Pseudomonas oryzae and application thereof
By introducing Pseudomonas oryzae and Myriophyllum sp. into constructed wetlands and optimizing operating parameters, the problems of limited microbial denitrification capacity and high cost were solved, achieving efficient and stable denitrification of aquaculture wastewater, which is suitable for the treatment of high-nitrogen aquaculture wastewater.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-14
AI Technical Summary
Existing constructed wetland technologies have limited denitrification capacity of microbial strains when treating aquaculture wastewater, resulting in unstable denitrification efficiency. Under high loads, they are prone to inhibiting microbial activity, leading to high operating costs and making it difficult to scale up in rural areas.
Pseudomonas oryzae was used as a microbial agent and statically colonized in a surface flow constructed wetland. Combined with the construction of Myriophyllum spicatum wetland and the regulation of influent load, the operating parameters were optimized to form a multi-barrier denitrification system.
It achieves efficient and stable removal rates of total nitrogen and ammonia nitrogen, with removal rates of 88.94% to 98.97% and 90.26% to 99.58%, respectively. It reduces operating costs, is suitable for treating high-nitrogen aquaculture wastewater, does not require a large amount of exogenous carbon sources and mature sludge, and is suitable for promotion in rural areas.
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Abstract
Description
Technical Field
[0001] This application relates to the field of microbial technology, and in particular to a rice Pseudomonas bacillus and its applications. Background Technology
[0002] Constructed wetlands (CWs), as a green and ecological wastewater treatment technology, have advantages such as low operating costs, simple maintenance, and eco-friendliness, and are one of the core technologies for treating aquaculture wastewater. Aquaculture wastewater generally exhibits high pH values and high ammonia nitrogen (NH4+). + Characterized by high nitrogen content (N), high total nitrogen (TN), and high chemical oxygen demand (COD), nitrogen pollutant emissions have become a major source of agricultural non-point source pollution, seriously threatening the safety of surface water and groundwater ecosystems.
[0003] Currently, the core of using constructed wetlands for nitrogen removal from aquaculture wastewater relies on the synergistic effect of plants and microorganisms. The denitrification-anammox (PD-A) coupling process is a key pathway for efficient biological nitrogen removal. However, existing treatment technologies still have significant limitations. The nitrogen removal capacity of microbial strains is limited, the carbon-to-nitrogen ratio (C / N) of the aquaculture wastewater itself is not fully utilized, and the nitrogen removal efficiency is unstable. High-load aquaculture wastewater can easily inhibit microbial activity, leading to the accumulation of nitrogen pollutants. In addition, the operating costs are relatively high, and they mostly rely on the addition of exogenous carbon sources or inoculation with mature sludge, making it difficult to promote on a large scale in rural areas. Therefore, there is an urgent need for a more efficient microbial strain and a more efficient method for treating aquaculture wastewater. Summary of the Invention
[0004] In view of this, the purpose of this application is to provide a rice Pseudomonas bacillus and its application to solve the above-mentioned technical problems.
[0005] A first aspect of this application provides a *Pseudomonas rice* strain (…). Pseudomonas oryzae The accession number is CGMCC No. 32915, deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on December 5, 2024. Its name is... Pseudomonas oryzae CN5, the 16S rDNA sequence of the rice Pseudomonas is shown in SEQ ID No: 1.
[0006] When *Pseudomonas rice* was inoculated at a concentration of 0.8% to 1.2% (v / v) and anaerobically cultured at 25°C to 28°C for 12 to 35 hours, it achieved a nitrate degradation rate of approximately 63%. This *Pseudomonas rice* strain can utilize existing nitrogen and carbon sources in aquaculture wastewater to degrade NO3-. - -N is converted to NO2 - -N, and has good NO2 --N cumulative effect, has the ability to stably accumulate nitrite converted from nitrate during the reaction process.
[0007] In a second aspect of this application, a microbial agent is provided, including *Pseudomonas rice* as described in the first aspect above. Using this agent in the treatment of aquaculture wastewater can improve the denitrification effect.
