정량 PCR을 이용한 비위생 매립지의 특정 세균 및 효소 유전자와 수질인자와의 상관관계 평가 = Comparative Assessment of Specific Genes of Bacteria and Enzyme over Water Quality Parameters by Quantitative PCR in Uncontrolled Landfill

국문 초록 (Abstract)

매립지를 직접 생태학적으로 모니터링하는 방법을 개발하고자, 매립지 내의 생화학적 반응에 관여하는 세균들과 효소의 양을 정량함과 동시에 지하수 수질인자와 상호 연관성을 조사하여 생태학적 인자와의 연계 이용 가능성을 평가하였다. 이를 위하여 4개의 매립 종료된 비위생 매립지(천안(C), 원주(W), 논산(N), 평택(P) 매립지)에서 계절별로 지하수 시료를 채취하였으며 동시에 16S rDNA 방법을 사용하여 미생물 다양성을 분석하였다. 이를 기반으로, 매립지에서 주로 발견되는 세균과 효소를 대표하는 유전자를 정량하기 위한 특이 프라이머 쌍을 제작하였으며 상관계수에 기초하여 수질인자와 유전자 지표 인자간의 정량적 관련성을 비교하였다. 그 결과 DSR(황환원 세균) gene과 BOD(생화학적 산소요구량)사이의 상관관계는 0.8 이상인데 반해 NSR(질산화 세균-Nitrospira sp.) gene과 질산성 질소는 0.9 이상이었다. 안정화지표(BOD/COD)와 MTOT(메탄 산화 세균), MCR(Methyl coenzyme M reductase), Dde(Dechloromonas denitrificans) gene들은 0.8 이상의 상관관계를 가졌으나 3가 철과 Fli(Ferribacterium limineticm) gene은 0.7로 낮았다. MTOT gene의 경우, BOD/COD과의 관련성이 100%에 가깝게 높았다. 또한, 혐기성 유전자들(nirS-아질산 환훤효소, MCR, Dde, DSR)과 DO 역시 0.8 이상으로 나타나 일반적인 매립지 혐기성 반응들이 DO에 크게 의존함을 보였다. 결론적으로 분자생물학적 조사와 수질인자가 높은 상호연관성이 있었으며 real-time PCR이 전통적인 모니터링 인자들과 동시에 상호 보완적으로 모니터링에 사용됨으로써 매립지안정화 및 주변 영향을 평가하는데 효율적으로 사용 될 수 있음을 알 수 있었다.

다국어 초록 (Multilingual Abstract)

As for the increasing demanding on the development of direct-ecological landfill monitoring methods, it is needed for critically defining the condition of landfills and their influence on the environment, quantifying the amount of enzymes and bacteria mainly concerned with biochemical reaction in the landfills. This study was thus conducted to understand the fates of contaminants in association with groundwater quality parameters. For the study, groundwater was seasonally sampled from four closed unsanitary landfills(i.e. Cheonan(C), Wonju(W), Nonsan(N), Pyeongtaek(P) sites) in which microbial diversity was simultaneously obtained by 16S rDNA methods. Subsequently, a number of primer sets were prepared for quantifying the specific gene of representative bacteria and the gene of encoding enzymes dominantly found in the landfills. The relationship between water quality parameters and gene quantification were compared based on correlation factors. Correlation between DSR(Sulfate reduction bacteria) gene and BOD(Biochemical Oxygen Demand) was greater than 0.8 while NSR(Nitrification bacteria-Nitrospira sp.) gene and nitrate were related more than 0.9. A stabilization indicator(BOD/COD) and MTOT(Methane Oxidation bacteria), MCR(Methyl coenzyme M reductase), Dde(Dechloromonas denitrificans) genes were correlated over 0.8, but ferric iron and Fli(Ferribacterium limineticm) gene were at the lowest of 0.7. For MTOT, it was at the highest related at 100% over BOD/COD. In addition, anaerobic genes(i.e., nirS-Nitrite reductase, MCR, Dde, DSR) and DO were also related more than 0.8, which showing anaerobic reactions generally dependant upon DO. As demonstrated in the study, molecular biological investigation and water quality parameters are highly co-linked, so that quantitative real-time PCR could be cooperatively used for assessing landfill stabilization in association with the conventional monitoring parameters.

참고문헌 (Reference)

1 "The organic geochemistry of a sanitary ladnfill leachate plume" 1986

2 "The effect of landfill leachate irrigation on soil gas composition : Methane oxidation and nitrous oxide formation" 164 : 295-313, 2005

3 "Status report on policy of environment conservation" 2003

4 "Standard Methods for the Examination of Water and Wastewater, 20th ed." 1988

5 "Sessitsch＂16S rRNA based T-RFLP analysis of methane oxidizing bacteria-Assessment, critical evaluation of methodology performance and applicationfor landfill site cover soils" 858 : 101-107, 2005

6 "Rhodoferax ferrireducens sp. nov., a psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the reduction of Fe(III)" 53 (53): 669-673, 2003

7 "Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays" 2423-2429, 2003

8 "Quantification of genetically modified soybean by quenching probe polymerase chain reaction" A-F : 2005

9 "Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR" 67 : 972-976, 2001

10 "Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR" 67 : 972-976, 2001

11 "Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction" 6597-6604, 2003

12 "Phylogeography of Sulfate-Reducing Bacteria among Disturbed Sediments, Disclosed by Analysis of the Dissimilatory Sulfite Reductase Genes(dsrAB)" 1004-1011, 2005

13 "Phylogenetic diversity of bacteria in the leachate of a full-scale recirculating landfill" 50 : 175-183, 2004

14 "Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill" 242 : 297-303, 2005

15 "Molecular diversity of denitrifying genes in continental margin sediments within the oxygen- deficient zone off the pacific coast of mexico" 69 (69): 3549-3560, 2003

16 "Molecular analysis of microbial community in a groundwater sample polluted by landfill leachate and seawater" 6B (6B): 165-170, 2005

17 "Methods for assessing the composition and diversity of soil microbial communities" 15 : 25-36, 2000

18 "Limitations of soil enzymes as indicators of soil pollution" 32 : 1867-1875, 2000

19 "Isolation and identification of methanogen-specific DNA feom blanket bog peat by PCR amplification and sequence analysis" 62 : 668-675, 1996

20 "Ferribacterium limneticum, gen. nov., sp. nov., an Fe(III)-reducing microorganism isolated from mining-impacted freshwater lake sediments" 171 : 183-188, 1999

21 "Enzymatic treatment of sanitary landfill leachate" 44 : 1103-1108, 2001

22 "DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria" 128-134, 2005

23 "Diversity of nitrite reductase gene fragments in forested upland and wetland soils" 68 : 1893-1900, 2002

24 "Distribution and Composition of Microbial Populations in a Landfill Leachate Contaminated Aquifer(Grindsted, Denmark)" 37 : 197-207, 1999

25 "Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa" 55 : 1255-1265, 2005

26 "Complex Microbial Communities Inhabiting Sulfide-rich Black Mud from Marine Coastal Environments" 8 : 1-16, 2000

27 "Characterization of microbial populations in landfill leachate and bulk samples during aerobic bioreduction" 5 : 285-294, 2001

28 "Behaviour of high-rate anaerobic processes treating landfill leachate" 6 (6): 73-79, 2001

29 "A molecular view of microbial diversity in a dynamic landfill in Qubec" 162 : 219-226, 1998