KR100350926B1 - A novel microorganism excellently decomposing ethyleneglycol - Google Patents
A novel microorganism excellently decomposing ethyleneglycol Download PDFInfo
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- KR100350926B1 KR100350926B1 KR1020000021577A KR20000021577A KR100350926B1 KR 100350926 B1 KR100350926 B1 KR 100350926B1 KR 1020000021577 A KR1020000021577 A KR 1020000021577A KR 20000021577 A KR20000021577 A KR 20000021577A KR 100350926 B1 KR100350926 B1 KR 100350926B1
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- ethylene glycol
- microorganism
- pseudomonas putida
- wastewater
- microorganisms
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 title claims abstract description 172
- 244000005700 microbiome Species 0.000 title claims abstract description 48
- 229940093476 ethylene glycol Drugs 0.000 title description 50
- 241000589776 Pseudomonas putida Species 0.000 claims abstract description 25
- 239000002351 wastewater Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000000354 decomposition reaction Methods 0.000 claims description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 9
- 238000004065 wastewater treatment Methods 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 5
- 235000011009 potassium phosphates Nutrition 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 235000002639 sodium chloride Nutrition 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 230000015556 catabolic process Effects 0.000 claims description 4
- 238000006731 degradation reaction Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 3
- 239000011609 ammonium molybdate Substances 0.000 claims description 3
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 3
- 229940010552 ammonium molybdate Drugs 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 3
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 239000002609 medium Substances 0.000 description 17
- 229920000728 polyester Polymers 0.000 description 9
- 230000008569 process Effects 0.000 description 8
- 230000004580 weight loss Effects 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 239000004753 textile Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 229910052697 platinum Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000006391 Luria-Bertani Medium Substances 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 241000123447 Solicoccozyma terreus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- SRBFZHDQGSBBOR-IOVATXLUSA-N Xylose Natural products O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- KKEYFWRCBNTPAC-UHFFFAOYSA-L terephthalate(2-) Chemical compound [O-]C(=O)C1=CC=C(C([O-])=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-L 0.000 description 1
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/40—Pseudomonas putida
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
본 발명은 에틸렌글리콜의 분해 능력이 우수한 신규한 미생물 슈도모나스 푸티다 씨제이-이지 에이(Pseudomonas putidaCJ-EG a)(기탁번호 제KFCC-11129호) 및 이것을 이용한 에틸렌글리콜이 함유된 폐수의 처리방법을 제공한다.The present invention relates to a novel microorganism Pseudomonas putida CJ-EG a (Accession No. KFCC-11129) and a method for treating wastewater containing ethylene glycol using the same. to provide.
Description
본 발명은 에틸렌글리콜 분해 능력을 지닌 미생물 슈도모나스 푸티다 씨제이-이지 에이(Pseudomonas putidaCJ-EG a) 및 이것을 이용한 폐수처리방법에 관한 것으로서, 보다 상세하게는 호기성 조건하에서 생육이 양호하고 탁월한 에틸렌글리콜 분해능력을 지닌 미생물, 슈도모나스 푸티다 씨제이-이지 에이(Pseudomonas putidaCJ-EG a) 및 이것을 이용하여 에틸렌글리콜이 함유된 폐수를 생물학적으로 처리하는 방법에 관한 것이다.The present invention relates to a microorganism Pseudomonas putida CJ-EG a having an ethylene glycol decomposing ability and a wastewater treatment method using the same, and more particularly, to excellent growth and excellent ethylene glycol decomposition under aerobic conditions. A microorganism with the capacity, Pseudomonas putida CJ-EG a, and a method for biologically treating wastewater containing ethylene glycol using the same.
현재 각종 산업폐수에는 통상의 유기물질 뿐만 아니라 분해가 어려운 난분해성 화합물도 포함되어 있으며, 이러한 난분해성 화합물 중에서도 에틸렌글리콜 화합물은 폴리에스터 감량가공 공정에서 다량으로 이용, 방출되고 있는 물질로서, 활성 오니 공정에서는 처리효율이 낮으며 자연분해가 어려워 환경오염의 대표적 원인물질로 대두되고 있다.Various industrial wastewaters include not only ordinary organic materials but also hardly decomposable compounds that are difficult to decompose. Among these hardly decomposable compounds, ethylene glycol compounds are used and released in a large amount in a polyester weight loss process. In Esau, its treatment efficiency is low and it is difficult to decompose naturally.
