KR100333440B1 - Strain for disintegrating gasoline Pseudomonas sp. HPLG-1 - Google Patents
Strain for disintegrating gasoline Pseudomonas sp. HPLG-1 Download PDFInfo
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- 239000003502 gasoline Substances 0.000 title claims abstract description 70
- 241000589774 Pseudomonas sp. Species 0.000 title 1
- 241000609869 Pseudomonas syringae pv. maculicola Species 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims 1
- 238000000354 decomposition reaction Methods 0.000 abstract description 13
- 239000003440 toxic substance Substances 0.000 abstract description 7
- 231100000614 poison Toxicity 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 239000003921 oil Substances 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 239000002689 soil Substances 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 239000011707 mineral Substances 0.000 description 7
- 235000010755 mineral Nutrition 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 241000589516 Pseudomonas Species 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- YNQLUTRBYVCPMQ-UHFFFAOYSA-N Ethylbenzene Chemical compound CCC1=CC=CC=C1 YNQLUTRBYVCPMQ-UHFFFAOYSA-N 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004710 electron pair approximation Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000009931 harmful effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- NNPPMTNAJDCUHE-UHFFFAOYSA-N isobutane Chemical compound CC(C)C NNPPMTNAJDCUHE-UHFFFAOYSA-N 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000007003 mineral medium Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 239000012855 volatile organic compound Substances 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- PAAZPARNPHGIKF-UHFFFAOYSA-N 1,2-dibromoethane Chemical compound BrCCBr PAAZPARNPHGIKF-UHFFFAOYSA-N 0.000 description 1
- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical compound CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101100283604 Caenorhabditis elegans pigk-1 gene Proteins 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000809 air pollutant Substances 0.000 description 1
- 231100001243 air pollutant Toxicity 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- -1 etc. Chemical compound 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000001282 iso-butane Substances 0.000 description 1
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000037323 metabolic rate Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- ODLMAHJVESYWTB-UHFFFAOYSA-N propylbenzene Chemical class CCCC1=CC=CC=C1 ODLMAHJVESYWTB-UHFFFAOYSA-N 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003168 reconstitution method Methods 0.000 description 1
- 239000003802 soil pollutant Substances 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000003403 water pollutant Substances 0.000 description 1
Classifications
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- 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
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- 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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- 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)
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- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
본 발명은 가솔린 분해균주 슈도모나스 마큘리콜라 HPLG-1(기탁번호: KCTC 0714 BP)에 관한 것으로, 각종 오염 장소에서 균주들을 분리하여, 가솔린 분해능을 많이 갖는 균주를 선별하였고, 또한, 가솔린 중 휘발성이 높은 비휘발된 가솔린과 유독성분이 많이 함유된 50% 휘발된 가솔린을 분해하는 효과를 살펴본 후, 다양한 환경조건에 따른 생장연구를 수행하였다. 그 결과, 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1은 가솔린에 의해 오염된 지역에서 기름의 휘발율을 낮추는데 효과가 있으며, 휘발된 후 독성성분이 많이 포함된 조건에서 기름의 분해도가 높은 특징이 있다.The present invention relates to a gasoline decomposing strain Pseudomonas maculicola HPLG-1 (Accession Number: KCTC 0714 BP), by separating the strains at various contaminated sites, to select a strain having a lot of gasoline resolution, and also has a high volatility in gasoline After examining the effects of decomposing non-volatile gasoline and 50% volatile gasoline containing a lot of toxic substances, growth studies were conducted according to various environmental conditions. As a result, Pseudomonas maculicola HPLG-1 according to the present invention is effective in lowering the volatilization rate of oil in the region contaminated by gasoline, and has a characteristic of high decomposition degree of oil under conditions containing a lot of toxic components after volatilization. .
Description
본 발명은 가솔린 분해균주 슈도모나스 마큘리콜라 HPLG-1에 관한 것으로,보다 상세하게는 가솔린의 휘발율을 낮추는데 효과적이며, 독성이 많은 휘발된 가솔린에 대해서 분해능이 뛰어난 슈도모나스 마큘리콜라 HPLG-1에 관한 것이다.The present invention relates to a gasoline decomposition strain Pseudomonas maculocola HPLG-1, and more particularly to lowering the volatilization rate of gasoline, Pseudomonas maculocola HPLG-1 excellent in resolution for toxic gasoline that is toxic.
