• Journal of Ecology and Environment
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• v.31 no.4
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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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• v.15 no.6
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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.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.247-259
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• 2008

Terminal-restriction fragment length polymorphism (T-RFLP) analysis, one of rapid culture-independent microbial community analysis methods, was used to determine the lactic acid bacterial complexity and dynamics during kimchi fermentation at $15^{\circ}C$ and $4^{\circ}C$. At both temperatures, the common presence of Leuconostoc mesenteroides, Lc. inhae, Lc. kimchi, Weissella koreensis, W. cibaria, Lactobacillus sakei, Lb. curvatus, Lb. plantarum, Lb. paraplantarum, Lb. pentosus, and Lb. brevis was predicted. Lc. citreum and Enterococcus faecalis were detected at $15^{\circ}C$ and $4^{\circ}C$, respectively. W. koreensis predominated during the mid stage of kimchi fermentation whereas lactobacilli were dominants during later stage. Lb. sakei and Lb. curvatus became dominants regardless of fermentation temperature but the growth of Lb. plantarum, Lb. paraplantarum, Lb. pentosus, and Lb. brevis was restricted at psychrophilic temperature. Some species of leuconostocs were maintained until the later stage of kimchi fermentation.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.346-350
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• 2009

Although salt is known to influence the performance of nitrification significantly, it has not been well reported on how salt affects ammonia-oxidizing bacterial(AOB) community compositions and dynamics in wastewater treatment bioreactors. In this study, these questions were evaluated in a full-scale bioreactor treating saline wastewater. Clone library analysis for the ammonia monooxygenase subunit A gene revealed that AOB belonging to the Nitrosomonas europaea and the N. oligotropha lineages inhabited in the bioreactor. Terminal restriction fragment length polymorphism analysis for monthly samples demonstrated a fluctuation pattern among AOB populations, although AOB within the N. europaea lineage were dominant during the test period. Correlation analysis between patterns of terminal restriction fragments and environmental variables suggested that sodium, chloride, and sulfate were less important; rather, temperature was the most significant factor affecting the AOB community in the bioreactor.

• Journal of Korean Society on Water Environment
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• v.24 no.2
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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.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1470-1474
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• 2008

Nitrospira is a dominant member of nitrite-oxidizing bacteria (NOB) in nitrifying bioreactors as well as in natural habitats. In this study, Nitrospira NOB were investigated in the two nitrifying reactors operated with high and low dissolved oxygen (DO) concentrations for a period of 300 days. Phylogenetic and terminal restriction fragment length polymorphism analyses based on 168 rRNA gene sequences revealed that the Nitrospira community compositions of the two reactors during the early period related to group 1 and half of the Nitrospira community composition shifted to group 2 in the high-DO reactor after day 179, although there was no significant change in the low-DO reactor. These results suggested that DO was an important factor affecting Nitrospira community compositions in the nitrifying reactors.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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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.

• Journal of Korean Society on Water Environment
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• v.22 no.4
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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.

• Journal of Korean Society on Water Environment
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• v.24 no.1
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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.

• Microbiology and Biotechnology Letters
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• v.40 no.4
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• pp.389-395
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• 2012

T-RFLP (terminal restriction fragment length polymorphism) analysis, one of the most highly adopted culture-independent microbial community analysis methods, was applied to evaluate the colonization of probiotics in experimental animal gut. Lactic acid bacteria that exhibited cinnamoyl esterase activity were isolated from Korean fermented vegetables and identified by 16S ribosomal RNA sequence analysis. Lactobacillus plantarum KK3, which demonstrated high chlorogenic acid hydrolysis by cinnamoyl esterase activity, and acid/bile salt resistances, was cultured, freeze-dried, and fed to mice and the microbiota in their feces were monitored by T-RFLP analysis. The T-RF of L. plantarum was detected in the feces of mice after the start of administration and lasted at least 31 days after the initial 7 day feeding. T-RFLP analysis was considered a useful tool to evaluate the gut colonization of probiotic L. plantarum. In order to prove that L. plantarum was from viable cells, we reisolated L. plantarum in the feces using cinnamoyl esterase activity media as the screening step. The colonization of L. plantarum KK3 in the mouse gut was confirmed by this research.