KR20130058089A - Stenotrophomonas sp. cw4-y capable of reducing odor and nitrification/de nitrification on aerobic condition and odor reduction mehod using it - Google Patents

Abstract

PURPOSE: Stenotrophomonas sp. CW-4Y(deposit number: KCTC 12050BP) which is capable of nitrification and denitrification under an aerobic condition is provided to reduce odor from ammonia gas, ammonia nitrogen, and nitrate nitrogen generated during a waste water treatment process. CONSTITUTION: Stenotrophomonas sp. CW-4Y(KCTC 12050BP) reduces odor and is capable of heterotrophic nitrification and denitrification under an aerobic condition. A deodorizing agent contains the strain as an active ingredient. Ammonia gas(NH_3) and ammonia nitrogen are removed using the strain. The strain is isolated from water which is stagnant in the river and sludge. [Reference numerals] (AA) Ammonia gas concentration(ppmv); (BB) Control group; (CC) Time(days)

Description

Odor-reducing Stenotropomonas spp. Strains capable of simultaneous heterotrophic nitrification and denitrification in aerobic state and odor reduction methods using the same {Stenotrophomonas sp. CW4-Y capable of reducing odor and nitrification / de nitrification on aerobic condition and odor reduction mehod using it}

The present invention relates to a specific novel Stenotrophomonas sp. Strain which can simultaneously nitrify and denitrify in a heterotrophic aerobic state when a carbon source is supplied, and a method for reducing odor using the same.

In the case of nitrification by microorganisms, ammonia (NH ₃ ) is oxidized by bacteria in the aerobic state to nitrite (NO ^{2 －} ) to form nitrate (NO ^{3 －} ). Denitrification occurs in the anaerobic state, where nitrates act as final electron acceptors instead of oxygen and are reduced to produce energy. Denitrification on the agricultural side can inhibit the growth of crops due to the loss of nitrogen sources in the soil. Nitrogen monoxide, an intermediate product generated during the destruction of the ozone layer and denitrification by nitrogen, on the natural ecosystem side. Toxicity and the production of nitrosoamine (carcinogen) by the reaction of nitrite and secondary amine have been studied a lot. In particular, wastewater treatment is used to reduce nitrous acid and nitric acid produced by aerobic nitrification bacteria to nitrogen gas using organic materials.

Ammonia nitrogen accounts for a large amount of nitrogen in the sewage, and in order to treat it with biological techniques, microorganisms involved in nitrification in aerobic conditions and microorganisms involved in denitrification in anaerobic conditions must be used. do. In addition, the representative nitrifying microorganisms known to date are isolated and treated as sewage treatment plant or silver Azotobacter , Azomonas , Clostridium , Nitobacter , Nitrosomonas . and and nitro Spira (Nitrospira) is known denitrification microorganism Agrobacterium mainly by microorganisms to survive in anaerobic conditions bakteo (Agrobacter), Kam Philo bakteo (Camphylobacter), this Kell Rera (Eikenella), Plastic beam bacteria (Flavobacter), Kingella , Moraxella , Morococcus , Ochrobacterium , Oligella , Sphingobacterium , Tsukamurella and the like are known.

In the technology of treating the influent wastewater by the multi-stage double cycle inflow method (Application No. 10-1999-7000103), the influent wastewater and denitrification were performed under anaerobic conditions, and the sludge was efficiently nitrified and denitrified in the shrimp farm. In a microbial isolation study (application number: 10-2005-0012593), microorganisms were used to control water quality in anaerobic conditions, but they did not have the ability to reduce odors such as ammonia. In addition, in the sewage and wastewater treatment process (application number: 10-2001-19607) for denitrification and sludge-free discharge, a patent was issued regarding the biological treatment step using anaerobic and aerobic microorganisms for the introduced sewage and wastewater. Reactors were installed to utilize microorganisms growing in two environments.

Deodorization and advanced treatment method and apparatus of sewage and wastewater (Registration No .: 10-446718) treats wastewater by making the structure as simple as possible, but uses aerobic and anaerobic reactors, wastewater treatment apparatus using microbial filter media, and The method (Registration No. 10-512686) undergoes nitrification under aerobic conditions followed by denitrification under anaerobic conditions. Nitrogen removal from wastewater (Registration No .: 10-242715) is carried out in the first and second stages to denitrify the fluid with methanol and sulfuric acid in the first stage anoxic denitrification tank to supply the carbon source by supplying the wastewater to the contact tank. It is a technique to remove nitrogen by passing the oxidized product to an aerobic nitrification tank.