그 동안 국내외적으로 미생물을 이용한 생물공학적 처리방법에 대한 다양한 연구가 진행되어 왔다. 즉, 특정한 방향족 화합물을 분해하는 능력을 지닌 균주를 선별하고 이용하는 것으로서 벤젠 분해방법(Eric 등, Appl. Environ. Microbiol., Vol. 55, p.3221, 1989), 슈도모나스 푸티다(Pseudomonas putida)와 크립토코커스 엘리노비(Cryptococcus elinovii)를 혼합배양한 후 활성탄에 고정화시켜서 페놀을 분해하는 방법(Rehm 등, Appl. Microbiol. Biotechnol.,Vol. 26, p.283, 1987), 및 슈도모나스 속을 이용한 나프탈렌 분해방법(Kobayashi 등, Environ. Sci. Technol., Vol. 16, p.170, 1982) 등과 같이 미생물을 이용하여 실제 산업현장의 폐수처리에 응용하려는 연구가 다각도로 진행되었다. 그러나 아직은 일부 국한된 난분해성 물질에 대해서만 분해능력을 가진 미생물이 개발되고 있어서 그 범위가 한정적이며, 실제 폐수에서의 분해효율 및 안정성이 미흡하기 때문에 실제 폐수에서도 그 분해효능이 효과적인 것으로 확인된 미생물의 개발이 긴요한 실정이다.Various researches on biotechnological treatment methods using microorganisms have been conducted at home and abroad. In other words, by selecting and using strains capable of degrading specific aromatic compounds, benzene decomposition methods (Eric et al., Appl. Environ. Microbiol., Vol. 55, p.3221, 1989), Pseudomonas putida and Cryptococcus elinovii is mixed culture and immobilized on activated carbon to decompose phenol (Rehm et al., Appl. Microbiol. Biotechnol. , Vol. 26, p. 283, 1987), and naphthalene using Pseudomonas genus. Various researches have been conducted to apply the microorganisms to the actual industrial wastewater treatment using decomposition methods (Kobayashi et al., Environ. Sci. Technol., Vol. 16, p. 170, 1982). However, microorganisms with degradability have been developed for only a limited amount of hardly decomposable materials, and the scope thereof is limited, and since the degradability and stability in actual wastewater are insufficient, the development of microorganisms proved effective in actual wastewater. This is a critical situation.
따라서, 본 발명의 목적은 호기성 조건하에 생육이 양호하고 에틸렌글리콜 및 이를 함유하는 수용액으로부터 에틸렌글리콜 성분을 효과적으로 분해할 수 있는 신규의 미생물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a novel microorganism which can grow well under aerobic conditions and can effectively decompose ethylene glycol components from ethylene glycol and an aqueous solution containing the same.
본 발명의 다른 목적은 상기 미생물을 이용하여 에틸렌글리콜이 함유된 폐수를 생물학적으로 처리하는 효과적인 방법을 제공하는 것이다.Another object of the present invention is to provide an effective method for biologically treating wastewater containing ethylene glycol using the microorganism.
폴리에스터 감량공정에 따르면, 일반적으로 110℃, 5% NaOH 수용액에 폴리에스터 섬유를 투입하여 반응시키면 테레프탈레이트와 에텔렌글리콜이 가수분해된다. 이때 감량 비율은 공정에 따라 20 내지 30% 정도이고 손실된 수산화나트륨을 보충하면서 10 내지 30회 정도 감량을 수행하여 폴리에스터 올리고머의 침전물이 생기면 전량 배출하게 된다. 가수분해된 감량폐액은 COD(Mn)가 20,000ppm 이상이고, BOD가 100,000ppm 정도이며, 때에 따라 5배 정도의 변화가 있는 고농도 유기성 폐수이다.According to the polyester weight loss process, terephthalate and ethylene glycol are hydrolyzed when polyester fiber is added to a 5% NaOH aqueous solution at 110 ° C. At this time, the reduction ratio is about 20 to 30% depending on the process, and the loss is performed 10 to 30 times while replenishing the lost sodium hydroxide, the total amount is discharged when a precipitate of the polyester oligomer is produced. The hydrolyzed weight loss wastewater is a high concentration organic wastewater having a COD (Mn) of 20,000 ppm or more, a BOD of about 100,000 ppm, and sometimes a change of about 5 times.
