Pseudomonas oleovorans qs-7 isolated from pig farm biogas slurry oxidation pond and its application

Abstract

The application belongs to the technical field of biology, and particularly relates to a Pseudomonas oleovorans QS-7 isolated from a pig farm biogas slurry oxidation pond and application of the Pseudomonas oleovorans QS-7 in denitrification treatment of pig breeding wastewater rich in ammonia nitrogen. The application discloses the Pseudomonas oleovorans QS-7, and the preservation number is CGMCC No.30323. The application also simultaneously provides the use of the Pseudomonas oleovorans QS-7: treating pig breeding wastewater rich in ammonia nitrogen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Pseudomonas oleovorans QS-7 isolated from a pig farm biogas slurry oxidation pond and application thereof in denitrification treatment of pig breeding wastewater rich in ammonia nitrogen. BACKGROUND

[0002] With the development of industry and agriculture and the improvement of people's living standards, the discharge of wastewater containing nitrogen compounds has increased dramatically, which has become a major source of environmental pollution and attracted much attention. Nitrogen exists in wastewater in various forms, such as organic nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, among which ammonia nitrogen is one of the most important forms. Ammonia nitrogen refers to free ammonia (or non-ionic ammonia, NH3) or ionic ammonia (NH4 + ) form; the higher the pH, the higher the proportion of free ammonia; otherwise, the proportion of ammonium salt is high. Ammonia nitrogen is a nutrient in water bodies, which can cause eutrophication and is a major oxygen-consuming pollutant in water bodies, which can easily cause the proliferation of algae and other microorganisms in water, making it difficult for water treatment plants to operate, causing drinking water to have an unusual odor, and in severe cases, the dissolved oxygen in water can decrease, resulting in the death of a large number of fish. In addition, ammonia nitrogen can also increase the amount of chlorine used in water disinfection and industrial circulating water sterilization treatment, and has corrosive properties to some metals (copper); under the action of nitrifying bacteria, it is oxidized to nitrite and nitrate, and nitrate can induce high iron hemoglobinemia in infants, while nitrosamine generated by the hydrolysis of nitrite has strong carcinogenicity, which directly threatens human health. China has strict requirements for the concentration of ammonia nitrogen in surface water quality and wastewater discharge. Therefore, finding an efficient method and technology for treating wastewater containing ammonia nitrogen is a current research hotspot, which is an urgent technical requirement for enterprises discharging wastewater containing ammonia nitrogen.

[0003] Current research shows that ammonia nitrogen removal methods in wastewater can be summarized into three categories, namely physical method, chemical method and biological method. Physical methods include stripping, zeolite denitrification, membrane separation technology, ammonia stripping, etc., chemical methods include MAP precipitation, catalytic oxidation, electrochemical oxidation, breakpoint chlorination, etc., and biological methods include anaerobic treatment, aerobic treatment and anaerobic-aerobic combined method, etc. Compared with the physical method, the ammonia produced by the physical method needs subsequent treatment and has high cost, and direct discharge exists the problem of secondary pollution, while the chemical method itself has high cost, and ammonia nitrogen is converted into oxidized state or other forms, and the total nitrogen in water body does not change substantially. However, the biological treatment method utilizes microorganisms to decompose and transform pollutants in wastewater, part of which is converted into nitrogen gas and part of which is assimilated into microbial biomass, which completely eliminates the potential harm of ammonia nitrogen in wastewater to the environment, and is an effective method for treating ammonia nitrogen wastewater with low cost and no secondary pollution, which has attracted widespread attention from researchers.

[0004] The invention of CN201910638848.X "Pseudomonas monteilii XZL-13 and its application" informs a Pseudomonas monteilii XZL-13, preservation number CGMCC No.17514, strain XZL-13 has corresponding degradation and utilization ability for corn oil, animal oil, peanut oil, sesame oil, coconut oil and olive oil.

[0005] The invention of CN202310323095.X "Pseudomonas monteilii SD-2 and its application in degrading organic pollutants" informs a Pseudomonas monteilii SD-2, preservation number CCTCC No.M2023241, strain SD-2 has a removal rate of up to 100% for ethanol within 400mg / L.

[0006] The invention of CN202211277741.5 "Pseudomonas chengduensis DMC-X1, immobilized bacterial agent and application" informs a Pseudomonas chengduensis DMC-X1, preservation number GDMCC No.62596, strain DMC-X1 has high degradation capacity for microcystins (MCs), and can be repeatedly used by embedding immobilization.

