A strain of Shewanella and its application in the treatment of black and smelly water

Abstract

The invention discloses a strain of Shewanella and application thereof in treating black and smelly water. The Shewanella is Baltic Shewanella ( Shewanella baltica ), named R-2, and deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on March 29, 2024, with the deposit number: CGMCC No.30187. The Shewanella R-2 strain of the present invention can effectively degrade dinitro-o-cresol, phosphorus, COD and ammonia nitrogen pollutants in black and odorous water bodies, has excellent degradation effect, and the strain is resistant to low temperature and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Shewanella and application thereof in the treatment of black and smelly water bodies. Background Art

[0002] There are currently a variety of treatment technologies and methods for the pollution of black and odorous water bodies. Source control and pollution interception are fundamental measures to control pollutants from entering the water body at the source. By strengthening the supervision of sewage outlets, limiting pollutant emissions, and building sewage treatment facilities, the input of exogenous pollutants can be reduced. At the same time, through dredging, ecological restoration, and active water circulation, the internal pollution of water bodies can be improved and the self-purification capacity of water bodies can be improved. The application of these treatment technologies and methods requires comprehensive consideration of multiple factors such as the degree of water pollution, treatment objectives, and economic conditions, and the formulation of scientific and reasonable treatment plans.

[0003] As a typical black and smelly water pollutant, the research and development of dinitro-o-cresol treatment technology faces many challenges, and an efficient and widely applicable treatment method has not yet been formed. This pollutant mainly comes from industrial production processes such as chemicals, dyes, and wood preservation. Once it enters the water body, it will cause long-term and serious pollution to the water quality. In black and smelly water bodies, the presence of dinitro-o-cresol not only aggravates the stench and blackening of the water body, but also poses a potential threat to aquatic organisms and human health due to its bioaccumulation and difficulty in degradation. Traditional water treatment methods, such as physical precipitation and chemical oxidation, are not effective in treating dinitro-o-cresol. The physical precipitation method is difficult to effectively separate it from the water body; the chemical oxidation method requires a large amount of oxidant and may cause secondary pollution. Therefore, it is particularly important to develop an efficient treatment technology for dinitro-o-cresol in black and smelly water bodies. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a low-temperature resistant Baltic Shewanella which has excellent degradation effects on dinitro-o-cresol, phosphorus, COD and ammonia nitrogen pollutants in black and odorous water bodies.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A strain of Shewanella, the Shewanella being Shewanella balticum ( Shewanella baltica ), named R-2, and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.30187.

[0007] Inoculum prepared from Shewanella spp.

[0008] The application of Shewanella or its bacterial agent in the degradation of dinitro-o-cresol in black and odorous water bodies, the concentration of dinitro-o-cresol in the black and odorous water bodies is 100-250 mg / L, the pH is 6.0-8.0, and the temperature is 8-30°C.

[0009] The method for degrading dinitro-o-cresol in black and smelly water by using a bacterial agent comprises the following specific steps: adding the bacterial agent into the black and smelly water at a volume ratio of 1% to 3%, stirring evenly, and performing degradation.

[0010] Application of Shewanella or bacterial agents in degrading phosphorus, COD and ammonia nitrogen in black and smelly water bodies.

[0011] A method for preparing a bacterial agent comprises the following specific steps:

[0012] S1. Cultivate strain R-2 in LB medium to obtain seed solution;

[0013] S2, inoculating the seed solution into a fermentation medium for expansion and culture to obtain a bacterial solution;

[0014] S3, mixing the bacterial solution with zeolite powder, and drying to obtain a solid bacterial agent.

[0015] Preferably, in the aforementioned step S1, the components of the LB medium are: 5 g yeast extract, 10 g peptone, 5 g NaCl and 1 L water; in the aforementioned step S2, the components of the fermentation medium are: 6% sucrose, 2.5% peptone, and 0.7% calcium carbonate; the inoculation amount of the seed liquid is 2%~5%, and the culture conditions are 20~30°C and the pH value is 6~8.

