G Microbial Ecology, Environmental Microbiology, and ... · PDF fileG_ Microbial Ecology, Environmental Microbiology, and Public Health ... Dept. of Microbiology, ... There were some

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G _ Microbial Ecology, Environmental Microbiology, and Public Health

Purification of Siderophore Produced by Bacillis licheniformis K11, a Plant Growth Promotimg Rhizobacterium (PGPR) Sang-Min WOO and Sang-Dal KIM*Dept. of Applied Microbiology, Yeungnam University, Kyeongbuk, 712-749. *Corresponding author: [email protected]

An auxin-producing plant growth promoting rhizobacterium(PGPR), Bacillus licheniformis K11 was isolated from the soil in Gyeongsan, Gyeongbuk. B. licheniformis K11 was identified to produce siderophore by the formation of orange halos zone on CAS(chrome azurol S) blueagar and also by CAS liquid assay. SiderophoreK11 was determined to bea catechol type siderophore that Bacillus spp. generally produce. The siderophoreK11 was highly produced in Sucrose-asparagine-MgSO4

(SAM) medium(pH 8.0) after 4days at 30℃. SiderophoreK11 was purifiedfrom culture broth of B. licheniformis K11 by using Ambelite XAD-2 , Sephadex LH-20 chromatography, and HPLC.

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Microbial Fe(III) Reduction Enhances Stabilization of As(III) into Fe(III)-oxyhydroxide Nanorods Ji-Hoon LEE and Hor-Gil HUR*Dept. of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712 Republic of Korea. *Corresponding author: [email protected]

Arsenite [As(III)] adsorption on diverse iron minerals has been attracting a lot of interest for the cleanup technology of arsenic contamination. It is well known that arsenite is more toxic, harmful and mobile than arsenate [As(V)]. In this study, arsenite adsorbed on the synthetic Fe(III)-oxyhydroxide was trapped inside the iron minerals by biotransformation of the initial FeOOH through Fe(III) reduction by Shewanella sp. HN-41. The surface adsorption and trapping inside the mineral structure were investigated by chemical sequential extraction andatomic environments around arsenic atoms with extended x-ray absorption fine structure spectra (EXAFS). Compared with normal sorption of arsenite on the FeOOH, arsenite supposedly trapped in the iron minerals by microbial iron reduction was extracted out with majority portion by the later step of sequential extraction. The atomic environments such as interatomic distances and coordination numbers around As and Fe atoms were different between the control adsorption of As on FeOOH and the trapped As into the iron minerals. Those results tell us that the supposedly trapped arsenite into the iron minerals has more stability than the normal adsorption and it could be a better technology for remediation or stabilization of arsenic.

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Genomic Diversity of ECOR Strains Determined by DNA Microarrays Kum-Ok HWANG and Jae-Chang CHO*Institute of Environmental Sciences & Department of Environmental Sciences, Hankuk University of Foreign Studies, KOREA 449-791. *Corresponding author: [email protected]

Prokaryotic genomes are highly diverse, and strains belonging to a single species show significant heterogeneity in genome size and DNA composition. Classical standard reference strains of Escherichi coli (ECOR strains) were analyzed by BOX-PCR genome fingerprinting and DNA microarrays. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to determine the same genotypes, 28 genotypes were identified in 71 ECOR strains. Chi-sequare test of goodness of fit showed statistically signigicant association between the genotypes defined by BOX-PCR fingerprinting and the groups defined by MLEE. Incident-based coverage estimator (ICE) estimated potentially 420 genotypes in E. coli strains. Highest diversity and equitability were observed for MLEE group B1 (H=2.19, J=0.92) and the second highest diversity and equitability for MLEE B2 (H=1.68) and ICE (40) were higher than those of group D (H=1.65, ICE=24). We are currently carrying out DNA microarray results analysisto investigate the heterogeneties in the genomes of ECOR strains.

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Isolation of Microbes from Kimchi by Centrifugation with Ficoll Seong Woon ROH, Young-Do NAM, Ho-Won CHANG and Jin-Woo BAE*Biological Resources Center, KRIBB, Daejeon 305-806, S. Korea. *Corresponding author: [email protected]

Centrifugation is a process used to separate or concentrate materials suspended in a liquid medium. The theoretical basis of this technique is the effect of gravity on particles in suspension. Sucrose, Cesium chloride, Percoll or Ficoll-density gradient centrifugation have been used to selectively isolate bacteria from soil in pedology or to separate blood to its components; erythrocytes, leukocytes etc. To devise and evaluate a method for selective isolation of bacteria from kimchi, we examined the applicability of the ficoll-density gradient centrifugation method. Every suspension of kimchi soup is centrifuged through the gradient consisting of various ficoll densities at diverse centrifugation speed and manifold time. Analysis of every ficoll layer and original kimchi soup sample based on PCR-denaturing gradient gel electrophoresis (DGGE) ofSSU rRNA genes suggested that bacterial diversity was conserved after bacteria-specific isolation with ficoll-density centrifugation under the certain specific condition. The present results indicated that the centrifugation by using ficoll could be used to successfully and efficiently isolate bacteria that are existed in kimchi.

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371The Korean Society for Microbiology and Biotechnology

2006 Annual Meeting and International Symposium

Alteration of Motility, Endospore Formation, Plant Growth Promotion, and Antagonism by Phenotype Variation of a Rhizobacterium Paenibacillus polymyxa E681 Yong Ho JEON1,2, Choong-Min RYU1, Hoon CHEONG1, Soo Young PARK1, Jihyun F. KIM1 and Seung-Hwan PARK*1

1Laboratory of Microbial Genomics, Systems Microbiology Research Center, KRIBB, 52 Eoeundong, Yuseong, Daejeon 305-600, S. Korea. 2Agro-tech ResearchGroup, KT&G Central Research Institute, 434 Dangsu-dong, Gwonsun-gu, Suwon441-480, S. Korea. *Corresponding author: [email protected]

Paenibacillus polymyxa strain E681 isolated from winter barley roots previously reported as a promising plant growth-promotion rhizobacterium(PGPR) that reduced disease incidence against soil-borne pathogenic fungi and promoted growth of several plant species. During functional study with the bacterium, we found that at least two different colony types were appeared when the bacteria were spread on the agar medium24 hrs after broth culture of P. polymyxa E681 at 30℃ but did not at 20℃. Original colony was milky white, round, shiny, and bold referred to as “opaque phase I colony”. Other major colony was round with a scalloped edge, not shiny, and flat referred to as “translucent phase II colony”. The translucent phase II colonies of P. polymyxa E681 showed defective major capacity as PGPR such as root colonization, promoting root elongation, and antagonism against Rhizoctonia solani. Our results obtained here showed that the phenotype variants (translucent type II colonies) decreased the capacity of endospore formation, plant growth promotion and antagonism while increased motility than those of opaquetype I colony. Our study suggests that phenotype variation of PGPR strains can be concerned prior to apply PGPR strain(s) into agriculture.

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Expression of the nirS Gene at Different C/N ratios under Aerobic Condition by Real-time PCR Su-Jeong YOON, Sun-Ja CHO and Sang-Joon LEE*Dept. of Microbiology, Pusan National University, Busan, Korea, 609-735. *Corresponding author: [email protected]

It is known that the rate-limiting parameters in aerobic denitrification may include dissolved oxygen concentration, C/N ratio, temperature and pH. However, the most suitable C/N ratio for aerobic denitrification is still unknown, especially for aerobic denitrifiers. In this study, we identified the optimal range of the C/N ratios for the aerobic denitrifierisolated from Su-young WWTP in Busan, Korea by real-time PCR. Thisisolated strain was previously identified as Pseudomonas fluorescens DN-9 based on 16S rDNA sequence and physiological characteristics. We used Pseudomonas stutzeri as positive control and Escherichia coli as negative control compared with the isolate DN-9. Efficiencies of denitrification under aerobic condition were studied under three differentculture medium conditions: (ⅰ) at the C/N ratio of 2, (ⅱ) at the C/Nratio of 10, (ⅲ) at the C/N ratio of 20. There were some differencesin growth rate at different C/N ratios. By the results, we suggest that insufficiency of carbon source at C/N ratio of 2 causes low level of cellyield. According to analysis of 15N ratio in cell mass, we supposed that N content in cell mass was nearly equal to NH4

+ consumption. Expression of the nirS gene at C/N ratio of 20 was higher than others by real-time PCR.

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Development and Validation of Saccharomyces cerevisiae-Specific PCR Primer Ho-Won CHANG1, Young-Do NAM1, Seong Woon ROH1, Jung-Hoon YOON2, Jung-Sook LEE2 and Jin-Woo BAE*1

1Biological Resources Center, KRIBB, Daejeon 305-806, S. Korea. 2Laboratory of Microbial Function, KRIBB, Daejeon 305-806, S. Korea. *Corresponding author: [email protected]

Saccharomyces cerevisiae have been used in baking and brewing industry for a long time. Although isolation of a novel strain of S. cerevisiae with higher productivity of ethanol, vitamins and citric acid is important, current diagnostics and determination of S. cerevisiae is known to be labor-intensive work that is involved with cultivation of yeast, genomic DNA extraction, PCR-amplification of model genes, sequencing and comparing the sequences to other strains in public database using BLAST. Thus, development of rapid and reliable diagnostic tool is warranted in order to explore the S. cerevisiae as a novel bioresource. In this study, we developed a S. cerevisiae species-specific PCR primer and several primers for phylogenetically and hierarchically higher yeast group including S. cerevisiae based on D1/D2 region of 26S rRNA and ITS region. Two species-specific primers and seven group-specific primers were constructed and their specificity was evaluated and validated with 21 yeast strains. With these primers developed in this study, we analyzed the quantities of S. cerevisiae and related species in several foods by real time quantitative PCR.

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Analysis Methods of Microbial Community and Their Application to Biofilters Yun-Seong JO and Kyung-Suk CHO*Ewha Womans University, Dept. of Environmental. Sci. and Eng. College of Engineering, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul, 120-750, Korea. *Corresponding author: [email protected]

There are four key factors for gas-phase biofilters; biocatalysts, packing materials, design/operating techniques, and diagnosis/management techniques. The efficiency of biofilters is significantly affected by the structure of microbial population as well as loading conditions. The compositions and concentrations of gases emitted from industries are changed irregularly according to process and operation conditions. For enhancing the treatment efficiency of biofilters in irregular loading condition, it is necessary to study on transient response of biofilters: the development of rapid and accurate detecting methods for air pollutants-degrading microorganisms; characterizing entire and specified microbial population dynamics in relation to changing operating conditions; monitoring removal efficiency of pollutant in biofilters; and monitoring physico-chemical parameters. The microbial studies on biofilters are mostly performed on the basis of culture-dependent methods. Recently, various methods have been proposed to characterize the structure of microbial community in environmental samples. In this study, molecular, biochemical and physiological methods for profiling microbial communities are reviewed, and their application to biofilters isdiscussed.