[0008] A third aspect of this application provides a method for preparing the microbial inoculant as described in the second aspect above, comprising: inoculating *Pseudomonas oryzae* as described in the first aspect above into a denitrifying medium and culturing it for 2 to 3 days to obtain a bacterial suspension (OD600 approximately 1.2), culturing at a temperature of 25°C to 28°C, centrifuging the bacterial suspension, removing the supernatant, and resuspending it in phosphate buffer to obtain the microbial inoculant.
[0009] The denitrification medium has a pH of 7.0 to 7.3. Each liter of denitrification medium contains 0.2 g of KNO3, 0.566 g of sodium citrate, 10.55 g of Na2HPO4·12H2O, 1.5 g of KH2PO4, 0.1 g of MgSO4·7H2O, and 2 mL of trace element solution. Each liter of trace element solution contains 50.0 g of EDTA-Na2, 5.06 g of MnCl2·4H2O, 2.2 g of ZnSO4, 5.0 g of FeSO4·7H2O, 5.5 g of CaCl2, 1.61 g of CoCl2·6H2O, and 1.57 g of CuSO4·5H2O.
[0010] A fourth aspect of this application provides the application of *Pseudomonas rice* as described in the first aspect above in denitrification of aquaculture wastewater, wherein using this strain in aquaculture wastewater treatment can improve the denitrification effect.
[0011] A fifth aspect of this application provides a method for treating aquaculture wastewater, comprising: constructing a surface flow constructed wetland; adding the microbial agent described in the second aspect above to the surface flow constructed wetland for static colonization; and diluting the aquaculture wastewater before injecting it into the surface flow constructed wetland for denitrification treatment.
[0012] Furthermore, the surface flow constructed wetland includes a three-tiered *Myriophyllum spicatum* wetland, each tier having a length of 5m and a width of 2m. *Myriophyllum spicatum* (… Myriophyllum aquaticum The planting density is 600 plants / m². By constructing a plant-microbe synergistic denitrification microenvironment, the allelochemicals secreted by the roots of *Myriophyllum spicatum* and the root attachment effect are utilized to promote the aggregation of functional bacterial communities. The *Myriophyllum spicatum* wetland includes a filler layer with a thickness of 40cm to 60cm. The filler layer is made of paddy soil or peat moss, providing a suitable colonization carrier for denitrifying bacteria.
[0013] Furthermore, based on the weight of the packing layer, the dosage of the microbial agent is 10. 5 copies / g to 10 6 Copies / g; During the initial operation or maintenance period of the wetland, the water level is drained to below the surface of the filler layer. The microbial agent is added by mixing it with a porous carrier (such as biochar powder or modified clay) and reinjecting it into the filler layer or at the inlet, or by injecting the microbial agent into the filler layer or at the inlet using a multi-point injection method. Static establishment includes closing the outlet of the surface flow constructed wetland and maintaining static flooding for 3 to 5 days. During the static establishment stage, no water is added or drained. The large specific surface area of the Myriophyllum spicatum roots and root exudates serve as a natural biofilm carrier, promoting rapid adsorption and film formation of functional bacteria on the filler and root surface, and preventing the loss of the microbial agent due to water erosion.
[0014] Furthermore, the ammonia nitrogen concentration of the aquaculture wastewater is 250 mg / L to 540 mg / L, the total nitrogen concentration is 300 mg / L to 600 mg / L, the pH value is 8.0 to 8.6, and the carbon-to-nitrogen ratio is 6 to 7.
