A strain of Brevibacterium epidermidis HRKJ-2 and its application in wastewater decontamination

Abstract

The present invention belongs to the technical field of microbial wastewater treatment, and discloses a strain of Brevibacterium epidermidis HRKJ-2, whose deposit number is CGMCC No.24349. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 20, 2022. The strain can achieve the simultaneous removal of ammonia nitrogen, total nitrogen and COD under full aerobic conditions, and at the same time has the characteristics of resistance to high concentrations of ammonia nitrogen.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial wastewater treatment, and specifically relates to a strain of Brevibacterium epidermidis HRKJ-2 and application thereof in wastewater decontamination. 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. The main pollutant indicators that have been detected to be excessive in my country's freshwater resources (including rivers, lakes, surface water from large-scale water conservancy projects, and groundwater, etc.) include chemical oxygen demand (COD), 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 elements exist in various forms of pollutants and are difficult to remove. If they are discharged into the environment without effective treatment, they will cause serious harm to the water ecosystem and human health.

[0003] 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. In recent years, a new type of denitrification microorganism, heterotrophic nitrification-aerobic denitrification bacteria, has been discovered and continuously isolated. This type of microorganism can simultaneously carry out nitrification and denitrification under aerobic conditions, complete 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. At present, many heterotrophic nitrification-aerobic denitrification strains have been isolated and screened, such as Pseudomonas stutzeri, Klebsiella pneumoniae, AcinetobacterJunii, Alcaligenes faecalis, etc. However, there has been no report on Brevibacterium epidermidis that can achieve the simultaneous removal of ammonia nitrogen, total nitrogen and COD under fully aerobic conditions and has the characteristics of resistance to high concentrations of ammonia nitrogen. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a strain of Brevibacterium epidermidis HRKJ-2, whose deposit number is CGMCC No. 24349. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 20, 2022, which is referred to as CGMCC, and its address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a strain of Brevibacterium epidermidis HRKJ-2, whose deposit number is CGMCC No.24349.

[0007] In another aspect, the present invention provides a microbial preparation, wherein the active ingredient of the microbial preparation is the above-mentioned Brevibacterium epidermidis HRKJ-2.

[0008] Another aspect of the present invention provides the use of Brevibacterium epidermidis HRKJ-2 or the microbial preparation in removing one or more pollutants including COD, ammonia nitrogen and total nitrogen in sewage.

[0009] Furthermore, the sewage is pig farming wastewater with high ammonia nitrogen concentration.

[0010] Furthermore, the ammonia nitrogen concentration of the sewage is 400-500 mg / L.

[0011] Compared with the prior art, the advantages of the present invention are:

[0012] 1. The Brevibacterium epidermidis HRKJ-2 provided by the present invention is reported for the first time in the field of sewage denitrification. It not only has a good removal effect on ammonia nitrogen and total nitrogen, but also can remove COD in the water body, thereby realizing simultaneous denitrification and carbon removal of sewage, and has good application potential in simplifying the denitrification process and saving processing space and cost.

[0013] 2. The strain HRKJ-2 provided by the present invention can achieve an ammonia nitrogen removal rate of 65% and a TN removal rate of 62.4% in the denitrification process of pig wastewater with an initial ammonia nitrogen concentration of 465 mg / L after aerobic treatment for 72 hours, and has an obvious bio-enhancement effect on the rapid denitrification of high-concentration ammonia nitrogen wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings required for describing the specific implementation manner are briefly introduced below.

[0015] Figure 1 This is a Gram-stained micrograph of Brevibacterium epidermidis HRKJ-2.

[0016] Figure 2 This is a graph showing the denitrification performance of Brevibacterium epidermidis HRKJ-2 in sterilized domestic sewage.

[0017] Figure 3 This is a graph showing the denitrification performance of Brevibacterium epidermidis HRKJ-2 in sterilized swine wastewater. DETAILED DESCRIPTION

[0018] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and the protection scope of the present invention cannot be limited by this. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.

[0019] The culture medium used in the examples is as follows:

[0020] LB medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1 L distilled water.

[0021] 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.

[0022] 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.

[0023] Example 1 Isolation and screening of strains

[0024] Enrichment culture: The sample was the activated sludge from the aerobic section of a sewage treatment plant in Urumqi collected in July 2020. 20 mL of fully mixed activated sludge was suspended in 180 mL of 0.2% sodium chloride solution, and 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 is 48 hours. After each cycle, 10 mL of the enrichment solution was added to a new 100 mL of heterotrophic nitrification medium to continue enrichment culture, and enrichment culture was continued for 5 cycles. During this period, the removal of ammonia nitrogen in the culture solution was detected.

[0025] 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.

[0026] Strain screening: Use an inoculation loop to pick the purified strains and inoculate them into the sterilized LB liquid medium. After culturing at 30°C and 180rpm for 24h, a certain amount of bacterial solution is drawn according to the inoculation amount of 5% (v / v), centrifuged at 4000rpm for 5min, and then the bacteria are collected. After washing with sterile water, they are inoculated into a 150mL conical flask containing 50mL heterotrophic nitrification medium, and cultured in a shaking table 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 medium. The strains with heterotrophic nitrification function are screened out.

