KR20020011252A - Benzene-degrading bacteria and its utiliztion method - Google Patents
Benzene-degrading bacteria and its utiliztion method Download PDFInfo
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Abstract
Description
본발명은 벤젠 분해균주 및 이의 이용방법에 관한 것이다.The present invention relates to a benzene decomposition strain and a method of using the same.
전세계적으로 석유탄화수소는 산업현장에 널리 쓰이고 있으며 많은 양의 원유 또는 정제된 제품들이 생산, 운반, 저장, 이용 등의 과정으로 환경에 유출되고 있다. 이것들은 계속되는 사회발전과 더불어 그 사용량이 증가하므로, 이에 따라 환경오염이 더욱 심화되고 있어, 이러한 유류에 의한 환경오염을 정화하는 것을 필수적이며, 해외에서는 수년 전부터 여러 가지 처리방법들이 연구되어 왔으며 국내에서도 활발히 개발되고 있는 실정이다.Petroleum hydrocarbons are widely used in industrial fields around the world, and large quantities of crude oil or refined products are being released into the environment through production, transportation, storage and use. As their consumption increases with the continuous development of society, environmental pollution is intensifying accordingly, and it is essential to clean up the environmental pollution caused by oil, and various treatment methods have been studied abroad for many years. It is being actively developed.
현재 주유소, 공장이나 각종 사업체 등에서 기름에 의한 환경오염 문제의 심각성이 국내외적으로 크게 대두되고 있다. 그런데 전체 기름 중 특히 가솔린에 많이 포함되어 있는 벤젠은 발암물질로 잘 알려져 있으며 비교적 높은 용해도와 휘발성으로 인해 대기와 수질에 매우 큰 오염을 일으키고 있다. 따라서 이를 분해할 수 있는 미생물의 개발이 절실히 필요한 실정이다.At present, the seriousness of oil pollution at gas stations, factories, and businesses is increasing at home and abroad. However, benzene, which is abundant in gasoline, is known as a carcinogen and causes very high pollution to air and water due to its relatively high solubility and volatility. Therefore, the development of microorganisms that can decompose it is an urgent need.
종래의 벤젠분해균주는 대부분 낮은 벤젠 농도에서 행해진 것으로서 이는 실험실 수준에서 행해질 수 있는 것이나 (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318; Heldeman 1994 Applied and environmental microbiology 60:2697-2703; Haack et al 1994 Applied and environmental microbiology 60:2480-2493; Robert et al. 1998 Applied and environmental microbiology 64:3961-3165; Norman et al. Plant diesease 1997 81:847-850; Jaunisses et al 97 Jaunisses 등 Phytoma la defence des vesetanx 49:17-22), 실제 오염지에서는 벤젠농도가 높아 직접 이들 균주를 접종하여 벤젠을 분해하는데 이용할 수 없다는 단점이 있다.Conventional benzene cracking strains are mostly done at low benzene concentrations, which can be done at the laboratory level (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318; Heldeman 1994 Applied and environmental microbiology 60: 2697-2703; Haack et. al 1994 Applied and environmental microbiology 60: 2480-2493; Robert et al. 1998 Applied and environmental microbiology 64: 3961-3165; Norman et al. Plant diesease 1997 81: 847-850; Jaunisses et al 97 Jaunisses et al. Phytoma la defence des vesetanx 49: 17-22), there is a disadvantage that the concentration of benzene in the contaminated place cannot be used to decompose benzene by directly inoculating these strains.
따라서 이를 분해할 수 있는 미생물의 개발이 절실히 필요하며, 유류오염지등 실제 필드에 적용하기 위해서는 고농도의 톨루엔을 함유한 경우에도 생육 및 분해가 충분히 높은 정도로 이루어지는 균주 및 이를 이용한 경제적, 효과적 및 환경친화적인 벤젠 분해방법이 필요하다.Therefore, it is urgently required to develop microorganisms capable of decomposing them, and to apply them to actual fields such as oil contaminated places, even if they contain high concentrations of toluene, the strains are sufficiently grown and decomposed enough to be economically effective and environmentally friendly. Phosphorous benzene decomposition method is required.