[0015] Furthermore, the dilution includes mixing the aquaculture wastewater with fresh water to obtain a diluted water body. A wastewater pond, a clear water pond, and a distribution pond can be set up at the wetland inlet. The wastewater pond stores the aquaculture wastewater, and the clear water pond stores fresh water from reservoirs, rivers, or lakes. The aquaculture wastewater and fresh water are mixed in the distribution pond for dilution, and the influent load is adjusted to 1 / 3 of the aquaculture wastewater volume. The influent load can be flexibly adjusted according to the concentration of the aquaculture wastewater. If the total nitrogen concentration of the aquaculture wastewater is too high, the influent load can be further reduced to 1 / 4 of the aquaculture wastewater volume. The total nitrogen concentration of the diluted water body is less than or equal to 200 mg / L, the ammonia nitrogen concentration is less than or equal to 180 mg / L, and the total nitrogen volumetric loading rate is 2.5 mg / (m³). 3 ·d) to 5.3 mg / (m 3 ·d); The carbon-to-nitrogen ratio of the diluted water injected into the surface flow constructed wetland is 5 to 8, which can avoid the inhibition of microbial denitrification activity by excessive load.
[0016] Furthermore, the denitrification treatment adopts an intermittent flow operation mode. The total hydraulic load of the denitrification treatment is 0.15 m³ / d to 0.18 m³ / d, and the hydraulic retention time (HRT) of each stage of the *Myriophyllum spicatum* wetland is 11 to 15 days. In spring, summer, and autumn, the water temperature is higher, and the microbial activity is strong, so it can be operated at full load (0.18 m³ / d) according to the design parameters. In winter, the water temperature is lower, and the microbial activity decreases, so the hydraulic load can be appropriately reduced to 0.15 m³ / d, and the hydraulic retention time can be extended to 13 to 15 days to compensate for the impact of low temperature on denitrification efficiency. The water level of the *Myriophyllum spicatum* wetland is 20 cm to avoid the increase in greenhouse gas emissions caused by excessively low water levels and the inhibitory effect of excessively high water levels on microbial denitrification. Two L-shaped drainage pipes can be installed at the outlet to adjust the water level in real time and ensure stable system operation.
[0017] The foxtail grass wetland requires regular routine maintenance. Every autumn, the foxtail grass residue should be harvested, leaving 10cm to 20cm above the ground to prevent the residue from decomposing and affecting the water quality. Every 3 to 5 years, the packing layer should be cleaned or partially replaced to prevent blockage that could affect water flow and microbial activity.
[0018] This aquaculture wastewater treatment method addresses the shortcomings of existing constructed wetlands for treating aquaculture wastewater, such as low nitrogen removal efficiency, high cost, and poor stability, through optimized construction of Myriophyllum spicatum wetlands, addition of functional microbial agents, control of influent load, and synergistic optimization of operating parameters. It achieves efficient, ecological, and low-cost removal of total nitrogen from aquaculture wastewater. Testing shows that this method achieves a total nitrogen removal rate of 88.94% to 98.97% and an ammonia nitrogen removal rate of 90.26% to 99.58%, making it suitable for treating aquaculture wastewater with high ammonia nitrogen levels. It requires no large additions of exogenous carbon sources or mature sludge and has low operating costs. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in this application or related technologies, the drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is an electron micrograph of a type of Pseudomonas rice in an embodiment of this application.
[0021] Figure 2 This is a comparison chart of the total nitrogen removal rates of Example 1 and Comparative Example 1 of this application.
[0022] Figure 3 This is a comparison chart of the relative abundance of Example 1 and Comparative Example 1 of this application.
[0023] Figure 4This is a comparison graph showing the changes in ammonia nitrogen in Example 1 and Comparative Example 2 of this application.
[0024] Figure 5 This is a comparison graph showing the total nitrogen changes between Example 1 and Comparative Example 2 of this application. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.
[0026] It should be noted that, unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.
[0027] The following describes specific embodiments in conjunction with... Figures 1 to 5 The technical solution of this application will be described in detail below.
[0028] rice Pseudomonas ( Pseudomonas oryzae The accession number is CGMCC No. 32915, deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on December 5, 2024. Its name is... Pseudomonas oryzae CN5.
