Pseudomonas aeruginosa HRKJ-4, microbial preparation and application thereof

Abstract

The present invention provides a Pseudomonas aeruginosa HRKJ-4, a microbial preparation and an application thereof. The deposit number of Pseudomonas aeruginosa HRKJ-4 is CGMCC No. 24351, and the strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 20, 2022. The strain can grow well and remove ammonia nitrogen in the salinity range of 5-40g / L, especially when the salinity is 40g / L, it can also show a promoting effect on bacterial growth, and has good application prospects in the denitrification treatment of high-ammonia nitrogen livestock and poultry breeding wastewater with medium and high salinity.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pseudomonas aeruginosa HRKJ-4, a microbial preparation and applications thereof. Background Art

[0002] With the rapid development of industrial and agricultural production and the improvement of people's living standards, the generation and discharge of various types of sewage have increased year by year. According to the "Environmental Bulletin" published by the Ministry of Ecology and Environment, the main pollutants detected in my country's freshwater resources that exceeded the standard include ammonia nitrogen (NH 4 -N), total nitrogen (TN) and total phosphorus (TP). Among them, nitrogen-containing pollutants have become the main source of environmental pollution, attracting widespread attention from all walks of life. Nitrogen exists in various forms of pollutants and is difficult to remove. If it is discharged into the environment without effective treatment, it will cause serious harm to the aquatic ecosystem and human health.

[0003] Biological denitrification, represented by the activated sludge method, is widely used in the treatment of various types of nitrogen-containing wastewater due to its economic, high efficiency, and no secondary pollution. Traditional biological denitrification refers to the removal of nitrogen through the two processes of aerobic autotrophic nitrification and anaerobic heterotrophic denitrification under the combined action of microorganisms. However, this process has the following disadvantages: (1) Autotrophic nitrifying bacteria grow slowly, have poor environmental adaptability, are weak in shock load resistance, and are easily inhibited by high concentrations of ammonia nitrogen and nitrite nitrogen. (2) The two reactions of nitrification and denitrification cannot be unified in time and space, which increases investment and operating costs.

[0004] In recent years, a new type of denitrifying microorganism, heterotrophic nitrification-aerobic denitrifying bacteria, has been discovered and continuously isolated. This type of microorganism can simultaneously perform nitrification and denitrification under aerobic conditions, completing the simultaneous removal of carbon and nitrogen pollutants, and has a fast growth rate, which can shorten the reaction cycle, save space and reduce operating costs. It has good application prospects in the field of sewage denitrification. However, most of the strains reported so far have poor tolerance and treatment effects on sewage with high salinity and high ammonia nitrogen concentration. Summary of the invention

[0005] The main technical problem solved by the present invention is to provide a Pseudomonas aeruginosa HRKJ-4, a microbial preparation and application thereof, so as to solve the problem that heterotrophic nitrification-aerobic denitrification bacteria in the prior art have poor tolerance to sewage with high salinity and high ammonia nitrogen concentration and poor treatment effect.

[0006] In order to solve the above technical problems, a technical solution adopted in the present application is: to provide a Pseudomonas aeruginosa HRKJ-4, whose preservation number is CGMCC No.24351.

[0007] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection on January 20, 2022, abbreviated as CGMCC. Its address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The strain deposit number is CGMCC No. 24351.

[0008] Another technical solution adopted in the present application is to provide a microbial preparation, including the above-mentioned Pseudomonas aeruginosa HRKJ-4.

[0009] Another technical solution adopted in the present application is to provide an application of the above-mentioned Pseudomonas aeruginosa HRKJ-4 or the above-mentioned microbial preparation in sewage treatment.

[0010] In one embodiment, the sewage treatment includes removing one or more pollutants including COD, ammonia nitrogen and total nitrogen in the sewage.

[0011] Preferably, the sewage treatment is to simultaneously remove COD, ammonia nitrogen and total nitrogen in the sewage.

[0012] In one embodiment, the sewage is sewage with high ammonia nitrogen concentration.

[0013] In one embodiment, the sewage is livestock and poultry breeding sewage with high ammonia nitrogen concentration.

[0014] In one embodiment, the sewage is pig farming sewage with high ammonia nitrogen concentration.