[0007] The invention of CN202310934714.9 "Pseudomonas hunanensis MGJ-2 and its application in degrading nicotine" informs a Pseudomonas hunanensis MGJ-2, preservation number CGMCC No.25121, strain MGJ-2 has high degradation capacity for nicotine, which can reduce the harm of smoking to the body, and can be used for tobacco waste treatment and improve tobacco utilization rate.

[0008] The invention of CN202211650598.X "Pseudomonas lurida WS43 with wide temperature range heterotrophic nitrification-aerobic denitrification and its application" informs a Pseudomonas lurida WS43, preservation number CGMCC No.22944, strain WS43 has heterotrophic nitrification-aerobic denitrification metabolic capacity under the condition of 2-40℃, and has good removal effect on nitrate nitrogen, nitrite nitrogen and ammonium nitrogen in wastewater.

[0009] The invention of CN202210926711.6, "Aerobic denitrifying Pseudomonas sp. ZZRD2, biological denitrification agent and application thereof", informs a Pseudomonas sp. ZZRD2, with the accession number CGMCC No. 16778, which can effectively remove nitrate in wastewater.

[0010] The invention of CN202111585008.5, "Pseudomonas stutzeri with aerobic denitrification and greenhouse gas emission reduction functions", informs a Pseudomonas stutzeri, with the accession number CGMCC NO. 23473, which has a high denitrification rate and strong N2O reduction capacity, resulting in less N2O emission and effective removal of nitrate in wastewater.

[0011] Li Q, Sun Z, Zhou H, Efficient Aerobic Denitrification of Pseudomonas gessardii YZ-7 and Its Denitrification Performance Research, South China Journal of Agriculture, 2023, 54(5): 1549-1558, found that Pseudomonas gessardii YZ-7 had a high NO3 - - removal rate of 99.71% to 99.85% when the carbon-nitrogen ratio (C / N) was 4.42 to 5.00.

[0012] The invention of CN202410306773.6, "Pseudomonas sp. NH-1, culture method and application thereof", informs a Pseudomonas sp. NH-1, with the accession number CCTCC NO: M 20232429, which can reduce ammonia nitrogen concentration in aquaculture water under heterotrophic aerobic conditions. However, it is more suitable for treating low-concentration ammonia-nitrogen-containing wastewater in aquaculture. The examples show that when the initial ammonia-nitrogen concentration is 6.654 mg·L -1 , the ammonia-nitrogen removal rate is 92.97%, and there is partial accumulation of nitrate and nitrite nitrogen.

[0013] CN201210269891.1 invention "A kind of denitrifying phosphorus bacteria and its application in wastewater treatment" informs a strain of denitrifying phosphorus bacteria (Pseudomonas oleovorans) CL-3, belongs to the oil-eating pseudomonas of pseudomonas genus, preservation number is: CCTCC NO:M 2012226. In wastewater treatment, it has the use of denitrification and dephosphorization. And CN201210269870.X invention "A kind of denitrifying phosphorus bacteria enhanced constructed wetland method for treating rural domestic sewage" also informs Pseudomonas oleovorans CL-3 can make the content indexes of COD, ammonia nitrogen and total phosphorus of effluent of rural domestic sewage treated by artificial wetland reach the first B standard of "Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant" (GB18918-2002). But it is more suitable for the treatment of low-concentration ammonia-nitrogen-containing wastewater, the example shows that when the initial concentration of ammonia nitrogen is 70 mg·L -1 , the ammonia nitrogen removal rate is 90%, and there is partial nitrate nitrogen and nitrite nitrogen accumulation, resulting in a total nitrogen removal rate of about 70%. SUMMARY

[0014] The technical problem to be solved by the present application is to provide a strain of Pseudomonas oleovorans QS-7 that can be used for denitrification of ammonia-nitrogen-rich pig breeding wastewater and its use.

[0015] To solve the above technical problems, the present application provides a strain of Pseudomonas oleovorans QS-7, preservation number: CGMCC No.30323.

[0016] It is a Pseudomonas oleovorans

[0017] As an improvement of the Pseudomonas oleovorans strain of the present application: the 16S rDNA gene sequence of the strain is shown in SEQ ID NO:1.

