A strain of denitrifying achromobacterium HRKJ-1 and its application in wastewater treatment

Abstract

The present invention belongs to the technical field of microbial wastewater treatment, and discloses a strain of Achromobacter denitrificans HRKJ-1, whose deposit number is CGMCC No.24348. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on January 20, 2022, referred to as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.24348. The strain can achieve the simultaneous removal of ammonia nitrogen, total nitrogen and COD under aerobic conditions, and has the characteristics of acid and alkali resistance, salt resistance and high concentration of ammonia nitrogen resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial wastewater treatment, and specifically relates to a strain of Achromobacter denitrifyingus HRKJ-1 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. According to the "Environmental Bulletin" 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 (including rivers, lakes, surface water such as 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 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 activated sludge, 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 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, Acinetobacter Junii, Alcaligenes faecalis, etc. However, there has been no report on denitrifying achromobacter that is acid- and alkali-resistant, high-salt-resistant, and has the ability to efficiently remove high-concentration ammonia nitrogen and COD. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a strain of Achromobacter denitrificans HRKJ-1, whose deposit number is CGMCC No. 24348. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on January 20, 2022, which is referred to as CGMCC. Its address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is CGMCC No. 24348.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a denitrifying Achromobacter (Achromobacter denitrificans) HRKJ-1, whose deposit number is CGMCC No.24348.

[0008] In another aspect, the present invention provides a microbial preparation, wherein the active ingredient of the microbial preparation is the above-mentioned Achromobacter denitrificans HRKJ-1.

[0009] Another aspect of the present invention provides the use of the Achromobacter denitrificans HRKJ-1 or the microbial preparation in removing one or more pollutants including COD, ammonia nitrogen and total nitrogen in sewage.

[0010] Furthermore, the sewage is pig farming wastewater with high ammonia nitrogen concentration.

[0011] Furthermore, the ammonia nitrogen concentration of the sewage is 400-500 mg / L.

[0012] Furthermore, the pH value of the sewage is in the range of 5-10.5.

[0013] Furthermore, the salinity of the sewage is in the range of 5-40 g / L.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] 1. The denitrifying colorless bacteria (Achromobacter denitrificans) HRKJ-1 provided by the present invention has a good effect in the microbial treatment of pig wastewater with high ammonia nitrogen concentration. 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 great application potential in simplifying the denitrification process and saving processing space and cost.

[0016] 2. The strain HRKJ-1 of the present invention has a wide pH adaptability and can grow well and remove ammonia nitrogen efficiently in the pH range of 5-10.5.

[0017] 3. The strain HRKJ-1 of the present invention has salt tolerance and can still achieve an ammonia nitrogen removal rate of more than 90% at a salt concentration of 40 g / L.

[0018] 4. The strain HRKJ-1 provided by the present invention can achieve an ammonia nitrogen removal rate of 93.9%, a TN removal rate of 68.2%, and a COD removal rate of 80.5% after aerobic treatment for 72 hours in the denitrification process of pig wastewater with an initial ammonia nitrogen concentration of 465 mg / L, thereby realizing rapid denitrification treatment of high-concentration ammonia nitrogen wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is a Gram-stained micrograph of Achromobacter denitrificans HRKJ-1.

[0021] Figure 2 The growth status of Achromobacter denitrificans HRKJ-1 at different pH values.

[0022] Figure 3 The removal of ammonia nitrogen and COD by Achromobacter denitrificans HRKJ-1 at different pH values.

[0023] Figure 4 This is the growth status of Achromobacter denitrificans HRKJ-1 under different salinities.

[0024] Figure 5 This is the ammonia nitrogen removal by Achromobacter denitrificans HRKJ-1 under different salinities.

[0025] Figure 6 This is a graph showing the denitrification performance of Achromobacter denitrificans HRKJ-1 in sterilized swine wastewater. DETAILED DESCRIPTION

[0026] 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.

[0027] The culture medium used in the examples is 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] 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.