가솔린의 성분은 휘발성분인 VOC(Volatile Organic Compounds: 휘발성 유기화합물질)로 구성되어 있으며, 이 가운데에서도 증기압(vapor pressure: VP)이 100∼102atm로 상대적으로 높은 물질인 프로필렌, 이소부탄, n-부탄, 메틸부탄 등과, 증기압이 10-3∼100atm로 상대적으로 낮은 물질인 n-펜탄, 1-펜탄, 사이클로펜타, 사이클로헥산, 벤젠, 1,2-디브로모메탄(1,2-dibromomethane), 에틸벤젠, 자일렌, 프로필벤젠, 옥탄, 디메틸부탄 등이 섞여 있어서, 휘발성이 매우 강하며, 독성이 강한 물질인 BTEX성분이 다량 함유되어 있는 물질이다. 따라서, 미 환경보호국(U.S. EPA)에서는 가솔린 및 가솔린에 포함된 성분을 일반적으로 대기오염물질로 분류하고 있으며, 이와 동시에 지표수 및 토양의 주된 오염원으로서 단지 몇 리터의 유출로도 수백만 리터의 수자원을 오염시킬 수 있음을 경고하고 있다.Gasoline is composed of volatile VOC (Volatile Organic Compounds), among which propylene and isobutane are relatively high vapor pressures (VP) of 10 0 to 10 2 atm. , n-butane, methylbutane, etc., n-pentane, 1-pentane, cyclopenta, cyclohexane, benzene, 1,2-dibromomethane, which have relatively low vapor pressures of 10 −3 to 10 0 atm. , 2-dibromomethane), ethylbenzene, xylene, propylbenzenes, octane, dimethylbutane, etc., which is very volatile and contains a large amount of BTEX, a highly toxic substance. Therefore, the US Environmental Protection Agency (US EPA) generally classifies gasoline and its components as air pollutants, while polluting millions of liters of water with just a few liters of runoff as a major source of surface water and soil. It warns you that you can.
가솔린은 오염된 매체에 따라 대기오염, 수질오염, 토양오염 등으로 나눌 수 있으며 특히 휘발성분으로 인하여 인축에 독성 원인이 되는 독성물질이 많이 포함된 유독성 물질이다. 따라서, 이들 물질이 환경에 유출되어 공기 중으로 휘발되면 강한 독성을 띤 물질들이 공기 중으로 휘발되어 인축에 매우 큰 피해를 주게 되고, 이 유류가 토양에서 유출되어 수질에 녹아들어 수류를 따라 흘러가게 되면 수질오염원으로 매우 큰 피해를 주게 되어 커다란 사회적 문제를 야기시킬 수 있고 오염범위도 넓고 지하심층으로 확산된다는 점에서 오염정화를 위한 처리가 상당히 어렵고 많은 비용과 장비를 필요로 하게 된다.Gasoline can be divided into air pollution, water pollution, soil pollution, etc., depending on the polluted medium. Especially, gasoline is a toxic substance that contains a lot of toxic substances that cause poisoning due to volatile content. Therefore, when these substances are released to the environment and volatilized into the air, highly toxic substances are volatilized into the air, causing a great damage to the human beings, and this oil flows out of the soil, melts into the water, and flows along the stream. As the source of pollution can cause great social problems, and the range of pollution is wide and spreads deep underground, the treatment for pollution purification is quite difficult and requires a lot of cost and equipment.
또한, 가솔린이 토양에 오염되었을 경우, 경유와 같은 다른 유류와는 달리 공기중에서 비교적 빨리 휘발되지만 이중 빨리 휘발되는 것보다 휘발이 늦은 물질에서 비교적 유독물질이 많으므로, 높은 기름 오염지에서 이 유독물질을 빠른 시간 내에 분해할 것이 요구된다.In addition, when gasoline is contaminated with soil, it is volatilized relatively rapidly in the air unlike other oils such as diesel, but it is more toxic in late volatilized materials than in volatilized ones. It is required to decompose in a short time.