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372 www.kormb.or.kr

Isolation, Identification, and Characterization of a Keratinolytic Bacterium Eun Ok WOO1, Hong Joo SON2 and Sang Joon LEE*1

1Dept. of Microbiology, Pusan National University, Busan, Korea. 2School of Applied Life Science, Pusan National University, Miryang, Korea. *Corresponding author: [email protected]

Feather wastes, almost pure keratin protein, are produced in large amounts as a waste by-product of poultry processing. Biodegradation of feather by microorganism processing represents an alternative method to improve the nutritional value of feather waste. In this report, we collected various samples from composting rice straws, and soil in Korea and screened keratinolytic microorganism. Especially, RS7, the isolate, ispresented the highest feather-degrading activity among the isolates and identified as Bacillus sp. The organism was grown with feathers as its primary source of carbon, nitrogen and energy. The optimum rate of growth in LB medium occurred at 37℃ and at pH 8.0 to 9.0. Growth on native feather medium of various temperature and pH was determined by plate colony count and keratinolytic activity assay was measured spectrophotometrically at 280 nm the liberation of soluble peptides or amino acids from the shattered feather. The growth and keratinolytic activity of RS7 was optimally active at pH 9.0 and temperature of 30℃.The culture medium that contained (%, w/v): NaCl 0.05%, K2HPO4

0.03%, KH2PO4 0.04%, MgCl2 0.01%, Urea 0.05% and 0.1% native whole chicken feather.

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World-wide Distribution Patterns of Acidobacterium spp. in Global Soil Samples Sang-Hoon LEE and Jae-chang CHO*Institute of Environmental Sciences & Department of Environmental Sciences, Hankuk University of Foreign Studies, KOREA 449-791. *Corresponding author: [email protected]

The members of genus Acidobacterium are previously uncultured bacteria. Although the genus includes three cultured strains, Acidobacterium spp. were still unknown bacteria in respect of their physiology and ecological roles. We studied the global distribution patterns of Acidobacterium sp. using subgroup- specific primers (31F, A,Y, O, G). Soil DNAs were extracted from global soil samples and acidobacterial 16S rRNA genes were amplified, and analyzed by terminalrestriction fragment length polymorphism analysis (T-RFLP). Twenty five samples (89%) showed positive PCR results for genus Acidobacterium, and acidobacterial subgroup A. T-RFLP patterns were consistent with the PCR results, and unpredicted T-RFs, which representas-yet-unpublished acidobacterial 16S rRNA gene sequences, were also found. This study showed that the genus Acidobacterum and Acidobacterum-like bacteria are ubiquitous in soil, and suggested that unrevealed high diversity resides in this bacterial group.

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Analysis of the Microbial Community in the Culture Enriched with Heavy Metals Joung Soo LIM2, Kyung Hwan OH1, Young Hoon KIM1, Jae Youn LEE1, Eun Young LEE2 and Keun KIM*1

1Dept. of Bioscience and Biotechnology, The University of Suwon, 445-743. 2Dept. of Envrionmental Engineering, The University of Suwon, 445-743. *Corresponding author: [email protected]

Heavy-metal absorbing microorganisms were enriched from the sediment of P lake. Heavy metal mixtures containing of Cu, Zn, Pb, Cr,and Ni was spiked to the culture medium inoculated with microorganisms.And then the concentrations of heavy metals were increased to 100 ppm step by step by 10 fold serial subculturing. Over ninety percent removal of heavy metals were obtained by the enriched culture. The microbial community of P lake was analyzed by DGGE (denaturing gradient gel electrophoresis). The enriched culture was changed from various strains to the one dominant strain by increased concentrations of different heavy metals. From this result, it was expected that the dominant strain can beapplied to the microbial refinement of lake polluted by various heavy metals.

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Factors Control Prodigiosin Biosynthesis of the Marine Bacterium Hahella chejuensis Soon-Kyeong KWON1,2, Yon-Kyoung PARK1, Choong-Min RYU1, Seung-Hwan PARK1 and Jihyun F. KIM*1,2

1Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB). 2Functional Genomics Field, School of Science, University of Science and Technology (UST). *Corresponding author: [email protected]

Hahella chejuensis produces a red pigment which has the lytic activityagainst red-tide organisms. The red pigment was identified as prodigiosin. Based on genome sequence information of H. chejuensis andfunctional analysis, the genomic region involved in biosynthesis of the red pigment was elucidated. We named this gene cluster hap after Hahella prodigiosin. To search for the factors that control the productionof prodigiosin, a plasmid library of the H. chejuensis was constructed and transformed into E. coli clones carrying the hap. Plasmids affecting the prodigiosin synthesis were isolated and transformed again into the E. coli clones. Suspected fifty-three clones were selected and sequenced. Seven of them contained hap genes or genes located next to the hapcluster, while others were expected to encode genes involved in the two-component signaling system. These genes are likely candidates for key regulators of prodigiosin synthesis. By uncovering factors involved in prodigiosin synthesis, not only can we give information on how prodigiosin synthesis pathway is controlled, but induce over-expression of prodigiosin in a heterologous host like E. coli or in H. chejuensisitself.

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Monitoring Microbial Densities of the Korean Cattle (Hanwoo) Farm-houses in Gyeongsanbuk-do Heeyoung PARK1, Eunseo PARK2, Boyoon SEO3, Daehyun KIM3, Tahkjoong SONG3 and Yongho KHANG*1

1School of Biotechnology, Yeungnam University, Gyeongsan, 712-749. 2Dept. of Applied Microbiology, Yeungnam University, Gyeongsan, 712-749. 3Dept. of Applied MIcrobiology, Yeungnam University, Gyeongsan, 712-749. *Corresponding author: [email protected]

The microbial densities of Korean cattle(Hanwoo) farm-houses locatedin the 20 cities of Gyeongsangbuk-do were investigated. Samples were collected from the air inside the farm-houses, the drinking water for Hanwoo cattles, and the bottom soils inside the farm-houses. Colonies grown on LB agar plates were counted automatically by use of Gel/Chemi Doc System(Bio-Rad, USA). The ranges of microbial densities were appeared as follows: 2.8~15.0 cfu/㎠ of plate in air; (0.6~8.4)x106 cfu/㎎ of soils, (0.1~8.7)x103cfu/㎖ of drinking water. PCR amplication and DGGE analysis were performed to detect pathogenic microorganisms such as Campylobacter jejuni, Campylobactercoli, Clostridium perfringens, Escherichia coli O157, Listeria spp., Salmonella sp., M. avium subsp. paratuberculosis, Yersinia spp..

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Effects of Cr(VI), CN, Hg and As on Electricity Generationof Electrochemically-active Bacteria in a Microbial Fuel CellMia KIM1, Gil Ju JIN1, Won Hui CHO1, Dong Kwon LEE1, Seok Min YOON2, Hyung Joo KIM2 and Moon Sik HYUN*1

1Research Institute, KORBI Co. Ltd., Seoul, 136-791. 2Dept. of Microbial Engineerging, Konkuk University, Seoul. *Corresponding author: [email protected]

Effect of toxic materials including Cr(VI), CN, Hg and As on electrochemically-active bacteria (EAB) in a microbial fuel cell (MFC) was investigated. Current generation characteristics of EAB were conveniently monitored using a microbial fuel cell format and a computer controlled potentiometer. When the Cr(VI) was added to the MFC, a rapid decrease in current was observed. Same result was observed when the Hg was added to the EAB in the MFC. In the presence of CN and As, however, increases in current were observed. The inhibition ratios were generally proportional to toxic substances concentration. The combined effects of toxic substances were also examined by adding various chemical combinations of Cr(VI)+Hg, As+CN and Cr(VI)+CN to the MFC. Current decreases were observed with all combinations regardless of whether an individual substance caused an increased current. More additive effects were observed at higher concentration. These experimental results showed that the microbial fuel cells can be used as biomonitoring biosensors because they are sensitive to current changes induced by known toxic substances.

G-14

Characterization of Serratia sp. K1RP-49: Natural ChelatorProductivity, Heavy Metal Resistance, and Plant Growth-Promoting Ability So-Yeon KOO and Kyoung-Suk CHO*Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, 120-750. *Corresponding author: [email protected]

In heavily industrialized areas, soil sites are contaminated with high concentrations of heavy metals. These pollutants are highly accumulated to the human body through the food web and cause serious diseases. Toremove heavy metals from the soil, a potential strategy is the environmental friendly and cost effective phytoremediation. Serratia sp. K1RP-49 was isolated from the rhizosphere of the barnyard grass(Echinochloa crus-galli) which had grown in oil contaminated soil, and its properties were studied. The Serratia sp. K1RP-49 could produce various organic acids (natural chelator, NC) such as oxalic acid (138.0 mg/mL), maleic acid (78.9 mg/mL), citric acid (0.160 mg/mL), malic acid (1.77 mg/mL), formic acid (0.088 mg/mL), acetic acid (3.40 mg/mL), and succinic acid (6.93 mg/mL). The isolate could grow in the 10% LB-agar medium supplemented with Cd2+ (2.0 mM), Zn2+ (4.0 mM), Cr6+ (5.0 mM), Pb2+ (7.5 mM), or Cu2+ (1.0 mM) respectively. This bacterium could produce a indole acetic acid (IAA, phytohormone) approximately 17.09 mg/L, display 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and produce a siderophore(s). These properties of the Serratia sp. K1RP-49 indicate that this bacterium can enhance the growth of plants in heavy metal contaminated soil.

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Production of Antifungal Compounds as Chitinase and Cellulase by Isolated Soil Microorganisms Jong-Ok JANG1, Jung-Bok LEE1, Chung-Sig CHOI2, Se-Won LEE1 and Gi-Seok KWON*1

1Major in Medical Plant Resources, School of Bioresource Sciences, Andong National University, Andong, Korea. 2HansBio Co., HansBio Co.,BI center 303, Andong National University, Andong, Korea. *Corresponding author: [email protected]

Chemical pesticide applied to increase plant production is a major input in agriculture. However they are costly, can cause environmental ruin and may induce pathogen resistance. Therefore, biopesticide have become an important approach to sustainable agriculture. Chitin and cellulose are the most abundant and renewable biopolymer on the Earth and they play major roles in degrading fungal cell walls. Our studies showed that chitinase and cellulase producing microorganisms were screened from soil samples and its production condition. Crude fungicideobtained from the culture broth of these strains grown in a defined medium containing 0.4% colloidal chitin minimal or 0.5% carboxymethylcellulose (CMC). Three strains were found to produce appreciable amount of both chitinase and cellulase for 3 days at 30℃ and 200rpm. Yellow halo was identified when the culture media supernatant was loaded onto 0.5% CMC agar plate using paper disc method. The results are 899, 919, 916 units and suggest that enzymes produced by three strains prevent of plant diseases by inhibiting growth of phythopathogenic fungi. Therefore, we should determination of enzymes and purification.

