MMPH-0 (Enterobacter aerogenes)에 의한 6가 크롬 오염 지하수의 생지화학적 정화

서현희,이성근,김강주,박은규,김영규,전철민,문지원,노열,Seo, Hyun-Hee,Rhee, Sung-Keun,Kim, Kang-Joo,Park, Eun-Gyu,Kim, Yeong-Kyoo,Chon, Chul-Min,Moon, Ji-Won,Roh, Yul 대한자원환경지질학회 2012 자원환경지질 Vol.45 No.2

Indigenous bacteria isolated from contaminated sites play important roles to remediate contaminated groundwater. Chromium has the most stable oxidation states. Cr(VI) is toxic, carcinogenic, and mobile, but Cr(III) is less toxic and immobile. In this study, indigenous microorganism (MMPH-0) was enriched from Cr(VI) contaminated groundwater, and identified by 16S rRNA gene analysis. Using MMPH-0, the effect of stimulating with e-donors (glucose, lactate, acetate, and no e-donor control), respiration conditions, biomass, tolerance, and geochemical changes on Cr(VI) reduction were investigated in batch experiments for 4 weeks. The changes of Cr(VI) concentration and geochemical conditions were monitored using UV-vis-spectrophotometer and Eh-pH meter. And the morphological and chemical characteristics of MMPH-0 and precipitates in the effluents were characterized by TEM-EDS and SEM-EDS analyses. MMPH-0 (Enterobacter aerogenes) was able to tolerate up to 2000 mg/L Cr(VI) and reduce Cr(VI) under aerobic and anaerobic conditions. MMPH-0 performed faster and higher efficiency of Cr(VI) reduction with electron donors (over 70% after 1 week with e-donor, 10-20% after 4 weeks without e-donor). The changes of Eh-pH in effluents showing the tendency from oxidizing to reducing condition and a bit of acidic change in pH due to microbial oxidation of organic matters donating electrons and protons suggested the roles of MMPH-0 on Cr(VI) in the contaminated water catalyzing to transit geochemical stable zone for more stable $Cr(OH)_3$ or Cr(III) precipitates. TEM/SEM-EDS analyses of MMPH-0 and precipitates indicate direct and indirect Cr(VI) reduction: extracellular polymers capturing Cr component outside cells. These results suggested diverse indigenous bacteria and their biogeochemical reactions might enhance more effective and feasible remediation technology of redox sensitive heavy metals in metal-contaminated in groundwater.

암모니아 산화 고세균의 독립성장에 필요한 결손 유전자 예측

한상수(Sang-Soo Han),이진영(Jin-Young Lee),이성근(Sung-Keun Rhee),김근중(Geun-Joong Kim) 한국생물공학회 2011 KSBB Journal Vol.26 No.3

As a number of archaea are ubiquitously found in non-extreme habitats, elucidation of their functional roles becomes currently an emerging issue. However, most of them are unable to grow in pure culture and so it remains to be established. In order to find genes lacking in the genome of an ammonia-oxidizing archaeon (AOA), we here report on the comparative analyses of an AOA genome with those of experimentally or theoretically established minimal genomes for independent growth. We assessed the genes lacking in AOA using logic of clusters of orthologous groups (COG), remote homology, consensus sequence weight matrix, function-based motif or domain, and then further excluded genes encoding hypothetical orarchaea-specific proteins. The results of these combination analyses revealed 19 candidate genes lacking in the genome of an AOA. Thus, our results provide a possibility of inducing independent growth of AOA when supplemented with product (s) of the lacking gene (s), and also give a chance for finding new proteins with novel sequence or structure space even if the predicted lacking-genes will be found using another algorithms or biochemical studies.