전 세계적으로 가솔린에 의한 오염은 생산, 운반, 저장, 사용 등에 의해 매우 큰 오염원으로 자연에 유출되고 있다. 이처럼 유해한 영향을 미치는 가솔린유를 정화시키는 방법으로는 물리적, 화학적, 생물학적 방법을 고려할 수 있으며 전자의 2가지 방법은 전통적으로 사용되어 왔던 반면, 미생물적인 생물학적 처리방법은 최근에 각광받기 시작하였다. 여기서 물리화학적인 방법은 토양오염물질을 근본적으로 파괴하는 것이 아니라 토양 외의 다른 매체로 옮긴 경우에 불과하며 궁극적인 해결방법은 아니다. 따라서 생물학적 처리방법을 이용한 광물화 방법이 처리비용과 환경정화 효과면에서 새로운 대안으로 각광받고 있다.Globally, pollution by gasoline is released into nature as a very large pollutant by production, transportation, storage and use. As a method of purifying gasoline oil which has such harmful effects, physical, chemical and biological methods can be considered. The former two methods have been traditionally used, whereas microbiological biological treatment methods have recently been in the spotlight. Here, the physicochemical method is not the ultimate destruction of soil pollutants, but only a case where it is transferred to a medium other than soil, and is not the ultimate solution. Therefore, mineralization method using biological treatment method has been spotlighted as a new alternative in terms of treatment cost and environmental purification effect.
따라서, 휘발이 높은 물질을 먼저 휘발시킨 후 독성이 많이 포함되어 있는 즉, BTEX 등이 포함된 물질을 남겨서 이를 매우 빨리 분해시키는 미생물 개발이 필요하다.Therefore, it is necessary to develop a microorganism which volatilizes a material having a high volatility first and then decomposes very quickly by leaving a substance containing a lot of toxicity, that is, BTEX.
이에 본 발명자들은 높은 기름오염지에서 가솔린유에 의한 유독물질을 빠른 시간내에 분해할 수 있고, 오염지에서의 휘발율을 효과적으로 낮출 수 있는 미생물을 선별하여 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by selecting microorganisms capable of decomposing toxic substances caused by gasoline oil in a high oil pollution place in a short time, and effectively lowering the volatilization rate at the contamination place.
본 발명은 가솔린 오염지역에서 가솔린의 휘발율을 낮추는데 효과적이며, 또한, 독성물질이 많이 포함된 휘발된 가솔린을 분해하는데 효과적인 슈도모나스 마큘리콜라 HPLG-1을 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a Pseudomonas maculicola HPLG-1 that is effective in reducing the volatilization rate of gasoline in a gasoline contaminated area and is also effective in decomposing volatilized gasoline containing a large amount of toxic substances.
또한, 본 발명은 상기 슈도모나스 마큘리콜라 HPLG-1가 가장 높은 활력을 가질 수 있는 최적의 생장환경 및 영양분을 제공하는 것을 그 목적으로 한다.In addition, an object of the present invention is to provide the optimum growth environment and nutrients that Pseudomonas maculocola HPLG-1 can have the highest vitality.
도 1은 상온에서 가솔린의 시간(일)에 따른 휘발량을 도시한 것이고,Figure 1 shows the volatilization according to the time (days) of gasoline at room temperature,
도 2는 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1의 질소원 변화에 따른 생장을 나타낸 그래프이고,Figure 2 is a graph showing the growth according to the nitrogen source change of Pseudomonas maculicola HPLG-1 according to the present invention,
도 3은 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1의 미네랄 영양원 감소에 따른 생장을 나타낸 그래프이고,Figure 3 is a graph showing the growth according to the reduced mineral nutrient source of Pseudomonas maculicola HPLG-1 according to the present invention,
도 4는 여러 pH에서 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1의 생장을 나타낸 그래프이고,Figure 4 is a graph showing the growth of Pseudomonas maculicola HPLG-1 according to the present invention at various pH,
도 5는 여러 완충조건의 pH에서 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1의 생장을 나타낸 그래프이다.FIG. 5 is a graph showing the growth of Pseudomonas maculicola HPLG-1 according to the present invention at pH of various buffer conditions.
상기한 바와 같은 목적을 달성하기 위한 본 발명의 특징은 다음과 같다.Features of the present invention for achieving the above object is as follows.
