KR100292332B1 - Novel Klebsiella Bacteria and Treatment Method of Waste Wood Using the Same - Google Patents
Novel Klebsiella Bacteria and Treatment Method of Waste Wood Using the Same Download PDFInfo
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- KR100292332B1 KR100292332B1 KR1019980020706A KR19980020706A KR100292332B1 KR 100292332 B1 KR100292332 B1 KR 100292332B1 KR 1019980020706 A KR1019980020706 A KR 1019980020706A KR 19980020706 A KR19980020706 A KR 19980020706A KR 100292332 B1 KR100292332 B1 KR 100292332B1
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- pcp
- waste wood
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- 235000010355 mannitol Nutrition 0.000 description 1
- LXKDFTDVRVLXFY-WQWYCSGDSA-N menaquinone-8 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 LXKDFTDVRVLXFY-WQWYCSGDSA-N 0.000 description 1
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- OOLLAFOLCSJHRE-ZHAKMVSLSA-N ulipristal acetate Chemical compound C1=CC(N(C)C)=CC=C1[C@@H]1C2=C3CCC(=O)C=C3CC[C@H]2[C@H](CC[C@]2(OC(C)=O)C(C)=O)[C@]2(C)C1 OOLLAFOLCSJHRE-ZHAKMVSLSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
본 발명은 신규한 클레브시엘라속 세균 Bu1 균주와 이를 이용한 폐목재의 처리방법에 관한 것이다.The present invention relates to a novel Klebsiella genus Bu1 strain and a method for treating waste wood using the same.
본 발명의 신규한 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주는 성장속도가 빠르고 배양이 용이하며 염소화 페놀화합물 특히, 고농도의 5염소화페놀(PCP)로 오염된 지하수, 대수층, 토양, 강이나 호수의 저니뿐만 아니라, 전신주, 철도침목이나 탄약상자 등의 폐목재에서 고농도의 5염소화페놀을 빠른시간내에 분해하므로 환경오염을 방지할 수 있는 뛰어난 효과가 있다.The novel Klebsiella bacteria Bu1 (KCTC 8852P) strain of the present invention is fast growing and easy to cultivate, and contaminated with chlorinated phenolic compounds, especially high concentrations of chlorinated chlorine (PCP), groundwater, aquifer, soil, river or In addition to the lake jersey, waste wood such as telephone poles, railway sleepers and ammunition boxes, high concentrations of chlorinated pentachloride are quickly decomposed, so it is possible to prevent environmental pollution.
Description
본 발명은 신규한 클레브시엘라속 세균과 이를 이용한 폐목재의 처리방법에 관한 것이다. 더욱 상세하게는, 본 발명은 토양으로부터 분리되고 염소화페놀 화합물, 특히 고농도의 5염소화페놀(penta-chlorophenol, 이하 PCP라 한다) 분해능이 있는 신규한 클레브시엘라속 세균 Bu1 균주와 이를 이용한 폐목재를 처리하는 방법에 관한 것이다.The present invention relates to a novel Klebsiella genus bacterium and a method for treating waste wood using the same. More specifically, the present invention is a novel Klebsiella genus Bu1 strain isolated from soil and capable of degrading chlorinated phenolic compounds, particularly high concentrations of penta-chlorophenol (PCP) and waste wood using the same. It is about how to process.
2염소화페놀(di-chlorophenol), 3염소화페놀(tri-chlorophenol), 4염소화페놀(tetra-chlorophenol) 및 PCP와 같은 염소화 페놀화합물은 염소화도가 높을수록 생물체의 성장을 억제하는 효과가 높다. 특히 염소화페놀 화합물중 가장 많이 염소로 치환된 PCP는 제초제, 살충제, 살균제, 목재보존제 등으로 폭넓게 사용된다(Wild et al., Chemosphere, 24, 833, 1992).Chlorinated phenolic compounds, such as di-chlorophenol, tri-chlorophenol, tetra-chlorophenol, and PCP, have higher chlorination effects to inhibit the growth of organisms. Among the chlorinated phenolic compounds, PCP, the most substituted with chlorine, is widely used as a herbicide, insecticide, fungicide, wood preservative, etc. (Wild et al., Chemosphere, 24, 833, 1992).