[0033] Take the culture fluid after 5 generations of enrichment, dilute it with sterile water in different ratios of 10-3-10-7, apply 100 μL of dilution to heterotrophic nitrification solid medium in each gradient, place it in a biochemical incubator at 30°C for 48 hours, observe the growth of the colonies, select single colonies with different morphologies, streak them on heterotrophic nitrification solid medium by plate streaking method, and culture them under the same conditions for 48 hours. Then select single colonies for multiple partition streaking purification until a pure strain is obtained, and place the purified strain in 20% glycerol in a -80°C refrigerator for storage.

[0034] 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.

[0035] Example 2 Identification of strains

[0036] After the above separation and purification process, a heterotrophic nitrification-aerobic denitrification bacterium was obtained. The morphological characteristics of the bacterium are rod-shaped, the size of the bacterium is (0.4-0.7) μm×(2-4) μm, the colony is round, the edges are neat and smooth, the colony on LB is off-white, and later it becomes light yellow, the surface is moist, and no spores are produced. It is a Gram-negative aerobic bacterium (such as Figure 1 shown).

[0037] 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.

[0038] The amplification primers are

[0039] 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2),

[0040] 1492R: TACGGYTACCTTGTTACGACTT(SEQ ID NO.3);

[0041] 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;

[0042] 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.

[0043] The PCR products were detected by agarose gel electrophoresis and then sequenced.

[0044] The 16S rDNA sequence of the bacterium was obtained after forward and reverse sequencing and splicing (SEQ ID NO.1). The sequence was subjected to Blast comparison in the NCBI database. The comparison results showed that the homology with Achromobacter denitrificans NRBC 15125 was the highest, reaching 99.78%. The strain was named Achromobacter denitrificans HRKJ-1, hereinafter referred to as strain HRKJ-1 in this application.

[0045] Achromobacter denitrificans HRKJ-1 was deposited in the General Microbiology Center of China Microorganism Culture Collection 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. 24348.

[0046] Example 3 Effect of pH on the growth of strain HRKJ-1

[0047] The pH of the heterotrophic nitrification medium was adjusted to 3, 4, 5, 7, 8.5, 9.5, 10.5 and 11.5, respectively. The strain HRKJ-1 was activated according to the conventional method, and a certain amount of bacterial liquid was taken according to the inoculation volume of 2% (v / v). The bacteria were collected after centrifugation at 4000 rpm for 5 min, washed with sterile water, and inoculated into the heterotrophic nitrification medium after pH adjustment. The culture was carried out in a shaker at 30°C and 180 rpm for 72 h, and the OD600 value under each pH condition was detected.

[0048] The results are as follows Figure 2 As shown. Strain FY1 can grow well in the pH range of 5-10.5. When the pH was 5, 7, 8.5, 9.5, and 10.5, the final OD600 values ​​were 1.217, 1.247, 1.203, 1.213, and 0.909, respectively. However, when the pH was lower than 5 or higher than 10.5, the growth of the strain was severely inhibited. The results show that strain HRKJ-1 can tolerate a strong acid-base environment and show good growth, indicating that strain HRKJ-1 can achieve good growth and colonization in acid-base wastewater.

[0049] Example 4 Effect of pH on Ammonia Nitrogen Removal by Strain HRKJ-1

[0050] The pH values ​​of the heterotrophic nitrification medium were 3, 4, 5, 7, 8.5, 9.5, 10.5, and 11.5, respectively. After the strain HRKJ-1 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 bacteria were collected after centrifugation at 4000 rpm for 5 min. The bacteria were washed with sterile water and inoculated into the heterotrophic nitrification medium after the pH was adjusted. The bacteria were cultured in a shaker at 30°C and 180 rpm for 72 h, and the COD and NH4-N contents in the water samples under the final pH conditions were detected.

[0051] The results are as follows Figure 3 As shown. Strain HRKJ-1 showed excellent ammonia nitrogen removal performance in the pH range of 4-11.5. When the pH was 5, 7, 8.5, 9.5, and 10.5, the ammonia nitrogen removal rates were 89.1%, 94.3%, 91.9%, 91.5%, and 91.9%, respectively, and the COD removal rates were 80.6%, 83.0%, 86.5%, 86.5%, and 84.9%, respectively. This shows that strain HRKJ-1 has broad application prospects in the denitrification treatment of acidic and alkaline wastewater.