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374 www.kormb.or.kr

Acidobacterial Sub-division is Dominant and Active Member of Microflora in Rhizosphere Soil SanG-Hoon LEE, Jae-Chang CHO and Jae-Chang CHO*Institute of Environmental Sciences & Department of Environmental Sciences, Hankuk University of Foreign Studies, KOREA 449-791. *Corresponding author: [email protected]

Culture-dependent and culture-independent molecular phylogenetic surveys were carried out on the bacterial community in chestnut rhizosphere soil. Clone libraries were constructed from the small-subunitrRNA genes amplified directly from the soil DNA and ribosomal RNA by PCR and reverse transcription (RT)-PCR, respectively. While most ofcultured bacteria were phylogenetically affiliated with division of Firmicutes, the cloned 16S rRNA gene sequences were primarily affiliated with one of three groups: division of Fibrobacter (ca. 60%), Proteobacteria (ca. 20%), and Firmicutes (ca. 5%). Acidobacteria-relatedsequences belonging to Fibrobacter division were the dominant member of the clone library generated by PCR as well as RT-PCR, and three deep branches, which mignt be novel clases-level taxaa in Acidobacteriasubdivision, were found. Although Acidobacteria subdivision has only one cultured member, Acidobacterium capsulatum, this study shows that bacteria belonging to Acidobacteria subdivision are the dominant and active members of microflora in the chestnut rhizosphere soil, suggestingthey might play an important role in rhizosphere soil ecosystem.

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Toxicity Assessment of Mmethyl tert-butyl ether (MTBE), Tert-butyl alcohol (TBA) and Formaldehyde (FA) by Psudomonas aeruginosa Wonsil CHO1, Hee Wook RYU2 and Kyung-Suk CHO*1

1Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, 120-750. 2Dept. of Chemical and Environmental Engineering, Soongsil university, Seoul, 156-743. *Corresponding author: [email protected]

Toxic effect of methyl tert-butyl ether (MTBE) and its metabolites such as tert-butyl alcohol (TBA) and formaldehyde (FA) on the growth rate of Psudomonas aeruginosa under the conditions of single, dual and triple mixtures was examined to propose the novel method for quantitatively estimating toxicity of these chemicals. The inhibition effects on the growth of P. aeruginosa by MTBE and its metabolites were evaluated using specific growth rate and growth inhibition index. Each compound inhibited the growth of P. aeruginosa noncompetitively,and the toxic effect was FA > TBA > MTBE. The inhibition constant (KI) of MTBE, TBA and FA is 1200, 375 and 2.5 mM, respectively. Based on KI of MTBE, the reduced inhibition constants (αi) for MTBE,TBA and FA were 1, 0.316 and 0.002, respectively. The synergic inhibition effect on the growth rate was significantly observed in the condition of binary or thrinary mixtures of MTBE, TBA and FA.

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Analysis of DEHP [Di-(2-EthylHexyl)-Phthalate] Biodegradation Using a Bacterial Cell Array Chip Jin Hyung LEE2, Javed H. NIAZI3, Byoung-In SANG3 and Man Bock GU*1

1College of Life Sciences and Biotechnology, Korea University. 2Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST). 3Hazardous Substances Research Centre, Korea Institute of Science and Technology (KIST). *Corresponding author: [email protected]

In this study, we present the application of the bacterial cell array chips for analysing biodegradation of DEHP [Di-(2-EthylHexyl)- Phthalate]. An anaerobic microbial culture system was adopted for biotransformation of DEHP [di-(2-ethylhexyl)-phthalate], a well known endocrine disrupting chemical and its byproducts were identified by using GC/MS analysis. Toxicity change due to the biodegradation was analysed by using bacterial cell array chip. The bacterial cell array chip is composed of numerous stress- or chemical-specific recombinant bioluminescent bacteria. Recombinant bioluminescent bacteria have different chemical- or stress-specific promoters fused with bacterial lux genes, thus enables toxicity analysis through their specific bioluminescentresponse. From the analysis, it was found that DEHP was degraded to phthalic acid as intermediate after 20-25 days. Regarding to toxicity, DEHP causes no significant toxic effect to E.coli although it is highly toxic to human. However, toxicity was increased by the anaerobic biodegradation process, especially membrane and protein damages. This study shows an application example of bacterial cell array chip biosensors in a field of environmental sciences and engineering.

G-19

Screening of PAHs-Resistant Plants for Rhizoremediation(Ⅰ)-Germination Test Jung Hee LEE, Da Hee YOON and Kyung-Suk CHO*Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, Korea, 120-750. *Corresponding author: [email protected]

For bioremediation of soil contaminated with polyaromatic hydrocarbons (PAHs), it is efficient to use PAHs-degrading-bacteria and plants simultaneously. Plants that can be used for this rhizoremediation should be somewhat resistant to PAHs. To select PAHs-resistant plants, 14 species of plants were preliminarily selected through literature survey.The germination rates of the plant seeds in PAHs-contaminated soil werecompared as follows. Soils were artificially contaminated with phenathrene 500mg/kg-soil and pyrene 250mg/kg-soil. Since PAHs (phenanthrene and pyrene) in acetone were added into soils, the soil samples added only acetone were prepared to investigate the effect of acetone on the germination rates. These soil samples were stabilized for 7 days, then 10 seeds were sown and cultivated at 20~25℃ for 30 days.Germination rates weren’t influenced by acetone. Four kinds of plants (kidney bean, corn, perennial ryegrass, barnyard grass) showed both relatively high germination rates and continuous growth in PAHs-polluted soils.

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Epidemiology and Molecular Characterization of Enterotoxigenic Escherichia coli Serogroup O6 Strains Isolated in Korea by Pulsed Field Gel Electrophoresis Jong-Chul KIM, Seung-Hak CHO, Hyun-Ho SHIN, Jae-Hwa LEE, Mi-Sun PARK and Bok Kwon LEE*Division of Enteric Bacterial Infections, Center for Infectious diseases, National Institute of Health (NIH), Nokbun-dong 5, Seoul 122-701, Korea. *Corresponding author: [email protected]

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence associated factors. In this work we show that theanalysis of band patterns generated by pulsed field gel electrophoresis(PFGE) of digested chromosomal DNA can be used detect genetic diversity among 37 ETEC strains expressing identical phenotypic traits. Nineteen different electrophoretic patterns(about 20 bands each) were distinguished. In most cases, strains with the same PFGE type belonged to the same serotype and similar pattern of antibiotic susceptibility. In this work we have shown that O6 ETEC(n=21) isolates from different area share a commom genetic origin. The broad range of PFGE types found in this study demonstrates the occurrence of similar genetic types among the ETEC O6 strains spread throughout Korea.

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Application of Real Time Quantitative PCR for Monitoringa BTX-degrading Rhodococcus sp. EH831 in biofilter Eun-Hee LEE2 and Kyung-Suk CHO*1

1Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, 120-750. 2Dept. of Environmental Science and Engineering, Ewha WomansUniversity, Seoul, 120-750. *Corresponding author: [email protected]

Biofiltration technology that is an economical and environmental- friendly method for the treatment of air pollutants has been widely used for the removal of benzene, toluene, and xylene (BTX). Because BTX removal efficiency was significantly influenced by the microbial activity in biofilter, it is very important to monitor the microbial behavior during biofilter operation. Rhodococcus sp. EH831 was isolated as an effective degrader of BTX, and it was proved as an applicable bacterium being able to enhance biofilter capacity. In this study, the real time quantitative PCR (RT-Q-PCR) methodology was investigated for the qualitative and quantitative monitoring of Rhodococcus sp. EH831 in biofilter. The effective method was optimized to recover genomic DNA from polyethylene parking materials, and the specific probe, that can selectively detect this bacterium, was designed. Moreover, the sensitivityof the probe was analyzed and derived relationship between threshold cycle (Ct ) and cell concentration of the bacterium. As a result, the sensitivity of probe was very high and correlation factor between Ct and cell concentration was very useful as 3.9.

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Growth Promotion of Chlorella by Brevundimonas nasdaeKyung-Woo JE, Sang-Eun JUNG and Tae-Jin CHOI*Department of Microbiology, Pukyong National University, Busan, Korea, 608-737. *Corresponding author: [email protected]

Chlorella is unicellular green alga which has been widely used in aquaculture and food industry. It can be inexpensively cultured in large scale because it requires only a limited amount of minerals and sunlight.Chlorella also has been mass cultured for basic research and have servedas model organisms for pioneering plant physiological and biochemical studies. Although symbiotic association of bacteria with microalgae has been suggested, there are limited information on the bacteria and their function in the association. During establishment of axenic culture of Chlorella ellipsoidea, eight bacteria have been isolated from the algal culture, and have been tested for their growth promoting activity. A strain named as P1 showing the highest activity was further characterized. The P1 strain was identified as Brevundimonas nasdae by the Vitek bacterial identification system and sequence analysis of the 16srRNA. The optimum conditions for B. nasdae were 25℃, pH 8.0, NaCl 0%. B. nasdae revealed the presence of bacteria on the surface of the algae and wrinkling of the cell surface. HPLC analysis of the bacterial culture filtrate showed the appearance of a new peak of polysaccharides.