[0029] Sources and isolation of *Pseudomonas oryzae* strains Sediment samples (10 cm depth) were collected from a pond in Shijiazhuang, Hebei Province, which had been long-term polluted by aquaculture wastewater. In a sterile anaerobic glove box, 10 g of sediment sample was weighed and placed into a serum bottle containing 90 mL of denitrification medium. A mixture of carbon dioxide and nitrogen was introduced to purge the air from the bottle. The anaerobic bottle was placed in a 30°C constant-temperature shaker and cultured at 150 r / min for 72 h. During the culture, an appropriate amount of glass beads was added to promote sufficient contact between the sludge and the medium. After the enrichment culture was completed, the supernatant bacterial solution was extracted from the anaerobic glove box and inoculated into freshly prepared denitrification medium at a 10% inoculum rate. The mixture of anaerobic gas was introduced, and the culture was repeated three times to obtain the enriched bacterial solution. The sample was then placed in sterile water within the anaerobic glove box, and the concentration was gradually diluted to 10% using a continuous dilution method. - ³ to 10 -6This promotes the uniform dispersion of single cells in the mixed bacterial solution in sterile water. 0.2 mL of the diluted bacterial suspension is spread onto denitrification solid medium, with three replicates for each concentration. The petri dishes are sealed with sealing film and inverted in a 30°C anaerobic incubator for 7 days. After visible colonies appear on the anaerobic plates, single colonies with good growth and significant morphological differences are selected in an anaerobic glove box based on their size, color, and morphology. These colonies are then streaked three times on fresh anaerobic solid medium to isolate and purify *Pseudomonas rice*. Figure 1 The image shown is an electron microscope image of this bacterial strain. The solid culture medium was prepared by adding 2% agar to liquid denitrification medium and sterilizing in an autoclave at 121°C for 20 min.
[0030] Preservation of Pseudomonas rice strains Inside the anaerobic glove box, use an inoculation loop to pick up a single colony and inoculate it onto the slant of a test tube, ensuring a strictly anaerobic environment. After inoculation, place the test tube in an anaerobic bag and then store it in a 4°C refrigerator. During the operation, pay attention to sealing the test tube to prevent contamination. This method can preserve the product for 1 to 3 months and is suitable for short-term storage and daily use.
[0031] For long-term storage (approximately 1 year), the bacterial strain can be inoculated into a liquid enrichment medium and cultured at 30°C and 150 rpm for 48 h. After collecting the bacterial solution into centrifuge tubes, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in 2 mL of sterile water, washing three times. Under aseptic conditions, mix the bacterial cells with 50% glycerol at a 1:1 ratio, adding 0.02% cysteine and 0.01% resazurin. Aliquot the mixture into cryovials, label them, and store at -80°C. For activation, thaw the cryovials at room temperature, spread 100 μL of the bacterial solution onto the surface of a solid culture medium, and incubate to restore bacterial activity.
[0032] Identification of physiological and biochemical characteristics of Pseudomonas oryzae strains Referring to Bergey's Manual of Bacterial Identification, 8th Edition, and the Manual of Systematic Identification of Common Bacteria, *Pseudomonas oryzae* underwent Gram staining, catalase, oxidase, citrate test, methyl red, VP assay, starch hydrolysis, nitrate reduction, and indole test. The test results are shown in Table 1, where "+" indicates a positive result and "-" indicates a negative result. *Pseudomonas oryzae* was positive for catalase, oxidase, citrate, methyl red, and nitrate reduction tests, but negative for Gram staining, VP assay, starch hydrolysis, and indole test.
[0033] Table 1. Physiological and biochemical characteristics of Pseudomonas oryzae.
[0034] sequencing of Pseudomonas rice strains Total DNA from the bacterial strain was extracted and purified using the Takara minibest bacterial genomic DNA extraction kit ver. 3.0 from Baori Biotechnology (Beijing) Co., Ltd. After the DNA concentration and purity were determined using a Nano Drop spectrophotometer, the species of the strain was identified. Universal primers for the bacterial 16S rDNA gene sequence, 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-GGTTACCTTGTTACGACTT-3′, were used to amplify the 16S rDNA fragment of the strain. The amplification products were prepared by Shanghai Sangon Biotech (Shanghai) Co., Ltd.