그러나, 에틸렌글리콜의 미생물적인 분해에 대한 연구는 거의 진행되지 않고 있다. 에틸렌글리콜은 활성 오니 공정에서는 처리효율이 낮으며 전체적인 폐수처리 효율을 낮추고 운전 불안의 원인이 되므로 신속히 처리할 수 있는 미생물의 개발이 시급하다. 따라서, 에틸렌글리콜을 분해하는 미생물을 선별하여 폴리에스터 감량 폐수의 처리에 응용하는 것이 필요하며 이러한 물질을 분해하는 미생물을 선택, 분리하기 위해서는 분리원의 선택과 대상 물질을 선정하는 것이 중요하다고 할 수 있다.However, little research has been conducted on the microbial degradation of ethylene glycol. Ethylene glycol has low treatment efficiency in active sludge process, and it is urgent to develop microorganisms that can be treated quickly because it lowers the overall wastewater treatment efficiency and causes driving anxiety. Therefore, it is necessary to select microorganisms that decompose ethylene glycol and apply them to the treatment of polyester weight loss wastewater.In order to select and isolate microorganisms that decompose such substances, it is important to select a source of separation and select a target material. have.
본 발명자들은 섬유공장의 폴리에스터 감량공정에서 배출되는 폐수로부터 에틸렌글리콜을 제거하는 능력이 우수한 미생물을 분리하는 시험을 하여 슈도모나스 푸티다 씨제이-이지 에이로 동정하였고, 이 미생물이 우수한 에틸렌글리콜분해 능력을 지닌 것으로 판명되어 본 발명을 완성하였다.The inventors of the present invention identified Pseudomonas putida CJ-E.A as a test for separating microorganisms having excellent ability to remove ethylene glycol from wastewater discharged from a polyester weight loss process of a textile mill, and the microorganisms showed excellent ethylene glycol decomposition ability. It turned out to have been completed the present invention.
도 1은 본 발명에 따라 순수 분리한 미생물 슈도모나스 푸티다 씨제이-이지 에이(Pseudomonas putidaCJ-EG a)에 대한 에틸렌글리콜 함유 최소배지에서의 성장을 도시한 그래프이다.1 is a graph showing growth in ethylene glycol-containing minimal medium for Pseudomonas putida CJ-EG a purely isolated microorganism Pseudomonas putida CJ-EG a according to the present invention.
도 2는 본 발명에 따른 상기 미생물 슈도모나스 푸티다 씨제이-이지 에이가 에틸렌글리콜 함유 최소배지에서 에틸렌글리콜을 제거하는 경향을 나타낸 그래프이다.Figure 2 is a graph showing a tendency to remove ethylene glycol in the microorganism Pseudomonas putida CJ-EJ A ethylene glycol containing minimal medium according to the present invention.
본 발명에 따르면, 호기성 조건에서 생육이 양호하며 에틸렌글리콜 분해능이 개선된 신규 미생물 슈도모나스 푸티다 씨제이-이지 에이(Pseudomonas putidaCJ-EG a)(기탁번호 제KFCC-11129호)가 제공된다.According to the present invention, there is provided a novel microorganism Pseudomonas putida CJ-EG a (Accession No. KFCC-11129) with good growth under aerobic conditions and improved ethylene glycol degradation.
상기 미생물은, 본 발명에 따라 염화나트륨, 황산암모늄, 인산 제 1 칼륨, 인산 제 2칼륨, 황산마그네슘, 황산 제 2철, 염화칼슘, 암모늄몰리브데이트, 황산망간을 기본으로 하고 유일한 탄소원으로서 에틸렌글리콜을 함유하는 에틸렌글리콜 함유 최소배지에서 에틸렌글리콜의 분해능력을 가진 미생물로서 선별된 것이다.According to the present invention, the microorganism is based on sodium chloride, ammonium sulfate, potassium phosphate, potassium phosphate, magnesium sulfate, ferric sulfate, calcium chloride, ammonium molybdate, manganese sulfate, and ethylene glycol as the only carbon source. It is selected as a microorganism having decomposing ability of ethylene glycol in the containing medium containing ethylene glycol.