G-23

Degradation of Organochlorine Pesticide Endosulfanin Artificial Slurry Reactor Jung-Bok LEE1, Ho-Yong SOHN2, Jong-Sik KIM3, Min-Seb JO1, Chung-SigCHOI4 and Gi-Seok KWON*1

1Major in Medical Plant Resources, School of Bioresource Sciences, Andong National University, Andong, Korea. 2Dept. of Food and Nutrition, Andong National University, Andong, Korea. 3Dept. of Biological Science Andong NationalUniversity, Andong, Korea. 43HansBio Co., HansBio Co.,BI center 303, Andong National University, Andong, Korea. *Corresponding author: [email protected]

Endosulfan has been ubiquitously detected in the atmosphere, soils, sediments, surface waters, rainwaters, and foodstuffs. Endosulfan comprised two parent isomers, the α- and β-endosulfan the ratio of technical endosulfan is about 7:3 and both are highly toxic to aqueous organisms. Endosulfan has been degraded by attack at the sulfite group via oxidation and/or hydrolysis to form the toxic endosulfan sulfate and the nontoxic endosulfan diol, respectively. While the endosulfan sulfate is stably maintained in soil and aqueous systems, endosulfan diol could further degrade to endosulfan ether, endosulfan hydroxyether and endosulfan lactone. This study assesses the role of the microorganism presents in the slurry reactor in the degradation of endosulfan and its metabolites in the soil and water environment. Klebsiella oxytoca KE-8 communites in the slurry reactor were spiked endosulfan concentrations (5mg/kg) for 4-day period and then monitored for 50 days. This study showed that K. oxytoca KE-8 could contribute in the degradation of endosulfan in the slurry (soil and water). [This work was supported by a grant(Code:2005031-034-421-006-01-00) from BioGreen 21 Program, Rural Development Administration, Republic of Korea]

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Use of 16S rRNA Gene Terminal Restriction Fragment Analysis to Evaluate the Impact of Geographic Location andPollution Level of Hydrocarbon on the Microbial Diversity of Soil Dae-Hyun CHO, Kyung-Hwa BAEK, Byung-Hyuk KIM, Young-Ki LEE, Byung-Dae YOON and Hee-Sik KIM*Environmental Biotechnology Research Center, KRIBB, Daejeon, 305-333. *Corresponding author: [email protected]

The importance and relevance of the geographical origin and the pollution level of hydrocarbons to microbial diversities of the soil samples were evaluated by using Terminal restriction fragment length polymorphism (t-RFLP). Several hydrocarbon-contaminated soils from same or different geographical locations in Korea were used in the study.The pollution level of petroleum hydrocarbon in soil samples was measured by gas chromatography (GC). t-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis for the bacterial community structure was performed by Ward's method using Jaccard distance and Hellinger distance. Principal components analysis (PCA) of TRF was also performed to characterize the associations among samples. PCA showed the difference of soil cluster according to locations and pollution level of soil samples. The soil communities according to locations were in close proximity but, those according to pollution level were not. The results of dendrogram analysis also corroborated the above PCA results. The results suggested that the geographical origin rather than the pollution level of the soil sample might be more important in determining the bacterial diversity within microbial communities.

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An Antifungal Activity of Streptomycetes sp. Strains againstPhytophthora cactorum Sanghyun LEE2, Joo-Hoon PARK1 and Yongsub YI*1

1Dept. of Herbal Medicine, Hoseo University, Chungnam 330-713. 2Department of Forest Diseases and Pests Korean Forest Research Institute. *Corresponding author: [email protected]

To measure antifungal activity of isolated strepyomycetes against Phytophthora cactorum, the isolated strepyomycetes were cultured on PDA plate inoculated with the pathogen. Growth inhibition of the pathogen was determined. Strains showing antifungal activity against P. cactorum were collected from soil of Chinju and Gyeongju area, Korea. Out of fifty strains, six strains showed antifungal activity in vitro againstP. cactorum. For strain identification, morphological characteristic and 16S rDNA sequences were determined. For the future work, in vivo test will be conducted to poted Aralia elata trees.

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Toluene-induced Accumulation of Trehalose by Pseudomonas sp. BCNU 106 through the Expression of otsAand otsB Homologs Hye Jung CHOI1,2, Min A LEE1,2, Seon-A KIM1,2, Su Dong CHO1, Ja Young MOON3, Domg-Wan KIM4, Jeoung Yoon SEO5, Yong Kee JEONG6, Dae-OhKWACK7, Jong Soo HEO8, Young-Guen LEE9 and Woo Hong JOO*1,2

1Institute of Genetic Engineering,. 2Department of Biology,Changwon National University, Changwon 641-773, Korea. 3Institute of Genetic Engineering, Department of Biochemistry and Health Sciences,. 4Institute of Genetic Engineering,Department of Microbiology,. 5Department of Environmental Engineering, Changwon National University, Changwon 641-773, Korea. 6Division of Applied Biotechnology, Dong-A University, Busan 604-714, Korea. 7Division of Biology Education, Gyeongsang National University,. 8Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea. 9School of Applied life Science, Pusan National University, Miryang 627-706, Korea. *Corresponding author: [email protected]

The objective of the present study was to investigate toluene-induced accumulation of trehalose in toluene-tolerant bacterium Pseudomonas sp.BCNU 106. Intracellular trehalose content and trehalase activity were determined in Pseudomonas sp. BCNU 106 by using cell-free extracts. Trehalose-biosynthetic genes were cloned and sequenced. Their RNA levels were also investigated by reverse transcriptase-PCR analysis. It was shown that mRNA levels of trehalose-biosynthetic trehalose-6- phosphate synthase and phosphatase genes peaked at 4 h after exposure to toluene and thereby trehalose levels peaked after 12 h of cultivation with 10%(v/v) toluene.In toluene-tolerant bacterium Pseudomonas sp. BCNU 106, trehalose was accumulated through the toluene-induced expression of trehalose-biosynthetic enzyme genes after exposure to toluene. Accumulation of high level of intracellular trehalose is responsible for the toluene tolerance in a toluene-tolerant bacteria.

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Advanced Remediation of Oil-contaminated Soil Using a Corn Plant and a Rhodococcus sp. U-Ri KO2, Sun Hwa HONG1, Jae Joon YOO2, Sung Hyun KIM3, Hye-Lim PARK4 and Kyung-Suk CHO*1

1Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, Korea, 120-750. 2Gyeonggi Science High School, Korea. 3Department of Life Science, Ewha Womans University. 4Department of Environmental Science and Engineering, Ewha Womans University. *Corresponding author: [email protected]

We researched on the advanced remediation of oil-contaminated soil through the exploration of bacterial symbiosis with plants. An oil-degrading rhizobacterium, Rhodococcus sp. 412, was isolated from oil- contaminated soil, and a plant species, Zea mays, being known as having ability to disintegrate oil was selected. Zea mays was seeded in oil-contaminated soil with or without Rhodococcus sp. 412, and then thefollowing factors were measured; seed germination rate, growth rate andbiomass of Zea mays, moisture content (MC), organic matter (OM), pH, soil microbial activity (Dehydrogenase ability: DHA), and the concentration of total petroleum hydrocarbon. Bacterial community was characterized using 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) fingerprinting method. After cultivating for 3 weeks, the growth of Zea mays in the contaminated soil inoculated with Rhodococcus sp. 412 was better than that in the soil without this bacterium. MC, OM, pH, and DHA were relatively higher in the contaminated soil with the bacterium and corn plant. These results indicate that the simultaneous use of corn plant and Rhodococcus sp. 412can give beneficial effect to the remediation of contaminated soil

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Preconcentration and Optical Detection of Copper (II) Usinga Copper Chelating Bead-packed Microfluidic Device with a Fluorescent Chemosensor Jeong-A KIM, Eun Jin JUN, Juyoung YOON, Seong-won NAM and Sungsu PARK*Division of Nano Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Ku, Seoul 120-750. *Corresponding author: [email protected]

Copper is an essential element needed to keep the skeletal, reproductive, and nervous systems healthy. However it causes gastrointestinal symptoms such as nausea, diarrhea, and vomiting in human when copper concentration reaches 4.0–6.0 mg/L in drinking water. Thus, the detection method of copper concentration is one of the prerequisite to control of Cu2+ in drinking water. We previously synthesized a fluorescent chemosensor effectively recognizing Cu2+ in solutions such as buffer and biological samples (0.5-50μM). In this study, for the practical applications of the fluorescent chemosensor, we have developed a microfluidic device for the fluorescent detection of Cu2+ in ppm range. In details, the microfluidic system was fabricated with a glass slide and polydimethyl siloxane, and copper-chelating alginate beads (BioScicence) were packed in the preformed device. The beads in the device were used to preconcentrate varied Cu2+

concentrations, and the preconcentrated Cu2+ was optically detected by eluting the preconcentrated metal ions with the fluorescent chemosensor in the microfluidic system. It is suggested that the microfluidic platform device is ready to develop for a optical biosensor which can preconcentrate and detect Cu2+ in water and/or other environmental samples.

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Biodegradation of 1,4-dioxane by Two Bacterial Consortia Isolated from Pre-exposed Activated Sludge Dong Yerp SUNG, Do Yun SHIN and kyoungphile NAM*School of Civil, Urban and Geosystem Engineering, Seoul National University, Seoul, 156-093. *Corresponding author: [email protected]

Two bacterial consortia who degrade 1,4-dioxane have been successfully isolated from pre-exposed activated sludge. 1,4-dioxane, which is resistant to microbial breakdown probably due to its ether bond,is classified as a probable human carcinogen by US EPA. Activated sludge continuously receiving 1,4-dioxane was enriched in an aqueous basal medium containing either 5 mM of 1,4-dioxane or tetrahydrofuran(THF) as a sole carbon and energy source. One consortium (designated as Dx 2) was obtained from 1,4-dioxane-added enrichment culture and the other (designated as Dx 4) from THF-added culture. 1,4-dioxane- utilizing bacterial colonies were successfully colonized on a selective agar medium in 4-5 days at 30°C. Each consortium consisted of two bacterial members. Analysis of 16S rDNA sequence of each consortium member and following BLAST search with NCBI database revealed thatthe consortium members were Microbacterium sp. and Klebsiella sp. for Dx 2 and Sphingomonas sp. and Aminobacter sp. for Dx 4. Their biodegradation kinetics of 1,4-dioxane and the physiological role of eachbacterium as a consortium member is under study.

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Induction of a Glucose-resistant Mutant of Arthrospira platensis Myoung-Sook BAE1, Gang-Guk CHOI1, Jeseop PARK2, Bokjun PARK2, Chi-Yong AHN1 and HEE-MOCK OH*1

1Environmental Biotechnology Center, Korea Research Institute of Bioscience andBiotechnology, Daejeon 305-333, Korea. 21Biotechnology Laboratory, R&D Center, Daesang Corporation, Gyeonggi-do 467-813, Korea. *Corresponding author: [email protected]

Arthrospira (Spirulina) platensis is one of the commercially important filamentous cyanobacteria. It can be used as feed and additive food around the world. A mutant of Arthrospira platensis PCC 9108 was isolated using a UV treatment, which was able to grow in SOT medium containing 4% glucose. The mutant designated as A. platensis M9108 showed an improved efficiency of glucose utilization because the mutantwas resistant to high glucose concentration. Although the specific growthrates of A. platensis (PCC 9108) and its mutant (M9108) were similar under autotrophic condition, the specific growth rate of A. platensisM9108 (0.025 h-1) was 2.2-fold higher than that of A. platensis PCC 9108 (0.013 h-1) under heterotrophic condition. The productivity of A. platensis M9108 under heterotrophic condition was 1.6 fold higher than that of A. platensis PCC 9108. In autotrophic and mixotrophic cultivation, however, the productivity of the mutant was slightly higher than that of A. platensis PCC 9108. These results showed that the mutantpossessed the facility to assimilate and metabolize glucose efficiently under heterotrophic condition.