PCP는 0.6ppm의 낮은 농도에서도 대부분의 어류에 대해 급성독성을 나타내고 페놀에 비하여 독성이 40배나 높으며(Liu et al., Bull. Environ. Contam. Toxicol., 27, 289, 1981), 광합성과 수중 조류(algae)의 생장도 억제한다(Gotham & Rhee, J. Great Lakes Res., 8, 328, 1982). 이러한 부작용 때문에 미국의 환경보호청은 PCP를 포함한 염소화페놀 화합물 10종을 유기유해화학물질(organic priority pollutants)로 선정하고, 그 사용을 제한하였다(Freeman, Standard Handbook of Harzardous Waste Treatment and Disposal, McGraw Hill, NY, 1989).PCP is acutely toxic to most fish at low concentrations of 0.6 ppm and 40 times more toxic than phenol (Liu et al., Bull. Environ. Contam. Toxicol., 27, 289, 1981). It also inhibits the growth of algae (Gotham & Rhee, J. Great Lakes Res., 8, 328, 1982). Because of these side effects, the US Environmental Protection Agency selected 10 chlorinated phenolic compounds, including PCPs, as organic priority pollutants and restricted their use (Freeman, Standard Handbook of Harzardous Waste Treatment and Disposal, McGraw Hill, NY, 1989).
그러나 PCP가 1930년대에 개발된 이래로 꾸준히 생산이 증가되어 1985년에만 10만톤이 생산되었으며 이들은 지하수, 대수층, 토양, 강이나 호수의 저니 등에 잔류하고 있다. 또한 전신주, 철도침목이나 탄약상자 등의 목재에도 다량의 PCP가 잔류되어 있어, 이들을 소각하면 2차 환경오염물질을 발생할 가능성이 높다. 따라서 PCP를 비롯한 염소화페놀 화합물의 분해능을 갖는 미생물을 이용한 생물학적 처리로 잔류량을 감소시키거나 무독화해야 한다. 종래의 PCP 분해방법으로는 슬러리 처리법등이 있다. 즉, 염소화페놀 화합물, 특히 PCP를 분해하는 미생물은 분해시 생성되는 염산으로 인하여 PCP-MS(mineral salts) 배지를 푸른색에서 노란색으로 변화시키므로, 이를 지표로 하여 PCP를 탄소원으로 이용하는 PCP 분해 미생물을 분리하고(Kennes & Lemma, Biotech. Lett., 16, 759, 1994), 분리된 미생물을 이용하여 PCP로 오염된 토양을 처리한다(미국특허 제 4,923,125호, 국제특허공개 94-22605호). 한편, 염소화페놀 화합물중 PCP 분해 미생물인 플라보박테리움속 미생물(Flavobacterium sp.,ATCC 39723)로부터 PCP 분해에 관련되는 유전자 pcpA, pcpB, pcpC 등이 클로닝되었음이 보고되었다(미국특허 제 5,364,787호). 또한 PCP 분해 미생물로 아트로박터(Arthrobacter)등의 세균과 백색부후균인 파네로카에테(Phanerochaete)가 보고되었으나, 이들의 PCP 처리농도는 400ppm에 불과하다(Topp & Hanon, Appl. Environ. Microbiol., 56, 541, 1990; Edgehill & Finn, ibid, 45, 1122, 1983; Lamar & Dietrich, ibid, 56, 3093, 1990; Otto et al., Appl. Microbiol. Biotechnol., 40, 926, 1990).However, since the PCP was developed in the 1930's, production has steadily increased, producing 100,000 tons in 1985 alone, which remains in groundwater, aquifers, soils, rivers and lakes. In addition, a large amount of PCP remains in timber such as telephone poles, railway sleepers and ammunition boxes, and incineration of them is likely to cause secondary environmental pollutants. Therefore, the residual amount should be reduced or detoxified by biological treatment using microorganisms capable of degrading chlorinated phenolic compounds including PCP. Conventional PCP decomposition methods include slurry treatment. In