[0018] The present application also provides the use of the above-mentioned Pseudomonas oleovorans strain: treating ammonia-nitrogen-containing (ammonia-nitrogen-rich, ammonia-nitrogen concentration of 750-1500 mg·L -1 ) pig breeding wastewater.

[0019] As an improvement of the use of the Pseudomonas oleovorans strain of the present application: removing nitrogen from ammonia-nitrogen-rich pig breeding wastewater.

[0020] The preservation information for strain QS-7 of this invention is as follows:

[0021] Preservation Name: Pseudomonas oleovorans, Preservation Institution: China General Microbiological Culture Collection Center, Preservation Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Preservation Number: CGMCC No. 30323, Preservation Date: April 12, 2024.

[0022] The colony characteristics of this strain are as follows: it is a short rod-shaped organism with a rough surface, pili, does not produce spores, has a small capsule, is Gram-negative, and has a cell size of (1.2–1.6) μm × (0.5–0.7) μm. On solid culture media, the colonies are round, with a raised, smooth surface, intact edges, and are grayish-white and opaque. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO: 1.

[0023] The strain of this invention was obtained by screening wastewater from a biogas oxidation pond in a pig farm in Pingxiang City, Jiangxi Province. Based on the fatty acid profile generated by the qualitative and quantitative analysis of strain QS-7 using the Sherlock MIS software system and compared with the Library database, strain QS-7 was preliminarily identified as *Pseudomonas oleovorans*, with a similarity index (SI) of 0.983. 16S rDNA sequencing also confirmed that the strain is *Pseudomonas oleovorans*, with a similarity index (SI) of 0.99.

[0024] The strain of this invention was inoculated into pig farming wastewater rich in ammonia nitrogen and subjected to treatment at 15–35°C and 150–200 rpm. -1 Cultivating under aerobic conditions for 2-3 days can effectively remove CODcr, BOD5 and ammonia nitrogen from wastewater.

[0025] The ammonia-rich pig farming wastewater (hereinafter referred to as ammonia-containing wastewater) described in this invention has a CODcr concentration of 6000–12000 mg·L⁻¹. -1 BOD5 concentration is 3000-5000 mg / L -1 and ammonia nitrogen concentration of 750–1500 mg·L -1 .

[0026] This strain can be used to treat ammonia-rich pig farm wastewater. Under aerobic conditions, at pH approximately 7.0 and temperature (25–30) °C, inoculating with 5% QS-7 bacteria at a volume ratio yields an OD of [missing value]. 415nm0.2, the CODcr concentration of the pig breeding wastewater rich in ammonia nitrogen was 11000 mg·L -1 , the BOD5 concentration was 5500 mg·L -1 , and the ammonia nitrogen concentration was 800 mg·L -1 , under the condition of rotating speed 180 r·min -1 for 3 days, filtration, the removal rates of CODcr, BOD5 and ammonia nitrogen in the wastewater were 98.54%, 98.73% and 98.17% respectively, and nitrate nitrogen and nitrite nitrogen were not detected, which means that no intermediate product is accumulated, the reduced ammonia nitrogen is completely converted by the microorganism, and the water quality can reach the limited standard (the CODcr concentration is 400 mg·L -1 , the BOD5 concentration is 150 mg·L -1 , and the ammonia nitrogen concentration is 80 mg·L -1 ) of the Discharge Standard of Pollution Discharge from Livestock and Poultry Breeding (GB 18596-2001).

[0027] The beneficial effects of the present application mainly reflect that the Pseudomonas oleovorans QS-7 for ammonia nitrogen wastewater denitrification treatment is screened, which provides a technical basis for controlling the influence of wastewater rich in ammonia nitrogen on the environment and has a wide application prospect.

[0028] In summary, the Pseudomonas oleovorans QS-7 separated from the pig farm biogas slurry oxidation pond is found, which has the potential of completely denitrifying high ammonia nitrogen wastewater with high efficiency and without nitrate nitrogen and nitrite nitrogen residues, but no related research has been reported at home and abroad, therefore, the present application relates to the Pseudomonas oleovorans QS-7 separated from the pig farm biogas slurry oxidation pond and the application thereof in the denitrification treatment of pig breeding wastewater rich in ammonia nitrogen, which is first discovered.