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Screening of PAH-tolerant plants for rhizoremediation (Ⅱ) - Growing test in PAH-contaminated soil So-Yeon KIM, Min-Kyung SONG, Sun Hwa HONG and Kyung-Suk CHO*Department of Environmental Science and Engineering, Ewha Womans University,Seoul, 120-750. *Corresponding author: [email protected]

Polycyclic aromatic hydrocarbons (PAHs) are indecomposable contaminants that come from natural and anthropological sources. Rhizoremediation using plants and rhizobacteria simultaneously is one ofattractive methods for the remediation of PAH-contaminated soil. In thisstudy, plants having resistant to PAHs, were selected by comparing the growth rates in PAH-contaminated soil. Echinochloa crus-galli(L.) Beauv., Lolium perenne L., Zea mays, and Phaseolus vulgaris var. humilis were seeded in soil contaminated with phenanthrene (phe) and pyrene (pyr) in dimethylformamide (DMF). The finial concentrations of PAHs in soil were phe 250 mg/kg-soil + pyr 125 mg/kg-soil, phe 500 mg/kg-soil + pyr 250 mg/kg-soil, and phe 750 mg/kg-soil + pyr 375 mg/kg-soil. Soil contaminated with only DMF was prepared to estimate the effect of DMF on the growth of plants. The germination and growthrates of plants in soils contaminated PAHs or DMF was compared with those in non-contaminated soil. Zea mays and Phaseolus vulgaris var. humilis grew well with 66∼100% of germination rate in soil contaminated with PAHs and DMF, indicating that these plants have relatively tolerance to PAHs.

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Bacterial Communities and Diversity During Composting Process by 16S rDNA Sequences Analysis Kye Man CHO1, Sun Mi LEE1, Su Young HONG1, Renukaradhya K MATH1, Yong Hee KIM1, Sun Joo HONG1, Hoon KIM3 and Han Dae YUN*1,2

1Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Korea. 2Institute of Agriculture and Life Sciences, Gyeongsang NationalUniversity, Chinju 660-701, Korea. 3Department of Agricultural Chemistry, Sunchon National University, Suchon, 540-742, Korea. *Corresponding author: [email protected]

The bacterial diversity and their communities during the composting process was examined using a PCR-based approach. The 16S rDNA sequences were amplified from the compost which was composted for following number of days (0 day, 1 day, 3 day, 7 day, 10 day, 14 day) and were cloned likely to be represent different bacterial niches. Base on temperature changes, the composting process could be divided into mesophilic (0 day, 1 day), thermophilic (3 day, 7 day, 10 day), and cooling-down stages (14 day). The predominant species at the different composting stages were: Agrobacterium tumefaciens (0 day, 40.0%), Flexibacter tractuosus (1 day, 20.0%), Bacillus thermocloaceae (3 day, 20.0%), Bacillus humi (7 day, 70.0%), Bacillus humi (10 day, 85.0%), and Bacillus humi (14 day, 82.5%). The predominant bacterial group waschanged from Agrobacterium tumefaciens in the meophilic stage (0 day) to Bacillus humi in the cooling-down stage (14 day). Bacillus humi was first appeared on the 3rd day of composting. This change in bacterial communities may be significant for the composting process. Especially, Bacillus humi which plays a very important role in the composting process.

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Accumulation of High Concentration of cis-Toluene Dihydrodiol by Pseudomonas putida DOT-T1E While Growing on Toluene Jiyoung SEO2 and Hor-Gil HUR*1

1Center for water Research of GIST, Oryong-dong, Buk-gu, Gwangju 500-712, Korea. 2Department of Environmental Science and Engineering, Gwangju Instituteof Science and Technology (GIST), Oryong-dong, Buk-gu, Gwangju 500-712, Korea. *Corresponding author: [email protected]

Pseudomonas putida DOT-T1E, a rifampin-resistant derivative of strain DOT-T1E, is highly resistant to solvents, with the logarithm of thepartition coefficient in a mixture of octanol and water being higher than 2.5. This aromatic compound serves as a carbon and energy source of the bacteria. The metabolic route for the mineralization of toluene by DOT-T1E is the Tod pathway, in which toluene is oxidized to cis-toluene dihydrodiol, which in turn is channeled to Krebs cycle intermediates via a meta-cleavage pathway. Burkholderia cepacia strain G4 contains the genes encoding for toluene 2-monooxygenase on a degradative plasmid pTOM which catalyzes the first 2 steps in which toluene is metabolized. Toluene 2-monooxygenase is three component enzyme systems and along with reduced NADH carries out consecutive hydroxylations at the ortho and meta positions of toluene under aerobic condition. P. putida strain, which contains a defect in the structural gene for cis-toluene dihydrodiol dehydrogenase, accumulates cis-dihydrodiol products in the culture medium containing toluene. Conventional chemical processes can not be used to inexpensively synthesize cis-dihydrodiols which are very versatile synthons to make sugars, alkaloids, pharmaceuticals and chiral compounds.

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Comparative Analysis of Microbial Communities by Denaturing Gradient Gel Electrophoresis during 2003 - 2005in Daechung Reservoir So-Ra KO, Chi-Yong AHN, Seung-Hyun JOUNG and Hee-Mock OH*Environmental Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea. *Corresponding author: [email protected]

The change of microbial communities during cyanobacterial bloom was comparatively analyzed by both microscopy and 16S rDNA PCR-DGGE in Daechung Reservoir during 2003 - 2005. Traditional morphological analysis showed that Cyanophyceae dominated microbial community in the cyanobacterial bloom. The genera of Microcystis, Oscillatoria, Phormidium, and Anabaena were dominant. We used DGGE profiles and phylogenetic trees to analyze the community structure and diversity of the predominant phytoplankton community. The DGGE banding patterns demonstrated that the most frequent bands were identified as Microcystis spp. during the experimental periods, Oscillatoria spp. were also identified and dominated on September 2, 2003 and September 14, 2004 whereas Anabeana sp. was showed a peakon September 21, 2005. The members of the Proteobacteria (α, β and γ subdivisions) was detected the DGGE profiles only in 2005. Over 50% bands in DGGE were identified to be uncultured species, suggestingthat further studies needed in this area. Molecular phylogenetic analysis indicated that the presence of variant organisms, several of which were related to the cyanobacterial bloom.

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Selection of the Functional Microbial Community from Diesel-Contaminated Soil Dae-Hyun CHO1, Kyung-Hwa BAEK1, Seung-Goo LEE2, Jung-Hoon YOON2, Jae-Jun SONG3, Byung-Dae YOON3 and Hee-Sik KIM*1

1Environmental Biotechnology Research Center, KRIBB, Daejeon, 305-333. 2Systems Microbiology Research Center. 3Molecular Bioprocess Research Center.*Corresponding author: [email protected]

The impacts of microbial community (MC) can be felt in every aspectof life and in every corner of the globe. However, there is only small information and technique to obtain the functional microbial community (FMC) and to preserve the MC. MC is naturally present in our environment, soil, sediment, ocean, etc, while FMC is endowed with thespecific function and composed with the filtered microbes from natural MC. In this work, we obtained the FMC degrading diesel and developed the technique for the preservation of MC. Microbial diversity in MC wasanalyzed by DGGE and t-RFLP, and the degradation activity of MC against diesel was measured by GC. FMC highly degrading diesel was selected from the diesel-contaminated soil by enrichment culture technique. FMC showed the more stable and reduced microbial composition and higher degradation activity to diesel oil than natural MCof soil sample. Zeolite was used as the adsorbent for the preservation of FMC. Zeolite enhanced the maintaining of microbial diversity in FMC. After 6 months" preservation at -190℃ in DMSO, it was confirmed that FMC maintained the degrading activity and microbial diversity more than 90%. The FMC highly degrading diesel was selected from soil and the effective method for the preservation of MC was developed.

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Bacteriostatic Effects of Dryopteris crassirhizoma Nakai Against Propionibacterium acnes Hyun Ju KIM, Shin Wook CHOI and Chang Soon YOON*RADIANT INC, #207, Bioindustry Innovation Center, Hi-Tech Venture Town, 198-53 Hupyeong, Chuncheon, Kangwon, Korea, 200-957. *Corresponding author: cro@eradiant co.kr

The development of acne appears to be influenced by not only genetic and environmental factors but also unbalanced hormone secretion, leading to hyperkeratinization, activation of Propionibacterium acnes andskin inflammation. Although various acne remedies have been used, most of them shows various side effects such as allergy and skin irritation. Recently, accumulated studies have been focused on screening natural anti-acne products without such side effects. Dryopteris crassirhizoma N.a perennial plant, is known to have various physiological effects including anti-bacterial and fungal effects. Therefore, in the present study, anti-acne effects of Dryopteris crassirhizoma N. against Propionibacterium acnes was examined for cosmetic application using minimum inhibitory concentration (MIC) and paper disk diffusion assay.The MIC value of Dryopteris crassirhizoma extracted with ethanol is 0.0078 mg/ml compared with that of triclosan (0.004 mg/ml) as a control, implying strong anti-acne effect even in crude extracts. This anti-acne effect is confirmed by paper disk diffusion assay. The inhibitory size of the extract is 34 mm in the presence of 500 ug/ml. The ethanol extract of Dryopteris crassirhizoma is stable in high temperatures (70, 80, 90, 100, 121℃) and pH2-pH11.

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Development of Bio-pesticide with Slow-releasing System for the Prevention of Soil-borne Disease Kyung Hwan OH1, Jae Pil CHOI2, Young Kwon KIM2 and Keun KIM*1

1Dept. of Bioscience and Biotechnology, The University of Suwon, 445-743. 2Institute of Biotechnology, Korea Bio co., Ltd, 445-964. *Corresponding author: [email protected]

Club root caused by Plasmodphora brassical is one of the major cabbage diseases known in Korea. Since Plasmodphora brassical is artificially unculturable in a laboratory, Phithium sp. which is culturable and has similar characteristics to Plasmodphora brassical was used to screen for the microbial strain having strong antifungal activity. One microbial strain isolated from soil showed strong antifungal activity against Phithium sp. and was identified as Bacilus sp. based on it"s 16SrDNA sequence. This strain produced high amount of spore in DSM media with shaking. Various biodegradable polymers were screened to find the best adhesive agent to be used in slow-releasing system. Slow-releasing activity of the mixture of two different polymers was lower that of each one polymer. To determine the survival ratio of the Bacillus spore entrapped in an adhesive agent after long time, the slow-releasing formulation was incubated at 50 ℃ rather than at room temperature. The results showed that the analysis of spore viability with the incubation of the slow-releasing system at 50 ℃ is an efficient method and could much shorten the incubation time.

