KR100828566B1 - 4 Pseudomonas fluorescens K4 having excellent ability of denitrification - Google Patents

Abstract

A Pseudomonas fluorescens K4 strain is provided to degrade simultaneously high concentration of nitrogen oxide and ammonia components through denitrification, so that industrial wastewater containing nitrogen oxide and ammonia components is biologically treated. A Pseudomonas fluorescens K4(KCCM 10841P) strain is isolated from the soil around a sewage disposal plant, has 16S rRNA(ribosomal RNA) sequence having the nucleotide sequence of SEQ ID NO:1, simultaneously denitrify nitric acid nitrogen and ammonia nitrogen, and has optimal hydrogen concentration of pH 5.0-9.0, optimal temperature of 25-40 deg. C and C/N ratio of 1.0. The wastewater is treated by culturing Pseudomonas fluorescens K4(KCCM 10841P) strain at 30 deg. C and 110 rpm for 24 hours and adding the cultured medium to the wastewater.

Description

Pseudomonas fluorescens K4 having excellent ability of denitrification

1 is a graph showing the results of investigating the nitric acid resolution and growth rate by temperature of the jeongjeongjung of the present invention.

Figure 2 is a graph of the results of investigating the nitrate nitrogen degradation ability and growth rate by pH of the identified strain of the present invention.

3 is a graph showing the results of investigating the nitrate nitrogen resolution and growth rate according to the C / N ratio of the identified strain of the present invention.

Figure 4 is a graph showing the results of investigation of the nitric acid resolution and growth rate by volume treated the strain of the present invention in the wastewater.

5 is a graph showing the results of investigating the resolution of nitrate inorganic salts and inorganic salts of the identified strains of the present invention.

6 is a graph showing the results of investigating the resolution of nitrate nitrogen and ammonia nitrogen of the identified strain of the present invention.

Figure 7 is a graph of the results of the investigation of the nitrate nitrogen resolution and growth rate of the identified strain of the present invention according to the C / N ratio.

The present invention relates to Pseudomonas fluorescens K4 having the ability to simultaneously decompose high concentrations of nitrogen oxides or ammonia components.

The most common method for removing nitrogen contained in wastewater in wastewater treatment is biological nitrogen removal, which is a denitrification by nitrate reduction or ammonia oxidation using heterotrophic microorganisms. Denitrification is carried out by a number of heterotrophic microorganisms and autotrophic micro-organisms that can be easily Dwarf the oxygen breathing and nitrogen breathing the microorganism most of Pseudomonas (Pseudomonas), Alcaligenes (Alcaligenes), Paracoccus (Paracoccus) and thio bacilli ( Thiobacillus ) is a microorganism belonging to the gram negative proteobacteria ( Proteobacteria ). Biological denitrification by the microorganisms is used for the treatment of wastewater containing nitrogen components, because nitrate and ammonia components are harmful substances and there are restrictions on the effluent.

On the other hand, in order to biologically denitrify the nitrate, the concentration of nitrate present in the wastewater must be diluted to an appropriate concentration (about 100 ppm) or less to allow biological treatment. Recently developed strain in France ( Pseudomonas The method using halodenitrificans ) was able to degrade nitrates present in the waste water at very high concentrations (62 g / L, 1 M), but decomposition to ammonia nitrogen could not be achieved using this strain.

In general, nitrates have been reported to degrade only nitrates (CW Francis and MW Callahan, Biological Denitrification and Its Application in Treatment of High-Nitrate Wastewater, J. Environmental Quality 4, pp153, 1975). Microorganisms that simultaneously decompose and ammonium are known to be rare. However, most of the wastewater contains ammonia nitrogen, and in particular, wastewater generated in the nuclear industry is more commonly included. Therefore, it is required to develop a strain capable of simultaneously decomposing nitrate and ammonia nitrogen.

The present inventors have identified a novel microorganism that decomposes nitrate nitrogen and ammonia nitrogen in soil microorganisms, and completed the present invention by confirming that the microorganism has an excellent effect in removing high concentration nitrate nitrogen as well as ammonia nitrogen. It was.