본 발명에 의한 균주의 분리 추출 및 재구성 방법은 가솔린의 휘발성 측정 단계, 유류에 의해서 오염된 해수 및 토양 분리단계, 균주 분리단계, 분리된 균주의 가솔린 휘발억제 및 가솔린 분해력 평가단계, 균주동정단계로 연구를 실시하였으며, 영양분에 따른 생장 검토를 통해 가솔린 분해 균주의 최적의 생장조건을 선정하였다.Separation extraction and reconstitution method of the strain according to the present invention is to measure the volatility of gasoline, seawater and soil separation step contaminated by oil, strain separation step, gasoline volatilization inhibition and gasoline decomposition ability evaluation step of the isolated strain, strain identification step The optimal growth conditions of gasoline digested strains were selected by examining growth according to nutrients.
이하에서 첨부한 도면을 참고로 하여 본 발명의 바람직한 실시예를 상세히 설명하고자 한다. 그러나, 하기한 실시예는 본 발명의 구성 및 효과를 입증하기 위한 본 발명의 일실시예일 뿐 본 발명이 하기한 실시예에 한정되는 것은 아니다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following examples are only examples of the present invention for demonstrating the constitution and effects of the present invention, and the present invention is not limited to the following examples.
먼저, 본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1의 분리 및 동정에 대해서 설명한 다음, 상기 HPLG-1의 최적의 생장조건에 대해서 설명하고자 한다.First, the separation and identification of Pseudomonas maculicola HPLG-1 according to the present invention will be described, and then the optimum growth conditions of the HPLG-1 will be described.
가솔린의 휘발성 측정 단계Volatile Measurement Steps for Gasoline
가솔린의 기름분해도를 평가하기 위하여 가솔린유를 500㎖ 실린더에 50㎖ 넣은 다음 상온에서 26일간 두고 가솔린의 휘발량을 평가하였다. 그 결과는 도 1과 같다.In order to evaluate the degree of oil decomposition of gasoline, 50 ml of gasoline oil was put in a 500 ml cylinder, and the volatilization amount of the gasoline was evaluated for 26 days at room temperature. The result is shown in FIG.
도 1에 의하면, 가솔린은 상온에서 4일 이후에 약 50% 이상 휘발됨을 알 수 있다.1, it can be seen that gasoline is volatilized about 50% or more after 4 days at room temperature.
또한, 가솔린이 토양에 오염되었을 경우에는 토양 내에 유류 중 흡착하는 물질이 존재하여 토양의 물리성에 의해 휘발이 이보다는 훨씬 늦을 것으로 판단된다. 이에 따라 톨루엔, 크실렌, 에틸벤젠, 벤젠 등은 공지되어 있는 바와 같이 증기압(vapor pressure ; VP)이 10-3∼100atm 범위의 비교적 낮은 물질이므로 휘발후에 남은 물질에는 벤젠, 톨루엔, 에틸벤젠, 크실렌이 비교적 많이 포함되어 있으리라 판단되며 이러한 물질들은 용해도가 1이상의 물질이므로 수질오염에도 심각한 문제를 야기할 수 있다.In addition, when gasoline is contaminated with soil, there is a substance adsorbed in oil in the soil, and volatilization may be much later than this due to the physical properties of the soil. Accordingly, toluene, xylene, ethylbenzene, benzene and the like are known to have relatively low vapor pressure (VP) in the range of 10 −3 to 10 0 atm. It is believed that xylene is relatively high and these substances are more than 1 solubility and can cause serious problems in water pollution.
유류에 의해서 오염된 해수 및 토양분리 단계Seawater and Soil Separation Stages Contaminated by Oil
하기의 표 1에서와 같이 폐기물 유출로 인하여 10년 이상 오랜 기간 동안 다양한 종류의 유류에 의해 오염·축적되어 있는 경기도 지역 일대의 기름오염지역에 존재하는 토양을 채취하였다. 채취한 샘플들을 비닐백 또는 플라스틱통에 투입한 후 -5∼0℃에서 보관하였다.As shown in Table 1 below, soils were collected from oil-contaminated areas around Gyeonggi-do, which were contaminated and accumulated by various types of oils for a long time due to waste spills. The collected samples were put in a plastic bag or plastic container and then stored at -5 to 0 ° C.
