새로운 2,4-D 분해미생물의 분리 및 특성 규명

가종억 서울대학교 농업생명과학대학 농업개발연구소 1998 농업생명과학연구 Vol.2 No.-

Nine numerically dominant 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from rice field soils. Most of the isolates were identified as Burkholderia or Sphingomonas species by fatty acid methyl ester (FAME) analysis, and they exhibited diverse chromosomal DNA patterns in polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. The isolates utilized 2,4-D as the sole source of carbon and two Sphingomonas species were capable of mineralizing both 3-chlorobenzoate (3-CB) and 4-chlorobenzoate (4-CB), in addition to 2,4-D. Plasmid DNAs were detected from all of the isolates, and conjugation analysis revealed that 2,4-D degradative genes were located on transferable plasmids in most of the isolates. PCR analysis with specific primers selected from tfd genes showed that 67% of the isolates had DNA sequences homologous to the five ftd genes of the 2,4-D degradative plasmid pJP4 of Alcaligenes eutrophus JMP134. Among the isolates, strain TFD7 appeared to be a new genotype in that it contained a transmissible 2,4-D degradative plasmid nonhomologous to the tfd genes. 2,4-D was persistent in natural paddy soils which contained no indigenous 2,4-D-degrading microorganisms, but the application of the 2,4-D-degrading isolates resulted in rapid decline of the soil 2,4-D residues.

페녹시계 제초제 분해미생물과 탈질화 세균에 대한 유전생태학적 연구

가종억 서울대학교 농업생명과학대학 농업개발연구소 1997 농업생명과학연구 Vol.1 No.-

DNA probes 에 의한 토양의 이사디 ( 2,4 - D ) 분해세균의 검출

가종억 ( Jong Ok Ka ) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.5

Total bacterial community DNA, which was extracted from microcosm soil and field soil after 2.4D amendments, was analyzed on Southern blots, using the tfdA gene probe derived from plasmid pJP4 and the Spa probe from Sphingomonas paucimobilis. Southern blot analyses with total bacterial DNA extracted from soils inoculated with Pseudomonas cepacia/pJP4 revealed that DNA probe method could detect the 2,4-D degrading bacteria down to 10^6 cells/g dry soil. In the microcosm experiment, there was a good correlation between 2,4-D degradation and banding patterns in hybridization analyses performed after each 2,4-D treatment using the two probes. When bacterial DNA extracted from microcosm soil was hybridized with the tfdA probe. a change in the position of hybrid bands was observed over time in a Southern blot, suggesting that population change or possibly genetic rearrangement in 2,4-D degrading microbial populations occurred in this soil. With the Spa probe, one hybrid DNA band was persistently observed throughout the five 2,4-D additions. When bacterial DNA isolated from the field soil was probed with the tfdA and Spa, strong hybridization signal was observed in the 100 ppm-treated subplot, weak signal in the 10 ppm-treated subplot, and no significant signal in the 1 ppm-treated and control subplots. The data show that DNA probe analyses were capable of detecting and discriminating the indigenous 2,4-D degrading microbial populations in soil amended with 2,4-D under laboratory and field conditions.

Growth Promotion of Xanthium italicum by Application of Rhizobacterial Isolates of Bacillus aryabhattai in Microcosm Soil

이솔,가종억,송홍규 한국미생물학회 2012 The journal of microbiology Vol.50 No.1

This study was conducted using rhizobacteria, which are able to exert beneficial effects upon plant growth in the infertile soil collected from barren lakeside areas. Four strains of plant growth promoting bacteria were isolated from the rhizosphere of a common wild plant, Erigeron canadensis. Isolated strains LS9, LS11, LS12, and LS15 were identified as Bacillus aryabhattai by 16S rDNA sequence analysis. B. aryabhattai LS9, LS11, LS12, and LS15 could solubilize 577.9, 676.8, 623.6, and 581.3 mg/L of 0.5% insoluble calcium phosphate within 2 days of incubation. Production of indole acetic acid, a typical growth promoting phytohormone auxin, by strain LS15 was 471.3 mg/L in 2 days with the addition of auxin precursor L-tryptophan. All the strains also produced other phytohormones such as indole butyric acid, gibberellins, and abscisic acid, and strain LS15 showed the highest production rate of gibberellin (GA3), 119.0 μg/mg protein. Isolated bacteria were used in a microcosm test for growth of wild plant Xanthium italicum, which can be utilized as a pioneer plant in barren lands. Seed germination was facilitated, and the lengths of roots, and shoots and the dry weights of germinated seedlings after 16 days were higher than those of the uninoculated control plants. Root lengths of seedlings of X. italicum increased by 121.1% in LS11-treated samples after 16 days. This plant growth-promoting capability of B. aryabhattai strains may be utilized as an environmentally friendly means of revegetating barren lands, especially sensitive areas such as lakeside lands.