북극 Svalbard 지역 해양 퇴적물의 고세균 amoA 유전자의 다양성 분석

박수제,이성근,Park, Soo-Je,Rhee, Sung-Keun 한국미생물학회 2014 미생물학회지 Vol.50 No.2

북극지역은 지구온난화로 인하여 생태계에 큰 영향을 받고 있다. 본 연구는 북극 Svalbard 지역에서 육지 빙하의 해빙(ice melt)의 영향을 받는 해양퇴적층에서 질산화 과정에 핵심역할을 하는 고세균의 질산화유전자(ammonia monooxygenase, AMO)의 공간적 분포의 변화를 조사하였다. 해빙으로 인한 퇴적물 퇴적속도와 고세균 AMO의 alpha subunit를 코딩하는 amoA 유전자와의 관계를 클론라이브러리 분석을 통하여 분석하였다. 육지와 근접하여 퇴적속도가 가장 빠른 정점(188)에서 고세균 amoA 유전자의 다양성이 육지에서 비교적 먼 지역의 정점(176과 184)에 비해 현저히 낮음을 알 수 있었다. 3 정점의 고세균 amoA 유전자 클론들의 평균 아미노산 서열의 상동성은 94%(염기서열 91%)로 나타났다. 176과 184 정점에서 분석된 고세균 amoA 유전자 클론들 중 약 45%가 Nitosopumilus clade와 근연관계에 있는 반면, 188 지역의 경우 낮은 염농도에서 발견되는 Nitrosoarchaeaum clade와 근연관계에 있는 클론들이 발견되었다. 토양 고세균유래 amoA 유전자는 육지에 근접하여 해빙에 의한 영향을 가장 많이 받는 188정점에서만 발견이 되었다. 본 연구를 통하여, 해빙으로 인하여 육지로부터 운반되는 퇴적물의 량이 증가함에 따라, 해양퇴적층의 질소순환관련 미생물 군집에 변화가 유발되는 것으로 추정되며, 고세균의 amoA 유전자가 해양퇴적층 질소순환생태계 변화의 지표로 이용될 수 있음을 알 수 있었다.

염전으로부터 농화배양된 호염 메틸영양미생물 군집의 특성

김종걸,박수제,이성근,Kim, Jong-Geol,Park, Soo-Je,Rhee, Sung-Keun 한국미생물학회 2010 미생물학회지 Vol.46 No.3

C-1 compounds are observed in anaerobic sediment of high salt environments. Thus, surface sediments and waters from these environments are therefore potential habitats for aerobic methylotrophic microorganisms. The soil samples collected from saltern and tidal flat as inoculums and methanol as carbon and energy source was supplied. After subculture depending on the salt concentration, methanol oxidizing bacteria growth condition investigated, the results of methanol oxidizing bacteria can grow in salt conditions, and the maximum concentration was 20%. Analysis based on denaturing gradient gel electrophoresis of 16S rRNA genes indicates that Methelyophaga-like bacteria were dominants of methylotrophs in the enrichment culture. Quantitative PCR showed that archaeal cells were about 1-10% of bacterial cells. Additionally archaea were assumed not to be involved in methanol oxidation since bacterial antibiotics completely blocked the methanol oxidation. Our results suggest that Methelyophaga-like bacteria could be involved in C-1 compounds oxidation in hypersaline environments although those activities are sensitive to salinity above 20%.

곰소 염전에서 분리한 호염성 고세균의 특성 분석

고현우,김소정,이성근,박수제,Koh, Hyeon-Woo,Kim, So-Jeong,Rhee, Sung-Keun,Park, Soo-Je 한국미생물학회 2015 미생물학회지 Vol.51 No.4

Most of halophilic archaea are found in the various hypersaline environments including solar saltern, salt lake with very high salt concentration. The present study is about isolation and characterization of halphilic archaea from Gomso solar saltern known as a representative high salt environment in Korea. In order to isolate the halophilic archaea, we prepared and used high salt medium. Finally, total 7 strains obtained were tentatively identified based on comparative similarity analysis for 16S rRNA gene sequence and physiological traits. All halophilic archaea belonged to Haloruburm, Halogeometriucm, Halobacterium, and Haloarcula genera. These isolates were all Gram-staining negative, and growth was not observed using nitrate as an alternative electron acceptor under anaerobic conditions. In addition, all isolates required about 12-30% (w/v, NaCl) salt. This case study might provide basic information on microbial isolation technologies and related research in halophilic microorganisms from domestic halophilic environments, and contribute to obtaining useful indigenous halophilic archaea in a variety of extreme environmental conditions.