[0029] It should be emphasized that: the known Pseudomonas oleovorans and its similar genera mainly degrade organic matter, denitrify nitrate nitrogen or remove low-concentration ammonia nitrogen, and there is a certain accumulation of nitrate nitrogen and nitrite nitrogen in the process of removing ammonia nitrogen, which means that part of the removed ammonia nitrogen does not really leave the water body (converted into nitrate nitrogen and nitrite nitrogen form). The Pseudomonas oleovorans QS-7 separated from the pig farm biogas slurry oxidation pond found in the present application can be used for denitrification treatment of high ammonia nitrogen wastewater, and there is no accumulation of nitrate nitrogen and nitrite nitrogen, and the removed ammonia nitrogen really leaves the water body, which is first discovered. BRIEF DESCRIPTION OF DRAWINGS

[0030] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0031] Figure 1 Pseudomonas oleovorans QS-7 (enlarged 30000 times) ;

[0032] Figure 2 16S rDNA PCR amplification results (1: strain QS-7; Marker: DNA standard molecular weight) ;

[0033] Figure 3 Strain QS-7 phylogenetic tree based on 16S rDNA sequence. DETAILED DESCRIPTION

[0034] The present application will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present application is not limited to this:

[0035] Example 1, Screening and identification of ammonia nitrogen removal strain

[0036] 1. Materials and methods

[0037] 1.1 Culture medium and reagent

[0038] Enrichment medium: ammonium sulfate (2, 5, 10, 20 or 30 g, gradually increased to add for acclimation enrichment), CaCl2 6H2O 0.1 g, MgCl2 0.25 g, K2HPO4 1.5 g, NH4Cl 1 g, peptone 9 g, H2O 500 mL, pH = 6.5-7.0;

[0039] Basic medium: (NH4)2SO4 1.5 g, MgSO4 7H2O 0.5 g, KH2PO4 0.5 g, NaCl 0.5 g, K2HPO4 1.5 g, H2O 1000 mL, pH 4.0;

[0040] The basic medium is added with 1.5% (w / v, 1.5 g per 100 mL) agar and 30 g L -1 of ammonium sulfate to prepare the corresponding solid basic medium;

[0041] Pig breeding wastewater rich in ammonia nitrogen: pH about 7.0, CODcr concentration of 11000 mg L -1 , BOD5 concentration of 5500 mg L -1 , ammonia nitrogen concentration of 800 mg L -1 .

[0042] 1.2 Determination of strain treatment efficiency on pig breeding wastewater rich in ammonia nitrogen

[0043] After the single strain was purified, the strain liquid of OD 415nm =0.2 was inoculated into 100 mL of the pig breeding wastewater rich in ammonia nitrogen (pH about 7.0, CODcrconcentration of 11000 mg·L -1 , BOD5concentration of 5500 mg·L -1 , ammonia nitrogen concentration of 800 mg·L -1 ) in a test tube (250 mL) at a volume inoculation of 5%, and un-inoculated mixed wastewater was used as a control, and the strain liquid was cultured in an aerobic way on a constant temperature shaker at 30℃ and 180 r·min -1 for 3 days. After the culture was finished, the mycelium was removed by filtering with a 0.20 μm membrane, and the filtrate was used for determination of CODcr, BOD5, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen (the method is described in: State Environmental Protection Administration of China, Water and Wastewater Monitoring and Analysis Methods (Fourth Edition), 2002, China Environmental Science Press).

[0044] Removal rate (%) = (residual amount of the control sample - residual amount of the treated sample) x 100 / residual amount of the control sample

[0045] 1.3 Selection of dominant ammonia nitrogen removal strain

[0046] 1.3.1 Strain source

[0047] The biogas slurry oxidation pond wastewater was collected from a pig breeding farm in Pingxiang City, Jiangxi Province.

[0048] 1.3.2 Isolation, purification and screening of ammonia nitrogen removal strain

[0049] 10 ml of the wastewater was removed from the biogas slurry oxidation pond of the pig breeding farm and added to a 100 mL sterile enrichment medium in a 250 mL test tube in a sterile environment. The test tube was cultured on a shaker at 30℃ and 180 r·min -1 for 7 days, and then transferred to the next batch of enrichment medium (sulfate concentration gradient was 2, 5, 10, 20 or 30 g·L -1 ) at a 10% inoculation amount, and cultured under the same condition for 7 days. Then, the test tube was transferred to a sterile liquid of the basic medium containing 30 g·L -1 of sulfate (i.e., the sulfate concentration of the basic medium was 30 g·L -1 ) at a 10% inoculation amount, and cultured for 7 days. After continuous transfer for 2 times, 0.1 mL of the obtained fermentation liquid was repeatedly plated and isolated on a solid basic medium, and the pure colonies were inoculated on a slope and stored in a refrigerator at 4℃.