pKT230 벡터를 이용한 Pseudomonas sp. P20 2,3-Dihydroxybiphenyl Dixygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

Biphenyl과 4-chlorobiphenyl을 분해하는 자연계 분리 균주인 Pseudomonas sp. P20의 chromosomal DNA로부터 pBluescript SK(+)를 이용하여 pcbABCD 유전자를 클로닝하여 재조합플라스미드 pCK1을 제조하였고, 또 pcbCD 유전자를 포함하여 pCK102을 제조하였다. 방향족 탄화수소 화합물의 생분해는 벤젠고리의 개환 과정이 중요하기 때문에, 2,3-dihydroxybiphenyl(2,3-DHBP)의 벤젠고리의 개환에 관여하는 2,3-DHBP dioxygenase(2,3-DHBD) 유전자를 pKT230 벡터를 이용하여 pCK102로부터 클로닝하였다. EcoRI으로 절단한 pCK102와 pKT230 벡터를 ligation시켜 13.8 kb의 hybrid plasmid pKK1을 제조하였다. 2,3-DHBD 유전자를 포함하는 pKK1을 E. coli XL1-Blue에 형질전환시켜 E. coli KK1 재조합 균주를 얻은 후, 2,3-DHBD의 활성을 측정하였다. E. coli KK1의 2,3-DHBD의 효소활성은 pBluescript SK(+)를 이용하여 제조한 재조합 균주인 E. coli CK102의 효소활성과 유사하였으나, Pseudomonas sp. DJ-12와 Pseudomonas sp. P20과 같은 자연계 분리균주보다 훨씬 높았다. Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2,3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2,3-DHBP dioxygenase activity. The specific 2,3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20

pKT230 벡터를 이용한 Pseudomonas sp. P20의 2,3-Dihydroxybiphenyl Dioxygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근,Kim, Ji-Young,Kim, Chi-Kyung,Ka, Jong-Ok,Min, Kyung-Hee,Park, Yong-Keun 한국미생물 · 생명공학회 1996 한국미생물·생명공학회지 Vol.24 No.6

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

Pilot 규모 고정생물막 BNR공정을 이용한 하수처리 및 상향류 바이오비드(R) 공법을 적용한 실증플랜트의 생활하수 처리시 생물막 특성

김미화,문선정,주동훈,박태주,가종억 대한환경공학회 2005 대한환경공학회 학술발표논문집 Vol.2005 No.12

중,소규모 하수처리 시스템으로 적용된 현장규모 상향류식 바이오비드(R) 반응기의 생물막 군집특성

김미화,주동훈,최원석,가종억 대한환경공학회 2004 대한환경공학회 학술발표논문집 Vol.2004 No.12

Effects of Phosphate Addition on Biofilm Bacterial Communities and Water Quality in Annular Reactors Equipped with Stainless Steel and Ductile Cast Iron Pipes

