Pseudomonas sp. and application thereof in preparation of pig breeding wastewater treatment agent

Abstract

The application discloses a pseudomonas and application of the pseudomonas in preparation of a pig farm breeding wastewater treatment agent. The strain screened by the application has excellent ammonia nitrogen degradation capacity and heavy metal removal capacity, and has certain removal effect on indexes such as COD and total phosphorus. The strain is stable in activity, easy to activate and proliferate, and has a lower use cost, and has very important application value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial wastewater treatment, specifically relating to a strain of Pseudomonas and its application in the preparation of a wastewater treatment agent for pig farming. Background Technology

[0002] In recent years, my country's livestock and poultry farming industry has developed rapidly, with intensive and high-density farming models being vigorously promoted. Trace elements are essential nutrients for livestock and poultry growth and development, but they are also a major source of pollution from the livestock industry. Wastewater generated by the livestock and poultry farming industry mainly consists of livestock and poultry excrement, feed residue, and farm runoff. Livestock and poultry farming wastewater is a highly concentrated and complex organic pollutant wastewater, and its discharge seriously threatens the aquatic ecological environment. The wastewater exhibits an extremely imbalanced carbon-to-nitrogen ratio (COD concentration 400-20000 mg / L, total nitrogen concentration 200-16000 mg / L), excessive heavy metal content (copper and zinc being the most serious), high treatment difficulty, and is rich in organic matter, nitrogen, phosphorus, and other nutrients and energy substances. Directly discharging livestock and poultry wastewater into water bodies will lead to eutrophication, disrupt the aquatic ecological balance, and pose a fatal threat to organisms. Therefore, treating livestock wastewater to meet discharge standards is an urgent priority.

[0003] Currently, wastewater denitrification methods mainly include air stripping, chemical precipitation, adsorption, breakpoint chlorination, biological methods, and membrane technology. Heavy metal treatment primarily involves physical composting, pyrolysis, chemical passivation, chemical leaching, and biological removal technologies. Compared to physical and chemical methods, biological methods offer advantages such as safety, economy, and no secondary pollution, thus leading to their wider application. Within biological methods, utilizing microorganisms for denitrification and heavy metal removal offers advantages such as ease of operation and strong sustainability, and has gradually become the primary method for treating livestock and poultry farm wastewater. However, livestock and poultry farm wastewater is an extremely polluting type of water, making the screening of microorganisms for highly efficient treatment of this wastewater of great significance. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a strain of Pseudomonas and its application in the preparation of livestock and poultry wastewater treatment agents.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0006] This invention obtained a strain of *Pseudomonas* from livestock and poultry wastewater collected in Rucheng County, Yongzhou City, Hunan Province. The colony morphology of this strain is characterized as follows: white, round colonies with a smooth, moist, and opaque surface. Figure 3The 16S rRNA sequence was BLAST-aligned and identified as Pseudomonas sp., named Pseudomonas A4. It was deposited on August 25, 2023, at the China Center for Type Culture Collection (Wuhan, China) with accession number CCTCC NO: M 20231542.

[0007] The Pseudomonas strain described in this invention can be used to prepare a wastewater treatment agent for pig farming, which degrades ammonia nitrogen and removes heavy metals, including copper and zinc, from pig farming wastewater.

[0008] This invention also provides a method for treating wastewater from pig farming, wherein the method involves activating the Pseudomonas strain A4 to an effective viable count of 1 × 10⁻⁶. 7 -5×10 7 A bacterial suspension of CFU / mL was prepared and then added to the pig farming wastewater at an inoculation rate of 5-10%. The wastewater was treated at 26-35℃ (preferably 28-35℃) and 120-180 rpm for 24-72 hours. The pH value of the wastewater was 5-9.

[0009] Studies have shown that *Bacillus agave* and *Bacillus subtilis*, isolated by Liu Jinghui from wastewater from a pig farm in Panyu District, Guangzhou City, Guangdong Province, achieved ammonia nitrogen degradation rates of 40.7% and 36%, respectively, after 72 hours of cultivation in simulated wastewater with an initial ammonia nitrogen concentration of 848 mg / L. *Bacillus cereus*, isolated and purified by Wang Chengmin et al. from soil surrounding a manganese mine in Guangxi, showed a similar degradation rate at 10 mg / L Cu. 2+ The removal rate was 73.3%. Huang et al. obtained samples from Guangzhou, China, from soil pollutants containing 1.4 mg Cd per kilogram of soil. 2+ Bacillus cereus was isolated from soil and its effect on Cu 2+ Zn 2+ The removal rates were 14-25% and 26-42%, respectively. In comparison, the *Pseudomonas* A4 strain described in this invention can achieve a degradation rate of 30-40% for 1000 mg / L ammonia nitrogen within 72 hours, and a degradation rate of 10 mg / L Cu within 48 hours. 2+ Removal can reach up to 75-80% for 10 mg / L Zn 2+ The strain can remove up to 85-90% of nitrogen, and it also has a certain removal effect on indicators such as COD and total phosphorus. It does not cause secondary pollution like physical or chemical denitrification methods, demonstrating its great potential and broad application prospects in livestock wastewater treatment. Furthermore, livestock wastewater mainly originates from pig farms and has a high ammonia nitrogen content. Therefore, this strain exhibits strong ammonia nitrogen tolerance and simultaneously possesses the characteristic of highly efficient removal of both ammonia nitrogen and heavy metals. Attached Figure Description