[0015] In one embodiment, the ammonia nitrogen concentration of the sewage is 400-500 mg / L.

[0016] In one embodiment, the salinity of the sewage is 5 to 40 g / L.

[0017] Different from the prior art, the beneficial effects of this application are:

[0018] 1. The Pseudomonas aeruginosa HRKJ-4 provided in the present application can grow well in the salinity range of 5-40g / L, especially when the salinity is 40g / L, it can also show a promoting effect on bacterial growth, can tolerate a salt concentration of up to 40g / L and show good growth, and can achieve good growth in sewage treatment with medium and high salinity; when the sewage salinity is 5, 20, 40, and 60g / L, the final ammonia nitrogen removal rates are 89.2%, 91.9%, 88.7%, and 9.4%, respectively, and it has good application prospects in sewage treatment with medium and high salinity;

[0019] 2. When the Pseudomonas aeruginosa HRKJ-4 provided in the present application is applied to the treatment of pig wastewater with an initial COD of 11700 mg / L and an initial ammonia nitrogen of 465 mg / L, the 72h ammonia nitrogen removal rate reaches 97.2%, the COD removal rate is 77.8%, and the TN removal rate reaches 84.2%. The effluent ammonia nitrogen concentration has reached the requirements of my country's "Pollutant Emission Standards for Animal Husbandry and Poultry Industry (GB18596-2001)", and has good application prospects in the denitrification treatment of livestock and poultry wastewater with medium and high ammonia nitrogen concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a Gram-stained micrograph of Pseudomonas aeruginosa HRKJ-4 of the present application;

[0021] Figure 2 This is a graph showing the growth status of Pseudomonas aeruginosa HRKJ-4 under different salinity conditions;

[0022] Figure 3 This is a graph showing the ammonia nitrogen removal performance of Pseudomonas aeruginosa HRKJ-4 under different salinity conditions;

[0023] Figure 4 This is a diagram showing the treatment effect of Pseudomonas aeruginosa HRKJ-4 on pig wastewater in the present application. DETAILED DESCRIPTION

[0024] The present application discloses a Pseudomonas aeruginosa HRKJ-4, a microbial preparation and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0025] The Pseudomonas aeruginosa HRKJ-4, microbial preparations and reagents or excipients used in their applications provided in the present application can be purchased from the market.

[0026] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0027] The components of the culture medium used in the following examples are as follows:

[0028] LB medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1 L distilled water.

[0029] Heterotrophic nitrification medium: 4 g sodium chloride, 2.66 g disodium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 4.41 g potassium citrate, 0.38 g ammonium chloride, 3 mL trace element solution, 1 L distilled water, natural pH.

[0030] Trace element solution: 3g magnesium sulfate heptahydrate, 3g manganese sulfate monohydrate, 3g zinc sulfate heptahydrate, 1.12g boric acid, 0.3g ferrous sulfate heptahydrate, 0.6g calcium chloride dihydrate, 1L distilled water.

[0031] Example 1: Isolation and screening of strains

[0032] (1) Isolation and purification of strains

[0033] In July 2020, samples were collected from the aerobic activated sludge of a sewage treatment plant in Urumqi. After the samples were fully mixed, 20 mL was suspended in 180 mL of 0.2% sodium chloride solution. 5 mL of the suspension was placed in a 250 mL conical flask containing 100 mL of heterotrophic nitrification medium and cultured at 30°C and 180 rpm for acclimatization and enrichment.

[0034] The acclimatization cycle was 48 hours. After each cycle, 10 mL of the enrichment solution was added to a new 100 mL heterotrophic nitrification medium for further enrichment culture. This was repeated for 5 cycles. During this period, the removal of ammonia nitrogen in the culture medium was tested. The culture medium after 5 generations of enrichment was taken and sterilized with water for 10 min. -3 ~10 -7 , and spread 100 μL of each dilution on a heterotrophic nitrification solid medium. Place it in a biochemical incubator at 30°C for 48 hours, observe the growth of the colonies, select single colonies with different morphologies, and streak them on a heterotrophic nitrification solid medium using the plate streak method, and culture them under the same conditions for 48 hours. Then select single colonies for multiple partition streak purification until a pure strain is obtained. The purified strain is placed in 20% glycerol and frozen at -80°C for later use.