other words, chlorinated phenolic compounds, in particular, microorganisms that degrade PCP, change the medium salts (PCP-MS) medium from blue to yellow due to the hydrochloric acid produced during decomposition. (Kennes & Lemma, Biotech. Lett., 16, 759, 1994), and treated the soil contaminated with PCP using isolated microorganisms (US Pat. No. 4,923,125, International Patent Publication No. 94-22605). Meanwhile, it has been reported that the genes pcpA, pcpB, pcpC, etc. related to PCP degradation have been cloned from Flavobacterium sp., ATCC 39723, a PCP degrading microorganism among chlorinated phenolic compounds (US Pat. No. 5,364,787). . In addition, PCP-degrading microorganisms, such as Arthrobacter and white rot, Panerochaete, have been reported, but their PCP concentration is only 400 ppm (Topp & Hanon, Appl. Environ.Microbiol). ., 56, 541, 1990; Edgehill & Finn, ibid, 45, 1122, 1983; Lamar & Dietrich, ibid, 56, 3093, 1990; Otto et al., Appl. Microbiol. Biotechnol., 40, 926, 1990) .
따라서, 본 발명은 고농도의 PCP 분해능을 갖는 미생물의 필요성을 절감하여 안출한 것으로, 본 발명의 목적은 고농도의 염소화합물, 특히 고농도의 PCP를 분해하는 우수한 능력을 가진 신규한 클레브시엘라속 세균 Bu1을 제공하는데 있다. 본 발명의 다른 목적은 본 발명의 신규한 상기 균주를 이용하여 PCP로 오염된 폐목재를 처리하여 환경오염을 방지하는데 있다.Therefore, the present invention is to reduce the need for microorganisms having a high concentration of PCP resolution, the object of the present invention is a novel Klebsiella genus bacteria having excellent ability to decompose high concentration of chlorine compounds, especially high concentration of PCP To provide Bu1. Another object of the present invention is to prevent environmental pollution by treating waste wood contaminated with PCP using the novel strain of the present invention.
본 발명의 상기 목적은 목재야적장에서 채취한 토양을 PCP 액체배지에 접종하여 진탕 배양하고, 진탕 배양액으로부터 미생물을 분리한 후 분리된 미생물을 PCP의 농도를 점차 증가시킨 배양액에서 반복배양하여 클레브시엘라속 미생물 Bul(KCTC 8852P)을 분리하고, 이를 PCP를 함유한 폐목재에 투여하여 PCP의 농도와 미생물의 수를 확인하므로써 달성하였다.The above object of the present invention is inoculated in a PCP liquid medium by inoculation of the soil collected from the wood yard shake shaking culture, and after separating the microorganism from the shaking culture medium, and repeatedly cultured in a culture medium gradually increasing the concentration of PCP Klebsi This was accomplished by isolating Ella genus Bul (KCTC 8852P) and administering it to the waste wood containing PCP to confirm the concentration of PCP and the number of microorganisms.
이하, 본발명의 구성 및 작용을 상세히 설명한다.Hereinafter, the configuration and operation of the present invention will be described in detail.