[0035] The obtained strain sequence was submitted to the NCBI database and analyzed using the BLAST tool. The sequencing results are shown in SEQ ID No: 1. The results indicate that the screened strain is... Pseudomonas oryzae Name it Pseudomonas oryzae CN5.
[0036] Example 1 Livestock and poultry farm wastewater was collected from Jinjing Town, Changsha City, Hunan Province. The wastewater quality indicators are as follows: ammonia nitrogen concentration is 320 mg / L to 480 mg / L, total nitrogen concentration is 380 mg / L to 545 mg / L, pH value is 8.4 to 8.6, and C / N ratio is 6 to 7, which meets the characteristics of high ammonia nitrogen farm wastewater.
[0037] A surface flow constructed wetland was constructed downstream of a fish farm in Jinjing Town, Changsha City, Hunan Province. The surface flow constructed wetland consisted of three levels of *Myriophyllum spicatum* wetlands, each level measuring 5m in length and 2m in width. Local paddy soil was used as the filler, with a thickness of 50cm. *Myriophyllum spicatum* was planted at a high density on the wetland surface. Myriophyllum aquaticum The planting density is controlled at 600 plants / m², utilizing its well-developed root network to provide attachment sites for microorganisms.
[0038] To prepare microbial inoculants, the frozen inoculants were added at a volume fraction of 0.8% to 1.2%. Pseudomonas oryzaeCN5 bacterial culture was inoculated into denitrification medium and cultured for 2 to 3 days to obtain a bacterial suspension (OD600 approximately 1.2). The culture temperature was 25°C to 28°C. The bacterial suspension was centrifuged to remove the supernatant. The precipitated bacterial cells were resuspended in a small amount of phosphate buffer and can then be used as a microbial agent for denitrification enhancement. The denitrification medium has a pH of 7.0 to 7.3. Each liter of denitrification medium contains 0.2 g of KNO3, 0.566 g of sodium citrate, 10.55 g of Na2HPO4·12H2O, 1.5 g of KH2PO4, 0.1 g of MgSO4·7H2O, and 2 mL of trace element solution. Each liter of trace element solution contains 50.0 g of EDTA-Na2, 5.06 g of MnCl2·4H2O, 2.2 g of ZnSO4, 5.0 g of FeSO4·7H2O, 5.5 g of CaCl2, 1.61 g of CoCl2·6H2O, and 1.57 g of CuSO4·5H2O.
[0039] Add microbial inoculants to the surface flow constructed wetland, adjusting the concentration of the microbial inoculants (cultivated to 10) to the logarithmic growth phase. 8 The bacterial solution (CFU / mL) was diluted at a ratio of 5% by the volume of the packing material. During the wetland startup phase, the water level was lowered to half the height of the packing material layer, and the bacterial solution was injected into the dense root zone of *Myriophyllum spicatum* in the packing material layer using a multi-point injection method. After addition, water was added to the normal water level (20cm), the inlet and outlet valves were closed, and the mixture was left to stand and aerated for 5 days. No hydraulic exchange was performed during this period, allowing the functional bacteria to fully utilize root exudates and the pores on the surface of the packing material to complete the initial colonization and biofilm formation, avoiding the problem of easy loss with water caused by conventional direct addition.
[0040] Wastewater ponds, clear water ponds, and distribution ponds are set up at the wetland inlet. The wastewater ponds store aquaculture wastewater, and the clear water ponds store freshwater from reservoirs, rivers, or lakes. The aquaculture wastewater is mixed with freshwater in the distribution ponds to dilute the wastewater. The influent load is adjusted to 1 / 3 of the wastewater volume to avoid the inhibition of bacterial activity by high concentrations of free ammonia.
[0041] The diluted aquaculture wastewater was injected into a surface flow constructed wetland for denitrification treatment. The intermittent flow operation mode was adopted, with a hydraulic load of 0.18 m³ / d and a hydraulic retention time (HRT) of 11 to 13 days for each level of Myriophyllum spicatum wetland.