[0016] The invention is beneficial in that: the invention screens out a Baltic Sea Shewanella R-2 strain capable of degrading black and odorous water bodies, the strain has excellent degradation effects on dinitro-o-cresol, phosphorus, COD and ammonia nitrogen pollutants, and the strain is resistant to low temperatures and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the colony morphology of strain R-2;

[0018] Figure 2 is the phylogenetic tree of strain R-2;

[0019] Figure 3 This is a graph showing the growth ability of strain R-2 at different temperatures;

[0020] Figure 4 This is the degradation effect diagram of strain R-2 on dinitro-o-cresol at different pH;

[0021] Figure 5 This is a graph showing the degradation effect of strain R-2 on dinitro-o-cresol at different temperatures;

[0022] Figure 6 This is the degradation effect diagram of strain R-2 under different initial concentrations of dinitro-o-cresol;

[0023] Figure 7 This is a diagram showing the effect of phosphorus removal by strain R-2;

[0024] Figure 8 This is a graph showing the degradation effect of strain R-2 on COD and ammonia nitrogen in black and smelly water. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1, Isolation and identification of strain R-2:

[0027] Water samples were extracted from black and smelly water bodies in multiple places. After dilution at a certain multiple, 50 μL was taken and spread on solid plates. The plates were inverted and cultured for 24 hours at a low temperature of 10°C. After that, single colonies were selected and streaked again to separate the purified strains. The isolated strains were inoculated into LB liquid culture medium and cultured at 25°C and 180rpm for 24 hours to obtain activated bacterial solution. The obtained bacterial solution was inoculated into simulated high-phosphorus wastewater at a ratio of 10%, and cultured at 25°C and 180rpm. The strain with the strongest phosphorus removal ability was selected and named R-2.

[0028] The above LB medium contains the following ingredients: 10 g / L peptone, 3 g / L yeast extract, and 10 g / L sodium chloride.

[0029] The components of the simulated high-phosphorus wastewater are: glucose 1.5 g / L, sodium acetate 0.75 g / L, magnesium sulfate heptahydrate 1.18 g / L, ammonium chloride 0.9 g / L, and potassium dihydrogen phosphate 0.2 g / L.

[0030] All culture media were sterilized at 121°C for 20 min before use; inoculation was carried out in a clean workbench; and the strains were stored at -80°C with glycerol.

[0031] The strain shows strong adaptability under a wide range of growth conditions. It can grow in a temperature range of 5°C to 35°C, in an acid-base environment with a pH value of 5.8 to 9.8, and in salinity conditions of 0% to 12%, and its growth process requires sufficient oxygen. It is particularly worth mentioning that the strain performs well in removing phosphorus and has a significant phosphorus removal effect. Morphologically, the strain is negative after Gram staining. Under an electron microscope, it is a bacillus with special structures such as capsules and flagella. After culturing on solid culture medium for 24 hours, the colony morphology is round and milky white. The colony morphology is shown in the figure below. Figure 1 shown.

[0032] The present invention determines the 16S rDNA sequence of the strain, and the sequence table thereof is shown in SEQ ID NO: 1. The DNA of the strain R-2 was extracted by a gene extraction kit and used as a PCR reaction template, and the primers for bacterial amplification were bacterial universal primers. 27F: (5-AGAGTTTGATC-CTGGCTCAG-3) and 1492R: (5- GGTTACCTTGTTACGACTT-3), 25 μL 2×Mix Taq was mixed with 2 μL of each forward and reverse primer and 2 μL of bacterial solution, and then PCR amplification was performed after adding water to a 50 μL system. The PCR reaction program was 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s; 55°C renaturation for 1 min; 72°C extension for 1 min, for a total of 30 cycles; 72°C extension for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and then sent to a sequencing company for sequencing, and the 16S rDNA sequence results of the strain were searched and compared in the NCBI database using the BLAST method. The 16S rDNA sequences of strain R-2 and its related strains were aligned using Mega (Version 7.0) software, and the phylogenetic tree was constructed using the Neighbor-joining method, as follows Figure 2 The strain R-2 was identified as belonging to the genus Shewanella and was named Shewanella Baltica. Shewanella baltica R-2.

