Ochrobactrum HRKJ-3, microbial preparation and application thereof

Abstract

The present invention provides a strain of Ochrobacterium HRKJ-3, a microbial preparation and an application thereof. The Ochrobacterium HRKJ-3 is deposited with CGMCC No. 24350. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 20, 2022, and the strain deposited with CGMCC No. 24350. The strain can simultaneously carry out heterotrophic nitrification and aerobic denitrification under aerobic conditions, has good application potential in simplifying the denitrification process, saving processing space and cost, and has good application prospects in the treatment of domestic sewage and the denitrification of livestock and poultry breeding sewage with high ammonia nitrogen concentration.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Ochrobactrum HRKJ-3, 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 "Bulletin on the State of the Environment" published by the Ministry of Ecology and Environment, the main pollutant indicators that have been detected to exceed the standard in my country's freshwater resources include ammonia nitrogen (NH4-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 a variety of pollutant forms 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. Summary of the invention

[0004] The main technical problem solved by the present application is to provide a Bacillus HRKJ-3, a microbial preparation and its application, so as to solve the problems existing in the activated sludge biological denitrification method in the prior art.

[0005] In order to solve the above problems, a technical solution adopted in the present application is: to provide an Ochrobactrum pituitosum HRKJ-3, whose preservation number is CGMCC No.24350.

[0006] 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, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The strain deposit number is CGMCC No. 24350.

[0007] Another technical solution adopted in the present application is to provide a microbial preparation, including the above-mentioned Ochrobactrum HRKJ-3.

[0008] Another technical solution adopted in the present application is to provide an application of the above-mentioned Ochrobactrum HRKJ-3 or the above-mentioned microbial preparation in sewage treatment.

[0009] In one embodiment, the sewage treatment includes removing one or more pollutants including COD, ammonia nitrogen and total nitrogen in the sewage.

[0010] Preferably, the sewage treatment includes the simultaneous removal of COD, ammonia nitrogen and total nitrogen in the sewage.

[0011] In one embodiment, the sewage is domestic 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 wastewater with high ammonia nitrogen concentration.

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

[0016] Different from the prior art, the beneficial effects of this application are:

[0017] 1. The Ochrobacterium HRKJ-3 provided in the present application can simultaneously carry out heterotrophic nitrification and aerobic denitrification under aerobic conditions, with a maximum ammonia nitrogen removal rate of 7.05 mg / L / h. It not only has a good removal effect on ammonia nitrogen and total nitrogen, but also can remove most of the COD in the water body, thereby realizing the simultaneous denitrification and carbon removal of sewage, and has good application potential in simplifying the denitrification process and saving processing space and cost;

[0018] 2. When the Ochrobacterium HRKJ-3 provided in the present application is applied to the treatment of domestic sewage with an initial COD of 2000 mg / L and an initial ammonia nitrogen of 100 mg / L, it can remove almost all ammonia nitrogen and COD in domestic sewage within 24 hours, with removal rates of 98% and 100%, respectively. Ochrobacterium HRKJ-3 has good application prospects in domestic sewage treatment;

[0019] 3. When the Ochrobacterium HRKJ-3 provided in this 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, it can remove 83% of ammonia nitrogen, 80.7% of COD and 79.6% of TN within 72 hours. Ochrobacterium HRKJ-3 can maintain treatment efficiency under high ammonia nitrogen conditions and has good application prospects in the denitrification treatment of livestock and poultry wastewater with high ammonia nitrogen concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a Gram-stained microscopic picture of Ochrobacterium HRKJ-3 of the present invention;

[0021] Figure 2 is a graph showing the relationship between the growth and heterotrophic nitrification characteristics of Ochrobacterium HRKJ-3 of the present invention;

[0022] Figure 3 This is a diagram showing the treatment effect of Ochrobactrum HRKJ-3 of the present invention on domestic sewage;

[0023] Figure 4 This is a diagram showing the treatment effect of Ochrobactrum HRKJ-3 of the present invention on piggery wastewater. DETAILED DESCRIPTION

[0024] The present application discloses a kind of Ochrobactrum HRKJ-3, 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 method and application 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 Ochrobactrum HRKJ-3, microbial preparations and reagents or excipients used in their applications provided in the present application can be purchased from the market.

[0026] The content of this application 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 identification of Ochrobacterium HRKJ-3

[0032] (1) Strain isolation and purification

[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 taken and 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 acclimated and enriched at 30 ° C and 180 rpm. The acclimation cycle was 48 hours. After each cycle, 10 mL of the enriched solution was added to a new 100 mL of heterotrophic nitrification medium to continue enrichment culture. This was continued for 5 cycles of enrichment culture. During this period, the removal of ammonia nitrogen in the culture solution was detected.

[0034] Take the culture medium after 5 generations of enrichment and wash with sterile water at 10 -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 stored at -80°C for later use.

