• Journal of Ecology and Environment
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• 제31권4호
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• pp.309-316
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• 2008

To develop a monitoring method for soil microbial communities in rice paddy fields, we used terminal restriction fragment length polymorphism (T-RFLP) to compare soil bacterial community structure in rice paddy fields experiencing different management practices: organic practices, conventional practices without a winter barley rotation, and conventional practices with a winter barley rotation. Restriction fragment length profiles from soils farmed using organic practices showed very different patterns from those from conventional practices with and without barley rotation. In principal component analyses, restriction fragment profiles in organic practice samples were clearly separated from those in conventional practice samples, while principal component analysis did not show a clear separation for soils farmed using conventional practices with and without barley rotation. The cluster analysis showed that the bacterial species compositions of soils under organic practices were significantly different from those under conventional practices at the 95% level, but soils under conventional practice with and without barley rotation did not significantly differ. Although the loadings from principal component analyses and the Ribosomal DNA Project II databases suggested candidate species important for soils under organic farming practices, it was very difficult to get detailed bacterial species information from terminal restriction fragment length polymorphism. Rank-abundance diagrams and diversity indices showed that restriction fragment peaks under organic farming showed high Pielou's Evenness Index and the reciprocal of Simpson Index suggesting high bacterial diversity in organically farmed soils.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1170-1177
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• 2005

Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor inoculated oil-degrading microorganisms during bioremedial treatability tests. A pair of universal primers, fluorescently labeled 521F and 1392R, was employed to amplify small subunit rDNA in order to simultaneously detect two bacterial strains, Corynebacterium sp. IC10 and Sphingomonas sp. KH3-2, and a yeast strain, Yarrowia lipolytica 180. Digestion of the 5'-end fluorescence/labeled PCR products with HhaI produced specific terminal-restriction fragments (T-RFs) of 185 and 442 bases, corresponding to Corynebacterium sp. IC10 and Y. lipolytica 180, respectively. The enzyme NruI produced a specific T-RF of 338 bases for Sphingomonas sp. KH3-2. The detection limit for oildegrading microorganisms that were inoculated into natural environments was determined to be $0.01\%$ of the total microbial count, regardless of the background environment. When three oil-degrading microorganisms were released into oil-contaminated sand microenvironments, strains IC10 and 180 survived for 35 days after inoculation, whereas strain KH3-2 was detected at 8 days, but not at 35 days. This result implies that T-RFLP could be a useful tool for monitoring the survival and relative abundance of specific microbial strains inoculated into contaminated environments.

• 한국물환경학회지
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• 제24권2호
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• pp.195-200
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• 2008

To analyze the riverine microbial community structure, genetic fingerprints and ecological indexes such as species abundances, diversity, evenness, dominance of targeted rivers in Gyeonggi Province were acquired and evaluated using terminal restriction fragment length polymorphism (T-RFLP) technique. Genetic fingerprinting technique such as T-RFLP, which is able to show the microbial community clearly unlike traditional culture-dependent techniques, was thought to be useful to analyse the riverine microbial ecosystem under various factors. Riverine ecosystem evaluation using visible organisms would give biased results with time, targeted organism and researcher. But, T-RFLP, which can exclude the subjected biases such as culture condition and identification, would be an option to understand natural ecosystem by including the microorganisms that defy culture but perform important functions.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.15-21
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• 2002

Terminal-restriction fragment length polymorphism (T-RFLP) analysis has been optimized by using in vitro model community composed of genomic DNAs of known bacterial strains and has been applied to assess the bacterial community structure in marine sediments. The specific fluorescence-labeled terminal restriction fragments (T-RFs) between 39 and 839 base long specifying each strain were precisely measured for known bacterial strains. The addition of a co-solvent (dimethylsulfoxide or glycerol) into PCR reactions has reduced differential PCR amplification. Comparative bacterial community structure was investigated for pristine and polluted sediments. A complex T-RFLP pattern showing complex bacterial community structure was obtained in the pristine sediment, whereas simple T-RFLP pattern (low bacterial diversity) was shown in polluted sediments where caged aquaculture has been conducted for several years. The results of T-RFLP analysis were compared with that of cloning and sequencing 16S rDNA clones from the same sediments. Sequence analysis of 16S rDNA clones (72) of the pristine sediment revealed a diverse collection of lineages, largely of the class Proteobacteria ($6\%$ alpha subdivision, $46\%$ gamma subdivision, $13\%$ delta subdivision, and $3\%$ epsilon subdivision), Nitrospina $(8\%)$, high G+C gram positive $(8\%)$, Verrucomicrobia $(7\%)$, and Planctomycetes $(6\%)$. In the contaminated sediments, 17 $(59\%)$ of the 16S rDNA clones (29) were related to Campylobacter and symbiont of Rimicaris exoculata belonging to epsilon subdivision of Proteobacteria. The results obtained indicated that T-RFLP analysis is a rapid and precise technique for comparative bacterial community analysis.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.247-259
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• 2008

미생물 구조 및 집단 구성원의 변화를 배양에 의존하지 않고, 빠르게 평가할 수 있는 방법의 하나인 terminal-restriction fragment length polymorphism (I-RFLP) 분석을 김치 유산균 연구에 적용하여, $15^{\circ}C$ 및 $4^{\circ}C$ 발효 김치에 관여하는 유산균의 다양성과 역동성을 검토하였다. 두 발효온도에서 공통적으로 Leuconostoc mesenteroides, Lc, inhae, Lc. kimchii, Weissella koreensis, W. cibaria, Lactobacillus sakei, Lb. curvatus, Lb. plantarum, Lb. paraplantarum, Lb. pentosus, 및 Lb. brevis의 존재가 예상되었고, Lc. citreum과 Enterococcus faecalis는 각각 $15^{\circ}C$와 $4^{\circ}C$에서 검출되었다. W. koreensis는 중기발효에 우점을 점하였고, Lactobacillus 속은 발효 후기의 우점종으로 나타났다. Lb. sakei, Lb. curvatus는 발효온도에 관계 없이 우점종을 형성하였지만, Lb. plantarum, Lb. paraplantarum, Lb, pentosus와 Lb. brevis의 생육은 저온에서 잘 일어나지 않았다. 몇 종의 Leuconostoc 속 유산균은 발효 후기까지 생장이 유지되었다.