It is an object of the present invention to provide a novel strain that can biologically denitrify a wastewater containing high concentrations of nitrate and / or ammonia that can be reduced or oxidized by bacteria and to provide a method for denitrifying the wastewater using the same. will be.

In order to achieve the above object, the present invention provides a novel Pseudomonas fluorescens K4 strain.

The present invention also provides a composition for treating nitric acid denitrification of wastewater containing the strain as an active ingredient.

In addition, the present invention provides a nitrogen denitrification method comprising the step of supplying the novel Pseudomonas fluorescein K4 strain to the wastewater.

Hereinafter, the present invention will be described in detail.

The present invention is a novel Pseudomonas fluorescens K4 ( Pseudomonas fluorescens K4) provides a strain.

In order to identify microorganisms with nitrogen removal ability, the present inventors collected samples from soil near sewage treatment plant to remove high concentrations of nitrate and ammonia, Pseudomonas fluorescens K4 ( Pseudomonas). fluorescens K4) was identified and the strain was deposited on February 13, 2007 with accession number KCCM10841P to the Korea Microorganism Conservation Center. The strain of the present invention is characterized by having a 16S rRNA sequence set forth in SEQ ID NO: 1 and has the ability to simultaneously denitrate nitrate nitrogen and ammonia nitrogen. Conditions for denitrifying both nitrate nitrogen and ammonia nitrogen at the same time pH 5.0 to 9.0, the temperature is preferably 25 to 40 ℃, the optimum conditions are 30 ℃, pH 7.0, and the ratio of C / N is 1.0.

The present inventors then investigated the resolution of the nitrate strain of the novel strain of the present invention according to the concentration of nitrate inorganic salts and inorganic salts. As a result, the nitrate in the culture treatment tank was completely decomposed while showing a normal growth rate (see FIG. 5 and Table 4). Thereafter, the ammonia resolution of the new strain of the present invention was examined. As a result, the nitrate was first decomposed and then ammonia was decomposed while showing a normal growth rate. The present inventors investigated the denitrification efficiency of the new strain of the present invention according to the ratio of C / N. As a result, it was confirmed that the denitrification performance for nitrate nitrogen was improved by increasing the ratio of C / N to 1.0 (see FIG. 7).

Through the above results, it can be seen that the novel strain Pseudomonas fluorescens K4 strain of the present invention can be usefully used to denitrify not only nitrate nitrogen but also ammonia nitrogen.

The present invention also provides a composition for nitrogen denitrification of wastewater containing the strain as an active ingredient.

The composition is characterized by denitrifying nitrate nitrogen and / or ammonia nitrogen. The composition should essentially include nitrates such as nitrogen source NH ₄ NO ₃ and sodium citrate as carbon source, and additionally MgSO ₄ · H ₂ O, FeCl ₂ · H ₂ O, KH ₂ PO ₄ and It is preferable to add CaCl ₂ · H ₂ O.

In addition, the present invention provides a nitrogen denitrification method comprising the step of supplying the novel Pseudomonas fluorescein strain to the waste water.

The present invention provides a nitrogen denitrification method comprising the following steps: 1) culturing a new Pseudomonas fluoresce K4 strain in medium and 2) adding the culture solution to the wastewater.

In the above method, step 1) is preferably incubated the strain for 24 hours at 30 ℃ 110 ℃.

In the above method, the amount of the culture solution added in step 2) is preferably 1 to 20 parts by weight, more preferably 5 to 15 parts by weight, most preferably 10 parts by weight.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following Examples illustrate the present invention in detail, and the content of the present invention is not limited by the Examples.

Example 1 Separation of Denitrification Microorganisms for Nitrate Treatment

The present inventors collected activated sludge from Gongju-si sewage treatment plant 1 g of the sample was mixed with 50 ml of 0.85% sterile saline, shaken for 2 hours, and allowed to stand for 30 minutes, and then 1.0 ml of the supernatant was prepared with 0.1% NH ₄ NO ₃ and a medium containing the elements shown in Tables 1 and 2 below. It was added to 9.0 ml and anaerobic culture at 30 ℃. The culture medium of the changed color (green → blue) in the test tube is plated on 0.1% NH ₄ NO ₃ agar medium again, and each colony selected here is observed for removal efficiency by nitrate removal efficiency in 0.1% NH ₄ NO ₃ liquid medium. The screening was performed repeatedly by taking the high one, and finally, new cells capable of removing high concentrations of nitrate and ammonia were isolated.