디지털 PCR을 응용한 특정 amoA유전자를 가진 질산화 Archaea 동정

박병준,박수제,이성근,Park, Byoung-Jun,Park, Soo-Je,Rhee, Sung-Keun 한국미생물학회 2007 미생물학회지 Vol.43 No.3

Mesophilic Crenarchaeota have been known to be predominant among ammonia-oxidizing microorganisms in terrestrial and marine environments. In this study, we determined the archaeal phylotypes carrying specific amoA by combining digital PCR and multiplex-nested PCR. Analysis of samples in which amoA and 16S rRNA gene were amplified showed that amoA gene diversity was relatively higher than that of 16S rRNA gene. Nitrifying archaeal group I.1a was dominant over I.1b group of crenarchaota and euryarchaeota. This approach could be applied for interrelating a functional gene to a specific phylotype in natural environments. 해양 및 토양에서의 암모니아 산화는 세균에 비해 Crenarchaeota 그룹의 archaea에 의해 우세하게 일어나고 있음이 알려졌다. 서해 갯벌에서, 배양에 의존하지 알고, 특정 암모니아 산화유전자(amoA)를 가진 archaea을 동정하고자 디지털 PCR법을 응용한 nested PCR법을 개발하였다. amoA와 16S rRNA유전자가 동시에 증폭된 샘플의 분석결과, 16S rRNA유전자에 비해 amoA 유전자의 다양성 이 높았으며, I.1a 그룹의 crenarchaea가 I.1b 그룹의 crenarchaea보다 갯벌지역에서 암모니아 산화에 우점적으로 기여하고 있음을 알 수 있었다. 본 연구에서 시도된, 디지털 PCR과 multiplex-nested PCR을 접목한 접근법을 이용하면 특정 기능유전자를 가진 미생물을 환경에서 검증하는데 응용할 수 있을 것이다.

6가 크롬 오염 지하수 수질의 계절변화와 자연저감 가능성

전철민,안주성,노열,이성근,서현희,김구영,고동찬,손영철,김지욱,Chon, Chul-Min,Ahn, Joo-Sung,Roh, Yul,Rhee, Sung-Keun,Seo, Hyun-Hee,Kim, Gue-Young,Koh, Dong-Chan,Son, Young-Chul,Kim, Ji-Wook 대한자원환경지질학회 2008 자원환경지질 Vol.41 No.6

The Cr(VI) concentrations at the shallow aquifer well (MPH-0-1) of the Moonpyung groundwater monitoring station were in the range of 0.5 to 3.1 mg/L exceeding 10 to 62 times the guideline for drinking-water quality, indicating continuous contamination. However, Cr was not detected at the deep bedrock well and the other subsidiary monitoring wells except for MPH-1 and 6. Cross-correlation analyses were conducted for rainfall and groundwater level time series, resulting in the mean time of recharge after precipitation events to be 5.6 days. For rainy season, the water level was raised and the Cr(VI) concentration was several times lower than that during dry season at well MPH-0-1 well. Correlation of the Cr(VI) concentration with the groundwater-level showed that the Cr(VI) reduction was closely related with the groundwater-level rise in the well. However, the groundwater level rise during high water season induced the lateral migration of the Cr(VI)-contaminated groundwater at well MPH-4. We enriched and isolated a chromium reducing bacteria, Enterobacter aerogenes, from the Cr(VI)-contaminated groundwater in the wells MPH-0-1 and MPH-1. The bacteria may play an important role for immobilizing Cr(VI) in the Cr(VI)-contaminated groundwater. Therefore, the migration of the contaminant (Cr(VI) must has been restricted because of the natural attenuation by microbial reduction of Cr(VI) in the groundwater. This research suggests that the bioremediation of the Cr(VI)-contaminated groundwater by the indigenous bacteria may be feasible in the Cr(VI) contaminated groundwater.