[0010] Figure 1 The results are the determination results of the production curve of Pseudomonas A4 in LB liquid medium;

[0011] Figure 2 These are microscopic images of Pseudomonas under an A4 microscope;

[0012] Figure 3 This is a photograph of Pseudomonas A4 colonies on LB solid medium;

[0013] Figure 4 These are images from a verification experiment showing the BTB plate turning blue.

[0014] Figure 5 This is the result of a Neighbor-Joining-based phylogenetic analysis of Pseudomonas A4. Detailed Implementation

[0015] To select the most efficient ammonia nitrogen-degrading bacterium Pseudomonas.A4 and apply it to actual aquaculture wastewater treatment, this invention has undergone repeated experiments. The following are specific preferred embodiments to enable those skilled in the art to better understand the technical solution and effects of this invention. The invention will be specifically described below in conjunction with specific embodiments.

[0016] Example 1: Screening, isolation, and purification of Pseudomonas sp. A4 strains

[0017] BromothymoL blue (BTB) medium (g / L): potassium nitrate 1.00, L-asparagine 1.00, trisodium citrate 8.50, potassium dihydrogen phosphate 1.00, ferric chloride 0.05, calcium chloride 0.20, magnesium sulfate heptahydrate 1.00, bromothymoL blue 0.005, agar 20.00, pH 7.0.

[0018] Method for isolating and purifying microbial flocculant-producing strains: Aquaculture wastewater samples were mixed with LB liquid culture medium at an 8:2 ratio for enrichment culture. The enriched bacterial culture was then serially diluted. -5 10 -6 Heating in a 65℃ water bath for 10 minutes, then spreading the mixture, and repeating the streak multiple times until uniform colonies are obtained. Finally, pick single colonies onto BTB medium and observe the color change. Figure 4 After purifying the blue-colored colonies, single colonies of A1, A2, A3, A4, A5, and A6 were obtained and inoculated into LB liquid medium. The culture was carried out at 150 r / min and 30℃ for 24 h with constant temperature shaking. The bacterial cells were collected in 50% glycerol and stored in a -80℃ refrigerator.

[0019] Example 2: Screening for dominant bacteria on ammonia nitrogen-containing culture medium

[0020] The culture medium for determining ammonia nitrogen degradation consisted of: 5 g / L glucose, 0.25 g / L ammonium sulfate, 1.0 g / L sodium chloride, 0.5 g / L dipotassium hydrogen phosphate, 0.25 g / L magnesium sulfate, and water as the solvent. The glucose and ammonium sulfate concentrations were adjusted to achieve a C / N ratio of 7 and an ammonia nitrogen concentration of 1000 mg / L. Activated bacterial strains A1, A2, A3, A4, A5, and A6 were inoculated at a rate of 1% into 20 mL of LB medium and cultured at 150 rpm / min and 30 °C with constant shaking for 18 h to achieve a bacterial concentration of 1.0 × 10⁻⁶. 7 CFU / mL, the above bacterial suspension was added to 20mL of ammonia nitrogen degradation separation medium at a volume ratio of 5:100, and then cultured at 150r / min and 30℃ for 72h with constant temperature shaking. Samples were taken at 24h intervals.

[0021] The residual ammonia nitrogen was determined using Nessler's reagent method. The ammonia nitrogen degradation rate was calculated using the formula: (ammonia nitrogen content in the 0h culture medium - ammonia nitrogen content in the 72h culture medium) / ammonia nitrogen content in the 0h culture medium × 100%.

[0022] Table 1 shows the ammonia nitrogen degradation effects of different strains.

[0023]

[0024] The strain with the best ammonia nitrogen degradation effect was identified as Pseudomonas.A4.

[0025] Pseudomonas A4 microscopic morphology as follows: Figure 2 As shown, a colony photograph of Pseudomonas A4 on LB solid medium is shown. Figure 3 As shown.

[0026] Example 3: Homologous sequence analysis of 16S rRNA gene in Pseudomonas A4 strain

[0027] Single colonies were picked from LB agar plates and inoculated into 20 mL of LB liquid medium. The culture was incubated at 30°C with shaking at 150 rpm for 24 h. Finally, 1 mL of the bacterial culture was collected, centrifuged at 9000 rpm for 3 min, and the supernatant was removed. The bacterial cells were sent to BGI Genomics. Sequencing results were analyzed using BLAST software, and the highest homology alignment was performed with known 16S rRNA genes in GenBank. The phylogenetic analysis results of Pseudomonas A4 based on Neighbor-Joining are shown below. Figure 5 As shown.