[0050] The above sulfate concentration was 30 g·L -1The basic medium is prepared by adding 30 g of ammonium sulfate into 1000 ml of basic medium.

[0051] 1.3.3 Strain identification

[0052] The strain identification uses the Sherlock microbial identity system (MIS) software system of the American MIDI company. The system extracts and analyzes the fatty acids of the purified single colony on the basic medium according to the operating specifications of the MIDI company. The fatty acid components of the microorganism are analyzed qualitatively (species) and quantitatively (content), and the fatty acid spectrum is generated. The generated spectrum is compared with the database (Library), and the unknown strain is identified according to the similarity index SI (similarity index). The similarity index SI is greater than 0.9, which can basically determine that it is a certain microorganism. The system is a relatively fast and rich microbial identification system with a library, which has been widely used (Wu YP, Xu JM, Wang HZ, et al. Application of Sherlock MIS system in soil bacterial identification. Acta Pedologica Sinica, 2006, 43(4): 642-647).

[0053] In addition, the 16S rDNA identification method is used for verification and comparison with the Sherlock microbial identity system (MIS). The total DNA of the QS-7 strain is used as a template, and the universal primer of 16S rDNA gene is used for PCR amplification. The size of the amplified fragment can be determined by recovery and sequencing. The sequencing results are compared with the sequences in GenBank for homology comparison using BLAST software.

[0054] 1.3.4 Observation of strain morphological characteristics and determination of physiological and biochemical characteristics

[0055] The strain is inoculated in solid basic medium, and the morphological characteristics of the strain are observed by electron microscopy after 48 h. The purified strain growing in the logarithmic phase is stained with Gram, crystal violet and simple capsule. The physiological and biochemical characteristics are determined according to the Common Bacterial System Identification Manual (Dong XZ, Cai MY).

[0056] 2. Results

[0057] 2.1 Isolation and screening of strains

[0058] A strain capable of effectively realizing the denitrification treatment of pig breeding wastewater rich in ammonia nitrogen is obtained through isolation, purification and screening, which is named QS-7. The ammonia nitrogen degradation ability of other strains is different degrees of recession.

[0059] 2.2 Strain identification

[0060] 2.2.1 Basic morphology and physiological and biochemical characteristics of strain QS-7

[0061] like Figure 1 As shown, QS-7 is a short bacillus with a rough surface, pili, and does not produce spores. It has a small capsule, is Gram-negative, and its cell size is (1.2–1.6) μm × (0.5–0.7) μm. On solid culture media, colonies are round, with a raised, smooth surface, intact edges, and are grayish-white and opaque. It is negative for acetylmethylethanol (VP) formation, positive for indole, and cannot liquefy gelatin. Other physiological and biochemical characteristics are shown in Table 1.

[0062] Table 1. Physiological and biochemical characteristics of Pseudomonas oleovorans QS-7

[0063] Test item Result Physiological and biochemical tests Result Gram stain - V-P test - Methyl red - Indole test + Lipase - Hydrogen sulfide - Lysine decarboxylase + Tryptophan deaminase - Ornithine decarboxylase + Oxidase - Citrate + Urease hydrolysis - Xylose + Arginine dihydrolase - Glucose gas production + Sucrose acid production - Malonate - Maltose acid production - Lactose + Dulcitol acid production - L-alanine + Denitrification + Salicylic acid - Liquefied gelatin -

[0064] Note: + Positive reaction; - Negative reaction

[0065] 2.2.2 Identification of strain QS-7

[0066] (1) Qualification of QS-7 by Sherlock MIS system

[0067] Based on the fatty acid profile generated by the Sherlock MIS software system through qualitative and quantitative analysis of the fatty acids of bacteria QS-7, and by comparing it with the Library database, bacteria QS-7 was preliminarily identified as Pseudomonas oleovorans, with a similarity index (SI) of 0.983.