[0035] (2) Screening of strains

[0036] Use an inoculation loop to pick the purified strains and inoculate them into the sterilized LB liquid medium. After culturing at 30°C and 180 rpm for 24 h, take 2.5 mL of the bacterial solution according to the inoculum volume of 5% (v / v), centrifuge at 4000 rpm for 5 min, collect the bacteria, wash with sterile water, and inoculate into a 150 mL conical flask containing 50 mL of heterotrophic nitrification medium. Cultivate in a shaker at 30°C and 180 rpm for 72 h, and take samples to detect NH in the culture medium at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. 4 -N, TN and COD content, and screen out strains with heterotrophic nitrification function.

[0037] Example 2: Identification of strains

[0038] (1) Morphological identification

[0039] After the above separation and purification process, a heterotrophic nitrification-aerobic denitrification bacterium HRKJ-4 was obtained. The morphological characteristics of the bacterium are rod-shaped, the size of the bacterium is (0.5-0.8) μm×(1.2-3) μm, it does not produce spores, the colony is round, the edges are neat and smooth, and it is yellow-green on the LB medium. It is a Gram-negative aerobic bacterium. Figure 1 shown.

[0040] (2) Molecular Biological Identification

[0041] The obtained strains were subjected to molecular biological identification, and the bacterial 16S rDNA sequence (SEQ ID NO.1) was amplified by PCR and then sequenced and compared.

[0042] The amplification primers were 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2), 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID NO. 3);

[0043] The reaction system was: 10× Buffer 2 μL, 2.5 mM dNTP 1.5 μL, Primer1 1 μL, Primer2 1 μL, template 1 μL, enzyme 0.3 μL, water 13.2 μL, total volume 20 μL;

[0044] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, extension at 72°C for 10 min, and insulation at 4°C forever.

[0045] The PCR products were detected by agarose gel electrophoresis and then sequenced.

[0046] The 16S rDNA sequence of the bacterium (SEQ ID NO.1) was obtained after forward and reverse sequencing and splicing. The sequence was compared with the NCBI database by Blast. The comparison results showed that it had the highest homology with Pseudomonas aeruginosa JCM5962, reaching 99.93%. The strain was named Pseudomonas aeruginosa HRKJ-4.

[0047] Pseudomonas aeruginosa HRKJ-4 was deposited in the General Microbiology Center of the China Culture Collection Administration on January 20, 2022, abbreviated as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 24351.

[0048] Example 3: Determination of the effect of salinity on the growth of Pseudomonas aeruginosa HRKJ-4

[0049] The salinity of the heterotrophic nitrification medium was adjusted to 5, 20, 40, 60, and 80 g / L using NaCl, and Pseudomonas aeruginosa HRKJ-4 was activated according to the conventional method. A certain amount of bacterial liquid was taken according to the inoculation volume of 2% (v / v), and the cells were collected after centrifugation at 4000 rpm for 5 min. The cells were washed with sterile water and inoculated into the heterotrophic nitrification medium after the salinity was adjusted. The cells were cultured in a shaker at 30°C and 180 rpm for 72 h, and the OD600 values ​​at each final salinity were detected.

[0050] The results are as follows Figure 2 As shown. Pseudomonas aeruginosa HRKJ-4 can grow well in the salinity range of 5-40g / L. When the salinity was 5, 20, 40, and 60g / L, the final bacterial density OD600 values ​​were 0.603, 0.617, 1.305, and 0.249, respectively. In particular, when the salinity was 40g / L, it could also promote bacterial growth. However, when the salinity reached 60g / L and above, the growth of the strain was severely inhibited. The above results show that HRKJ-4 can tolerate salt concentrations as high as 40g / L and show good growth, indicating that Pseudomonas aeruginosa HRKJ-4 can achieve good growth and colonization in medium and high salinity sewage treatment.