본 발명은 목재야적장에서 채집한 토양 샘플을 PCP 액체배지에서 진탕배양하여 미생물들을 분리하는 단계; 분리된 미생물을 PCP 농도를 점차 증가시킨 배지에서 배양하여 클래브시엘라속 세균 Bul을 분리하는 단계; 분리된 클래브시엘라속 미생물 Bul을 PCP 함유 배지에 투여하여 PCP의 농도와 분해속도를 확인하는 단계; 및 클래브시엘라속 미생물 Bul을 PCP를 함유한 폐목재에 투여한 후 PCP의 농도와 미생물의 수를 확인하는 단계로 이루어진다.The present invention comprises the steps of separating the microorganisms by shaking culture the soil samples collected in the timber yard in PCP liquid medium; Culturing the isolated microorganisms in a medium gradually increasing the PCP concentration to isolate the genus Clavsilia Bul; Confirming the concentration and degradation rate of the PCP by administering the isolated clab ciella microorganism Bul to the PCP-containing medium; And the step of confirming the concentration of the PCP and the number of microorganisms after administering the clab ciella microorganism Bul to the waste wood containing PCP.
이하, 본 발명의 구성 및 작용은 실시예 및 실험예를 통하여 상세히 설명하지만 본 발명의 권리범위가 이들에 의해 제한되는 것은 아니다.Hereinafter, the configuration and operation of the present invention will be described in detail through Examples and Experimental Examples, but the scope of the present invention is not limited thereto.
실시예 1. PCP 분해 세균의 분리Example 1 Isolation of PCP Degrading Bacteria
부산, 여수, 군산, 인천에 소재하는 목재야적장에서 채집한 토양 각각 1g을 채취하고, 이를 PCP 100ppm을 함유하고 있는 PCP 액체배지(K2HPO40.65g, KH2PO40.17g, MgSO40.1g, NaNO30.5g, PCP(Fluka사 제품) 100 ppm을 증류수 1리터에 녹여 형성한 배지, pH 7.3)에 접종하고, 30℃에서 10-30일간 진탕 배양한다. 이때 사용된 상기의 PCP는 목재시료 5g을 99.9% methanol 60mL에 4시간 이상 침륜 혼합한 다음, 헥산 30mL을 첨가하여 추출하는 과정을 3회 반복한 후, 추출된 PCP를 진공 농축하여 개스크로마토그래프(Gas Chromatograph, GC)로 분석하여 얻었다(GC 분석 조건은 내경 0.32mm, 길이 32m인 칼럼(Supelco, capillary DB-1)을 3분간 80℃를 유지시킨 후 분당 8℃씩 증가시키면서 150℃에서 15분간 유지시키고, 주입온도 200℃, 검출온도 250℃에서 FID로 검출하였으며 주입개스는 수소 40psi, 공기 55 psi, 질소 70psi로 흘려주었고, 잔류농도는 PCP의 표준방정식에 따라 결정하였다). 이어서, 상기 진탕배양액 1mL을 취하여 PCP-MS 고체배지(PCP 액체배지에 BTB(bromo-thymol blue) 20mg, agar 1.5%를 첨가하여 형성한 배지)에 접종하고, 30℃에서 3~20일간 배양하였다. PCP가 분해되면 염산이 생성되므로 집락이 형성되는 부근에는 노란색을 띄게 된다. 순수 분리한 노란색의 집락을 PCP-MS 액체배지(PCP 액체배지에 BTB 20mg을 첨가하여 형성한 배지)에 접종하여 30℃에서 진탕 배양하여 PCP 100ppm을 분해하는 미생물 70종을 분리하였다.1g of soil collected from wood yards in Busan, Yeosu, Gunsan, and Incheon, and PCP liquid medium containing 100 ppm of PCP (K 2 HPO 4 0.65g, KH 2 PO 4 0.17g, MgSO 4 0.1 g, 0.5 g of NaNO 3 and 100 ppm of PCP (Fluka Co., Ltd.) were inoculated into a medium formed by dissolving in 1 liter of distilled water (pH 7.3), followed by incubation for 10-30 days at 30 ° C. In this case, the PCP used was mixed with 5 g of wood samples in 60 mL of 99.9% methanol for 4 hours or more, and then extracted by adding 30 mL of hexane three times, and then the extracted PCP was concentrated in vacuo to obtain a gas chromatograph ( Gas Chromatograph (GC) was used for the analysis (GC analysis conditions: the column diameter 0.32mm, 32m length column (Supelco, capillary DB-1) was maintained at 80 ℃ for 3 minutes and increased by 8 ℃ per minute 15 minutes at 150 ℃ It was maintained and injected with FID at the injection temperature of 