[0052] Example 5 Effect of salinity on the growth of strain HRKJ-1

[0053] The salinity of the heterotrophic nitrification medium was adjusted to 5, 20, 40, 60, and 80 g / L using NaCl. The strain HRKJ-1 was activated according to the conventional method, and a certain amount of bacterial liquid was taken according to the inoculation volume of 2% (v / v). The bacteria were collected after centrifugation at 4000 rpm for 5 min, washed with sterile water, and 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, and the OD600 values ​​at each final salinity were detected.

[0054] The results are as follows Figure 4 As shown. The strain HRKJ-1 can grow well in the salinity range of 5-40g / L. When the salinity was 5, 20, 40, and 60g / L, the final OD600 values ​​were 1.115, 1.117, 1.540, and 0.266, respectively. However, when the salinity reached 60g / L and above, the growth of the strain was severely inhibited. The above results show that HRKJ-1 can tolerate salt concentrations up to 40g / L and show good growth, indicating that the strain HRKJ-1 can achieve effective growth and colonization in sewage with medium and high salinity.

[0055] Example 6 Effect of salinity on ammonia nitrogen removal by strain HRKJ-1

[0056] The salinity of the heterotrophic nitrification medium was adjusted to 5, 20, 40, 60, and 80 g / L using NaCl. The strain HRKJ-1 was activated according to the conventional method, and a certain amount of bacterial liquid was taken according to the inoculation volume of 2% (v / v). The bacteria were collected after centrifugation at 4000 rpm for 5 min, washed with sterile water, and 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 the NH4-N content in the water sample.

[0057] The results are as follows Figure 5 As shown. The strain HRKJ-1 showed 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 93.5%, 91.4%, 91.2%, and 20.1%, respectively. When the salinity reached 60g / L and above, although HRKJ-1 still had an ammonia nitrogen removal rate of 20% at a salinity of 60g / L, the overall ammonia nitrogen removal efficiency of the strain was severely inhibited. The above results show that HRKJ-1 can tolerate salt concentrations as high as 40g / L and exert efficient ammonia nitrogen removal, indicating that the strain HRKJ-1 has good application prospects in sewage treatment with medium and high salinity.

[0058] Example 7 Denitrification performance of strain HRKJ-1 in sterilized pig wastewater

[0059] 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.

[0060] 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 at 115°C for 30 minutes. After conventional activation, the strain HRKJ-1 was inoculated into the sterilized piggery 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 detected every 36 hours. The results are as follows: Figure 6 shown.

[0061] The results showed that 36 hours after the inoculation of strain HRKJ-1, the ammonia nitrogen content dropped rapidly from 465mg / L to 258.9mg / L, and the COD dropped from 11700mg / L to 7509mg / L, indicating that HRKJ-1 can quickly adapt to the actual pig wastewater with high ammonia nitrogen concentration and achieve rapid removal of pollutants; by the 72nd hour, the ammonia nitrogen quickly dropped to 28.5mg / L, and the COD dropped to 2283.8mg / L. The ammonia nitrogen removal rate reached 93.9% in 72 hours, the COD removal rate reached 80.5%, and the TN removal rate reached 68.2%. 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 strain HRKJ-1 has good application prospects in the denitrification treatment of livestock and poultry wastewater with medium and high ammonia nitrogen concentrations.

[0062] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments may have different values.

Claims

1. A denitrifying achromobacterium ( Achromobacter denitrificans ) HRKJ-1, characterized in that Its deposit number is CGMCC No. 24348.

2. A microbial preparation, characterized in that: The active ingredient of the microbial preparation is the denitrifying Achromobacterium ( Achromobacter denitrificans )HRKJ-1.

3. The denitrifying Achromobacterium according to claim 1 ( Achromobacter denitrificans ) Use of HRKJ-1 or any one of the microbial preparations 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 livestock and poultry breeding wastewater with high ammonia nitrogen concentration, and the ammonia nitrogen concentration of the sewage is 400~500 mg / L.

5. 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.

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

7. The use according to claim 3, characterized in that: The pH value of the sewage is in the range of 5-10.

5.

8. The use according to claim 3, characterized in that: The salinity of the sewage is in the range of 5-40 g / L.

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