[0068] (2) Validation of QS-7 by 16S rDNA identification method

[0069] I. DNA Extraction Results

[0070] Using total DNA from QS-7 strain as a template, PCR amplification was performed using the universal gene for 16S rDNA, yielding a fragment approximately 1 kb in size. Figure 2 The amplified fragment was recovered and sequenced, and its size was determined to be 1421 bp (SEQ ID NO: 1).

[0071] II. 16S rDNA gene PCR amplification and sequence analysis

[0072] Homology comparison of the sequencing results with those in GenBank using BLAST software revealed that the QS-7 strain has high homology with Pseudomonas oleovorans, with a homology of nearly 99%, indicating a close genetic distance.

[0073] According to the physiological and biochemical properties of QS-7, Sherlock MIS (MIDI identification system) and 16S rDNA phylogenetic analysis, QS-7 is identified as Pseudomonas oleovorans. Figure 3 It can be seen that the bacteria belong to Pseudomonas oleovorans.

[0074] 3 Conclusion

[0075] A high-efficiency strain QS-7 (CGMCC No. 30323) capable of effectively treating ammonia-nitrogen-rich pig breeding wastewater was isolated from the oxidation pond wastewater of a pig farm in Pingxiang City, Jiangxi Province. It was identified as Pseudomonas oleovorans by Sherlock MIS system and 16S rDNA.

[0076] QS-7 was preserved, and the preservation information is as follows:

[0077] Preservation name: Pseudomonas oleovorans, preservation unit: China General Microbiological Culture Collection Center, preservation address: No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard, preservation number: CGMCC No. 30323, preservation date: April 12, 2024.

[0078] Example 2, strain treatment efficiency on ammonia-nitrogen-rich pig breeding wastewater

[0079] 1. Materials and methods

[0080] 1.1 Culture medium and reagents

[0081] Basic medium: MgSO4.7H2O 0.5g, (NH4)2SO4 1.5g, KH2PO4 0.5g, NaCl 0.5g, K2HPO4 1.5g, H2O 1000mL, pH 4.0;

[0082] Ammonia-nitrogen-rich pig breeding wastewater: pH about 7.0, CODcr concentration 11000mg·L -1 , BOD5 concentration 5500mg·L -1 , ammonia nitrogen concentration 800mg·L -1 .

[0083] 1.2 Determination of strain treatment efficiency on ammonia-nitrogen-rich pig breeding wastewater

[0084] The purified single strain QS-7 was expanded in the basic medium, and the expansion was as follows: 1 inoculation ring of QS-7 was added to 100mL basic medium at 30℃, 180r·min -1on a constant temperature shaker until the bacterial amount OD 415nm =0.2 (the culture time is about 2 days) is obtained; the bacterial strain QS-7 culture solution is obtained.

[0085] According to a 5% volume inoculation amount, the bacterial amount OD 415nm =0.2 of the bacterial strain QS-7 culture solution is inoculated into 100 mL of the ammonia-nitrogen-rich pig breeding wastewater (pH is about 7.0, CODcr concentration is 11000 mg·L -1 , BOD5 concentration is 5500 mg·L -1 , and ammonia-nitrogen concentration is 800 mg·L -1 ) in a 250 mL triangular flask, and the ammonia-nitrogen-rich pig breeding wastewater without inoculation is used as a control, and the aerobic culture is carried out at 30℃, 180 r·min -1 on a constant temperature shaker for 3 days. After the culture is finished, the CODcr, BOD 5、 ammonia-nitrogen, nitrate-nitrogen and nitrite-nitrogen of the pig breeding wastewater are determined according to the method in Example 1.