균주분리 단계Strain Separation Step
상기 채취된 오염시료에 가솔린과 미네랄을 첨가하여 3일간 배양한 후 다음과 같은 실험을 수행하였다. 즉, 상기 시료내의 미생물 중 미네랄원을 넣어 생장이 용이하도록 하였으며, 가솔린 분해력이 큰 미생물의 생장을 돕기 위하여 가솔린 30%를 첨가하여 실험을 수행하였다. 즉, 상기 시료내의 균주들 중 미네랄원을 넣어 생장이 용이하도록 하였으며, 가솔린분해력이 큰 균주의 농화배양을 위하여 가솔린 30%를 첨가하였다.After the incubation for 3 days by adding gasoline and minerals to the collected contaminated sample was carried out the following experiment. That is, by putting the mineral source of the microorganisms in the sample to facilitate the growth, the experiment was performed by adding 30% gasoline to help the growth of microorganisms with high gasoline decomposition ability. That is, the mineral source of the strains in the sample was added to facilitate growth, and gasoline 30% was added for the enrichment culture of the strain having high gasoline decomposition ability.
그 후, 가솔린 분해가 가능한 세균을 LA(luria bertani broth + Agar, Difco, USA)에 도말하여 순수분리하였고, 다음으로 유일한 탄소원으로 가솔린을 30% 포함하는 미네랄 배지(표 2 참조)에 분리한 세균을 30℃에서 10일간 각각 생장시킨 후 가솔린의 분해도를 검정하였다. 이들 가운데 생장이 가장 우수하며 가솔린의 분해도가 가장 높은 균주를 최종적으로 선발하여 보관하였다.After that, bacteria capable of decomposing gasoline were plated on LA (luria bertani broth + Agar, Difco, USA) and separated purely, and then bacteria separated on mineral medium containing 30% gasoline as the only carbon source (see Table 2). Were grown at 30 ° C. for 10 days and then assayed for decomposition of gasoline. Among them, the best growth and the highest decomposition rate of gasoline were finally selected and stored.
가솔린 휘발억제 및 기름분해력 평가Gasoline Volatilization Inhibition and Oil Degradation Evaluation
가솔린의 분해력은 실험구내의 미생물 대사활동에 의해 소모된 가솔린의 소모량을 측정하고, 대조구의 자연소모량을 구해 보정하여 평가하였다. 본 발명에 의한 실험구와 대조구의 내용은 다음과 같다.The degradability of gasoline was evaluated by measuring the consumption of gasoline consumed by the microbial metabolic activity in the experimental zone, and calculating the natural consumption of the control. The contents of the experimental and control groups according to the present invention are as follows.
첫째, 실험구 1은 유일한 탄소원으로 휘발하지 않은 가솔린이 30%(v/v) 함유된 20㎖의 변형된 미네랄 배지(Modified minerals broth),First, Experiment 1 was the only carbon source, 20 ml of modified minerals broth containing 30% (v / v) of unvolatile gasoline,
둘째, 실험구 2는 유일한 탄소원으로 50% 휘발한 잔여가솔린이 30%(v/v) 함유된 20㎖의 변형된 미네랄 배지.Second, Experiment 2 was a 20 ml modified mineral medium containing 30% (v / v) of residual gasoline, 50% volatilized as the only carbon source.
이러한 총 2가지의 배지를 준비한 후, LB에서 하룻밤 생장된 상기 균주를 각각 1%(v/v) 농도로 접종하였고, 상기의 2가지 배지조건에 미생물을 접종하지 않은것으로 대조구 1, 2를 설정하였다. 그런 다음 30℃±1, 150rpm으로 진탕배양하였으며, 각각 3일 후와 10일후에 실험구와 대조구의 가솔린 잔량을 측정하였다. 그 결과는 표 3과 같다.After preparing a total of these two media, the strains grown overnight in LB was inoculated at a concentration of 1% (v / v), respectively, and control groups 1 and 2 were set as not inoculating the microorganisms in the two medium conditions. It was. Then, shaking was incubated at 30 ℃ ± 1, 150rpm, and after 3 days and 10 days, respectively, the residual gasoline in the experimental and control groups was measured. The results are shown in Table 3.