본발명은 유류 및 유독성분 중 벤젠 (benzene)을 1500 ppm이상의 고농도 함유배지에서 빠른 시간 내에 많은 양을 분해시킬 수 있고, 혐기성 조건 및 호기성 조건에서 생장가능하며, 넓은 범위의 생육 PH를 가지며 1500ppm이상의 높은 농도에서도 효과적인 벤젠분해능을 갖는 슈도모나스 푸티다 HPLB-1(Pseudomonas putidaHPLB-1) 및 이의 이용방법에 관한 것이다.The present invention can decompose a large amount of benzene in oil and toxic components at a high concentration of more than 1500 ppm in a short time, can grow under anaerobic and aerobic conditions, has a wide range of growth pH, and has more than 1500 ppm. Pseudomonas putida HPLB-1 ( Pseudomonas putida HPLB-1) having an effective benzene decomposition at high concentrations and a method of using the same.
도 1a 및 도 1b는 본균주의 16s rRNA 분석법을 이용한 동정결과를 나타내는 데이터이다.1A and 1B are data showing the results of identification using the 16s rRNA assay of the present strain.
본발명은 벤젠분해균주 및 이의 이용방법에 관한 것이다.The present invention relates to a benzene decomposition strain and a method of using the same.
일반적으로 미생물에 의한 벤젠오염 분해제품은 벤젠농도가 1000ppm이하의 농도에서 분해연구을 수행하고 있다. 그러나 본 미생물은 높은 1500ppm의 고농도 벤젠이 함유된 배지에서 실험을 수행하여 균주를 분리 및 실험하였다. 따라서 기타 분리미생물에 비하여 훨씬 효과적인 높은 벤젠분해율을 보이는 것으로 생각된다.In general, decomposition products of benzene pollution by microorganisms have been conducted at a concentration of benzene of less than 1000 ppm. However, the microorganism was tested in a medium containing a high concentration of 1500 ppm high benzene to isolate and test the strain. Therefore, it is thought to show high benzene decomposition rate which is much more effective than other isolated microorganisms.
오염농도의 수준은 자연환경에서 다양하게 나타날 수 있으므로 낮은 농도와 높은 농도에서도 분해활성이 높아야 효율적인 유독 폐가스 제거미생물이라고 할 수 있으며 또한 호기조건뿐만 아니라 혐기조건에서도 효율적인 생장이 이루어져야만 분해할 세균의 밀도가 증가하게 된다. 그리고 토양내 또는 배양기상에서 독성가스형태의 휘발물질인 벤젠을 효율적으로 분해하려면 미생물의 분해속도가 중요하며 미생물이 생장하는 장소의 pH도 매우 주요한 요소이다.The level of pollutant concentration can vary in the natural environment. Therefore, it is effective to remove toxic waste gas from low and high concentrations. Will increase. In order to efficiently decompose benzene, which is a toxic gas in the soil or in the incubator, the decomposition rate of microorganisms is important, and the pH of the place where microorganisms grow is also a very important factor.
현재의 세균 분리는 실험실 조건에서 즉, 낮은 벤젠농도와 미생물 생장에 매우 적합한 조건에서 실시되었지만 본 연구는 오염지의 환경과 유사한 벤젠농도가 높은 상태에서 오염을 줄이기 위한 균주가 필요한 것이다. 따라서 이와 같은 방법으로 실제오염상황과 비슷한 조건에서 감소율을 측정하기 위한 벤젠 분해 균주의 선발, 균주 동정과 가시적 세균활력검정으로 벤젠을 오염원으로 하는 지역에 사용함으로써 목적를 달성하고자 한다. 또한 넓은 범위의 pH와 혐기성조건에서도 벤젠을 분해할 수 있는 우수한 균주를 선발하는데 본발명의 또다른 목적이 있다.Current bacterial isolation has been carried out under laboratory conditions, i.e. under conditions suitable for low benzene concentrations and microbial growth, but this study requires strains to reduce contamination at high concentrations of benzene similar to the environment of contaminated sites. Therefore, this method aims to achieve the objective by selecting benzene degrading strains to identify the reduction rate under conditions similar to the actual pollution situation, identifying the strains, and using the benzene as a contaminant as a visible bacterial activity test. In addition, it is another object of the present invention to select excellent strains capable of decomposing benzene in a wide range of pH and anaerobic conditions.