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Biodegradation Kinetics of Cyclodione Insecticide Endosulfan by Isolated Fungi Jung-Bok LEE1, Ho-Yong SOHN2, Kee-Sun SHIN3, Jong-Sik KIM4, Min-Seb JO1, Chung-Sig CHOI5 and Gi-Seok KWON*1

1Major in Medical Plant Resources, School of Bioresource Sciences, Andong National University, Andong, Korea. 2Dept. of Food and Nutrition, Andong National University, Andong, Korea. 3Biological Resource Center, Korea ResearchInstitute of Bioscience and Biotechnology (KRIBB), Korea. 4Dept. of Biological Science, Andong National University, Andong, Korea. 5HansBio Co., HansBio Co.,BI center 303, Andong National University, Andong, Korea. *Corresponding author: [email protected]

Endosulfan, classified as an organochlorine pesticide, is rated by the U.S. EPA as a Category 1 pesticide with extremely high acute toxicity.This study describes the biodegradation kinetics of endosulfan and the metabolic pathway utilized by isolated fungi. Complete disappearance of both α- and β-endosulfan were observed during 16 days of incubation with isolated fungi in flasks containing 10 mg L-1 of endosulfan. The rate constants (K) for biodegradation of endosulfan by fungi using zero-order kinetic was 1.397 mg L-1 day-1. The Fungal degraded about 95% and 100% of α- and β-endosulfan, respectively, in 10 days of incubation in flask spiked with 10 mg L-1 of endosulfan. The results of this study suggest that these novel strains may be used for the bioremediation of endosulfan-contaminated site. [This work was supported by a grant (Code: 2005031-034-421-006-01-00) from BioGreen 21 Program, Rural Development Administration, Republic of Korea]

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A Solvent-tolerant Isolates of Pseudomonas putida Min A LEE1,2, Hye Jung CHOI1,2, Seon-A KIM1,2, Su Dong CHO1, Ja Young MOON1,3, Domg-Wan KIM1,4, Jeoung Yoon SEO5, Yong Kee JEONG6, Dae-Oh KWACK7, Jong Soo HEO8, Young-Guen LEE9 and Woo Hong JOO*1,2

1Institute of Genetic Engineering,. 2Department of Biology,Changwon National University, Changwon 641-773, Korea. 3Department of Biochemistry and Health Sciences,. 4Department of Microbiology,. 5Department of Environmental Engineering, Changwon National University, Changwon 641-773, Korea. 6Division of Applied Biotechnology, Dong-A University, Busan 604-714, Korea. 7Division of Biology Education,. 8Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, Korea. 9School of Applied life Science, Pusan National University, Miryang 627-706, Korea. *Corresponding author: [email protected]

Three solvent-tolerant bacteria were isolated from river sediment samples. An analysis of the 16S rDNA gene sequence and physiologicalcharacteristics showed that these strains are a member of the species Pseudomonas putita, and they were designated as strain BCNU106, BCNU154, and BCNU171. These organisms were also characterized several different experimental procedures including organic solvent tolerance range, and survival after solvent exposure. Especially, these three strains were able to utilize toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene. The degradation of three xylene isomers by these strains are particularly important, since very few microorganisms have been reported to utilize three xylene isomers as a sole carbon source. The unique properties of these strains, such as solvent-tolerance and the ability to degrade three xylene isomers, may have important implications for the treatment of solvent wastes.

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Study of Halophilic Bacteria Isolated from Solar Saltern SoilSeog Ju LEE1, Sung Koo KANG1, Young Sup CHO1, Chan Ki HONG2 andBong Geum CHO*1

1Dept. of Applied Biochemistry, Konkuk University, ChungJu, 380-701. 2Genicos,313-3 Songdae-ri, Ochang, Cheongwon, ChungBuk, KOREA. *Corresponding author: [email protected]

Halophilic Bacteria obtained from solar saltern soil and closed saltern soil in YeongJong-Do, Inchen, Korea. We acquired 36 strains using medium inclusive of 10% NaCl for isolated halophilic bacteria. Bacterialdiversity were investigated by phylogenetic analysis of the partial 16S rDNA sequence. A neighbor joining tree of the partial 16S rDNA sequence divided the 36 isolated into 2 major group, 34 strains of Firmicutes and 2 strains of Gammaproteobacteria. The Firmicutes group consisted of several minor group of the Bacillus sp.(38.8%), Halobacillussp.(19.4%), Virgibacillus sp.(19.4%), Oceanobacillus sp.(8.3%), Paraliobacillus sp.(2.7%). The Gammaproteobacteria group consisted of theHalomonas sp.. Bacillus megaterium(30.5%) was the most predominant species among all the isolated, followed by Halobacillus trueperi(16.6%).Recently, halophilic bacteria make a study of industrial utilization but also study of the polar microorganisms and a salts waste water treatment, a heavy metal recovery, polymer and fermentation of salted fish using extreme halophilic enzyme. This study keep these utilityvalue in view, we tried an experiment from among isolated strains into Halobacillus sp. and Halomonas sp., a part of moderately holophilic bacteria.

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Screening Bacilli for the Growth Promotion of Corn at Low Temperature Jung Wook YANG, Soo Hyun LEE, Hyun Hee LEE, Soon Kyeong KWON,Choong Min RYU and Seung-Hwan PARK*Systems Microbiology Research Center, KRIBB, Daejeon, 305-600, S. Korea. *Corresponding author: [email protected]

Plant growth-promoting rhizobacteria (PGPR) are a group of root–colonizing bacteria that have been utilized as microbial inoculants to enhance plant growth and to control plant diseases. The plant growth promotion by PGPR has reported on the numerous crops. Many studies were particularly concentrated on Bacillus species as PGPR for large-scale application due to formation of endospores that are durable under several stress conditions. To screen Bacilli that enhanced corn growth at low temperature, five corn seeds inoculated with each 1000 Bacilli strains isolated from diverse plants in Korea were placed in a commercial paper cup. The seedlings were grown in the growth chamberwith regulation of temperature and humidity. In the first screening, out of 1000 strains, corn seedlings treated with 37 Bacilli strains were increased fresh weight and shoot length compared to water control. Fromthe result of second screening, seed treatment of eight strains was consistently promoted fresh weight and shoot length. The fresh weight of corn treated with the selected strains was increased upto 103 % and shoot length did upto 23 % compared with water control. Our results show that eight Bacillus strains can use as microbial inoculants to inducegrowth promotion of corn at low temperature.

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Archaeal Diversity Analysis during Kimchi Fermentation using 16S rRNA Gene-based Approaches Young-Do NAM, Ho-Won CHANG, Seong Woon ROH and Jin-Woo BAE*Biological Resources Center, KRIBB, Daejeon 305-806, S. Korea. *Corresponding author: [email protected]

Kimchi is a traditional Korean food fermented by microorganisms from a variety of vegetables. Many bacteria have been elucidated as related to kimchi fermentation and among these bacteria, Lactic acid bacteria are known to mainly perform significant roles. However, archaea have never been studied in kimchi fermentation. In this study, we have characterized the archaeal dynamics of kimchi during fermentation by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. DGGE band patterns showed that most of bands persisted throughout the fermentation, and only minor changes occurred. Halobacterium sp. and Halorhabdus sp. were the most predominant archaea and there was no crenarchaea. The culture independent method used here showed that the archaeal communities certainly exist and somewhat change throughout the fermentation of kimchi.

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Addition Effect of Thermophilic Bacteria for Food Waste Degradation Hwe-Su YI, Keyung-Jo SEUL, Yu-Mi PARK, Kyoung-Hwa PARK, Ji-HyunJEONG and Sa-Youl GHIM*Dept. of Microbiology, Kyungpook National University, Daegu, 702-701. *Corresponding author: [email protected]

Food waste is one of the largest components of daily generated wastesby weight. Therefore, various methods including landfill, incineration, compost and extinction to treat the food waste have been developed. Besides, the best way for nature might be extinction degraded by microorganisms. In this study, food wastes were applied to decompose into the Mugri (Isung engineering, Korea), a food waste reduction machine, added sawdust of cryptomeria. Degradation effects were better when the machine was worked at high temperature than low temperature.Thermophilic bacteria were isolated from cryptomeria sawdust and the food waste products degraded by the machine. Acinetobacter baumannii,Enterobacter sp. and Erwinia cypripedii were partially identified from cryptomeria sawdust and mostly Bacilluis sp. from the degraded products. The isolated bacteria turned out to have degradative enzyme activities. About 33%, 20%, 21% and 23% of the bacteria showed amylase, cellulase, protease, and lipase activities, respectively. When 30 isolates of the thermophilic bacteria were added into the machine for food waste degradation, a rather positive effect was detected comparing the case without bacteria addition.

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Inactivation of Bacteria by Photocatalyst PolyoxometalatesJae Won LEE1, Eunyoung BAE2 and Hyung Joon CHA*1

1Department of Chemical Engineering Pohang University of Science and Technology, Pohang 790-784, Korea. 2Department of Environmental Science andEngineering Pohang University of Science and Technology, Pohang 790-784, Korea. *Corresponding author: [email protected]

Polyoxometalates (POMs) as a homogeneous photocatalyst and semiconductor oxide TiO2 as a heterogeneous photocatalyst share many aspects of similarity in their operating mechanisms. In this study, we compared photocatalytic inactivation of pathogenic E. coli using POM and TiO2 in aqueous solution. Almost all the initial E. coli (5 X 107

cell/ml) were inactivted with 40 min in the presence of both POM and TiO2, but the POM-mediated inactivation was faster than that with TiO2

under the experimental conditions employed in this study. Photocatalyticinactivation of E. coli was more efficient by free H4O40SiW12 or Mo12O40P than by TiO2. Kinetic studies using tert-butyl alcohol or methanol as an OH radical scavenger suggested that OH radicals are dominant photooxidant in photocatalyst inactivation. In particular, the biocidal action of the photocatalyst has been accepted that reactive oxygen species (ROS) and OH radicals play the role.