[0028] Example 4: Growth curve of Pseudomonas A4

[0029] LB medium ( / L): 10g sodium chloride, 10g peptone, 5g yeast extract, 20g agar (not added to liquid medium), pH 6.8-7.2;

[0030] For Pseudomonas A4 from Example 2 of this invention, a single colony was picked from an LB agar plate and transferred to 20 mL of LB liquid medium. The culture was incubated at 30°C with shaking at 150 rpm for 24 h. Then, 1% of the culture was transferred to 20 mL of liquid medium and incubated at 30°C with shaking at 150 rpm. Samples were taken every 2 h to measure the OD value. 600 Value. The specific growth curve measurement results are as follows: Figure 1 As shown in the figure, the horizontal position is h.

[0031] Example 5: Determination of the ammonia nitrogen removal capacity of Pseudomonas A4

[0032] The culture medium used to determine the ammonia nitrogen degradation effect consisted of: glucose 5 g / L, ammonium sulfate 0.25 g / L, sodium chloride 1.0 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 0.25 g / L, and water as the solvent. The glucose and ammonium sulfate concentrations were adjusted to achieve a C / N ratio of 7 and an ammonia nitrogen concentration of 1000 mg / L. The activated bacterial strain was inoculated at a rate of 1% into 20 mL of LB medium and cultured at 150 rpm and 30 °C with constant shaking for 18 h to achieve a bacterial concentration of 1 × 10⁻⁶. 7 -5×10 7 CFU / mL, the bacterial culture was added to 20 mL of ammonia nitrogen degradation separation medium at a volume ratio of 5:100, and then cultured at 150 rpm / min and 30℃ for 72 h with constant temperature shaking. Samples were taken every 24 h.

[0033] The residual ammonia nitrogen was determined using Nessler's reagent method. The ammonia nitrogen degradation rate was calculated using the formula: (ammonia nitrogen content in the 0h culture medium - ammonia nitrogen content in the 72h culture medium) / ammonia nitrogen content in the 0h culture medium × 100%.

[0034] Table 2 shows the ammonia nitrogen degradation rate (%) of Pseudomonas A4 in the culture medium at different time periods.

[0035]

[0036] Example 6: Determination of the heavy metal removal capacity of Pseudomonas A4

[0037] LB medium ( / L): 10g sodium chloride, 10g peptone, 5g yeast extract, 20g agar (not added to liquid medium), pH 6.8-7.2.

[0038] The seed culture of the strain was inoculated into LB medium at a rate of 1% and cultured at 30°C and 150 rpm for 18 h on a shaker. This liquid was then used as activated bacterial cells. Under specific pH and temperature conditions, the activated bacterial cells were added at a volume of 5% to a solution containing 10 mg / L Cu. 2+ 10mg / L Zn 2+ The sample was placed in 20 mL of LB solution and shaken at 150 rpm. Samples were taken every 12 hours and centrifuged at 9000 rpm for 10 min. The concentration of residual heavy metals in the supernatant was determined by complexometric colorimetry and zinc mercury colorimetry using copper ion assay kits and zinc ion assay kits (catalog numbers E010-1-1 and E011-1-1) sold by Nanjing Jiancheng Bioengineering Institute, and the removal rate was calculated.

[0039]

[0040] Where R is the removal rate of heavy metals, C0 is the initial concentration of heavy metals (mg / L), and C e This represents the concentration (mg / L) of heavy metals in the solution at equilibrium.

[0041] Table 3 shows the removal rates (%) of copper and zinc by Pseudomonas A4 at different time points in the culture medium.

[0042]

[0043] Example 7: Application of Pseudomonas A4 in actual aquaculture wastewater

[0044] Wastewater from a large pig farm in Liuyang City, Hunan Province, was tested, and the ammonia nitrogen content was found to be 1048 mg / L. The strain Pseudomonas sp. A4 was activated using the method described above. The activated bacterial solution was added to the wastewater at a volume ratio of 5:100 and cultured at 150 rpm / min and 30℃ for 72 hours with constant temperature shaking. Samples were taken every 24 hours, and the final ammonia nitrogen content was determined to be 895 mg / L, with an ammonia nitrogen removal rate of 10%-20%.

Claims

1. A strain of Pseudomonas, characterized in that, The Pseudomonas strain was named Pseudomonas A4, accession number: CCTCC NO: M 20231542.

2. The application of the Pseudomonas strain as described in claim 1 in the preparation of a treatment agent for livestock and poultry wastewater that degrades ammonia nitrogen and removes copper and zinc.

3. A method for treating livestock and poultry wastewater, characterized in that, The method involves using the Pseudomonas strain described in claim 1. Pseudomonas A4 The effective viable bacteria count after activation is 1×10⁻⁶. 7 -5×10 7 Prepare a bacterial solution of CFU / mL, and then add the bacterial solution at an inoculation rate of 5-10% to the livestock and poultry wastewater, and treat it at 26-35℃ and 120-180rpm for 24-72h.

4. The method as described in claim 3, characterized in that, The pH value of the livestock and poultry wastewater is 5-9.

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