In addition, the biological sewage and wastewater treatment apparatus combined with nitrogen and phosphorus removal and its treatment method (Registration No .: 10-109181) are apparatuses for biologically treating organic sewage and wastewater, and have a median layer to secure high concentrations of denitrifying bacteria and denitrification. An anaerobic tank for maximizing phosphorus release by securing a large number of dephosphorus microorganisms returned from an anoxic tank and an inflow or a phosphorus absorption tank to induce the above, is installed in the upper and lower portions of the separation reaction tank, and induces smooth phosphorus absorption and nitrification in the aeration tank (aerobic tank). It is a way to remove nitrogen.

Biological nitrogen and phosphorus removal method of organic sewage and wastewater (Registration No .: 10-90903) supplies organic sewage and wastewater from the sedimentation tank to the biological treatment tank to carry out anaerobic treatment, aerobic treatment, and semi-treatment. In a method for removing biological nitrogen and phosphorus from organic sewage and wastewater, which simultaneously removes organic matter, nitrogen, and phosphorus from wastewater, organic acids are produced and purified from sludge of sedimentation tank and surplus sludge of biological treatment tank, and the activity of microorganisms is reduced. A method for removing nitrogen and phosphorus from sewage and wastewater, wherein the purified organic acid is supplied to a wastewater and wastewater treatment process in a biological treatment tank so as to promote biological treatment of sewage and wastewater by microorganisms.

Wastewater treatment apparatus and method using an anaerobic-aerobic fluidized bed (Registration No .: 10-510406) is installed on the top of an anaerobic tank and an aerobic tank and desorbs microorganisms in the media to keep the size of bioparticles constant. Nitrogen is removed using biomembrane separation means, and sewage and wastewater treatment method by advanced biological treatment (Registration No.: 10-438323) is introduced into anaerobic tank by fermentation by hydrolysis and some fermentation reaction in anaerobic tank. The product is returned to the total denitrification tank, which is an anoxic tank, used as an organic source for denitrification, and a part of the return sludge returned from the settling tank is introduced into the anoxic tank and denitrified.

As such, the patented wastewater treatment process involves nitrification in an aerobic state and denitrification in an anaerobic state when biological treatment is performed using a structure change of a wastewater treatment device, heat treatment, or a filtration membrane. Most of the removal of nitrogen through. Therefore, in the case of wastewater treatment using biologically known microorganisms, aerobic and anaerobic reactors must be present in order to remove nitrogen from the sewage treatment process. It has a problem.

In addition, wastewater treatment studies using microorganisms have been conducted by simply removing nitrogen sources by adding nitrifying and denitrifying microorganisms to each reactor. However, a large amount of odor is generated in this process, and this odor is a representative household pollutant that directly or indirectly affects the inhabitants' lives according to the degree of irritation. It is an urgent matter.

The present invention has been made to solve the above problems, the inventors of the present invention is a genus of Stenotrophomonas isolated from the standing water and sludge around the stream long contaminated with factory wastewater (Stenotrophomonas sp ) The present invention was completed by discovering that the CW-4Yn strain is capable of simultaneously nitrifying and denitrifying in an environment provided with a carbon source. At the same time, the strain has the unique property of reducing ammonia gas, ammonia nitrogen and nitrate nitrogen.

An object of the present invention is a genus of Stenotrophomonas which can treat organic nitrogen with only an aerobic reactor in a wastewater treatment process in which an aerobic reactor and an anaerobic reactor are installed to remove organic nitrogen. sp ) To provide a CW-4Yn strain.

Another object of the present invention is to provide a method for eliminating odors and nitrogenous odors of ammonia generated in a sewage treatment plant as well as simplifying the wastewater treatment process using the strain.

In order to achieve the above object, the present invention provides a strain of Stenotrophomonas sp CW-4Y (Accession No .: KCTC 12050 BP) isolated from sewage and sludge accumulated around a stream contaminated with plant wastewater for a long time. do.

Stenotrophomonas genus according to the invention sp ) CW-4Y (Accession No .: KCTC 12050 BP) strain is a heterotrophic microorganism, a specific microorganism capable of simultaneous nitrification and denitrification in aerobic state, and can be used for biological treatment in sewage treatment. In addition, it has the ability to reduce odor when supplying a carbon source of ammonia gas, ammonia nitrogen and nitrate nitrogen which can be generated in the wastewater treatment process.

