnology Co., Ltd. for 16S rRNA gene PCR amplification and sequencing. The 16S rRNA gene sequence phylogenetic tree was constructed using MEGA7.0 software (see Figure 3 ), the 16S rRNA gene sequencing results of Q-13 strain were compared with the homologous sequences in NCBI, and it was most closely related to strain NR_113730.1 Sphingobium yanoikuyae. Combined with the culture characteristics of the strain, strain Q-13 was identified as Sphingobium yanoikuyae (Sphingobium yanoikuyae).

[0046] Example 2

[0047] Verification of the effect of strain Q-13 on simultaneous removal of COD, ammonia nitrogen and total phosphorus in simulated sewage

[0048] The strain Q-13 seed solution was centrifuged and washed three times with 0.85% saline, and the concentration was adjusted to 1×10 8CFU / mL, inoculated at a 1% inoculum size into 500mL Erlenmeyer flasks containing 200mL of simulated sewage containing 70mg / L, 7.0mg / L and 0.3mg / L of COD, ammonia nitrogen and total phosphorus respectively, and cultured in a constant temperature shaking incubator at 28℃ and 180r / min for 2 days. After the culture was completed, the supernatant was allowed to stand for 20min, and the COD, ammonia nitrogen and total phosphorus concentrations were measured. The simulated sewage without inoculation of strain Q-13 seed liquid was used as the control to calculate the removal rates of COD, ammonia nitrogen and total phosphorus. The results are shown in the table. Figure 4 . Figure 4 The results showed that the removal rates of COD, ammonia nitrogen and total phosphorus of strain Q-13 for simulated sewage were 81.98%, 78.12% and 83.56%, respectively.

[0049] Example 3

[0050] Comparison of the effects of strain Q-13 and Sphingomonas (BNCC335865, Beijing Beina Chuanglian Biotechnology Research Institute) on simultaneous removal of COD, ammonia nitrogen and total phosphorus

[0051] Q-13 and Sphingomonas (BNCC335865, Beijing Beina Chuanglian Biotechnology Research Institute) were activated and the concentration was adjusted to 1×10 8 CFU / mL, and inoculated at a 5% inoculum size into 500mL Erlenmeyer flasks containing 200mL of simulated sewage containing 70mg / L, 7.0mg / L and 0.3mg / L of COD, ammonia nitrogen and total phosphorus, respectively. The culture was carried out in a constant temperature shaking incubator at 28℃ and 180r / min for 2 days. After the culture was completed, the culture was allowed to stand for 20min, and the COD, ammonia nitrogen and total phosphorus concentrations of the supernatant were determined. The simulated sewage without inoculation of strain Q-13 seed liquid was used as a control to calculate the removal rates of COD, ammonia nitrogen and total phosphorus. The results are shown in Table 1. Table 1 shows that strain Q-13 and Sphingomonas achieved comparable removal efficiencies for ammonia nitrogen in simulated wastewater, both reaching approximately 77%. Strain Q-13 achieved a total phosphorus removal rate of 83.33%, while Sphingomonas achieved a 58.33% removal rate. Strain Q-13 also achieved a COD removal rate of 81.43%, while Sphingomonas achieved a 68.57% removal rate. Strain Q-13 demonstrated a balanced removal of COD, ammonia nitrogen, and total phosphorus, while Sphingomonas demonstrated poor COD and total phosphorus removal efficiencies.

[0052] Table 1

[0053]

[0054] Example 4

[0055] Verification of the effect of strain Q-13 on simultaneous removal of COD, ammonia nitrogen and total phosphorus from eutrophic river water

[0056] The eutrophic river water used for the test was taken from the Shengze River in Suzhou. After measurement, the initial COD concentration in the water was 53 mg / L, the initial ammonia nitrogen concentration was about 4.25 mg / L, and the initial total phosphorus content was about 0.350 mg / L.

[0057] The strain Q-13 seed solution was centrifuged and washed three times with 0.85% saline, and the concentration was adjusted to 10 8 CFU / mL, 5% of the inoculum was inoculated into 500mL triangular flasks containing 200mL of eutrophic river water, and cultured in a constant temperature shaking incubator at 28℃ and 180r / min for 3 days. The COD, ammonia nitrogen and total phosphorus concentrations of the eutrophic river water were measured at different treatment times, and the removal rates of COD, ammonia nitrogen and total phosphorus were calculated. The results are shown in the table. Figures 5 to 7 The results showed that after 60 hours of treatment, the removal rates of COD, ammonia nitrogen and total phosphorus indicators of eutrophic river water by strain Q-13 were 77.36%, 80.71% and 77.71%, respectively, while taking into account the removal effects of COD, ammonia nitrogen and total phosphorus.

[0058] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A sphingobacterium strain, characterized in that The sphingobacterium strain is the Sphingobium yanoikuyae strain, which was deposited in the China Center for Type Culture Collection on May 27, 2022, with a deposit number of CCTCC No: M 2022746.

2. The sphingobacterium strain according to claim 1, characterized in that The sphingobacterium strain grows well at 25-30°C.

3. The sphingobacterium strain according to claim 1, characterized in that The sphingobacterium strain is non-motile and aerobic. When cultured on a plate, the bacteria are yellow, round, and have a moist and smooth surface.

4. The use of the sphingobacterium strain or its fermentation liquid in river water treatment according to claim 1, characterized in that: The river water treatment is to use the sphingomyelinase strain or its fermentation liquid described in claim 1 to remove COD, ammonia nitrogen and total phosphorus in the river water.

5. A method for regulating river water bodies, characterized in that: The sphingomyelinase bacteria strain or its fermentation liquid according to claim 1 is used to remove COD, ammonia nitrogen and total phosphorus in river water.

6. The river water treatment method according to claim 5, characterized in that: The Sphingobium yanoikuyae strain is inoculated into the river water to be treated and cultured for 2 to 4 days.

7. The method for regulating river water bodies according to claim 5, characterized in that: The concentration of the Sphingobium yanoikuyae seed solution was adjusted to 1×10 8 ~9×10 8 CFU / mL, inoculated into river water at an inoculation rate of 1% to 5%, the temperature of the river water treatment is 25 to 30°C, and the pH value is 6.0 to 9.

0.

8. The river water treatment method according to claim 5, characterized in that: The COD concentration in the river water to be treated is 50-70 mg / L, the ammonia nitrogen concentration is 4-10 mg / L, and the total phosphorus content is 0.2-0.5 mg / L.

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