[0086] 2. Results

[0087] After the bacterial strain QS-7 is cultured for 3 days under the conditions of pH about 7.0, 5% volume of the bacterial solution inoculation amount (the bacterial concentration OD 415nm =0.2 of the QS-7 mother solution), 30℃, 180 r·min -1 and aerobic environment, the removal rates of CODcr, BOD5 and ammonia-nitrogen in the pig breeding wastewater are 98.54%, 98.73% and 98.17% respectively, and the residual concentrations of CODcr, BOD5 and ammonia-nitrogen are 160.6 mg·L -1 , 69.8 mg·L -1 and 14.6 mg·L -1 respectively, and the nitrate-nitrogen and nitrite-nitrogen are not detected, which means that there is no intermediate product accumulation, and the reduced ammonia-nitrogen is completely converted by the microorganism. It can be seen that the QS-7 has a high removal capacity for the ammonia-nitrogen in the ammonia-nitrogen-rich pig breeding wastewater, and there is no accumulation of intermediate product. The water quality can reach the limited standard (CODcr concentration is 400 mg·L -1 , BOD5 concentration is 150 mg·L -1 and ammonia-nitrogen concentration is 80 mg·L -1 ) of the pollutant discharge standard (GB18596-2001) of the livestock and poultry breeding industry, which shows that the Pseudomonas oleovorans QS-7 separated and screened in the application is a high-efficiency strain which can effectively realize the denitrification of the ammonia-nitrogen-rich pig breeding wastewater, and has a certain application potential for controlling the influence of the ammonia-nitrogen-rich wastewater discharge on the environment.

[0088] Comparative experiment, the rest of the strains obtained in the screening process and the current existing similar genus (as described in Table 2), after replacing QS-7, according to the method described in Example 2, the results obtained are compared with the results of QS-7 as described in Table 2. It can be seen that the Pseudomonas oleovorans QS-7 discovered in the present application is a high-efficiency strain that can effectively achieve denitrification of pig breeding wastewater rich in ammonia nitrogen. It can simultaneously and efficiently remove CODcr, BOD5 and ammonia nitrogen in wastewater, and there is no accumulation of nitrate and nitrite nitrogen. It has certain application potential for controlling the impact of ammonia nitrogen-rich wastewater discharge on the environment, while other similar genera do not have this ability or have weak ability.

[0089] Table 2

[0090]

[0091]

[0092] Example 3: Strain treatment performance test on low-concentration ammonia nitrogen-containing breeding wastewater produced by water flushing pen breeding method

[0093] 1.1 Culture medium and reagents

[0094] Basic medium: MgSO4.7H2O 0.5g, (NH4)2SO4 1.5g, KH2PO4 0.5g, NaCl 0.5g, K2HPO4 1.5g, H2O 1000mL, pH 4.0;

[0095] Ammonia nitrogen-containing pig breeding wastewater produced by water flushing pen breeding method: pH about 7.0, CODcr concentration 5650mg·L -1 , BOD5 concentration 2830mg·L -1 , ammonia nitrogen concentration 407mg·L -1 .

[0096] 1.2 Strain treatment performance on low-concentration ammonia nitrogen-containing breeding wastewater produced by water flushing pen breeding method

[0097] After the purified single strain was expanded in the basic medium, the strain QS-7 culture solution with OD 415nm =0.2 was obtained, and the above QS-7 culture solution was inoculated into 100mL ammonia nitrogen-containing pig breeding wastewater (pH about 7.0, CODcr concentration 5650mg·L -1 , BOD5 concentration 2830mg·L -1 , ammonia nitrogen concentration 407mg·L -1)250mL triangular flask, with non-bacterial water flush column breeding mode to produce ammonia nitrogen containing pig breeding wastewater as control, in 30℃, 180r·min -1 constant temperature shaker for 3 days. After the culture, the CODcr, BOD 5、 ammonia nitrogen, nitrate nitrogen and nitrite nitrogen of the strain QS-7 in the ammonia nitrogen containing pig breeding wastewater were determined according to the method of example 1.

[0098] 2. Results

[0099] The strain QS-7 in pH about 7.0, 5% wastewater volume of bacteria inoculation amount (QS-7 mother liquor bacteria concentration OD 415nm =0.2) 30℃, 180r·min -1 and aerobic environment conditions, the removal rate of CODcr, BOD5 and ammonia nitrogen of the water flush column breeding mode to produce ammonia nitrogen containing pig breeding wastewater was 97.89%, 98.45% and 98.3% respectively. The residual concentration of CODcr, BOD5, total phosphorus and ammonia nitrogen was 119.2mg·L -1 , 43.8mg·L -1 and 6.9mg·L -1 , nitrate nitrogen and nitrite nitrogen was not detected, and the water quality met the limit standard of the pollutants discharge standard of livestock and poultry breeding industry (GB18596-2001) (CODcr concentration was 400mg·L -1 , BOD5 concentration was 150mg·L -1 and ammonia nitrogen concentration was 80mg·L -1 ), which indicated that the Pseudomonas oleovorans QS-7 separated and screened by the application was a high efficient strain which could effectively realize the denitrification of the ammonia nitrogen containing pig breeding wastewater, and had certain application potential for controlling the influence of the ammonia nitrogen containing wastewater discharge on the environment.