Tofua Arc의 열수구환경으로부터 호열성 혐기성 고세균(Thermococcus)의 농화배양 및 동정

차인태,김소정,김종걸,박수제,정만영,주세종,권개경,이성근,Cha, In-Tae,Kim, So-Jeong,Kim, Jong-Geol,Park, Soo-Je,Jung, Man-Young,Ju, Se-Jong,Kwon, Kae-Kyoung,Rhee, Sung-Keun 한국미생물학회 2012 미생물학회지 Vol.48 No.1

Hydrothermal vents (HTV) provide special environments for evolution of lives independent on solar energy. HTV samples were gained from Tofua arc trench in Tonga, South Pacific. We investigated archaeal diversity enriched using combinations of various electron donors (yeast extract and $H_2$) and electron acceptors [Iron (III), elemental sulfur ($S^0$) and nitrate. PCR amplification was performed to detect archaeal 16S rRNA genes after the cultures were incubated $65^{\circ}C$ and $80^{\circ}C$ for 2 weeks. The cultures showing archaeal growth were transferred using the dilution-to-extinction method. 16S rRNA gene PCR-Denaturing Gradient Gel Electrophoresis was used to identify the enriched archaea in the highest dilutions where archaeal growth was observed. Most of cultured archaea belonged to genus of Thermococcus (T. alcaliphilius, T. litoralis, T. celer, T. barossii, T. thoreducens, T. coalescens) with 98-99% 16S rRNA gene similarities. Interestingly, archaeal growth was observed in the cultures with Iron (III) and nitrate as an electron acceptor. It was supposed that archaea might use the elemental sulfur generated from oxidation of the reducing agent, sulfide. To cultivate diverse archaea excluding Thermococcus, it would be required to use other reducing agents instead of sulfide.

제주도 용암동굴 대섭이굴 미생물 막의 독특한 원핵미생물 군집

문종근,정만영,김종걸,박수제,김대신,김종식,이성근,Moon, Jong-Geun,Jung, Man-Young,Kim, Jong-Geol,Park, Soo-Je,Kim, Dae-Shin,Kim, Jong-Shik,Rhee, Sung-Keun 한국미생물학회 2013 미생물학회지 Vol.49 No.2

Cave environment provides special ecosystems for evolution of lives distant from surface environments. We investigated bacterial and archaeal communities of wall biofilm obtained from of a volcanic cave (Daesubee) in Jeju, Republic of Korea. Bacterial and archaeal 16S rRNA genes were PCR-amplified and sequenced using pyrosequencing technologies. Unique prokaryotic communities with low diversities were observed. The main bacterial sequences (ca. 83% of total reads) were affiliated with Pseudonocardia mongoliensis of phylum Actinobacteria and clustered with clones obtained from various caves. Reflection of light on the wall surface of cave might be caused by formation of beads of water caused by hydrophobic filaments of actinobacterial colonies. Main archaeal sequences (ca. 65.7% of total reads) were related with those of I.1a-Associated group of phylum Thaumarchaeota. The sequences were related with that of Candidatus Nitrosotalea devanaterra which was known to oxidize ammonia under acidic condition (ca. pH 5.0). Nutrients leached through volcanic soils contribute formation of unique microbial communities of wall biofilm of cave Daesubee.

우포늪, 순천만, 서해 갯벌에서부터 분리한 황산염/황-환원 세균의 특성 분석

김소정,민의기,홍희지,김종걸,정만영,차인태,이성근,Kim, So-Jeong,Min, Ui-Gi,Hong, Heeji,Kim, Jong-Geol,Jung, Man-Young,Cha, In-Tae,Rhee, Sung-Keun 한국미생물학회 2014 미생물학회지 Vol.50 No.3

황화합물은 혐기성환경에서 혐기성호흡을 위한 매우 중요한 전자수용체이다. 본 연구를 통하여 한국의 다양한 습지에서 배양을 통한 황산염/황-환원세균의 특성연구를 실시하였다. 이를 분리하기 위하여 혐기성 roll tube법을 통해 총 11개의 순수 배양체를 확보하였다. 16S rDNA를 이용한 계통분석 및 상동성 분석을 실시하여 Desulfovibrio 속의 세균 8종, Sulfurospirillum 속의 세균 2종, Desulfitobacterium 속의 세균 1종을 얻을 수 있었다. 이들 황산염/황-환원세균은 모두 lactate와 pyruvate를 전자공여체로 이용하였으며, sulfite and thiosulfate를 전자수용체로 이용할 수 있었다. 앞으로, 다양한 전자공여체와 배양조건을 통하여 유용한 절대혐기성 황산염/황-환원세균의 생물자원 확보에 기여할 것으로 기대된다.