가솔린 분석을 위하여 FID(Flame Ionized Detector)를 장착한 가스크로마토그라피(Gas Chromatography, HD6890, Hewlett Packard, USA)를 사용하였으며, 분석조건으로 미 환경부 지정 분석법(EPA Method 8015B)에 준하여 실시하였다.For gasoline analysis, gas chromatography equipped with Flame Ionized Detector (FID) (Gas Chromatography, HD6890, Hewlett Packard, USA) was used, and the analysis was conducted according to the US Department of Environment-designated analysis method (EPA Method 8015B).
상기 표 3에 의하면, 휘발전의 가솔린의 경우에는 3일후에 대조구에 비해 부피가 7.003% 증가하였고, 10일후에는 28.86% 증가하였음을 알 수 있다. 이는 휘발전 가솔린의 경우에는 휘발성이 매우 큰데, 상기 균주가 첨가된 가솔린의 경우에는 균주가 가솔린을 분해하기 위하여 가솔린의 휘발을 억제시키기 때문에 대조구에 비하여 그 휘발정도가 낮아 부피가 증가한 것이다. 한편, 3일후 보다 10일후에 그 증가량이 더 큰 것은 초기에는 가솔린의 휘발속도가 균주의 대사활동 속도보다 훨씬 크기 때문에 대조구에 비해 큰 차이가 없으나, 시간이 지남에 따라 균주가 대사활동을 하기 위하여 가솔린의 휘발을 억제하는 정도가 커지기 때문에 대조구에 비하여 그 증가량이 커진 것이다.According to Table 3, in the case of gasoline of the power plant, the volume increased by 7.003% compared to the control after 3 days, it can be seen that after 10 days increased by 28.86%. This is very volatility in the case of volatilized gasoline, in the case of the gasoline added strain is suppressed the volatilization of gasoline in order to decompose gasoline, the volatility is lower than the control, the volume is increased. On the other hand, the increase was greater after 10 days than after 3 days, since the volatilization rate of gasoline is much higher than the metabolic rate of the strain at first, but there is no big difference compared to the control, but the strain has a metabolic activity over time. Since the degree of suppressing the volatilization of gasoline is increased, the increase is larger than the control.
50% 휘발된 가솔린의 경우에는 대조구에 비하여 부피가 3일후에 48.04%, 10일후에 12.87% 감소하였음을 알 수 있는데, 이는 이미 50% 정도가 휘발된 후이므로 휘발속도가 크지 않은 반면에, 상기 균주의 가솔린 분해속도가 크므로 대조구에 비하여 부피가 감소하게 된 것이며, 또한, 초기에는 균주가 성장을 많이 하기 때문에 10일후보다 3일후의 감소량이 더 크게 나타난 것이다.In the case of 50% volatilized gasoline, the volume decreased by 48.04% after 3 days and 12.87% after 10 days, compared to the control, since the volatilization rate was not large since the volatilization rate was about 50%. Since the gasoline decomposition rate of the strain is large, the volume is reduced compared to the control, and also, since the strain grows a lot in the early stage, the decrease after 3 days is greater than that after 10 days.
따라서, 상기 균주는 가솔린에 의해 오염된 지역에서 가솔린의 공기 중 확산을 억제하는데 매우 유용할 것으로 판단되며, 또한 가솔린의 분해도가 시간별 및 초기에 매우 높은 것으로 판단된다. 따라서, 가솔린 오염지역에서 빠른 시간에 많은 양의 가솔린을 분해할 것으로 판단된다.Therefore, the strain is considered to be very useful in suppressing the diffusion of gasoline in the air in the region contaminated by gasoline, and also the decomposition rate of gasoline is judged to be very high in time and in the beginning. Therefore, it is believed that a large amount of gasoline will be decomposed quickly in a gasoline contaminated area.
기름 분해력 검정 후 균주 동정단계Strain Identification after Oil Degradation Test
상기 균주를 다음과 같이 동정하였다. 균주 동정결과는 선정된 그람 테스트(gram test)와 혐기·호기성 실험과 운동성 실험에서 그람(gram)음성, 호기성으로 나타났고, 형태적으로 짧은 막대모양이며, 운동성은 양성으로 나타났다. 또한, biolog.실험에 의한 균주의 생리적, 생화학적 특성을 조사한 결과 슈도모나스 마큘리콜라(P. maculicola)로 높게 나타났다.The strain was identified as follows. The results of the strain identification were gram negative and aerobic in the selected gram test, anaerobic and aerobic and motility experiments. In addition, the physiological and biochemical characteristics of the strains by biolog experiments were high as P. maculicola.