Figures 1a and 1b is a graph showing the ammonia gas concentration and ammonia gas removal rate is discharged from the oil inoculated the strain of the strain CW-4Y of the genus Stenotropomonas of the present invention.
Figure 2 is a graph showing the change of ammonia nitrogen, nitrogen nitrate concentrations in the ammonium nitrate medium inoculated stenotropomonas genus CW-4Y strain of the present invention.
Figure 3 is a graph showing the change of ammonia nitrogen, nitrate nitrogen concentration when anaerobic culture of the strain CW-4Y of the genus Stenotropomonas of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Steenotrophomonas of the present invention sp . ) The CW-4Y strain is a strain isolated from stagnant water and sludge around the polluted stream, and can perform nitrification and denitrification at the same time in aerobic state, and has a characteristic of reducing ammonia odor.

Steenotrophomonas of the present invention sp ) The CW-4Y strains collected rotten water and sludge around rivers contaminated with factory wastewater for a long time, inoculated 10 g of the sample into 100 mL of LB Broth medium, and incubated shaking culture at 180 rpm at 30 ° C for 24 hours. 1 g of strain was inoculated in 10 mL of distilled water to which 0.2 g of oil foil and 0.1% of glucose, which were generated gas, were concentrated at 30 ° C. and 180 rpm. The ammonia gas generated in the culture solution was analyzed using an ammonia gas detector tube (KITAGAWA, Japan), and the culture medium to reduce the emission of ammonia gas was smeared on LB Aagar medium and purified.

The inventors of the strain Stenotrophomonas sp. Obtained by the above method Stenotrophomonas sp. It was named CW-4Y and was deposited on November 7, 2011 at the Korea Institute of Bioscience and Biotechnology, a depository institution, and was given accession number KCTC 12050 BP.

Example 1 Isolation, Identification and Specific Activity of Stenotrophomonas sp. CW-4Y

(1) Isolation of Heterotrophic Nitrification-Denitrification Bacteria

The sewage and sludge from the surroundings of the stream contaminated with the factory wastewater were collected, and heterotrophic nitrification-denitrification bacteria were isolated from the sludge as follows. Inoculate 10g of the above sludge into 100mL of LB Broth medium and incubate for 24 hours at 180 rpm at 30 ℃ for 24 hours, and then inoculate 1 mL of strain into 10mL of distilled water containing 0.2g of ammonia and 0.1% glucose. The thickening was carried out at 30 ℃, 180rpm. The ammonia gas generated in the culture solution was analyzed using an ammonia gas detector tube (KITAGAWA, Japan), and the culture medium to reduce the emission of ammonia gas was smeared on LB Aagar medium and purified.

The components of CHF and CHNS composition are as follows. Oil foil: Castor gourd 57%, Rapeseed gourd 25%, Rice bran gourd 18% (C: 40.24 wt.%, H: 5.75 wt.% N: 5.28 wt.%, S: no detection) The strain was selected to reduce the ammonia gas through.

(2) Identification of Heterotrophic Nitrification-Denitrification Bacteria

The colonies of pure bacteria observed to have an ammonia gas reducing effect in (1) above were commissioned by Macrogen (MACROGEN) and analyzed PCR and nucleotide sequences. In this case, primers F27 (5'-AGA GTT TGA TCM TGG CTC AG-3 ') and R1492 (5'-TAC GGY TAC CTT GTT ACG ACT T-3') were used. The results of analyzing 16S rDNA nucleotide sequence of strain CW-4Y having ammonia gas reduction effect are shown in Table 1 below (NCBI accession number: JN995250). The DNA base sequence of CW-4Y (base sequence analysis results are shown in SEQ ID NO: 1) was 1451 bp in total, and NCBI blast search showed that CW-4Y was M2T2B9 (NCBI accession number: Stenotropomonas). GQ246699) is 1447/1450 (99%), and 1420/1422 (99%) with M2 (NCBI accession number (FJ357297)), which is a stenotropomonas genus that decomposes methyl parathion, an organophosphorus insecticide. It is 1449/1453 (99%) of YC-1 (NCBI accession number: DQ537219) in the Stenotropomonas genus that decomposes chlorpyrifos, a toxic substance that emits chlorides, nitrogen oxides, sulfur oxides, and phosphorous oxides. Most similar (Table 1).