또한, 본 발명의 다른 일면에 따르면, 본 발명에 따른 상기 미생물을 사용하여 에틸렌글리콜이 함유된 합성폐수를 처리하는 방법이 제공된다. 본 발명의 상기 방법을 적용하기에 적합한 폐수로서는 에틸렌글리콜이 1000ppm 이하로 함유된 것이 바람직하다.Further, according to another aspect of the present invention, there is provided a method for treating synthetic wastewater containing ethylene glycol using the microorganism according to the present invention. As wastewater suitable for applying the above method of the present invention, ethylene glycol is preferably contained at 1000 ppm or less.
보다 상세하게 설명하자면, 본 발명은 에틸렌글리콜 분해 능력이 우수한 슈도모나스 푸티다 씨제이-이지 에이 (Pseudomonas putidaCJ-EG a) 미생물에 관한 것이며, 이 신규한 미생물은 직접 폴리에스터 감량공정에서 배출되는 폐수에서 엄밀히 분리, 선별한 균주로서 제 3자의 분양을 위해 2000년 1월 29일자로 대한민국 서울시 서대문구 홍제동 소재의 한국종균협회(KFCC)에 기탁번호 제KFCC-11129호로 기탁, 보관되어 있다.More specifically, the present invention relates to a Pseudomonas putida CJ-EG a microorganism having excellent ethylene glycol degrading ability, and the novel microorganism is directly discharged from wastewater discharged from a polyester weight loss process. It is strictly isolated and screened as a deposit No. KFCC-11129 with the Korean Bacillus Association (KFCC) in Hongje-dong, Seodaemun-gu, Seoul, Korea on January 29, 2000.
본 발명자들은 대구지역의 섬유, 염색공장의 폴리에스터 감량공정의 폐수를채취하여 균주분리용 시료로 사용하여 미생물들을 순수분리 하였다. 상기 분리한 미생물들을 에틸렌글리콜을 유일한 탄소원으로 함유하는 최소배지를 사용하여 일정기간 순응시켜 적응시킨 후, 에틸렌글리콜을 함유한 고체배지에서 우수한 성장을 보인 단일 군락의 미생물들을 1차 선별하였다. 각각의 미생물들을 다시 에틸렌글리콜이 함유된 액체 최소배지에서 배양할 때 우수한 생육을 보이는 미생물을 최종 선별하였으며, 각종 시험을 거쳐 슈도모나스 푸티다 씨제이-이지 에이로 동정하였다. 또한, 선별된 미생물들은 우수한 에틸렌글리콜의 분해능력을 지닌 것으로 판명되었다.The present inventors collected the waste water of the polyester weight loss process of the textile, dyeing factory in Daegu area and used as a sample for strain separation to separate the microorganisms pure. After the isolated microorganisms were acclimated for a certain period of time using a minimum medium containing ethylene glycol as the only carbon source, a single colony of microorganisms showing excellent growth in the solid medium containing ethylene glycol was first selected. Each microorganism was finally screened for microorganisms showing excellent growth when incubated in a liquid minimal medium containing ethylene glycol and identified as Pseudomonas putida CJ-E. In addition, the selected microorganisms were found to have excellent decomposition ability of ethylene glycol.
상기에서 설명한 바와 같이, 에틸렌글리콜의 분해는 활성 오니에서는 제한되어 있으므로 에틸렌글리콜의 분해능력이 우수한 미생물을 선별하는 것은 중요한 의미를 갖는다.As described above, since the decomposition of ethylene glycol is limited in active sludge, it is important to select microorganisms having excellent decomposition ability of ethylene glycol.
본 발명에 따른 신규 미생물의 성상은 다음과 같다.The characteristics of the novel microorganism according to the present invention are as follows.
슈도모나스 푸티다 씨제이-이지 에이는 복합 배지상에서 둥글고 볼록한 외견의 콜로니를 형성하며 이의 특성은 표 1, 표 2, 표 3에 제시되었다.Pseudomonas putida CJ-E. A. forms round and convex outward colonies on the composite medium and its properties are shown in Tables 1, 2, and 3.
+: 양성, -: 음성+: Positive,-: negative
+: 양성, -: 음성+: Positive,-: negative
+: 양성, -: 음성+: Positive,-: negative
이상과 같이 에틸렌글리콜 분해 능력이 우수한 슈도모나스 푸티다 씨제이-이지 에이 미생물이 최종 선별되었다.As described above, Pseudomonas putida CJ-EJ microorganisms excellent in ethylene glycol decomposition ability were finally selected.