G-45

Bacteriostatic Effects of Dryopteris crassirhizoma Nakai Against Propionibacterium acnes Hyun Ju KIM, Nae Im KANG, Shin Wook CHOI and Chang Soon YOON*RADIANT INC, #207, Bioindustry Innovation Center, Hi-Tech Venture Town, 198-53 Hupyeong, Chuncheon, Kangwon, Korea, 200-957. *Corresponding author: [email protected]

The development of acne appears to be influenced by not only genetic and environmental factors but also unbalanced hormone secretion, leading to hyperkeratinization, activation of Propionibacterium acnes andskin inflammation. Although various acne remedies have been used, most of them shows various side effects such as allergy and skin irritation. Recently, accumulated studies have been focused on screening natural anti-acne products without such side effects. Dryopteris crassirhizoma N.a perennial plant, is known to have various physiological effects including anti-bacterial and fungal effects. Therefore, in the present study, anti-acne effects of Dryopteris crassirhizoma N. against Propionibacterium acnes was examined for cosmetic application using minimum inhibitory concentration (MIC) and paper disk diffusion assay.The MIC value of Dryopteris crassirhizoma extracted with ethanol is 0.0078 mg/ml compared with that of triclosan (0.004 mg/ml) as a control, implying strong anti-acne effect even in crude extracts. This anti-acne effect is confirmed by paper disk diffusion assay. The inhibitory size of the extract is 34 mm in the presence of 500 ug/ml. The ethanol extract of Dryopteris crassirhizoma is stable in high temperatures (70, 80, 90, 100, 121℃) and pH2-pH11.

G-46

Characterization of a Novel Gene Encoding an Amylolytic Enzyme from Metagenomic Libraries Daewon SEO, Jiae YUN, Sunhee LEE and Sangryeol RYU*Laboratory of Molecular Food Microbiology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea. *Corresponding author: [email protected]

Metagenomics employed as a means of investigating the entire geneticmaterial isolated from environmental samples can provide valuable sources of catalytic molecules. We constructed a metagenomic DNA library using BAC vector with DNA extracted directly from bovine rumen contents. Enzyme activity-based screening of the library resulted in the isolation of one amylase positive clone out of 8,000 clones with insert size ranging from 10 to 30 kb. The clone had a 750 bp ORF encoding a protein with 250 amino acids whose molecular weight is 28.3 kDa. Analysis of the sequence showed 32 % identities to a glycosyltransferase of Porphyromonas gingivalis W83 that belongs to the glycosyltransferase group family 2. The purified protein could synthesizemaltooligosaccharides from maltose and was also able to hydrolyze maltooligosaccharides to maltose and glucose even though the hydrolysisactivity was low. These results suggest that the protein has enzymatic activity associated with glycosyltransferase and may have specific substrate that should be characterized for elucidation of the mechanism of enzyme action.

G-47

Algicidal Activity of Red Pigment of Hahella chejuensis against Red Tide and Algal Blooming Species Sung Jin KIM, Joung Han YIM, Jae Eun LEE, In Hee KIM and Hong KumLEE*Polar Biocenter, Korea Polar Research Institute, KORDI, Songdo Techno Park, Incheon, 406-840. *Corresponding author: [email protected]

We reported that H. chejuensis red pigment showed algicidal effect onred tide microalga Cholodinium polykrikoides : More than 97.4 % of C. polykrikoides was lyzed within 1 h, at 10 ㎍/ℓ of red pigment. To extend the applicable microlagal species, Algicidal effect of red pigmentwas tested against 10 species of Dinophyceae, 1 species of Raphidophyceae, 2 species of Bacillariophyceae and 4 species of Cyanophyceae. Among them Chatonella sp.(Raphidophyceae) showed strong cell lysis in the presence of red pigment. In the f/2 medium containing 10 ㎍/ℓ ethanol-soluble purified red pigment, more than 92.1% Chatonella sp. was lysed within 1 h. As NFRI reported in 2005 that Chatonella sp. occurred red tide mixed with C. polykrikoides, Chatonellasp. is expected to become one of the important species causing red tide in coastal region. So H. chejuensis red pigment will be used for the bioremediation of Chatonella sp.

G-48

382 www.kormb.or.kr

Antibacterial Effects of Sophora flavescens Aition Extracts Against Staphylococcus aureus Causing Bovine Mastitis Hyun Ju KIM, Shin Wook CHOI and Chang Soon YOON*RADIANT INC, #207, Bioindustry Innovation Center, Hi-Tech Venture Town, 198-53 Hupyeong, Chuncheon, Kangwon, Korea, 200-957. *Corresponding author: [email protected]

Staphylococci and Streptococci have been known to cause bovine mastitis, one of chronic disease in dairy cattle. Although antibiotics havebeen used as a typical treatment, a plenty of recent studies have screenedplant extracts having strong anti-bacterial effects without side effects for the prevention or treatment of bovine mastitis. Sophora flavescensAition, a perennial plant has been used as a traditional remedy for eczema or a vermicide and known to have anti-allergic and anti-trichomonas effects. In the present study, the anti-bacterial effect of Sophora flavescens Aition against Staphylococcus aureus was examined by minimum inhibitory concentration (MIC) and paper disk diffusion assay. The MIC value of Sophora flavescens Aition extracted with ethanol and inhibition zone is 0.0625 mg/ml and 23 mm, respectively. Considered that the inhibition zone is 25 mm in the presence of 50 unitof erythromycin (control), the ethanol crude extract of Sophora flavescens Aition seems to have strong anti-bacterial effect The ethanol extract of Sophora flavescens Aition is stable in high temperatures (70-121℃) and pH2-pH11.

G-49

Genome Probing Microarrays Versus Subtracted Genome Microarrays Jin-Woo BAE, Young-Do NAM, Ho-Won CHANG, Hee-Mock OH, Jung-Sook LEE, Jung-Hoon YOON, Yong-Ha PARK and Jin-Woo BAE*Biological Resources Center (BRC), KRIBB, Eundong 52, Yusong, Daejeon,305-333. *Corresponding author: [email protected]

The generation of microarray probes with specificity below the specieslevel is an ongoing challenge, not least because the high throughput detection of microorganisms would be an efficient means of identifying environmentally relevant microbes. Here, we describe how suppression subtractive hybridization (SSH) can be applied to the production of microarray probes that are useful for microbial differentiation at the sub-species level. SSH was used to initially isolate unique genomic sequences of 9 Salmonella strains, and these were validated in quadruplicate by microarray analysis. The results obtained indicate that a large group of genes subtracted by SSH could serve together, as one probe, for detecting a microbial subspecies. Similarly, the whole microbial genome (not subjected to SSH) of the genome-probingmicroarrays (GPM) can be used as a species-specific probe. The detailedmethods described herein could be used and adapted for the estimation of any cultivable bacteria from different environments.

G-50

Detection of Bacillus anthracis Spores using Peptide-quantum Dot Conjugates Tae Jung PARK1 and Sang Yup LEE*1,2

1Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering, BioProcess Engineering Research Center, and Center for Ultramicrochemical Process Systems, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea. 2Department of BioSystems and Bioinformatics Research Center, KAIST, Daejeon 305-701, Korea. *Corresponding author: [email protected]

The detection method of Bacillus anthracis spores was developed by employing specific capture peptides conjugated with fluorescent quantum dots (QDs). It was possible to distinguish B. anthracis spores from the spores of Bacillus thuringiensis and Bacillus cereus using these peptide-QD conjugates by flow cytometry and confocal laser scanning microscopy. For more convenient high-throughput detection of B. anthracis spores, spectrofluorometric analysis of spore-peptide-QD conjugates was performed. B. anthracis spores could be detected in less than 1 h using this method. In order to avoid any minor yet false positive signal caused by the presence of B. thuringiensis spores, the B-Negative peptide, which can only bind to B. thuringiensis, conjugated with another type of QD that fluoresces at different wavelength was alsodeveloped. In the presence of mixed B. anthracis and B. thuringiensis spores, the BABA peptide conjugated with QD525 and the B-Negative peptide conjugated with QD585 were able to bind to the former and the latter, specifically and respectively, thus allowing the clear detection of B. anthracis spores by using two-QD-labeling system. This capture peptide-conjugated QD system should be useful for the detection of B. anthracis spores.

G-51

Characterization of Plant Growth Promoting Rhizobacteria in Soil Contaminated with Petroleum Hydrocarbons Sun Hwa HONG, So-Yeon KOO and Kyung-Suk CHO*Dept. of Environmental Science and Engineering, Ewha Womans University, Seoul, Korea, 120-750. *Corresponding author: [email protected]

Phytoremediation using PGPR has been attended for the remediation of soil contaminated petroleum hydrocarbons. In this study, 4 species of indigenous plants (Echinochloa crus galli, K1; Cyperaceae, K2; Oplismenus undulatifolius, H1; Persicaria nodosa Opiz, H2) in soil contaminated with diesel were collected with soil samples. Two samplesof rhizosphere soil and rhizosplane were prepared from each plant sample, and then approximate 40 colonies from each rhizosphere soil andrhizosplane were isolated. The plant growth promoting ability of isolatedbacteria were studied by testing siderophore synthesis ability and aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme activity. The ratio of the bacteria having ACC deaminase activity in the rhizosplane was higher than rhizosphere soil samples. On the contrary, the ratio of bacteria having siderophore synthesis ability was higher in rhizosphere soil samples rather than rhizosplane samples. The bacterial community in the rhizosphere soil of each plant was characterized using16S rDNA-PCR DGGE fingerprinting. The similarity of DGGE fingerprints between plant samples ranged from 27.6% to 39.4%. Actinobacteria, Proteobacteria, Firmicutes and Flavobacteria were identified as dominant bacteria in rhizosphere soils associated with plants.

G-52



Two-electrode System to Treat Lake Sediment Contaminated with Organics Materials and HydrocarbonGi Su KANG1,2, In Seop CHANG1,3, Hyunsoo MOON1,4, Yeng Fung CHOO1,Jae Kyung JANG1, Yong Keun PARK2 and Byung Hong KIM*1

1Bioelectronichemistry Lab., Water Environment and Remediation Research Center, Korea Institute of Science and Technology (KIST), 39-1 Hawolgok-dong,Sungpook-ku, Seoul 136-791, Korea. 2School of Life Sciences and Biotechnology,Korea University, 5-1 Anam-dong, Sungpook-ku, Seoul 136-701, Korea. 3Dept. Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong, Pook-ku, 136-701, Korea. 4Department of Biochemical Engineering, Yanbian University of Science and Technology, BeishanSt., Yanji City, Jilin Province, 133000, China. *Corresponding author: [email protected]

A two-electrode system was studied to oxidize organic matters in freshwater sediment. Cylindrical reactors with the diameter of 20 cm and 60 cm high were filled with sediment to 15 cm and filled with overlaying water. Graphite electrodes were installed at the bottom (anode), and the surface (cathode) of the reactors. A series of reactors was maintained under closed-circuit conditions while the others under open-circuit conditions as thecontrol. Power output increased with DO and proton concentration increases in cathode. The open circuit potential of the control reactors reached to 0.85 V. The oxidation-reduction potential of the anode (ORPanode) was calculatedas 290.3 mV (vs NHE) in the test reactors, and that of control reactors was -546.4 mV (vs NHE). After 1 year operation, 7.36±3.89 g (as volatile solid) was degraded in the sediment in the test reactors (n=3). In control reactors (n=3), the degradation of organic matter was 2.84±0.62 g. The columbic yield of the test reactors was 54.6±21.9(%). The critical DO concentration value observed was 4.044± 0.281 mg/L with the current of 2.521 ± 0.33 mA. Molecular ecological analyses showed different bacterial populations betweenthe test and control reactors not only in the anode but also in the cathode.