Strain Phylogenically related fungi Similarity
CW-4Y Stenotrophomonas sp. M2T2B9 (GQ246699) 1447/1450 (99%) Stenotrophomonas sp. M2 (FJ357297) 1420/1422 (99%) Stenotrophomonas sp. YC-1 (DQ537219) 1449/1453 (99%)

Experimental Example 1: Confirmation of ammonia gas reduction of heterotrophic nitrification-denitrification bacteria

In order to determine the ammonia gas reduction ability of the bacterial Stenotropomonas CW-4Y strain having the ammonia gas reducing ability of the present invention, 0.2 g of oil was added to a 70 mL vial, and 1 mL of the strain was added to 10 mL of distilled water having a glucose concentration of 0.1%. After inoculation, the concentration of ammonia was measured using ammonia gas detector tubes (KITAGAWA, Japan) on day 2 and day 7. In this experiment, the culture temperature was 30 ℃, shaking culture was carried out at 180rpm. The results are shown in FIGS. 1A and 1B. Figure 1a is a result of measuring the concentration of ammonia gas according to the inoculation of the CW-4Y strain of the genus Stenotropomonas of the present invention, Figure 1b is in accordance with the inoculation of CW-4Y strains of the genus Stenotropomonas of the present invention Removal rate of ammonia gas (black: control group not inoculated with CW-4Y; white: experimental group inoculated with CW-4Y).

As shown in Figure 1a, 1b, when the ammonia gas reduction capacity of the CW-4Y strain was examined as a result of the ammonia gas concentration of 17ppm on the 2nd day of the control group did not generate ammonia gas in the experimental group showed a 100% removal rate, on the 7th day The ammonia concentration of the control group was 31.4ppm, and in the experimental group inoculated with CW-4Y, it was 6.6ppm, indicating that 79% of the ammonia gas could be reduced.

Experimental Example 2: Confirmation of ammonia nitrogen and nitrate nitrogen reduction in heterotrophic nitrification-denitrification bacteria

Stenotrophomonas sp. Of the present invention. After incubating the culture solution of CW-4Y for 24 hours, inoculate 5 mL of the culture solution into 100 mL of ammonium nitrate medium containing ammonia nitrogen and ammonium nitrate, and incubate at 30 ° C. and 180 rpm to incubate ammonia nitrogen and nitrate nitrogen over time. The concentration of was confirmed and shown in FIG.

The composition of ammonium nitrate medium is as follows. Ammonium nitrate medium: K ₂ HPO _{4 ,} 14 g / L; KH ₂ PO ₄ 6 g / L; glucose 17 g / L; NH ₄ NO ₄ 1.9 g / L; MgSO ₄ H ₂ O 0.2 g / L; tracemineralsolution 2 mL / L. Tracemineral solution: ethylenediamine-N, N, N ', N'-tetraacetic acid, isodium salt, dihydrate (EDTA.2Na) 57.1 g / L; ZnSO ₄ 7H ₂ O 3.9 g / L; CaCl ₂ · 2H ₂ O 7 g / L; MnCl ₂ 4H ₂ O 5.1 g / L; 5.0 g / L of FeSO ₄ 7H ₂ O; (NH ₄ ) 6 Mo ₇ O ₂₄ .4H ₂ O 1.1 g / L; CuSO ₄ .5H ₂ O 1.6 g / L; CoCl ₂ .6H ₂ O (pH = 6.0) 1.6 g / L.

After incubation, 3mL of the sample was collected, and the ammonia nitrogen and the nitrate nitrogen were analyzed by using a reagent of C-MAC Co., Ltd. (C-MAC) and confirmed the absorbance by DR5000 of HACH, and Stenotrophomonas sp. Changes in concentrations of ammonia nitrogen and nitrate nitrogen over time by CW-4Y are shown in the graph of FIG. 2.

As shown in Figure 2, CW-4Y strain according to the present invention was confirmed to remove ammonia nitrogen and nitrate nitrogen at the same time (●: ammonia nitrogen; ▽: nitrogen nitrate).

Experimental Example 3: Confirmation of ammonia nitrogen and nitrate nitrogen reduction by shaking culture (aerobic state) and political culture (anaerobic state)

Stenotrophomonas sp. Of the present invention. The aerobic environment was incubated at 30 ° C and 180 rpm using shaking culture to confirm the attenuation of ammonia nitrogen and nitrate nitrogen according to the aerobic and anaerobic environment of CW-4Y. Injected into artificial anaerobic conditions, and then allowed to stand and incubated at 30 ° C. to change in time (days) are shown in Table 2, Stenotrophomonas sp. The change of ammonia nitrogen and nitrate nitrogen when CW-4Y was incubated was shown in the graph of FIG. 3 (●: ammonia nitrogen; ▽: nitrogen nitrate).