따라서, 본 균주는 슈도모나스 마큘리콜라와 가장 유사한 것으로 나타났으며, HPLG-1(기탁번호 : KCTC 0714BP)로 명명하여, 한국과학기술원 유전자 은행에 1999. 12. 15.자로 기탁하였다.Therefore, this strain was found to be the most similar to Pseudomonas macuricola, named as HPLG-1 (Accession No .: KCTC 0714BP), and was deposited on December 15, 1999 to the Korea Advanced Institute of Science and Technology Gene Bank.
상기와 같은 슈도모나스 마큘리콜라 HPLG-1의 생장에 있어서 질소원, 미네랄 영양원, pH 등이 미치는 영향을 검토하기 위하여 다음과 같은 실험을 하였다.In order to examine the effect of nitrogen source, mineral nutrient source, pH and the like on the growth of Pseudomonas maculicola HPLG-1, the following experiment was performed.
질소원에 따른 생장 검토Growth review by nitrogen source
가솔린 분해가 계속되는 경우 필요한 영양분을 검정하기 위하여 질소원 투여량에 따른 생장량을 비교하여 보았다. 상기 표 2의 영양분 중 NH4Cl 1g(N: 0.261g)대신에 (NH4)2SO41g(N: 0.21g), (NH4)2SO42g (N: 0.42g), (NH4)2SO44g (N: 0.84g), (NH4)2SO48g, (NH4)2SO416g (N: 1.68g)을 첨부한 후의 균의 생장량을 측정하여 질소원이 충분한 상태에서 균주의 생육력을 검정하였으며, 그 결과를 도 2에 나타내었다.In order to test the nutrients required when gasoline digestion continues, the growth rate according to the nitrogen source dose was compared. In the nutrients of Table 2, instead of NH 4 Cl 1g (N: 0.261g), (NH 4 ) 2 SO 4 1g (N: 0.21g), (NH 4 ) 2 SO 4 2g (N: 0.42g), (NH 4 ) Sufficient nitrogen source by measuring the growth of bacteria after 2 SO 4 4g (N: 0.84g), (NH 4 ) 2 SO 4 8g, (NH 4 ) 2 SO 4 16g (N: 1.68g) The growth ability of the strain was assayed in, and the results are shown in FIG. 2.
상기 도 2에서 A1∼A6는 각각 A1 : NH4Cl 1g, A2 : (NH4)2SO41g, A3 : (NH4)2SO42g, A4 : (NH4)2SO44g, A5 : (NH4)2SO48g, A6 : (NH4)2SO416g을 첨가한 경우를 나타낸 것이다.In Figure 2, A1 to A6 are each A1: NH 4 Cl 1g, A2: (NH 4 ) 2 SO 4 1g, A3: (NH 4 ) 2 SO 4 2g, A4: (NH 4 ) 2 SO 4 4g, A5 : shows the (NH 4) the addition of 2 SO 4 16g: (NH 4 ) 2 SO 4 8g, A6.
상기 도 2에 의하면 1g의 질소(NH4Cl)가 함유된 처리구보다 1g/L의 (NH4)2SO4가 함유된 영양원에서 가장 생육이 좋았으며, 그 이상의 질소원 투여는 다소 생육을 억제함을 알 수 있다.According to FIG. 2, growth was best in the nutrient source containing 1 g / L of (NH 4 ) 2 SO 4 than the treatment group containing 1 g of nitrogen (NH 4 Cl), and further nitrogen source administration suppressed growth. It can be seen.