200 ° C. and the detection temperature of 250 ° C., and the injection gas was flowed into 40 psi of hydrogen, 55 psi of air, and 70 psi of nitrogen, and the residual concentration was determined according to the standard equation of PCP. Subsequently, 1 mL of the shake culture solution was taken and inoculated into a PCP-MS solid medium (a medium formed by adding 20 mg of BTB (bromo-thymol blue) and 1.5% agar to the PCP liquid medium) and incubated at 30 ° C. for 3 to 20 days. . Decomposition of PCP produces hydrochloric acid, which is yellow in the vicinity of colony formation. Purely separated yellow colonies were inoculated in PCP-MS liquid medium (medium formed by adding BTB 20mg to PCP liquid medium), shaken and cultured at 30 ° C. to isolate 70 microorganisms that degrade 100 ppm of PCP.
실시예 2. 클레브시일라속 세균 Bul 균주의 분리Example 2 Isolation of the Klebsiila Bacterial Bul Strain
상기 실시예 1에서 분리한 미생물들을 PCP 1000ppm을 첨가한 PCP-MS 배지에 접종하여 상기 실시예 1과 동일한 방법을 반복하여 미생물 12종을 분리한 후, 상기 PCP-MS 배지의 PCP 농도를 2000ppm으로 증가시켜 미생물 2종을 분리하였고 이들을 각각 Bu1, Bu34로 명명하였다. 이중 Bu34는 이미 슈도모나스 푸티다 Bu34(KCTC 8719P)로 명명하여 특허로 출원하였다(한국특허출원 96-4305). 한편, 본 발명 균주는 Klebsiella sp. Bul로 명명하여 1997년 11월 17일 한국과학기술연구원 생명공학연구소내 유전자원센타에 기탁번호 KCTC 8852P로 기탁하였다.The microorganisms isolated in Example 1 were inoculated in PCP-MS medium to which PCP 1000ppm was added, and the same procedure as in Example 1 was repeated to isolate 12 microorganisms, and then the PCP concentration of the PCP-MS medium was 2000 ppm. Two microorganisms were isolated and named Bu1 and Bu34, respectively. Bu34 has already been filed as a patent named Pseudomonas putida Bu34 (KCTC 8719P) (Korean Patent Application 96-4305). On the other hand, the present invention strain Klebsiella sp. It was named as Bul and was deposited on November 17, 1997 at KCTC 8852P at the Genetic Resources Center in the Korea Institute of Science and Technology.
실시예 3. 클레브시엘라속 세균 Bu1 균주의 이화학적 특성 조사 및 동정Example 3. Investigation and Identification of Physicochemical Properties of Klebsiella Bacteria Bu1 Strains
TSA 배지(Tryptic Soy Agar, BBL사 제품)에 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주를 접종하여 30℃하에 24시간 배양하였다. 배양결과, 배지에는 백색의 집락이 형성되었고, 세포 크기는 0.6-6㎛(in length) 였다. 운동성은 없었으며 그람 염색은 음성이었고 생장온도는 4℃~40℃ 범위로, 최적온도는 37℃ 였다. 또한 그람음성세균에 적용하는 바이오로그 시스템(미국 Micro Station사 제품)을 이용하여 생리·생화학적인 특성을 검토하였다. 실험결과, 하기 표1과 같은 생리·생화학적 특성을 나타내었다.Klebsiella spp. Bu1 (KCTC 8852P) strains were inoculated in TSA medium (Tryptic Soy Agar, manufactured by BBL) and incubated at 30 ° C for 24 hours. As a result of culture, a white colony was formed in the medium, and the cell size was 0.6-6 탆 (in length). Gram staining was negative and growth temperature ranged from 4 ℃ to 40 ℃ with optimum temperature of 37 ℃. In addition, physiological and biochemical characteristics were examined using a biolog system (product of US Micro Station) applied to Gram-negative bacteria. As a result of the experiment, the physiological and biochemical properties shown in Table 1 are shown.