[0042] The surface flow constructed wetland operated stably for 304 days, with water samples collected 2 to 3 times per month. After passing through a 0.45 μm filter membrane, NH4 was measured using a continuous flow analyzer. + -N concentration was measured, TN was detected using a water quality kit, and the pH value of the water was monitored simultaneously. Microbial community analysis was also conducted on the wetland sediment during the mid-term operation to test whether functional bacteria had successfully colonized the wetland system.
[0043] Comparative Example 1 The treatment method was the same as in Example 1, except that no microbial agents were added, relying solely on indigenous microorganisms. Other process parameters were the same as in Example 1, and the surface flow constructed wetland was continuously operated for 304 days, with water quality indicators being monitored simultaneously.
[0044] Comparative Example 2 The treatment method was the same as in Example 1, except that the aquaculture wastewater was not diluted and raw water (180L / d) was directly injected into the surface flow constructed wetland. The surface flow constructed wetland was continuously operated for 304 days, and water quality indicators were monitored simultaneously.
[0045] Compare the test results of Example 1 and Comparative Example 1, such as Figure 2 As shown, the average total nitrogen removal rate of Example 1 (with sterile agent) was 97.11%, while the average total nitrogen removal rate of Comparative Example 1 (without sterile agent) was 88.86%. Figure 3 As shown, microbial community analysis was conducted on the wetland sediment during the mid-term operation. The results indicated that in Example 1... Pseudomonas oryzae The relative abundance of CN5 increased from less than 1% initially and remained between 10% and 15%, with the highest abundance in the rhizosphere soil of Myriophyllum spicatum. This demonstrates that through the aforementioned "static colonization" process, exogenous functional bacteria successfully established a dominant population in the wetland without significant loss.
[0046] Although both Example 1 and Comparative Example 1 were low-load (influent TN volumetric loading range of 2.6 mg / (m³) 3 ·d) to 5.1 mg / (m 3 •d)) Operating environment, but the start-up speed of sterile agents is relatively slow. By adding microbial agents in a targeted manner, the accumulation of functional microbial communities can be significantly promoted, the denitrification coupled nitrogen removal process can be started, and the total nitrogen removal rate can be improved.
[0047] Comparing the test results of Example 1 and Comparative Example 2, as follows: Figure 4 and Figure 5 As shown, in Example 1 (low load), the total nitrogen removal rate remained stable at 88.94% to 98.97%, with an average total nitrogen removal rate of 97.11%, and the ammonia nitrogen removal rate remained stable at 90.26% to 99.58%, with an average ammonia nitrogen removal rate of 98.23%; Comparative Example 2 (high load, influent TN volumetric loading range of 7.5 mg / (m³)) 3 ·d) to 15.1 mg / (m 3 The average total nitrogen removal rate was 83.57%, and the average ammonia nitrogen removal rate was 84.60%.
[0048] Example 1 shows that low-load regulation can effectively avoid the inhibition of microbial denitrification activity by high-load aquaculture tailwater, maintain a stable C / N environment, promote the enrichment of denitrifying bacteria, and the denitrification efficiency is significantly better than that of Comparative Example 2. In Comparative Example 2, due to the excessively high concentration of ammonia nitrogen in the influent, free ammonia with biotoxicity is produced in a high pH environment, the microbial activity is inhibited, nitrogen pollutants accumulate in the bottom sediment, and the denitrification efficiency is lower and fluctuates more.
[0049] In summary, the aquaculture wastewater treatment method of this application has the following significant advantages: High nitrogen removal efficiency: Through process integration and optimization, the total nitrogen removal rate can reach 88.94% to 98.97%, and the ammonia nitrogen removal rate can reach 90.26% to 99.58%, which is more than 16% higher than the uncontrolled process, and can efficiently treat aquaculture wastewater with high ammonia nitrogen.
[0050] Highly innovative in process: It integrates four major process modules for the first time: construction of Myriophyllum spicatum wetland, addition of denitrification functional bacteria, and regulation of influent load and operating parameters, breaking through the limitations of existing single processes and forming a multi-barrier denitrification system.