다음의 실시예에서 본 발명을 더욱 구체적으로 설명할 것인 바, 본 발명이 이들 실시예에 의해 제한되는 것은 아니다.The present invention will be described in more detail in the following examples, which should not be construed as limiting the invention thereto.
실시예 1Example 1
균주의 분리Isolation of Strains
에틸렌글리콜의 분해 능력과 생육이 우수한 미생물을 분리하고자, 경상도 지역 섬유공장의 폐수처리장에서 폐수 및 폐수가 유출되는 하천 주위의 물과 토양을 채취하여 일정기간 순응시킨 후, 순응된 미생물을 멸균 식염수에 현탁하여 루리아-버타니(Luria-Bertani) 배지(LB배지; 트립톤 0.1g, 효모 추출물 0.05g, 염화나트륨 0.05g, 포도당 0.01g 및 한천 0.2g)를 상기 폐수 혼합액(0.2㎛ 무균필터로 여과) 1리터에 녹여 만든 평판배지에 도말하였다.In order to separate the microorganisms having excellent decomposition ability and growth of ethylene glycol, water and soil around the stream where the wastewater and wastewater flows out from the wastewater treatment plant of the Gyeongsang-do textile factory are acclimated for a certain period of time, and then the acclimated microorganisms are sterilized in sterile saline. Suspended Luria-Bertani medium (LB medium; tryptone 0.1g, yeast extract 0.05g, sodium chloride 0.05g, glucose 0.01g and agar 0.2g) was mixed with the wastewater mixture (filtered with a 0.2㎛ sterile filter) It was plated on a flat plate made by melting in 1 liter.
도말한 평판 배지를 25 내지 30℃의 배양기에서 1 내지 3일간 배양하여 평판 배지상에서 우수한 성장성을 보이는 20여종의 단일군락 미생물들을 분리하였다.The plated plate medium was incubated for 1 to 3 days in an incubator at 25 to 30 ° C. to isolate 20 kinds of single colony microorganisms showing excellent growth on plate medium.
분리한 미생물들에 대한 에틸렌글리콜 분해능력을 측정하기 위하여 에틸렌글리콜 함유 최소배지(에틸렌글리콜 500mg 내지 1000mg, 염화나트륨 0.1g, 황산암모늄 2g, 인산 제 1 칼륨 4.5g, 인산 제 2 칼륨 5g, 황산마그네슘 0.2g, 황산 제 2철 0.02g, 염화칼슘 0.02g, 암모늄몰리브데이트 0.002g 및 황산망간 0.01g을 각각 1리터에 녹여 만든 액체배지)에 본 발명의 미생물을 각각 한 백금이씩 접종한 후 일정기간 순응시켰다. 그 후, 성장이 이루어졌을때 한천을 포함한 상기 조성의 에틸렌글리콜 함유 최소배지에 도말하여 단일 군락을 보인 10여종을 1차 선별하였다.Minimum medium containing ethylene glycol (ethylene glycol 500mg to 1000mg, sodium chloride 0.1g, ammonium sulfate 2g, potassium first potassium phosphate 4.5g, potassium diphosphate 5g, magnesium sulfate 0.2) g, ferric sulfate 0.02 g, calcium chloride 0.02 g, ammonium molybdate 0.002 g and 0.01 g of manganese sulfate in a liquid medium), each of which was inoculated with one platinum each of the microorganisms of the present invention for a certain period of time. Acclimation. Thereafter, when growth was made, 10 kinds of single colonies showing a single colony were first screened by spreading on a minimal medium containing ethylene glycol containing the agar.
한편 상기 1차 선별한 미생물을 상기 조성의 5ml 에틸렌글리콜이 함유된 최소배지가 들어 있는 각 배양 시험관에 한 백금이씩 접종한 후, 진탕회전 배양기에서 25 내지 30℃로 24시간동안 배양하고 그 배양액을 원심분리기(Vision, VS15000CFN)에서 5분간 원심분리하여 균체를 회수하였다. 회수한 미생물을 일부는 다음의 계속적인 실험에 사용하고 일부는 동결건조하여 보관하였다.Meanwhile, after inoculating the first selected microorganisms into each culture tube containing a minimum medium containing 5 ml ethylene glycol of the composition, each platinum was inoculated, and then cultured at 25 to 30 ° C. for 24 hours in a shaking rotation incubator. Cells were recovered by centrifugation for 5 minutes in a centrifuge (Vision, VS15000CFN). Some of the recovered microorganisms were used in subsequent experiments and some were lyophilized and stored.