상기의 결과 데이터는 대조군으로 Klebsiella pneumoniae KCTC 2208을 사용하였을 때의 생리·생화학적인 결과와 일치하였다. 한편, 오야이주와 코마가타의 방법(Oyaizu & Komagata, J. Gen. Appl. Microbiol., 29, 17, 1983)에 따라 신균주의 퀴논과 세포내 지방산 조성을 비교하였다. 퀴논은 ubiquinone-8 + menaquinone-8, 세포내 지방산 조성은 C12:0, C14:0, C16:0, C16:1, C17:0 Cyclo, C14:0 3OH로 나타났다. 따라서, 본 발명의 신균주는 클레브시엘라속으로 동정되었다.The above data were consistent with the physiological and biochemical results when Klebsiella pneumoniae KCTC 2208 was used as a control. Meanwhile, the quinone and intracellular fatty acid composition of the new strain were compared according to the method of Oyaizu & Komagata, J. Gen. Appl. Microbiol., 29, 17, 1983. Quinone was found to be ubiquinone-8 + menaquinone-8, and the intracellular fatty acid composition was C 12 : 0, C 14 : 0, C 16 : 0, C 16 : 1, C 17 : 0 Cyclo, C 14 : 0 3OH. Thus, the new strain of the present invention was identified as genus Klebsiella.
실시예 4. 클레브시엘라속 미생물 Bu1의 PCP 분해속도 측정Example 4 Determination of PCP Degradation Rate of Klebsiella Microorganism Bu1
K2HPO40.065g, KH2PO40.017g, MgSO4·7H2O 0.1g, NaNO3·0.5g, BTB 20mg을 증류수 1L에 녹여만든 최소배지(pH 7.2)에 PCP를 200, 500, 1000, 2000ppm로 점차 증가하여 투여하고 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주를 접종한 뒤, 시간별로 흡광도와 PCP 농도를 측정하였다. 실험결과, 시간당 PCP 분해속도는 하기 표 2와 같이 PCP 농도가 최저 200ppm일때 분해속도는 0.86(㎎/L/h) 였고, PCP 농도가 최고 2000ppm일때 분해속도는 4.0(㎎/L/h) 였다.0.02 g of K 2 HPO 4 , 0.017 g of KH 2 PO 4 , 0.1 g of MgSO 4 · 7H 2 O, 0.1 N of NaNO 3 , 0.5 g, and BTB 20 mg were dissolved in 1 L of distilled water in a minimum medium (pH 7.2). After gradually increasing to 1000, 2000ppm and inoculating the Klebsiella bacteria Bu1 (KCTC 8852P) strain, the absorbance and PCP concentration were measured over time. As a result, the decomposition rate of PCP per hour was 0.86 (mg / L / h) when the PCP concentration was at least 200ppm, and the decomposition rate was 4.0 (mg / L / h) when the PCP concentration was 2000ppm. .