[0100] Example 4, strain treatment performance detection of low carbon nitrogen ratio ammonia nitrogen containing pig breeding wastewater

[0101] 1. Materials and methods

[0102] 1.1 Culture medium and reagent

[0103] Basic culture medium: MgSO4.7H2O 0.5g, (NH4)2SO4 1.5g, KH2PO4 0.5g, NaCl 0.5g, K2HPO4 1.5g, H2O 1000mL, pH4.0;

[0104] Low carbon nitrogen ratio ammonia nitrogen containing pig breeding wastewater: pH about 7.0, CODcr concentration was 2197mg·L -1, BOD5 concentration is 1328 mg·L -1 , ammonia nitrogen concentration is 423 mg·L -1 .

[0105] 1.2 Strain treatment performance on low carbon-nitrogen ratio pig breeding wastewater rich in ammonia nitrogen

[0106] After the single strain is purified and expanded in the basic medium, the strain QS-7 culture solution with OD 415nm =0.2 is obtained, the QS-7 culture solution is inoculated into 100 mL low carbon-nitrogen ratio pig breeding wastewater rich in ammonia nitrogen (pH is about 7.0, CODcr concentration is 2197 mg·L -1 , BOD5 concentration is 1328 mg·L -1 , ammonia nitrogen concentration is 423 mg·L -1 ) 250 mL triangular flask at 5% volume inoculation amount, and the low carbon-nitrogen ratio pig breeding wastewater rich in ammonia nitrogen without inoculation is used as a control, and the aerobic culture is carried out at 30℃, 180 r·min -1 on a constant temperature shaker for 3 days. After the culture is finished, the CODcr, BOD 5、 ammonia nitrogen, nitrate nitrogen and nitrite nitrogen of the low carbon-nitrogen ratio pig breeding wastewater rich in ammonia nitrogen treated by the strain QS-7 are determined according to the method in the embodiment 1.

[0107] 2. Results

[0108] The removal rates of CODcr, BOD5 and ammonia nitrogen of the low carbon-nitrogen ratio pig breeding wastewater rich in ammonia nitrogen treated by the strain QS-7 are 96.37%, 97.28% and 94.87% respectively under the conditions of pH about 7.0, 5% wastewater volume inoculation amount (the QS-7 mother liquor bacterial concentration OD415nm=0.2), 30℃, 180 r·min -1 and aerobic environment, the residual concentrations of CODcr, BOD5 and ammonia nitrogen are 79.7 mg·L -1 , 36.1 mg·L -1 and 21.7 mg·L -1 , the nitrate nitrogen and nitrite nitrogen are not detected, the water quality meets the limited standard of the pollutant discharge standard for livestock and poultry breeding industry (GB18596-2001) (CODcr concentration is 400 mg·L -1 , BOD5 concentration is 150 mg·L -1 and ammonia nitrogen concentration is 80 mg·L -1 ), which indicates that the Pseudomonas oleovorans QS-7 separated and screened in the application is a high-efficiency strain which can effectively realize the denitrification of the pig breeding wastewater rich in ammonia nitrogen, and has certain application potential for controlling the influence of the wastewater rich in ammonia nitrogen on the environment.

[0109] Finally, it should be noted that the above enumeration is only several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or inferred from the disclosure of the present application by those of ordinary skill in the art should be considered within the scope of the present application.

Claims

1. Pseudomonas oleovorans QS-7, characterized in that Deposit No. CGMCC No. 30323.

2. The Pseudomonas oleovorans QS-7 of claim 1, characterized in that: The 16S rDNA gene sequence of the strain is shown as SEQ ID NO:

1.

3. Use of Pseudomonas oleovorans QS-7 according to claim 1 or 2, characterized in that: Treatment of pig breeding wastewater containing ammonia nitrogen.

4. Use of Pseudomonas oleovorans QS-7 according to claim 3, characterized in that: Removal of CODcr, BOD5 and ammonia nitrogen in pig breeding wastewater rich in ammonia nitrogen. Removal of CODcr, BOD5 and ammonia nitrogen in pig breeding wastewater rich in ammonia nitrogen.

Images