실시예 2Example 2
에틸렌글리콜 분해능력을 지닌 균주의 선별 및 에틸렌글리콜 제거능력 확인Selection of strains with ethylene glycol degradation ability and confirmation of ethylene glycol removal ability
상기의 1차 선별된 10종의 미생물들을 멸균증류수에 현탁한 후, 에틸렌글리콜을 500ppm으로 조정한 에틸렌글리콜함유 최소배지에 대하여 각 미생물을 1%(v/v)씩 접종하고, 진탕회전 배양기에서 25 내지 30℃로 배양하였다. 이후 에틸렌글리콜을 유일한 탄소원으로 이용하는 능력이 우수한 균주를 선별하기 위하여 생육도를 비교 관찰하였다.After suspending the first 10 selected microorganisms in sterile distilled water, each microorganism was inoculated by 1% (v / v) to the ethylene glycol-containing minimum medium adjusted to 500 ppm of ethylene glycol, and in a shaker incubator. Incubated at 25-30 ° C. Since growth rate was compared to select strains excellent in the ability to use ethylene glycol as the only carbon source.
에틸렌글리콜을 500ppm으로 조정한 에틸렌글리콜 함유 최소배지에 대하여 48시간 생육을 관찰한 결과, 도 1에 나타낸 것과 같이 슈도모나스 푸티다 씨제이-이지 에이로 동정된 미생물이 기타 미생물에 비하여 생육이 우수하여 최종 우수균주로 선별하였다.As a result of observing the growth for 48 hours on the ethylene glycol-containing minimum medium adjusted to 500 ppm of ethylene glycol, as shown in Fig. 1, the microorganisms identified as Pseudomonas putida CJ-E. Strains were selected.
실시예 3Example 3
에틸렌글리콜 분해 균주에 의한 에틸렌글리콜 함유 폐수의 처리결과 확인Confirmation of treatment result of ethylene glycol-containing wastewater by ethylene glycol decomposition strain
또한, 선별된 슈도모나스 푸티다 씨제이-이지 에이 미생물을 이용하여, 에틸렌글리콜의 농도를 1000ppm으로 조정한 에틸렌글리콜 함유 합성폐수에 대하여 에틸렌글리콜의 제거경향을 실험하였다. 즉, 진탕회전 배양기에서 25 내지 30℃로 배양하면서 일정량을 무균적으로 채취하여 적당량 희석한 후 원심분리하고, 그 상등액에 함유된 에틸렌글리콜의 농도를 측정하였다. 그 결과는 도 2와 같으며 1000ppm의 에틸렌글리콜 함유 최소배지에서 본 발명의 미생물 슈도모나스 푸티다 씨제이-이지 에이에 의한 에틸렌글리콜의 제거경향을 알 수 있다.In addition, the removal tendency of ethylene glycol was examined for the synthetic wastewater containing ethylene glycol adjusted to the concentration of 1000 ppm using the selected Pseudomonas putida CJ-E. That is, while culturing at 25 to 30 ℃ in a shaker incubator, a predetermined amount was aseptically collected, diluted appropriately, centrifuged, and the concentration of ethylene glycol contained in the supernatant was measured. The results are shown in Figure 2 and the removal tendency of ethylene glycol by the microorganism Pseudomonas putida CJ-EJ of the present invention in a minimum medium containing ethylene glycol of 1000ppm.
섬유공장의 폴리에스터 감량공정의 폐수처리장에서 분리, 선별된 에틸렌글리콜의 분해가 우수한 신규의 슈도모나스 푸티다 씨제이-이지 에이 미생물은 실제 현장에서 폐수처리에 이용이 가능하며 이 미생물을 이용한 환경처리제의 개발 및 다양한 생물학적 환경정화기술에 응용할 수 있을 것으로 기대된다.The new Pseudomonas putida CJ-E.A microorganism, which is excellent in the decomposition of ethylene glycol selected and separated in the wastewater treatment plant of the polyester weight loss process of a textile factory, can be used for wastewater treatment in the field and the development of an environmental treatment agent using the microorganism. And it is expected to be applicable to various biological environmental purification techniques.
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