G-53

Effect of Azide on Electrochemically Active Microbial Consortium Using Fuel Cell Yeng Fung CHOO1, Gi Su KANG1, Ji Young LEE1, Jae Kyung JANG1, HoIl PARK1, In Seop CHANG2 and Byung Hong KIM*1

1Bioelectrochemistry Lab., Water Environment and Remediation Research Center,Korea Institute of Science and Technology (KIST), 39-1 Hawolgok-dong, Sungpook-ku, Seoul 136-791, Korea. 2Dept. Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong, Pook-ku, 136-701, Korea. *Corresponding author: [email protected]

Azide (0.2mM) was added into acetate (100 mg L-1) fuel to enrich electrochemically active microbes. This is to eliminate the aerobic respiratory bacteria in the microbial populations during the enrichment process. The dissolved O2 (DO) concentration was higher (0.18±0.02ppm) in the anode effluent of the azide added MFCs than that of the control MFCs (0.08± 0.02ppm). These results show that azide inhibits the aerobic bacteria, and gives rise to a higher DO concentration.MFCs enriched with azide generated lower current (4 ± 0.5 mA) than the control MFCs (4.5± 0.2 mA). The Coulomb efficiency of the azide added MFCs was 51.2 ± 5%, lower than that of the control MFCs (73.3± 5a%). Thus, it is hypothesized that azide is used as an electron acceptor in the MFCs enriched in the presence of the terminal oxidase inhibitor. The analysis of 16S rDNA library showed that less diverse bacteria have been enriched in the presence of azide. Restriction fragment length polymorphism of the 16S rDNA library showed a clonewith a similarity of 98% with Geobacter sulfurreducens occupies 98.0 % of the population in the MFCs enriched with azide. Works on isolating these bacteria are on-going.

G-54

Isolation of a Novel Mycovirus OMIV in Pleurotus ostreatusand its Detection using a Triple Antibody Sandwich-ELISA Nam-Ju LEE, Hyun-Sook LEE and Hyeon-Su RO*Dept. of Microbiology, GyeongSang National University, Chinju, 660-701. *Corresponding author: [email protected]

A novel mycovirus was isolated from a diseased mushroom, Pleurotusostreatus, using a purification procedure involving PEG-NaCl precipitation, differential centrifugation, and equilibrium centrifugation ina CsCl gradient. The virion was a 43 nm isometric virus encapsulating double-stranded RNA genomes of 2.1, 2.0, 1.9, and 1.7 Kbp with a coatprotein of 58 KDa. The new mycovirus was named Oyster Mushroom Isometric Virus (OMIV). To detect OMIV in the mushroom, we constructed a triple antibody sandwich-ELISA (TAS-ELISA) system using anti-OMIV mouse monoclonal and anti-OMIV rabbit polyclonal antibodies. The TAS-ELISA system was sensitive enough to allow detection of OMIV in the mushroom with the naked eye. It successfully detected virus particles from 0.6 mg of diseased tissue as well as 0.4 mg/ml purified virus solution.

G-55

Annual Variation of Microcystis Community from Sedimentto Water Using Molecular Techniques in Daechung Reservoir in Korea Seung-Hyun JOUNG, Chi-Yong AHN and Hee-Mock OH*Environmental Biotechnology Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. *Corresponding author: [email protected]

The aim of this study is to confirm of annual changes of Microcystiscommunity from sediment to water. This study was conducted from March, 2005 to February, 2006 in Daechung Reservoir in Korea. The primer set (cpc57F-cpc356R) for the detection of Microcystis communitywas developed. Moreover, potential toxic Microcystis specific primer set (mcy117F- mcy359R) was made from mcy J gene sequences. Microcystis and potential toxic Microcystis were detected in all samples both sediment and water during the study period by multiplex PCR method. This result showed that the intensity of detected band displayeda difference as the sampling date in sediment, and band intensity was higher in winter than other seasons. Also, the genotype pattern of Microcystis was analyzed by DGGE. Microcystis and potential toxic Microcystis strains displayed a high diversity during a bloom-forming period in water. However, Microcystis strains displayed a high diversity at winter period in sediment, and potential toxic Microcystis strains did not changed in sediment. In conclusion, toxic and non-toxic strains always existed in both water and sediment, and these strains migrated between water and sediment as the change of season. We confirmed that Microcystis was deposited in sediment during the winter season.

G-56

384 www.kormb.or.kr

Heavy Metal Remediation with Hydrogen Sulfide Produced by Recombinant Bacteria Escherichia coli Tatsuya UNNO and Hor-Gil HUR*Dept. of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712 Republic of Korea. *Corresponding author: [email protected]

An aerobic sulfate reduction pathway for the secretion of sulfides was metabolically engineered by expressing two unique enzymes, serine O-acetyl transferase (SAT) and cysteine desulfhydrase (cdl), in recombinant bacteria E.coli. In cysteine biosynthesis, SAT catalyzes the acetylation of serine to form O-acetylserine, the final precursor to cysteine. In addition, SAT receives feed back inhibition by cysteine. Hiroshi Takagi et.al found that SAT from Arabidopsis thaliana showed feedback insensitive when expressed in E.coli, which resulted in overproduction of cysteine in E.coli. Cysteine desulfhydrase is an aminotransferase that converts cysteine into pyruvate, ammonia, and hydrogen sulfide. Especially active L-cysteine desulfhydrase was isolatedfrom Fusobacterium nucleatum by Haruka Fukamachi et.al. In this study,two unique enzymes, cdl and SAT, were constitutively expressed in E.coli and nonspecific heavy metal precipitation with the metabolically produced hydrogen sulfide was studied.

G-57

H _ Food Microbiology

Probiotic Activities of Lactobacillus johnsonii IDCC 9203 Seung-Hun LEE, Eun-Hee YANG, Hyuk-Sang KWON, Byung-Hwa KANGand Tae-Yong KIM*Research Laboratories, Ildong Pharmaceutical Co., Ltd. Yongin, 446-910. *Corresponding author: [email protected]

Here we report studies on a native Lactobacillus johnsonii IDCC 9203 used as a probiotic strain. L. johnsonii IDCC 9203 from infant feces is identified and investigated in vitro for functional characteristics as a potential new probiotic strain. In case of strain L. johnsonii IDCC 9203,its tolerance toward gastric juice (pH 2.3 and 2.5) was higher than that of strain L. rhamnosus GG. Also, L. johnsonii IDCC 9203 exhibited resistance to 0.3% bile salt. The in vitro antimicrobial effects of L. johnsonii IDCC 9203 were assessed by MIC test. The in vivo antimicrobial effects of L. johnsonii IDCC 9203 against Salmonella and Streptococci were also assessed. After oral administration of L. johnsoniiIDCC 9203, both viable bacteria in feces were decreased. The ICR-micereceived the L. johnsonii IDCC 9203 were reported no adverse effects and the strain could be isolated from their feces at a relatively high levelthan L. rhamnosus GG. Additionally, the oral administration of L. johnsonii IDCC 9203 led to an improvement of parameters such as the increases of weight, fecal moisture and the volume of the stools. The studies reveal that L. johnsonii IDCC 9203 may exert probiotic effects on human.

H-1

Thermal Inactivation of Enterobacter sakazakii in Rehydrated Dried Infant Formula Soo-Hwan KIM, Eun-Jin LEE, Ji-Heon HWANG and Jong-Hyun PARK*Dept. of Food Science and Biotechnology, Kyungwon University, Korea 461-701. *Corresponding author: [email protected]

Enterobacter sakazakii, designated as an unique microbial species in 1980, linked to outbreaks of meningitis, septicemia, and necrotizing enterocolitis mainly in neonates. One E. sakazakii type strain and two isolated strains were used to determine the heat resistance of this organism at 52, 56, 60℃ in saline, rehydrated dried infant formula and dried baby food. In saline, D-values of 7.64-9.87, 3.36-4.67, 0.87-1.24 min were obtained for each temperature. D-values were increased to 8.43-12.62, 3.91-4.67, 2.08-3.78 min in rehydrated dried infant formula and 11.24-13.32, 5.17-6.32, 2.09-2.76 min in dried baby food. The overall calculated z-value was 7.24-11.43 in saline and 11.40-22.99 in dried infant formula, and 9.95-13.12 in dried baby food. Thermal inactivation of E. sakazakii during the rehydration of dried infant formula with 50, 60, and 70℃ water was investigated. At 50℃, inactivation was not occurred for 20 min. At 60℃, inactivation by about1 log CFU/g occurred and E. sakazakii decreased 5 log CFU/g during 20 min at 70℃. Considering that the levels of E. sakzakii observed in dried infant formula have generally been 1 CFU/100 g of dry formula or less, comtamination with Ent. sakazakii can be reduced or eliminated by the use of water with a temperature of 60℃ or more.

H-2

Anti-Helicobacter pylori Activity of Lactic Acid Bacteria from Kimchi. Ji Young SON and Hae Choon CHANG*Department of Food and Nutrition, Chosun University, Gwangju 501-759. *Corresponding author: [email protected]

Lactic acid bacteria(LAB) isolated from Kimchi were screened for ihibition activity against Helicobacter pylori. Among 55 isolates, three rod-shaped and one coccus-shaped LAB showed strong ihibition activity against H. pylori. Inhibition activity were examined by using a paper disk diffusion method. Culture supernatants of 4 strains of Lactobacilli showed a strong inhibitory activity on the urease activity of Helicobacterpylori KCCM 41756. The four selected strains were identified and named Lactobacillus sakei ML5, Lactobacillus sakei CL1, Pediococcus pentosaceus MD1, Lactobacillus sakei CH2 based on biolog test and 16S rDNA sequences. Acid and bile tolerance of these LAB strains was evaluated. The result of viability test in artificial gastric juice and bile the isolate showed high viability(108-9CFU/ml) in 0.05M sodium phosphate buffer(pH 3.0), artificial gastric juice and 0.5%oxgal solution

H-3

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