장현정,최영준,노희명,가종억 한국미생물학회 2012 The journal of microbiology Vol.50 No.1

The impact of orthophosphate addition on biofilm formation and water quality was studied in corrosion-resistant stainless steel (STS) pipe and corrosion-susceptible ductile cast iron (DCI) pipe using cultivation and culture-independent approaches. Sample coupons of DCI pipe and STS pipe were installed in annular reactors, which were operated for 9 months under hydraulic conditions similar to a domestic plumbing system. Addition of 5 mg/L of phosphate to the plumbing systems, under low residual chlorine conditions,promoted a more significant growth of biofilm and led to a greater rate reduction of disinfection by-products in DCI pipe than in STS pipe. While the level of THMs (trihalomethanes) increased under conditions of low biofilm concentration, the levels of HAAs (halo acetic acids) and CH (chloral hydrate)decreased in all cases in proportion to the amount of biofilm. It was also observed that chloroform, the main species of THM, was not readily decomposed biologically and decomposition was not proportional to the biofilm concentration; however, it was easily biodegraded after the addition of phosphate. Analysis of the 16S rDNA sequences of 102 biofilm isolates revealed that Proteobacteria (50%) was the most frequently detected phylum, followed by Firmicutes (10%) and Actinobacteria (2%), with 37% of the bacteria unclassified. Bradyrhizobium was the dominant genus on corroded DCI pipe, while Sphingomonas was predominant on non-corroded STS pipe. Methylobacterium and Afipia were detected only in the reactor without added phosphate. PCR-DGGE analysis showed that the diversity of species in biofilm tended to increase when phosphate was added regardless of the pipe material, indicating that phosphate addition upset the biological stability in the plumbing systems.

서울시 상수계통에서 병원성균 Aeromonas (감마-프로테오박테리아) 분포연구

이은숙,이목영,한선희,가종억,Lee, Eun-Sook,Lee, Mok-Young,Han, Sun-Hee,Ka, Jong-Ok 한국미생물학회 2007 미생물학회지 Vol.43 No.2

The detection and distribution of Aeromonas in water supplies were investigated by using the USEPA Method 1605. Water samples were collected from the Han River, finished waters and tap waters supplied from Water Treatment Plants in Seoul monthly from July 2002 to December 2003. Aeromonas species in each water sample were quantified based on the development of yellow colonies on the surface of membrane filter using a selective medium (Ampicillin-Dextrin Agar with Vancomycin). The Quality Control (QC) for this study met the acceptance criteria of Method 1605. The concentrations of Aeromonas species in surface water samples ranged from $1.0{\times}10^{0}\;to\;9.8{\times}10^{3}\;CFU/ml$. Aeromonas species were found only in one tap water sample with concentration of 1 CFU/500 ml. No Aeromonas species were found in any finished water samples. Aeromonas species detected here were identified as A. salmonicida(51%), A. caviae(4.7%), A. schubertti(3.4%), A. sobria(3.8%), A. hydrophila(2.1%), and A. ichithiosmia(0.4%). A. salmonicida was the dominant species, which is of no significance to human health. Chlorine resistance of A. salmonicida was evaluated and as a result, 99.99% of A. salmonicida decreased after 30 seconds exposure at residual free chlorine 0.2 mg/L. These suggest that the waters supplied in Seoul may be safe against the pathogenic agent Aeromonas. USEPA 1605 방법을 이용하여 서울시 상수도계통에서 Aeromonas를 조사하였다. 2002년 7월부터 2003년 12월까지 매월 한강수계 하천수와 서울시 정수장에서 공급되는 정수, 수돗물에서 시료를 채취하였다. Aeromonas는 선택배지(ADA-V)를 사용하여 membrane 필터 표면에 성장한 노란색 집락을 계수하여 측정하였다. 하천수에서는 Aeromonas가 $1.0{\times}10^{0}-9.8{\times}10^{3}\;CFU/ml$의 농도로 검출되었으며, 정수에서는 검출되지 않았다. 수돗물에서는 141개 중 1개의 시료에서 1 CFU/500 ml의 농도로 검출되었다. 확인된 Aeromonas는 대부분 비병원성인 A. salmonicida(51%)였고, 이 외에도 A. caviae(4.7%), A. schubertti(3.4%), A. sobria(3.8%), A. hydrophila(2.1%), A ichithiosmia(0.4%)등이 동정되었다. A. salmonicida에 대한 염소 저항성을 평가한 결과, 0.2 mg/L 염소농도에서 30초 접촉 후, 99.99% (일부)가 제거되었다. USEPA 1605 방법에서 제시한 정도관리를 수행한 결과, 정도관리 허용기준을 만족하였다. 본 연구를 통해 서울시 상수도계통에서 Aeromonas에 대한 안전성이 확보되었다.