실시예 5. 본 발명 균주를 사용한 슬러리 상(Slurry phase) 처리Example 5 Slurry Phase Treatment Using the Strain of the Invention
실험예 1 : 본 발명의 균주를 사용한 박편형 폐목재의 처리Experimental Example 1 Treatment of Flakes Waste Wood Using Strain of the Present Invention
박편 형태의 폐목재 5g에 상기 실시예 4의 최소액체배지 65mL을 첨가하고, 본 발명의 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주 1.4 x 107/mL과 슈도모나스 푸티다 Bu34(KCTC 8719P) 2.1 x 107/mL를 접종한 다음 90-100rpm으로 진탕하면서 30℃에서 90시간 동안 배양하였다. 실험결과, 표3에서 볼 수 있듯이 90시간 배양한 후의 PCP 농도는 27(㎍/g dry wood)이었으며 이때의 세균수는 5.0 x 108(CFU/㎖) 이었다.To 5 g of flake wood, 65 mL of the minimum liquid medium of Example 4 was added, and the strain Klebsiella bacteria Bu1 (KCTC 8852P) strain 1.4 x 10 7 / mL and Pseudomonas putida Bu34 (KCTC 8719P) of the present invention were added. Inoculated with 2.1 x 10 7 / mL and incubated for 90 hours at 30 ℃ shaking at 90-100rpm. As a result, as shown in Table 3, the concentration of PCP after incubation for 90 hours was 27 (㎍ / g dry wood) and the bacterial count was 5.0 x 10 8 (CFU / mL).
실험예 2. 본 발명의 균주를 사용한 톱밥형 폐목재 처리Experimental Example 2 Sawdust Waste Wood Treatment Using Strain of the Present Invention
상기 실험예 1과 동일한 방법으로 폐목재를 처리하되, 박편 형태 대신에 톱밥 형태의 폐목재를 사용하여 PCP 농도와 미생물수의 변화를 측정하였다. 실험결과, 하기 표4와 같이 90시간 배양후의 PCP 농도는 27(㎍/g dry wood), 세균수는 5.0x108(CFU/mL) 이었다.Waste wood was treated in the same manner as in Experimental Example 1, but the change in PCP concentration and the number of microorganisms were measured using waste wood in the form of sawdust instead of flakes. As a result, as shown in Table 4, after 90 hours of incubation, the concentration of PCP was 27 (µg / g dry wood) and the number of bacteria was 5.0x10 8 (CFU / mL).
이상에서, PCP를 분해하는 본 발명의 균주는 90시간 동안 톱밥형 폐목재의 경우 98%, 박편형 폐목재의 경우 99%의 PCP를 각각 분해하였다. 그러나 본 발명의 균주를 접종하지 않은 대조군에서는 PCP가 전혀 감소하지 않았다.In the above, the strain of the present invention decomposing PCP decomposed PCP of the sawdust-type waste wood 98%, 99% of flaky waste wood, respectively for 90 hours. However, in the control group not inoculated with the strain of the present invention, no PCP was reduced.
실시예 6. 본 발명의 균주를 이용한 Composting 처리Example 6 Composting Treatment Using Strains of the Invention
실험예 1 : 클레브시엘라속 세균 Bu1 균주를 사용한 톱밥형 폐목재 처리Experimental Example 1 Sawdust Waste Wood Treatment Using Klebsiella Bacteria Bu1 Strain
톱밥 형태의 폐목재 200g에 상기 실시예 4의 최소액체배지 250mL을 첨가하고, 본 발명의 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주 4.8 x 108/g wood를 접종한 다음, 0-25℃ 온도하에 composting 처리법(McBain et al., Biodegradation, 6, 47-55, 1995)으로 200일간 처리하였다. 실험결과, 하기 표5와 같이, 최고 200일 동안 배양시 PCP의 농도는 150(㎍/g wood), 세균수는 1.0 x 1010(CFU/mL) 였다.200 ml of sawdust-type waste wood was added 250 mL of the minimum liquid medium of Example 4, and then inoculated with 4.8 x 10 8 / g wood of the genus Klebsiella bacterium Bu1 (KCTC 8852P) of the present invention, followed by 0-25 Treatment was carried out for 200 days using a composting method (McBain et al., Biodegradation, 6, 47-55, 1995) under the temperature of ℃. As a result, as shown in Table 5, the concentration of PCP was 150 (㎍ / g wood), the bacterial count was 1.0 x 10 10 (CFU / mL) when incubated for up to 200 days.