Medium component per 1000 ml of distilled water NH ₄ NO ₃ 1 g Asparagin 1 g Sodium citrate 8.5 g

 Distilled water 1000 ml Trace ingredients entering the sugar MgSO ₄ H ₂ O 1 g FeCl ₂ H ₂ O 0.005g KH ₂ PO ₄ 1 g CaCl ₂ H ₂ O 0.2 g

< Example  2> Identification of denitrifying microorganisms for nitrate treatment

The present inventors identified the strain isolated in Example 1 above. The isolates were identified by morphological, physical, biochemical properties and 16S rRNA sequencing methods. The identification method is as follows. DNA was isolated using the solgent purification beads, and PCR was performed using the following primers: 27F primer (5′-AGA GTT TGA TCC TGG CTC AG-3 ′): SEQ ID NO: 2) and 1492R primer (5′-GGT TAC CTT GTT ACG ACT T-3 ′: SEQ ID NO: 3). Then PCR was performed using Solgent's EF-Tag. PCR conditions are as follows. The cells were denatured at 95 ° C. for 15 minutes, 20 seconds at 95 ° C., 40 seconds at 50 ° C., and 1 minute 30 seconds at 72 ° C., and the cycle was repeated 30 times, followed by extension reaction at 72 ° C. for 5 minutes. Then, PCR products were obtained using Solgent's PCR purification kit, and confirmed by electrophoresis and confirmed the sequence (SEQ ID NO: 1). As a result, the strain is Pseudomonas fluorescein sense (Pseudomonas fluorescens ), and the taxonomic properties of the strain are shown in Table 3. In order to identify the optimum conditions for denitrification of the strain against nitrate nitrogen and ammonia nitrogen, the present inventors measured the denitrification capacity and growth ability of the strain according to temperature, pH, C / N ratio and microbial inoculation amount. As a result, temperature was 30 degreeC (FIG. 1), pH was 7.0 (FIG. 2), and C / N ratio was 1.0 (FIG. 3). In addition, it was confirmed that the optimal amount of microbial inoculation was 10% (FIG. 4). The present inventors referred to the strain as "Pseudomonas fluorescens K4 ( Pseudomonas). fluorescens K4) ”and was deposited with the Korea Microorganism Conservation Center on February 13, 2007 (Accession No. KCCM10841P).

Contents Characteristics morphological characteristics   Cell shape Gram stain Spore formation Motility  rod--+ Cultural characteristics Colony shape Colony surface Colony color  circular, undulate, convex smooth white Biochemical characteristics Gorwth in air Growth anaerobically Acid from glucose Oxidase Catalase O / F test Yellow pigmentation Arginine dihydrolase Nitrate reduction Indole production Urease β-Glucosidase β-Galactosidase Gelatin hydrolysis Assimilation: glucose, arabinose, mannose, mannitol, gluconate, N-acetyl-glucosamine, malate Assimilation: maltose, caprate, adipate, citrate  ---+ + Oxidation-+ +--+--+-

< Example  3> Investigation of strain performance according to nitrate inorganic salt and inorganic salt concentration

Potassium nitrate (KNO ₃ ), sodium nitrate (NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ) or calcium nitrate [Ca (NO ₃ ) ₂ ] Treated wastewater containing 0.1 to 0.6% of each concentration was prepared, and then added so that the concentration ratio of sodium citrate to nitrate was 1, and then composed of the new strain isolated and identified in Example 1 and the composition of Tables 1 and 2 above. The denitrification medium to which the medium component and the trace component were added was supplied to the culture tank. Nitrate resolution was examined after 2, 4, 6, 24, 36, 48, 56, 72, 80, 94 and 104 hours. The nitrate resolution investigation method is as follows. The microbial concentration was measured at 650 nm using a spectrophotometer (Lamda 2S, Perkin Elmer), the concentration of nitrate was measured using a DR-400 spectrophotometer (spectrophotometer, Hach, Co.). Cultures were sampled before nitrate measurement, centrifuged at 1,000 rpm for 10 minutes, and the supernatant from which the cells were removed was analyzed using TEST KIT (Nitrate Nitrogen Standard Solution).