본 발명의 유류분해 균주 분리방법은 유류로 오염된 토양을 채취하여 벤젠을 포함하는 배지에서 배양하여 벤젠분해능을 높은 균주를 선발하여 동정한다. 벤젠분해균주의 선발에 있어서 자연상황의 조건을 쉽게 인위적으로 부여해 줄 수 있는 기본영양배지(MSM : minimum salt medium; Na2HPO4·12H2O 9g/ℓ, KH2PO4 1.5g/ℓ, MgSO4·7H2O 0.2g/ℓ, NH4Cl 1.0g/ℓ, FeSO4·7H2O 0.1g/ℓ, CaCl2·2H2O 0.1 g/ℓ, 미량원소 용액(ZnSO4·7H2O 10mg/ℓ, MnCl·4H2O 3mg/ℓ, H3BO3 30mg/ℓ, CoCl2·6H2O 20mg/ℓ, CuCl·6H2O 0.56mg/ℓ, NiCl·6H2O 2mg/ℓ, NaMoO4·2H2O 10mg/ℓ) 3㎖/ℓ)에 탄소원 및 에너지원으로 벤젠을 첨가하여 분해미생물을 분리하였다. 상기단계 3)은 배양 후 많은 밀도를 가지는 우점균을 선별하여, 벤젠을 단일 탄소원으로 사용하는 기본배지에서 1∼3일간 호기조건으로 배양한 후 벤젠 분해도가 우수한 균주를 선택하였다. 선별된 균주가 다양한 pH와 무산소 상태에서도 잘 생장할 수 있는지의 여부를 검정하고 균주동정은 16s rRNA Gene에 기초하여 MicroSeq 16s rRNA sequencing kit를 이용하여 이루어졌으며, ATCC 표준균주의 16s rRNA 분석결과 라이브러리와 비교하여 동정하였으며, 그 결과는 도 1에 나타낸 바과 같이, 본발명의 균주는 슈도모나스 푸티다인 것으로 확인되었으며, 이 균주를 슈도모나스 푸티다 HPLB-1로 명명하였으며, 5월 18일자로 서울특별시 서대문구 홍제 1동에 소재하는 한국미생물보존센터에 기탁하여 수탁번호 KCCM-10187를 받았다.In the oil degradation strain separation method of the present invention, the soil contaminated with oil is collected and cultured in a medium containing benzene to identify and identify a strain having high benzene decomposition ability. Basic nutrient medium (MSM: minimum salt medium; Na2HPO4 · 12H2O 9g / ℓ, KH2PO4 1.5g / ℓ, MgSO4 · 7H2O 0.2g / ℓ) NH4Cl 1.0g / l, FeSO4 · 7H2O 0.1g / l, CaCl2 · 2H2O 0.1g / l, trace element solution (ZnSO4 · 7H2O 10mg / l, MnCl · 4H2O 3mg / l, H3BO3 30mg / l, CoCl2 · 6H2O 20mg / l Decomposing microorganisms were isolated by adding benzene as a carbon source and an energy source to 1 l, CuCl 6H 2 O 0.56 mg / l, NiCl 6H 2 O 2 mg / l, NaMoO 4 2H 2 O 10 mg / l). In step 3), after culturing, dominant bacteria having a high density were selected, and cultured under aerobic conditions for 1 to 3 days in a basic medium using benzene as a single carbon source, and then a strain having excellent benzene decomposition was selected. The strains were tested for growth at various pH and anaerobic conditions. Strain identification was performed using the MicroSeq 16s rRNA sequencing kit based on the 16s rRNA Gene. As a result, as shown in FIG. 1, the strain of the present invention was confirmed to be Pseudomonas putida, which was named Pseudomonas putida HPLB-1, and as of May 18, Hongje, Seodaemun-gu, Seoul It was deposited with the Korea Microbiological Conservation Center in Dong-A and received accession number KCCM-10187.