[0051] Example 4: Determination of the effect of salinity on ammonia nitrogen removal performance of Pseudomonas aeruginosa HRKJ-4

[0052] The salinity of the heterotrophic nitrification medium was adjusted to 5, 20, 40, 60, and 80 g / L using NaCl. Pseudomonas aeruginosa HRKJ-4 was activated according to the conventional method. A certain amount of bacterial liquid was collected according to the inoculation volume (v / v) of 2%, and centrifuged at 4000 rpm for 5 min to collect the bacteria. After washing with sterile water, it was inoculated into the heterotrophic nitrification medium after the salinity was adjusted. The culture was carried out in a shaker at 30 ° C and 180 rpm for 72 h. The samples were collected and centrifuged at 10000 rpm for 10 min to remove the bacteria, and the supernatant was taken to detect NH in the water sample. 4 -N content.

[0053] The results are as follows Figure 3As shown. Pseudomonas aeruginosa HRKJ-4 can achieve good ammonia nitrogen removal effect in the salinity range of 5-40g / L. When the salinity was 5, 20, 40, and 60g / L, the final ammonia nitrogen removal rates were 89.2%, 91.9%, 88.7%, and 9.4%, respectively. When the salinity reached 60g / L and above, the ammonia nitrogen removal efficiency of the strain was severely inhibited, dropping to about 10%. The above results show that Pseudomonas aeruginosa HRKJ-4 can tolerate salt concentrations up to 40g / L and exert efficient ammonia nitrogen removal, and has good application prospects in medium and high salinity sewage treatment.

[0054] Example 5: Denitrification performance of Pseudomonas aeruginosa HRKJ-4 in pig wastewater treatment

[0055] Fresh pig urine collected from a pig farm in Daxing, Beijing was mixed with tap water in a ratio of 1:4, and then 5% fresh pig manure was added and stirred evenly. After standing for 24 hours, the upper liquid was taken, and its initial ammonia nitrogen was 465 mg / L. Citrate was added to adjust the carbon-nitrogen ratio to 20, and then sterilized to obtain experimental pig wastewater with an initial COD content of 11700 mg / L.

[0056] After conventional activation, Pseudomonas aeruginosa HRKJ-4 was inoculated into the sterilized pig wastewater at a ratio of 2% (v / v), and aerobic treatment was carried out in a shaker at 30°C and 180 rpm. The ammonia nitrogen, TN and COD contents in the wastewater were tested every 36 hours. The results are as follows: Figure 4 shown.

[0057] The results showed that 36 hours after the inoculation of Pseudomonas aeruginosa HRKJ-4, the ammonia nitrogen content dropped rapidly from 465mg / L to 225.4mg / L, and the COD dropped from 11700mg / L to 8040mg / L, indicating that HRKJ-4 can quickly adapt to the high ammonia nitrogen concentration of piggery wastewater and achieve rapid removal of pollutants; by the 72nd hour, the ammonia nitrogen quickly dropped to 13mg / L, and the COD dropped to 2597mg / L. The ammonia nitrogen removal rate reached 97.2% in 72 hours, the COD removal rate reached 77.8%, and the TN removal rate reached 84.2%. The effluent ammonia nitrogen concentration has reached the requirements of my country's "Pollutant Emission Standards for Livestock and Poultry Breeding Industry (GB18596-2001)". This shows that Pseudomonas aeruginosa HRKJ-4 has a good application prospect in the denitrification treatment of livestock and poultry breeding wastewater with medium and high ammonia nitrogen concentrations.

[0058] The above description is only an implementation mode of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) HRKJ-4, characterized in that, Its deposit number is CGMCC No. 24351.

2. A microbial preparation, characterized in that: It includes the Pseudomonas aeruginosa HRKJ-4 described in claim 1.

3. Use of the Pseudomonas aeruginosa described in claim 1 or the microbial preparation described in claim 2 in sewage treatment.

4. The use according to claim 3, characterized in that: The sewage treatment is to remove one or more pollutants including COD, ammonia nitrogen and total nitrogen in the sewage.

5. The use according to claim 4, characterized in that The sewage is livestock and poultry breeding sewage with high ammonia nitrogen concentration, and the ammonia nitrogen concentration of the sewage is 400~500 mg / L.

6. The use according to claim 5, characterized in that The sewage is pig farming sewage with a high ammonia nitrogen concentration, and the ammonia nitrogen concentration of the sewage is 400-500 mg / L.

7. The use according to claim 4, characterized in that: The ammonia nitrogen concentration of the sewage is 400-500 mg / L.

8. The use according to claim 4, characterized in that: The salinity of the sewage is 5-40 g / L.

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