비교실험예 1 : 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주에 슈도모나스 푸티다 Bu34(KCTC 8719P)의 추가 접종에 따른 톱밥형 폐목재 처리Comparative Experimental Example 1: Sawdust-type Waste Wood Treatment by Additional Inoculation of Pseudomonas Putida Bu34 (KCTC 8719P) to Klebsiella Bacteria Bu1 (KCTC 8852P) Strain
클레브시엘라속 세균 Bul(KCTC 8853P) 균주에 슈도모나스 푸티다 Bu34(KCTC 8719P) 균주 6.5 x 106/g wood을 추가접종하고 상기 실험예 1과 동일한 방법으로 실험을 수행하여 톱밥형태의 폐목재를 처리하였다. 실험결과, 하기 표 6과 같이 최고 200일 동안 배양시 PCP 농도는 145(㎍/g wood), 미생물의 수는 3.6 x 109(CFU/g wood) 였다.Klebsiella genus Bul (KCTC 8853P) strains were further inoculated with Pseudomonas putida Bu34 (KCTC 8719P) strain 6.5 x 10 6 / g wood and the experiment was carried out in the same manner as in Experimental Example 1 sawdust type waste wood Was treated. As a result, as shown in Table 6, the PCP concentration was 145 (μg / g wood) and the number of microorganisms was 3.6 x 10 9 (CFU / g wood) when cultured for up to 200 days.
상기의 실시예 및 실험예를 통하여 알 수 있는 바와 같이 톱밥형태의 폐목재에 본 발명 클레브시엘라속 세균 Bu1(KCTC 8852P) 균주를 접종하여 200일간 처리한 경우 PCP 분해효율은 86%이며 슈도모나스 푸티다 Bu34(KCTC 8719P) 균주를 추가로 접종한 경우 PCP 분해효율은 87% 였다. 따라서, 본 발명의 클레브시엘라속 세균 Bul(KCTC 8852P) 균주는 성장속도가 빠르고 고농도의 PCP를 빠른속도로 분해함을 알 수 있다.As can be seen from the examples and experimental examples described above, the inoculation of the present invention, Clebsiella bacteria Bu1 (KCTC 8852P) strain inoculated sawdust-type waste wood 200 days treatment PCP degradation efficiency is 86% and Pseudomonas The additional inoculation of Putida Bu34 (KCTC 8719P) strain was 87% PCP degradation efficiency. Therefore, it can be seen that the Klebsiella genus Bul (KCTC 8852P) strain of the present invention has fast growth rate and rapidly degrades high concentrations of PCP.
본 발명은 상기의 실시예 및 실험예를 통하여 알 수 있는 바와같이, 고농도의 염소화 페놀화합물을 분해하는 신균주 클레브시엘라속 세균 Bu1(KCTC 8852P)균주를 토양에서 분리 제공하는 효과가 있다. 또한 본 발명의 신규한 클레브시엘라속 세균 Bu1(KCTC 8852P)균주를 이용하여 염소화된 페놀화합물, 특히 고농도의 PCP로 오염된 폐목재를 빠른시간내에 분해하여 환경오염을 방지할 수 있는 효과가 있으므로 산업미생물 및 환경보전산업상 매우 유용한 발명인 것이다.As can be seen from the above examples and experimental examples, there is an effect of separating and providing a new strain Klebsiella bacteria Bu1 (KCTC 8852P) strain in soil to decompose high concentration chlorinated phenolic compounds. In addition, by using the novel Klebsiella bacteria Bu1 (KCTC 8852P) strain of the present invention, it is possible to quickly decompose waste wood contaminated with chlorinated phenolic compounds, especially high-density PCP, to prevent environmental pollution. Therefore, it is a very useful invention for industrial microorganisms and environmental conservation industry.
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