실시예Example
실시예 1: 벤젠분해균주의 분리Example 1 Isolation of Benzene Strains
시료채취를 위하여, 경기도 일대에서 장기간 폐기물이 존재했던 지역을 조사하여 토양을 선택하고, 지하 0~50cm에서 기름지 토양을 채취하여 비닐봉투에 투입한 후 4℃에서 냉장보관 하였다.For sampling, soil was selected by surveying the area where wastes existed for a long time in Gyeonggi-do, and oily soil was collected from 0-50cm underground and put in plastic bags, and refrigerated at 4 ℃.
상기에서 채취한 시료 1g을 MSB(minimal salt broth) 50 ㎖가 포함된 250 ㎖ 삼각플라스크에 접종하고, 각각 1000ppm의 벤젠을 첨가한 후 테플론으로 처리한 고무마개로 막아 2∼3일간 30 ℃, 170rpm으로 집적배양하였다. 균주가 자라 배지가 탁하게 되면 2∼3회 계대배양한 후 루리아스 아가배지(Luria's agar media)에 평판도말하였다. 이틀 후 집락이 많이 형성된 균주를 다시 루리아스 아가배지에 선형 도말하여 배양한 후, 각각 500, 1000, 및 1500ppm 벤젠이 포함된 MSB에 접종시켰다. 24시간 진탕배양 (30℃, 170rpm)한 후 각각의 벤젠분해정도를 측정하였다.1 g of the sample collected above was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of MSB (minimal salt broth), and 1000 ppm of benzene was added thereto, followed by Teflon-treated rubber stopper at 30 ° C and 170 rpm for 2-3 days. Accumulation was carried out. When the strain grew and the medium became turbid, it was passaged 2-3 times and plated on Luria's agar media. Two days later, the colony-strains were linearly plated on Luria agar medium and cultured, followed by inoculation into MSB containing 500, 1000, and 1500 ppm benzene, respectively. After 24 hours shaking culture (30 ℃, 170rpm) the degree of decomposition of each benzene was measured.
상기 최소영양성분에 벤젠이 포함 배지에서 106∼107cfu/㎖의 범위 콜로니를 선택하여 각각 순수분리하고, 이 순수분리한 균주를 500, 1000, 1500ppm의 벤젠을 포함한 각 배지에서 24∼48시간 배양한 후에 잔존 벤젠양을 측정하였다. 벤젠분해도가 높은 배양기상의 세균을 평판도말법으로 분리하여 균주동정은 16s rRNA Gene에 기초하여 MicroSeq 16s rRNA sequencing kit를 이용하여 이루어졌으며, ATCC 표준균주의 16s rRNA 분석결과 라이브러리와 비교하여 동정하였다. 동정된 균주는 슈도모나스 푸티다(Pseudomonas putida) HPLB-1로 명명하였으며, 5월 18일자로 서울특별시 서대문구 홍제 1동에 소재하는 한국미생물보존센터에 기탁하여 수탁번호KCCM-10187를 받았다. 유전자 검색에 따른 동정결과를 도 1에 나타냈으며, 벤젠분해능에 관한 가스크로마토그래프는 도 2에 나타내었다.Selected colonies ranging from 10 6 to 10 7 cfu / mL in the medium containing benzene as the minimum nutrients were purified separately, and the purified strains were separated from the medium containing 500, 1000 and 1500 ppm of benzene in 24 to 48 After incubation for a time, the amount of benzene remaining was measured. Bacteria on the high benzene degradation culture were separated by platelet smear and identified using the MicroSeq 16s rRNA sequencing kit based on the 16s rRNA Gene, and compared with the 16s rRNA analysis library of the ATCC standard strain. The identified strain was named Pseudomonas putida HPLB-1, and was deposited on May 18 at the Korea Microorganism Conservation Center in Hongje 1-dong, Seodaemun-gu, Seoul and received accession number KCCM-10187. Identification results according to gene search are shown in FIG. 1, and gas chromatographs for benzene decomposition ability are shown in FIG. 2.