[0027] Example 2 Identification of strains

[0028] A heterotrophic nitrifying bacterium HRKJ-2 was obtained through the above separation and purification process. The morphological characteristics of the bacterium are short rods, the size of the bacteria is (0.8-1.0) μm×(1.0-2.5) μm, the colonies are round, the edges are neat and smooth, the colonies on LB are brown-yellow, the surface is moist and shiny, no spores are produced, and it is a Gram-positive aerobic bacterium.

[0029] The obtained strains were subjected to molecular biological identification and sequencing comparison after PCR amplification of the bacterial 16S rDNA sequence (SEQ ID NO.1).

[0030] 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2),

[0031] 1492R: TACGGYTACCTTGTTACGACTT(SEQ ID NO.3);

[0032] 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;

[0033] 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.

[0034] The PCR products were detected by agarose gel electrophoresis and then sequenced.

[0035] 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 Brevibacterium epidermidis P159 was the highest, at 99.57%. The strain was named Brevibacterium epidermidis HRKJ-2, hereinafter referred to as strain HRKJ-2 in this application.

[0036] Brevibacterium epidermidis HRKJ-2 was deposited in the General Microbiology Center of China Culture Collection Administration on January 20, 2022, abbreviated as CG MCC, and its address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] Example 3 Denitrification performance of strain HRKJ-2 in sterilized domestic sewage

[0038] The C / N ratio of actual domestic sewage (initial ammonia nitrogen content of about 100 mg / L) was adjusted to about 20 (initial COD content of about 1980 mg / L) by adding citrate and then sterilized. The strain HRKJ-2 was conventionally activated and inoculated into the sterilized domestic sewage at a ratio of 2% (v / v). The aerobic treatment was carried out in a shaking incubator at 30°C and 180 rpm. The ammonia nitrogen and COD contents in the sewage were detected every 24 hours. The results are shown in FIG. Figure 2 shown.

[0039] The results showed that within 24 hours after inoculation, strain HRKJ-2 was able to remove almost all ammonia nitrogen and COD in domestic sewage, with removal rates of 97.8% and 93.7%, respectively; further, the COD removal rate reached 100% after 48 hours, indicating that strain HRKJ-2 has good application prospects in domestic sewage denitrification treatment.

[0040] Example 4 Denitrification performance of strain HRKJ-2 in sterilized pig wastewater

[0041] The pretreatment method for laboratory pig wastewater is to mix fresh pig urine collected from a pig farm in Daxing, Beijing with tap water in a ratio of 1:4, then add 5% fresh pig manure by mass and stir evenly. After standing for 24 hours, take the upper layer of liquid as laboratory pig wastewater for use.

[0042] The C / N ratio of actual piggery wastewater (initial ammonia nitrogen content of about 465 mg / L) was adjusted to about 20 (initial COD content of about 11700 mg / L) by adding citrate and then sterilized. The strain HRKJ-2 was conventionally activated and inoculated into the sterilized piggery wastewater at a ratio of 2% (v / v). The aerobic treatment was carried out in a shaking incubator at 30°C and 180 rpm. The ammonia nitrogen, TN and COD contents in the wastewater were detected every 36 hours. The results are as follows: Figure 3 shown.

[0043] The results showed that 36 hours after the inoculation of strain HRKJ-2, the ammonia nitrogen content decreased slowly, from 465 mg / L to 386.9 mg / L, and at the 72nd hour, the ammonia nitrogen content dropped to 162.7 mg / L, and the COD content dropped to 6223 mg / L. The ammonia nitrogen removal rate was 65% at 72 hours, the COD removal rate was 46.9%, and the TN removal rate reached 62.4%. This indicates that strain HRKJ-2 has good application prospects in the denitrification treatment of livestock and poultry wastewater with medium and high ammonia nitrogen concentrations.

[0044] The above description is only an illustrative embodiment of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by any person skilled in the art without departing from the concept and principle of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A Brevibacterium epidermidis ( Brevibacterium epidermidis )HRKJ-2, whose deposit number is CGMCC No.24349.

2. A microbial preparation, characterized in that: The active ingredient of the microbial preparation is Brevibacterium epidermidis described in claim 1 ( Brevibacterium epidermidis )HRKJ-2.

3. The Brevibacterium epidermidis of claim 1 ( Brevibacterium epidermidis ) Use of HRKJ-2 or any microbial preparation described in claim 2 in removing one or more pollutants including COD, ammonia nitrogen and total nitrogen in sewage.

4. The use according to claim 3, characterized in that: The sewage is domestic sewage.

5. The use according to claim 3, characterized in that: The sewage is livestock and poultry breeding wastewater with high ammonia nitrogen concentration, and the ammonia nitrogen concentration of the sewage is 400~500 mg / L.

6. The use according to claim 3, 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.

7. The use according to claim 3, characterized in that: The ammonia nitrogen concentration of the sewage is 400-500 mg / L.

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