[0051] Low operating cost: No need to add large amounts of external carbon sources and mature sludge. Relying on the carbon source of the aquaculture wastewater itself and the targeted addition of functional bacteria, the cost per ton of water treated is extremely low. It is easy to maintain and can be promoted on a large scale in rural areas.
[0052] Eco-friendly: It adopts ecological treatment technology without the addition of chemical disinfectants and external pollutants, thus avoiding secondary pollution. At the same time, the planting of Myriophyllum spicatum can improve the wetland ecological environment, achieving the dual effect of water purification and ecological restoration.
[0053] High stability: Through precise control of load and operating parameters, the denitrification efficiency of the system fluctuates little during continuous operation, which can adapt to the treatment needs of aquaculture wastewater with different concentrations and is suitable for complex aquaculture wastewater discharge scenarios.
[0054] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of this disclosure (including the claims) is limited to these examples; within the framework of this disclosure, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of this disclosure as described above, which are not provided in detail for the sake of brevity.
[0055] This disclosure is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.
Claims
1. A type of rice Pseudomonas ( Pseudomonas oryzae ), characterized in that, The accession number is CGMCC No. 32915.
2. A microbial inoculant comprising Pseudomonas rice as described in claim 1.
3. A method for preparing the microbial inoculant according to claim 2, comprising: The *Pseudomonas rice* strain described in claim 1 was inoculated into a denitrification medium to obtain a bacterial suspension. The bacterial suspension was centrifuged, the supernatant was removed, and the suspension was resuspended in phosphate buffer to obtain a microbial agent.
4. The application of the rice Pseudomonas as described in claim 1 in denitrification of aquaculture wastewater.
5. A method for treating aquaculture wastewater, characterized in that, include: Constructing surface flow artificial wetlands; The microbial agent of claim 2 is added to the surface flow constructed wetland for static colonization; The aquaculture wastewater is diluted and then injected into the surface flow constructed wetland for denitrification treatment.
6. The method for treating aquaculture wastewater according to claim 5, characterized in that, The surface flow constructed wetland includes a three-level Myriophyllum spicatum wetland. Each level of the Myriophyllum spicatum wetland is 5m long and 2m wide, with a planting density of 600 plants / m². The Myriophyllum spicatum wetland includes a filler layer with a thickness of 40cm to 60cm. The filler layer is made of paddy soil or peat soil.
7. The method for treating aquaculture wastewater according to claim 6, characterized in that, The dosage of the microbial agent is 10g based on the weight of the packing layer. 5 copies / g to 10 6 copies / g; the microbial agent is added by mixing the microbial agent with a porous carrier and reinjecting it into the packing layer, or by injecting the microbial agent into the packing layer at multiple points; the static establishment includes closing the inlet and outlet of the surface flow constructed wetland and maintaining static flooding for 3 to 5 days.
8. The method for treating aquaculture wastewater according to claim 5, characterized in that, The aquaculture wastewater has an ammonia nitrogen concentration of 250 mg / L to 540 mg / L, a total nitrogen concentration of 300 mg / L to 600 mg / L, a pH value of 8.0 to 8.6, and a carbon-to-nitrogen ratio of 6 to 7.
9. The method for treating aquaculture wastewater according to claim 5, characterized in that, The dilution includes mixing the aquaculture wastewater with fresh water to obtain a diluted water body; the total nitrogen concentration of the diluted water body is less than or equal to 200 mg / L, and the ammonia nitrogen concentration is less than or equal to 180 mg / L; the carbon-to-nitrogen ratio of the diluted water body injected into the surface flow constructed wetland is 5 to 8.
10. The method for treating aquaculture wastewater according to claim 6, characterized in that, The denitrification treatment adopts an intermittent flow operation mode, the total hydraulic load of the denitrification treatment is 0.15 m³ / d to 0.18 m³ / d, the hydraulic retention time of each stage of the Myriophyllum spicatum wetland is 11 days to 15 days, and the water level is 20 cm.