실시예 2: 벤젠분해능 측정Example 2: Determination of Benzene Resolution
멸균된 7 ㎖ 최소영양배지와 단일 탄소원으로 첨가된 벤젠을 포함하는 혈청병(serum bottle)에 파장 660nm에서 흡광도 0.12로 조절된 배양균주 3 ㎖를 접종하고, 24∼48시간 동안 30℃에서 170rpm으로 진탕배양 하였다. 그런 후에 혈청병 상부공간의 벤젠을 500 ㎕ 가스 실린지를 사용하여 100 ㎕를 취하여 가스크로마토그래피로 잔류 벤젠양을 분석하고, 미생물의 유류소비율을 평가하기위해서 미생물를 넣지않고 유류와 미네랄성분만을 포함한 대조구와 투여한 대조구와 비교하였다. 본발명 균주의 시간에 따른 벤젠농도별 벤젠분해율을 표 2에 나타냈다.Inoculate 3 ml of the cultured strain with absorbance 0.12 at 660 nm in a serum bottle containing sterile 7 ml minimal nutrient medium and benzene added as a single carbon source, and shake at 170 rpm for 24 to 48 hours at 30 ° C. Incubated. Then, 100 μl of the benzene in the upper space of the serum bottle was taken using a 500 μl gas syringe, and the residual benzene amount was analyzed by gas chromatography.In order to evaluate the oil consumption rate of the microorganism, the control and administration containing only the oil and mineral components without microorganisms were administered. Compared to one control. Table 2 shows the benzene decomposition rate according to benzene concentration over time of the present invention strain.
미네랄영양분과 물을 넣고 거기에 각각의 농도(ppm)로 벤젠을 첨부시킨 후 그 안에 세균을 접종시켰을 경우 세균이 벤젠을 이용하여 첨가된 벤젠이 감소한 정도인 감소율을 보면, 500ppm의 경우 1시간이후부터 급격한 감소율을 보이며 4시간이후 100% 감소율을 보였다. 100ppm의 경우에 6시간에 100% 감소율을 보였고 500ppm에서는 7.5시간이후에 100% 감소율을 보였다. 또한, 첨가된 벤젠의 농도를 500, 1000, 1500ppm인 경우에 균주를 접종한 후 24시간배양하면 모든 농도에서 100%벤젠이 분해되었다.In case of adding mineral nutrients and water and attaching benzene to each concentration (ppm) and inoculating bacteria therein, the rate of reduction of the amount of benzene added by using benzene is reduced to 1 hour for 500 ppm. After 4 hours, it showed a sharp decrease, and after 4 hours, it showed a 100% decrease. In the case of 100 ppm, the reduction rate was 100% at 6 hours, and at 500 ppm, the reduction rate was 100% after 7.5 hours. In addition, 100% benzene was decomposed at all concentrations when the concentration of added benzene was 500, 1000, and 1500 ppm for 24 hours after inoculation of the strain.
실시예 3: 고농도벤젠의 분해율Example 3 Degradation Rate of High Concentration Benzene
주입된 벤젠의 농도가 1000 내지 5000 ppm범위인 것을 제외하고는 상기 실시예 2와 동일한 조건에서 본발명의 균주를 상기 농도의 벤젠을 포함하는 밴지에서 48시간동안 배양한 후에 벤젠분해율을 표 3에 나타냈다. 1500ppm까지의 벤젠농도에서는 48시간동안 배양한 후에 100%을 분해하였다. 벤젠분해율은 3000ppm에서 가장 높았으며 효율은 낮으나 분해가능한 최고농도는 4000ppm이며 5000ppm이상의 농도에서는 벤젠이 분해능이 관찰되지 않았다. 따라서 본발명의 균주는 4000ppm정도의 높은 벤젠농도에서도 생육가능하며 우수한 벤젠분해력을 나타냈다.Benzene decomposition rate is shown in Table 3 after incubating the strain of the present invention for 48 hours in the benzene containing the concentration of benzene under the same conditions as in Example 2 except that the concentration of the injected benzene ranges from 1000 to 5000 ppm. Indicated. Benzene concentration up to 1500ppm was incubated for 48 hours and then 100% decomposed. Benzene decomposition rate was the highest at 3000ppm, the efficiency was low but the highest degradable concentration was 4000ppm. Therefore, the strain of the present invention was able to grow at a high benzene concentration of about 4000 ppm and showed excellent benzene decomposition ability.