미네랄 영양원 감소에 따른 생장 비교Growth Growth with Reduced Mineral Nutrients
각종 영양분의 과부족에 대해서도 검정을 실시하였다. 표 2의 영양분에서 다량 첨가물질의 양을 각각 1/10씩 감소시켜 첨가하였을 때의 생장을 검토하였다. 도 3에서,The test was also carried out for the lack of nutrients. The growth of the nutrients in Table 2 was added by decreasing the amount of large amounts of additives by 1/10 each. In Figure 3,
B1 : 원래의 양대로 첨가한 경우 B2 : Na2HPO4·12H2O를 0.9g 첨가한 경우B1: When added in the original amount B2: When 0.9g of Na 2 HPO 4 · 12H 2 O is added
B3 : KH2PO4를 0.15g 첨가한 경우 B4 : MgSO4·7H2O를 0.02g 첨가한 경우B3: When 0.15 g of KH 2 PO 4 is added B4: When 0.02 g of MgSO 4 · 7H 2 O is added
B5 : NH4Cl를 0.1g 첨가한 경우 B6 : FeSO4·7H2O를 0.01g 첨가한 경우B5: When 0.1 g of NH 4 Cl is added B6: When 0.01 g of FeSO 4 · 7H 2 O is added
B7 ; CaCl2·2H2O를 0.01g 첨가한 경우를 나타낸다.B7; CaCl 2 · 2H 2 O shows a case where the addition of 0.01g.
도 3에 의하면 Na2HPO4의 경우 1/10첨가시 미생물 생장에 현격한 감소를 보였고, MgSO4의 경우 1/10첨가시 약간의 생장감소를 보였고, 기타 다른 영양분의 1/10투여시는 거의 생장 감소를 볼 수 없었음을 알 수 있다.According to FIG. 3, Na 2 HPO 4 showed a significant decrease in microbial growth at 1/10 addition, and MgSO 4 showed a slight decrease in growth at 1/10 addition, and at 1/10 administration of other nutrients. It can be seen that almost no growth was seen.
pH에 따른 생장 검토Review of growth by pH
여러 pH에서 본 발명에 의한 HPLG-1의 생장을 실험한 결과 도 4와 같았다. 도 4에 의하면, 초기에는 pH 5, 6, 7에서 생장이 가장 좋았고 36시간후는 pH4, 5, 6 7에서 생장이 일률적으로 좋았다. 따라서 본 발명에 의한 균주는 실험전체 pH에서 매우 활력이 높음을 알 수 있다.The growth of HPLG-1 according to the present invention at various pHs was as shown in FIG. 4. According to Figure 4, initially the growth was the best at pH 5, 6, 7, and after 36 hours the growth was uniformly good at pH 4, 5, 6 7. Therefore, the strain according to the present invention can be seen that the very high vitality at the pH of the experiment.
완충용액에서 pH에 다른 생장Different growth in pH in buffer
여러 완충조건의 pH에서 본 발명에 의한 HPLG-1의 생장을 조사하였으며, 그 결과를 도 5에 나타내었다. 도 5에 의하면, 완충용액에서의 pH에 따른 생장은 pH 5, 6, 7에서 거의 일률적으로 좋은 결과를 나타냄을 알 수 있다.The growth of HPLG-1 according to the present invention was investigated at various buffer conditions, and the results are shown in FIG. 5. According to Figure 5, it can be seen that the growth according to the pH in the buffer solution almost uniformly good results at pH 5, 6, 7.
따라서, pH에 따른 처리는 pH 5이상으로 처리해 주는 것이 가장 효율적이며, 또한, 국내의 토양은 대부분 약산성의 상태로 존재함으로 토양 중 큰 pH의 조절이 없더라도 토양가솔린을 분해하는데 효과적이다.Therefore, the treatment according to pH is the most efficient to treat the pH above 5, and also, since most of the domestic soil is in a slightly acidic state, it is effective to decompose the soil gasoline even without a large pH control of the soil.
본 발명에 의한 슈도모나스 마큘리콜라 HPLG-1은 휘발전후의 가솔린을 현저하고 효과적으로 분해하여 오염된 수질, 토질을 정화하는데 그 우수성이 있다.Pseudomonas maculola coli HPLG-1 according to the present invention remarkably and effectively decomposes gasoline after volatilization, and is excellent in purifying contaminated water and soil.
또한, 본 발명은 상기 가솔린 분해균주 HPLG-1의 최적의 생장조건을 제공하여 빠른 시간내에 그리고 지속적으로 유해물질을 제거할 수 있는 방법을 제공함으로써 시간과 비용면에서 보다 경제적인 잇점이 있다.In addition, the present invention is more economical in terms of time and cost by providing a method for providing the optimum growth conditions of the gasoline decomposition strain HPLG-1 to remove harmful substances quickly and continuously.
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