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.389-395
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• 2012

배양 비의존적 미생물 군집분석법의 하나로 활발하게 이용되고 있는 T-RFLP (terminal restriction fragment length polymorphism) 분석과 실험동물을 이용한 생균제의 장관 정착 평가를 시도해 보았다. 장관 정착 평가를 위한 생균제는 cinnamoyl esterase 활성 보유 유산균을 발효 침채류로부터 분리하였고, 16S ribosomal RNA 유전자 염기서열 분석을 통해 동정하였다. 분리 균주 중, cinnamoyl esterase에 의한 chlorogenic acid 분해활성이 높고, 내산성 및 담즙산 내성의 평가 결과가 우수한 Lactobacillus plantarum KK3 균주를 생균제로 선정하였다. 배양 후, 냉동건조하여 제조한 생균제를 생쥐에게 투여한 다음, T-RFLP 분석을 이용하여 생쥐 분변의 미생물상을 모니터링하였다. 생균제 투여에 따라 검출되기 시작한 L. plantarum의 T-RF가 투여 중지 후 24일까지 지속됨을 확인하여 T-RFLP 분석법이 생균제로 사용한 L. plantarum의 장관 정착 평가에 유용한 것으로 나타났다. 생균제 투여 전 생쥐의 분변에서 검출되지 않았던 cinnamoyl esterase 활성 보유 L. plantarum이 투여 중지 후 24일차 분변에서 배양법으로 검출되어 L. plantarum KK3 균주의 생쥐 장관 정착이 확인되었다.

• 한국물환경학회지
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• 제22권4호
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• pp.590-598
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• 2006

In-situ Air sparging (IAS) is a groundwater remediation technique, in which organic contaminants volatilize into air form the saturated to vadose zone. This study was carried out to evaluate the effect of sludge and soil microbial community structure on air sparging of Total Petroleum Hydrocarbons (TPH) contaminated groundwater soils. In the laboratory, diesel (10,000 mg TPH/kg) contaminated saturated soil. The Air was injected in intermittent (Q=1500 mL/min, 10 minute injection and 10 minute idle) modes. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for experiment with sludge soil samples that were closely related to Agrococcus, Flavobacterium, Thermoanaerobacter, Flexibacter and Shewanella, etc, in the clone library. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil the fate of microorganisms in natural microbial community.

• 한국물환경학회지
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• 제24권1호
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• pp.19-29
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• 2008

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air ($34.9{\pm}2.7^{\circ}C$) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, ${\gamma}$-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.

• 대한환경공학회지
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• 제32권4호
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• pp.369-378
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• 2010

폐기물매립장의 안정화에는 미생물이 중요한 역할을 수행한다. 폐기물매립장에서 미생물군집 변화 모니터링에 말단 제한절편다형성(Terminal Restriction Fragment Length Polymorphism; T-RFLP)법의 활용 가능성을 평가하고자 박테리아의 16S rDNA 서열에 기초한 T-RFLP법으로 4개의 폐기물매립장 내부에서 채취한 침출수의 미생물군집 구조를 조사하였다. T-RFLP법을 사용하여 해석한 침출수 내 우점 미생물군집 구조와 일반적으로 널리 사용되고 있는 16S rDNA 클론 해석법에 의한 우점 미생물군집구조는 유사하였다. 또한, T-RFLP법을 이용하여 폐기물매립장의 구조, 매립 폐기물 종류, 운영기간이 다른 폐기물매립장 침출수의 우점 미생물군집 구조가 서로 다르게 나타나는 것을 확인 할 수 있었다. 따라서 T-RFLP법을 사용하여 폐기물매립장 침출수내 미생물군집 구조를 장기적으로 모니터링 한다면 많은 비용과 시간이 소요되는 클론해석법의 반복적인 수행 없이도 비교적 간단하게 폐기물매립장의 안정화 정도를 평가할 수 있을 것으로 기대한다.

• 한국미생물·생명공학회지
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• 제42권2호
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• pp.121-130
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• 2014

Bacteria 군집 차이를 이용한 신속한 한국산 및 중국산 김치 원산지 판별 가능성의 검토를 위하여 Terminal Restriction Fragment Length Polymorphism (T-RFLP) 분석법을 적용하였다. T-RFLP 분석은 김치발효에 관여하는 주요 유산균의 빠르고, 재현성 있는 검출에는 효과적이었지만, 종(species) 수준에서의 미생물 확인에는 한계를 가지고 있어 한국산 및 중국산 김치에 특이적으로 존재하는 bacteria의 검출에는 부적합한 것으로 평가되었다. T-RFLP를 적용한 발효과정 중의 한국산 및 중국산 김치에 존재하는 bacteria 군집 천이 분석은 비슷한 양상으로 나타났고, Bacillus 속이 발효 후기까지 검출되었다. 또한 Bacillus 속은 발효 후기에 포자를 형성하는 것으로 확인되었다.