Shake Culture (mg-N / L / day) Political Culture (mg-N / L / day) Ammonia Nitrogen Reduction Rate 28.7 0 Nitric Nitrate Reduction Rate 53.1 51.1

As shown in Table 2 and Figure 3, the CW-4Y strain of the present invention was examined for the reduction rate according to the static culture and shaking culture, the rate of reduction of ammonia nitrogen in the stationary culture was 0mg-N / L / day, The rate of reduction of nitrate nitrogen was 51.1 mg-N / L / day (Table 2). On the other hand, in the shaking culture, the reduction rate of ammonia nitrogen was 28.7 mg-N / L / day and the reduction rate of nitrate nitrogen was 53.1 mg-N / L / day. In other words, it was confirmed that CW-4Y improved nitrification and denitrification ability under aerobic conditions.

Korea Biotechnology Research Institute KCTC12050BP 20111107

<110> LEE, EUN YOUNG          University-Industry Collaboration & Consulting Foundation          (Suwon University Industry-Academic Cooperation Foundation) <120> Stenotrophomonas sp. CW4-Y capable of reducing odor and          nitrification / de nitrification on aerobic condition and odor          reduction mehod using it <130> KDP11-945K <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1451 <212> DNA <213> Stenotrophomonas <400> 1 catggctcag agtgaacgct ggcggtaggc tacacatgca gtcgaacggc agcacagtaa 60 gagcttgctc ttacgggtgg cgagtggcgg acgggtgagg aatgcatcgg aatctactct 120 gtcgtggggg ataacgtagg gaaacttacg ctaataccgc atacgaccta cgggtgaaag 180 caggggatct tcggaccttg cgcgattgaa tgagccgatg cccgattagc tagttggcgg 240 ggtaagagcc caccaaggcg acgatcggta gctggtctga gaggatgatc agccacactg 300 gaactgagac acggtccaga ctcctacggg aggcagcagt ggggaatatt ggacaatggg 360 cgcaagcctg atccagccat accgcgtggg tgaagaaggc cttcgggttg taaagccctt 420 ttgttgggaa agaaaagcat tcggttaata cccgattgtt ctgacggtac ccaaagaata 480 agcaccggct aacttcgtgc cagcagccgc ggtaatacga agggtgcaag cgttactcgg 540 aattactggg cgtaaagcgt gcgtaggtgg ttgtttaagt ctgtcgtgaa agccctgggc 600 tcaacctggg aattgcgatg gaaactgggc gactagagtg tggcagaggg tagtggaatt 660 cctggtgtag cagtgaaatg cgtagagatc aggaggaaca tccgtggcga aggcgactgc 720 ctgggccaac actgacactg aggcacgaaa gcgtggggag caaacaggat tagataccct 780 ggtagtccac gccctaaacg atgcgaactg gatgttgggt gcaatttggc acgcagtatc 840 gaagctaacg cgttaagttc gccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcggtggag tatgtggttt aattcgatgc aacgcgaaga 960 accttacctg gccttgacat gcacggaact ttccagagat ggattggtgc cttcgggaac 1020 cgtgacacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg tccttagttg ccagcacgta atggtgggaa ctctaaggag 1140 accgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcatcatg gcccttacgg 1200 ccagggctac acacgtacta caatggtagg gacagagggc tgcaagccgg cgacggtgag 1260 ccaatcccag aaaccctatc tcagtccgga ttggagtctg caactcgact ccatgaagtc 1320 ggaatcgcta gtaatcgcag atcagcattg ctgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccatg ggagtttgtt gcaccagaag caggtagctt aaccttcggg 1440 agggcgctgc c 1451

Claims (3)

Odor-reducing Stenotrophomonas capable of simultaneous heterotrophic nitrification and denitrification in aerobic conditions sp.) CW-4Y strain (KCTC: 12050 BP).
Stenotrophomonas sp. ( CCTC : 12050 BP) odor preventing agent containing the active ingredient according to claim 1 (KCTC: 12050 BP).
A method of removing ammonia gas and ammonia nitrogen using a Stenotrophomonas sp. CW-4Y strain (KCTC: 12050 BP) according to claim 1.

Images