실시예 4: 생육 PH 측정Example 4: Growth PH Measurement
본발명의 슈도모나스 푸티다 HPLB-1의 생육 및 벤젠 분해 적정 pH범위측정을 위해서, 1.0M HCl과 NaOH를 이용하여 LB배지의 PH를 4, 5, 6, 7로 각각 조절한 4가지 배지에 하룻밤동안 배양한 본발명의 균주를 접종시켰다. 48 시간후에 자외선 스펙트로포토미터(Hewlett Packard 8453)을 사용하여 550nm파장에서 흡광도를 측정하면 다음 표 4와 같았다. pH 4에서의 흡광도가 0.1611로 상대적으로 낮은 값을 나타내지만 매우 낮은 pH라는 것을 감안한다면, 본균주는 PH4-7의 범위, 즉 자연계에서 일어날 수 있는 것의 모든 PH조건하에서 생장할 수 있음을 알 수 있다.In order to measure the pH range for growth and benzene decomposition of Pseudomonas putida HPLB-1, the pH of LB medium was adjusted to 4, 5, 6, and 7 using 1.0 M HCl and NaOH overnight. The strain of the present invention incubated during the inoculation was inoculated. After 48 hours, the absorbance at 550 nm was measured using an ultraviolet spectrophotometer (Hewlett Packard 8453). Given that the absorbance at pH 4 is relatively low at 0.1611, but at a very low pH, it can be seen that the strain can grow under the range of PH4-7, ie all PH conditions of what can occur in nature. have.
실시예 5: 호기 조건 및 혐기 조건에서의 생육실험Example 5: Growth experiment in aerobic and anaerobic conditions
혐기조건과 호기조건에서의 균주 생육실험을 수행하기 위하여, 브로모티몰불루(bromothylmol blue)가 첨가된 루리아스 아가 배양액 5 ㎖에, 하룻밤 배양한 균주 50㎕를 접종시켰다. 그런 후에 살균된 2 ㎖의 액체파라핀을 투여한 후 30 ℃에서 4일간 정치배양한 후에, 배지의 색깔 변화로 호기 또는 혐기적 조건을 평가하였다. 그 결과 슈도모나스 푸티다 HPLB-1를 배양한 배지는 혐기적 조건과 호기적 조건 모두에서 노란색으로 변화하여 호기와 혐기의 조건에서도 벤젠을 탄소원으로 하여 생장가능함을 확인하였다.In order to perform strain growth experiments under anaerobic and aerobic conditions, 50 µl of overnight cultured strains were inoculated into 5 ml of a Lurias agar medium containing bromothylmol blue. Thereafter, after sterilized 2 ml of liquid paraffin was administered and left incubated at 30 ° C. for 4 days, aerobic or anaerobic conditions were evaluated by color change of the medium. As a result, the medium in which Pseudomonas putida HPLB-1 was cultured was changed to yellow under both anaerobic and aerobic conditions, and it was confirmed that benzene can be grown as a carbon source even under aerobic and anaerobic conditions.
본 발명은 유류 및 유독성분 중 벤젠 (benzene)을 빠른 시간 내에 많은 양을 분해시킬 수 있고, 혐기조건 및 호기조건에서의 생장가능하며, 넓은 범위의 생육 PH를 갖는 도모나스 푸티다 HPLB-1(Pseudomonas putidaHPLB-1) 및 이의 이용방법에 관한 것이다. 본발명의 균주는 1500ppm이상의 높은 농도에서도 효과적인 벤젠분해 능력을 가지므로 벤젠으로 오염된 지역의 효과적인 정화방법이며, 환경친화적인 정화방법을 제공한다.The present invention is capable of decomposing large amounts of benzene in oil and toxic components in a short time, and is capable of growing under anaerobic and aerobic conditions, and having a wide range of growth PH. Pseudomonas putida HPLB-1) and a method of using the same. Since the strain of the present invention has an effective benzene decomposition ability even at a high concentration of more than 1500ppm, it is an effective purification method of the area contaminated with benzene, and provides an environmentally friendly purification method.
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