[0035] (2) Strain screening

[0036] Use an inoculation loop to pick up the purified strains and inoculate them into the sterilized LB liquid culture medium. After culturing at 30°C and 180rpm for 24h, a certain amount of bacterial solution is taken according to the inoculation volume of 5% (v / v), and the bacteria are collected after centrifugation at 4000rpm for 5min. After washing with sterile water, they are inoculated into a 150mL conical flask containing 50mL heterotrophic nitrification medium, and cultured in a shaker at 30°C and 180rpm for 72h. Samples are taken at 12h, 24h, 36h, 48h, 60h and 72h to detect the NH4-N, TN and COD contents in the culture solution, so as to 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 nitrifying bacterium HRKJ-3 was obtained. The morphological characteristics of the bacterium are short rods, the size of the bacterium is (0.3-0.5) μm×(0.8-2.0) μm, the colonies are round, the edges are neat and smooth, the colonies on LB are milky white to light yellow, the surface is moist and shiny, no spores are produced, and it is a Gram-negative aerobic bacterium. Figure 1 shown.

[0040] (2) Molecular Biological Identification

[0041] The obtained strains were identified by molecular biology and sequenced after PCR amplification of bacterial 16S rDNA sequences.

[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 the homology with Ochrobactrum pituitosum CCUG50899 was the highest, at 98.77%. The strain was named Ochrobactrum pituitosum HRKJ-3.

[0047] Ochrobactrum pituitosum HRKJ-3 was deposited in the General Microbiology Center of 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. 24350.

[0048] Example 3: Determination of heterotrophic nitrification characteristics of Ochrobacterium HRKJ-3

[0049] After activating and culturing Bacillus subtilis HRKJ-3 in an LB liquid test tube for 24 hours, 2 mL of bacterial liquid was aspirated according to an inoculation volume of 2% (v / v), and the bacteria were collected after centrifugation at 4000 rpm for 5 minutes. The bacteria were washed with sterile water and inoculated into 50 mL of heterotrophic nitrification medium (initial ammonia nitrogen concentration of 100 mg / L, C / N=20), and cultured in a shaker at 30°C and 180 rpm for 24 hours. During this period, samples were taken every 4 hours to measure the OD600 value, and the samples were centrifuged at 10000 rpm for 10 minutes to remove the bacteria, and the supernatant was taken to detect the COD, NH4-N, NO2-N, NO3-N, and TN contents in the water sample.

[0050] The results are as follows Figure 2 As shown. Ochrobacterium HRKJ-3 began to grow at 8h after inoculation, and the maximum growth rate was observed between 12h and 16h, which was 0.094 / h. After 32h, the strain growth ended and reached a plateau phase, with a maximum OD600 of 1.288. During this period, the contents of NH4-N, TN and COD decreased rapidly. The maximum ammonia nitrogen removal rate was 7.05mg / L / h. By the 32nd hour, almost all ammonia nitrogen was removed, and the maximum NH4-N, COD and TN removal rates were 99.3%, 84.7% and 84.8%, respectively, and no nitrite nitrogen and nitrate nitrogen were produced during the entire treatment process. This shows that Ochrobacter HRKJ-3 can carry out heterotrophic nitrification and aerobic denitrification under aerobic conditions.

[0051] Example 4: Denitrification performance of Ochrobacterium HRKJ-3 in domestic sewage treatment

[0052] Take domestic sewage with an initial ammonia nitrogen content of about 100 mg / L, add citrate to adjust the carbon-nitrogen ratio of the domestic sewage to 20, and then sterilize it to obtain experimental domestic sewage with an initial COD of about 2000 mg / L.

[0053] After conventional activation, Ochrobacterium HRKJ-3 was inoculated into the experimental domestic sewage 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 and COD content in the sewage was tested every 24 hours. The results are as follows: Figure 3 shown.

[0054] The results showed that within 24 hours after inoculation, Ochrobacterium HRKJ-3 was able to remove almost all ammonia nitrogen and COD in domestic sewage, with removal rates of 98% and 100%, respectively, indicating that Ochrobacterium HRKJ-3 has good application prospects in denitrification treatment of domestic sewage.

[0055] Example 5: Denitrification performance of Ochrobacterium HRKJ-3 in pig wastewater treatment

[0056] 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.

[0057] After conventional activation, Ochrobacterium HRKJ-3 was inoculated into the experimental pig wastewater at a ratio of 2% (v / v), and aerobic treatment was carried out in a shaker at 30°C and 180rpm. The ammonia nitrogen, TN and COD contents in the wastewater were tested every 36 hours. The results are as follows: Figure 4 shown.

[0058] The results showed that 36 hours after the inoculation of Ochrobacterium HRKJ-3, the ammonia nitrogen content dropped from 465mg / L to 313.7mg / L, and the COD dropped from 11700mg / L to 8791.5mg / L. At 72 hours, the ammonia nitrogen dropped to 79.1mg / L, and the COD dropped to 2260mg / L. The ammonia nitrogen removal rate reached 83% in 72 hours, the COD removal rate reached 80.7%, and the TN removal rate reached 79.6%. This shows that Ochrobacterium HRKJ-3 has a good application prospect in the denitrification treatment of livestock and poultry wastewater with medium and high ammonia nitrogen concentrations.

[0059] 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 species of Ochrobacterium Ochrobactrum pituitosum ) HRKJ-3, characterized in that Its deposit number is CGMCC No. 24350.

2. A microbial preparation, characterized in that: It includes the Ochrobacterium HRKJ-3 described in claim 1.

3. Use of the Ochrobactrum HRKJ-3 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 includes removing 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 domestic sewage.

6. 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.

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

8. The use according to any one of claims 4 to 5, characterized in that: The ammonia nitrogen concentration of the sewage is 400-500 mg / L.

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