[0033] The above strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024, and its classification name is Baltic Shewanella ( Shewanella baltica )R-2, the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is: No.30187.

[0034] Example 2: The preparation of the microbial agent comprises the following steps:

[0035] The strain Shewanella baltica R-2 was inoculated into 100 mL LB medium and cultured in a shaker at 25°C and 180 rpm for 24 h to obtain seed solution. The seed solution was then inoculated into a fermentation medium in a fermenter at a volume ratio of 2-5% for expansion culture. The culture was continued until 10 9 When the concentration reached CFU / mL, the bacterial sludge was collected by centrifugation at 4000 rpm; the bacterial sludge was mixed with zeolite powder at a volume ratio of 1:3 and dried at 25°C to obtain a solid bacterial agent.

[0036] The components of the LB medium are: 5 g yeast extract, 10 g peptone, 5 g NaCl and 1 L water, and the culture conditions are: temperature 25° C., time 24 h, pH 6-8.

[0037] The components of the fermentation medium are: sucrose 6%, peptone 2.5%, calcium carbonate 0.7%, and the culture conditions are: temperature 25°C, time 36 h, pH 6-8.

[0038] Example 3, Growth ability test of strain R-2 at different temperatures:

[0039] The temperature was set at 0℃, 4℃, 8℃, 15℃, 20℃, 25℃, 30℃, and 40℃, and the strain R-2 was inoculated into 8 groups of liquid culture media at an inoculation rate of 1% (v / v). The liquid culture media components were: peptone 10g / L, yeast extract 3g / L, and sodium chloride 10g / L. Each group was repeated three times and cultured at 180rpm. Samples were taken every 4h to measure the OD600 value. The growth curve is shown in the figure. Figure 3 shown.

[0040] Depend on Figure 3 It can be seen that the most suitable growth temperature for strain R-2 is 25℃, its temperature tolerance is 4~30℃, and it can grow well at 8℃, showing low temperature resistance.

[0041] Example 4, Degradation effect of strain R-2 at different pH:

[0042] Eight groups of liquid culture media with an initial concentration of dinitro-o-cresol of 200 mg / L were taken respectively. Different pH conditions (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively) were set for each group of liquid culture media. The strain R-2 was inoculated into the liquid culture media with different pH values ​​at an inoculation amount of 1% (v / v). The speed was 180 r / min and the temperature was 25°C. The culture was cultured for 48 h. The culture fluid at the end of the above culture was sampled to detect the degradation of dinitro-o-cresol. Figure 4 shown.

[0043] Depend on Figure 4 It can be seen that the strain can adapt to a wide range, the optimal treatment effect is between pH 6.0 and 8.0, and the degradation rate of dinitro-o-cresol is higher than 80%.

[0044] Example 5, Degradation effect of strain R-2 at different temperatures:

[0045] Six groups of liquid culture media with an initial concentration of dinitro-o-cresol of 200 mg / L were taken respectively. Different temperatures were set for each group of liquid culture media, namely 4°C, 8°C, 15°C, 20°C, 25°C, 30°C, and 40°C. The strain R-2 was inoculated into the liquid culture media at different temperatures at an inoculation amount of 1% (v / v). The pH was 6.0 and the rotation speed was 180 r / min. The culture was cultured for 48 h. The culture fluid at the end of the above culture was sampled to detect the degradation of dinitro-o-cresol. Figure 5 shown.

[0046] Depend on Figure 5 It can be seen that the strain can adapt to a wide range. At 8°C, the degradation rate of dinitro-o-cresol is 67%, and the degradation effect is good. When the temperature is 15-30°C, the treatment effect is best, and the degradation rate of dinitro-o-cresol can reach more than 80%.

[0047] Example 6, Degradation effect of strain R-2 at different initial concentrations of dinitro-o-cresol:

[0048] Under the conditions that the initial concentration of dinitro-o-cresol was 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L, the strain R-2 was inoculated into the liquid culture medium at an inoculation rate of 1% (v / v), pH 6.0, rotation speed 180 r / min, temperature 25°C, and cultured for 48 h. Samples were taken to detect the degradation of dinitro-o-cresol. Figure 6 shown.