As a result, as a result of examining the nitrate resolution according to the initial nitrate nitrogen concentration (0.1, 0.2, 0.3, 0.4, 0.5, 0.6%), the nitrates present in the culture treatment tank were completely decomposed 1 to 4 days after the culture (Table 1 4 and FIG. 5).

Nitrate Degradation Performance Test Results Implementation (NO ₃ ) Handout (%) Residence time NO ₃ ^- Degradation Rate (%) Mall / ℓday One 0.1 23 99.9 0.02 2 0.2 36 99.9 0.03 3 0.3 48 99.9 0.04 4 0.4 78 99.9 0.05 5 0.5 78 99.9 0.06 6 0.6 104 99.9 0.07

< Example  4> Isolate  Ammonia Resolution Investigation

In order to investigate the ammonia decomposition ability of the isolated strain of the present invention, the culture solution was collected during the culture period and the degree of ammonia degradation was examined. Ammonia decomposition degree measuring method is as follows. Microbial concentration was measured at 650 nm using a spectrophotometer (Lamda 2S, Perkin Elmer), the concentration of ammonia was measured using a DR-400 spectrophotometer (spectrophotometer, Hach, Co.). The culture solution was sampled before ammonia measurement, centrifuged at 1,000 rpm for 10 minutes, and the supernatant from which the cells were removed was analyzed using TEST KIT (NH4-N Standard Solution).

Isolation strains were cultured in the medium mentioned in Example 1, and in the initial stage of culture, nitrates were first decomposed, and after nitrates were decomposed, it was confirmed to decompose ammonia (FIG. 6).

< Example  5> Degradation of Nitrate Degrading Strains According to C / N Ratio Denitrification efficiency  Research

In order to investigate the optimum degradation conditions of nitrate for the isolated strain of the present invention, the nitrate resolution according to the C / N ratio was investigated. Nitrate resolution was investigated according to the method of Example 3.

The isolate strain Pseudomonas fluorescens K4 improved the denitrification performance for nitrate nitrogen as the C / N ratio was increased from 0 to 1.0 (Fig. 7).

Pseudomonas fluorescens K4 strain of the present invention is a strain that simultaneously denitrates nitrate nitrogen and ammonia nitrogen using carbon as an electron donor under anaerobic conditions, and particularly, has excellent denitrification performance for nitrate and ammonia wastewater present in high concentrations. It is particularly useful for treating wastewater.

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Claims (8)

Novel Pseudomonas fluorescens K4 strain deposited with accession number KCCM10841P, having the following bacteriological characteristics: 1) anaerobic bacteria; 2) use of nitrates and ammonium salts as inorganic nitrogen sources; And 3) Optimum condition for denitrification is pH 5 ~ 9 and temperature 25 ~ 40 ℃, C / N ratio 0 ~ 1.0. The Pseudomonas fluorescens K4 strain according to claim 1, wherein the optimum denitrification condition is pH 7.0, the temperature is 30 ° C, and the C / N ratio is 1.0. delete The Pseudomonas fluorescens K4 strain according to claim 1, which has a 16S rRNA nucleotide sequence as set forth in SEQ ID NO: 1. The composition for nitrogen denitrification of wastewater containing the strain of claim 1 as an active ingredient. 6. A composition according to claim 5, characterized by denitrifying nitrate nitrogen or ammonia nitrogen. Nitrogen denitrification treatment method comprising the step of supplying the wastewater Pseudomonas fluorescens K4 strain of the industrial wastewater. 8. The method for treating nitrogen denitrification according to claim 7, wherein nitrate nitrogen or ammonia nitrogen is denitrified.

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