[0049] Depend on Figure 6 It can be seen that when the initial concentration of dinitro-o-cresol is 100~250 mg / L, the degradation rate is above 85%, and the degradation effect is excellent.

[0050] Example 7, phosphorus removal effect of strain R-2:

[0051] The strain R-2 was cultured in LB liquid culture medium at 25°C and 200rpm for 48 hours to obtain an activated bacterial solution. The strain R-2 was inoculated into liquid culture medium with phosphorus concentrations of 5mg / L, 10mg / L, 20mg / L, 30mg / L, 40mg / L, and 50mg / L at an inoculation rate of 1% (v / v), and cultured in a shaker at 25°C and 200rpm. The phosphorus concentration in the supernatant was measured at different times, and the biomass was measured at a wavelength of 600nm to obtain the phosphorus removal efficiency of the strain and the growth curve of the strain at different phosphorus concentrations. The results are shown in FIG. Figure 7 shown.

[0052] Depend on Figure 7 It can be seen that the phosphorus removal rate of strain R-2 can be close to 100% after growing in low-concentration phosphorus wastewater for 48 hours, while the effect is reduced in high-phosphorus wastewater.

[0053] Example 8, Treatment effect of strain R-2 on black and odorous water bodies:

[0054] Take 10m 3The black and smelly water in a river channel has concentrations of 200 mg / L, 5 mg / L, 100 mg / L and 100 mg / L of dinitro-orthocresol, phosphorus, COD and ammonia nitrogen, respectively. The bacterial agent is evenly added to the black and smelly water at an inoculum rate of 1% (v / v), stirred evenly, and degraded at 25°C for 48 hours. The degradation effect is as follows: Figure 8 shown.

[0055] Depend on Figure 8 It can be seen that the degradation efficiency of the bacterial agent on dinitro-o-cresol, phosphorus, COD and ammonia nitrogen are 91.5%, 93.7%, 72.4% and 64.3% respectively. The experimental data show that the bacterial agent can not only degrade dinitro-o-cresol in black and smelly water bodies, but also effectively degrade phosphorus, COD and ammonia nitrogen in black and smelly water bodies, and has good application prospects in the field of black and smelly water treatment.

[0056] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A strain of Shewanella, characterized in that The Shewanella is Baltic Shewanella ( Shewanella baltica ), named R-2, and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.30187.

2. A bacterial agent prepared using the Shewanella described in claim 1.

3. Use of the Shewanella described in claim 1 or the bacterial agent described in claim 2 in degrading dinitro-o-cresol in black and smelly water, characterized in that: The concentration of dinitro-o-cresol in the black and smelly water is 100-250 mg / L, the pH is 6.0-8.0, and the temperature is 8-30°C.

4. A method for degrading dinitro-o-cresol in black and smelly water using the bacterial agent according to claim 2, characterized in that: The specific steps are: add the bacterial agent into the black and smelly water at a volume ratio of 1% to 3%, stir evenly, and degrade it.

5. Use of the Shewanella described in claim 1 or the bacterial agent described in claim 2 in degrading phosphorus, COD and ammonia nitrogen in black and smelly water.

6. A method for preparing the bacterial agent according to claim 2, characterized in that: The specific steps include: S1. Cultivate strain R-2 in LB medium to obtain seed solution; S2, inoculating the seed solution into a fermentation medium for expansion and culture to obtain a bacterial solution; S3, mixing the bacterial solution with zeolite powder, and drying to obtain a solid bacterial agent.

7. The method for preparing the bacterial agent according to claim 6, characterized in that: In step S1, the components of the LB medium are: 5 g yeast extract, 10 g peptone, 5 g NaCl and 1 L water; in step S2, the components of the fermentation medium are: 6% sucrose, 2.5% peptone, and 0.7% calcium carbonate; the inoculation amount of the seed liquid is 2%-5%, and the culture conditions are 20-30° C. and the pH value is 6-8.

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