Denaturing gradient gel electrophoresis를 이용한 한국의 논 토양 미생물 다양성 분석 방법

최명은 ( Myeongeun Choe ),홍성준 ( Sung Jun Hong ),임종희 ( Jong Hui Lim ),곽윤영 ( Yun Young Kwak ),백창기 ( Chang Gi Back ),정희영 ( Hee Young Jung ),이인중 ( In Jung Lee ),신재호 ( Jae Ho Shin ) 한국응용생명화학회 2013 Journal of Applied Biological Chemistry (J. Appl. Vol.56 No.2

Soil microbes are important integral components of soil ecosystem which have significant and diverse role in organic matter decomposition, nitrogen cycling, and nitrogen fixation. In this study an effective denaturing gradient gel electrophoresis (DGGE) method was employed for paddy soil microbial diversity survey. For optimum paddy soil microbial DNA extraction, different methods such as Lysis buffer, skim milk bead, sodium phosphate buffer, Epicentre Soil Master DNA extraction kit (Epicentre, USA) and Mo Bio Power Soil DNA kit (MO BIO, USA) methods were utilized. Among all the method, using Mo Bio Power Soil kit was most effective. DGGE analysis of Bacteria was carried out at 6% polyacylamide gel and 45-60% denaturing gradient in the optimal conditions. Whereas DGGE analysis of fungi was done at 6% polyacrylamide gel and 45-80% denaturing gradient in the optimal conditions. By applying the above assay, it was found that variation within the microbial community of paddy soil occurs by a factor of time. DGGE say used in this study through for a variety of soil microbial analysis suggests the potential use of this method.

Benzene, toluene 및 xylene 혼합가스의 혐기적 생분해

조경숙,김윤형,심은화,류희욱 한국냄새환경학회 2004 실내환경 및 냄새 학회지 Vol.3 No.3

Degradation characteristics of the mixtures of benzene, toluene and xylene (BTX) coupled to nitrate reduction were investigated in enrichment culture and the microbial community of the enriched BTX-degrading consortium was characterized by 16S rDNA PCR (polymerase chain reaction) and denaturing gradient gel electrophoresis (DGGE). The degradation rates of benzene, toluene and xylene in the mixtures by the enriched consortium were 113.6, 59.5, and 35.2 μmol L-1 d-1, respectively. Among sixteen clones isolated from the consortium through DGGE, five clones (BTX 1, BTX 8, BTX10, BTX 11, and BTX 12) showed numerical dominance. The clones BTX 10 and BTX 11 were most closely related to uncultured bacteria inhabiting aquatic environments contaminated with solvent or monochlorobenzene. These clones are expected to give a contribution to BTX degradation in the consortium. Five clones (BTX 2, BTX 6, BTX 14, BTX 15, and BTX 16) were affiliated with Pseudomonas sp. These clones may play an important role on BTX degradation although it is not clear how the clones function on the biodegradation mechanisms of BTX under anaerobic condition. 최종 전자수용체로 nitrate를 이용하여 benzene, toluene 및 xylene 혼합가스(BTX)를 분해하는 미생물을 집식배양하여 얻은 consortium의 BTX 분해 특성을 조사하였다. 또한, 혐기성 BTX 분해 consortium의 미생물 군집특성을 16S rDNA polymerase chain reaction (PCR)과 denaturing gradient gel electrophoresis (DGGE)를 이용하여 분석하였다. BTX 혼합조건하에서 consortium에 의한 benzene, toluene 및 xylene의 분해속도는 각각 113.6, 59.5, 및 35.2 μmol L-1 d-1이었다. DGGE를 통해 consortium으로부터 분리한 16개의 clones 중에서, BTX 1, BTX 8, BTX10, BTX 11, 및 BTX 12의 5개 clones이 우점종임을 알 수 있었다. BTX 10과 BTX 11는 용매 혹은 monochlorobenzene으로 오염된 수환경에서 서식하는 uncultured bacteria와 가장 유사성이 높은 것으로 동정되었는데, 이러한 결과는 이들 clones이 BTX의 혐기적 생분해에 중요하게 기여하고 있는 것을 시사한다. 또한, 5개의 clones(BTX 2, BTX 6, BTX 14, BTX 15, BTX 16)이 Pseudomonas sp.와 유사성이 높게 분석되었다. 비록 이들 clones이 혐기적 조건하에서 BTX 분해에 어떻게 관여하는지는 밝히지 못했지만, 이 clones도 BTX 생분해에 중요한 역할을 담당할 것으로 사료되었다.

DGGE 방법과 배양법을 이용한 강원지역 전통 발효 청국장에서 미생물의 다양성 분석

홍성욱(Sung Wook Hong),임인규(In Kyu Lim),김용우(Yong Woo Kim),신승미(Seung-Mee Shin),정건섭(Kun Sub Chung) 한국식품과학회 2013 한국식품과학회지 Vol.45 No.4

전통적인 방법으로 제조한 청국장과 볏짚시료로부터 배양방법과 비배양방법인 DGGE를 이용한 청국장의 발효미생물 군집을 분석하였다. 배양방법에서는 미륵산농원의 볏짚과 청국장 시료에서 P. agglomerans (65%)와 B. subtilis (60%)가 우점미생물로 나타내었고, 흥업토속 청국장의 볏짚과 청국장 시료에서는 B. amyloliquefaciens (57%와 52%)가 우점미생물로 확인하였다. DGGE 분석에서는 미륵산농원의 볏짚과 청국장 시료에서 P.agglomerans (Band intensity 20.6%)와 B. subtilis (Band intensity 25.7%)가 우점미생물로 나타내었고 흥업토속 청국장의 볏짚과 청국장 시료에서는 B. amyloliquefaciens (Band intensity 27.3%, 26.4%)가 우점미생물로 확인하였다. 그 외에도 볏짚에서는 Pantoea sp. Enterococcus durans, Enterobacter sp, P. ananatis, Bacillus sp., Rhodococcus sp. Pseudomonas fulva, Acinetobacter sp., B.licheniformis, B. amyloliquefaciens 등의 미생물을 확인하였고 청국장에서는 B. licheniformis, B. subtilis, Bacillus sp., B. circulans, Acinetobacter sp. 등의 미생물을 확인이 되었으며, 비배양 방법을 이용한 청국장 발효미생물 균총 조사는 배양이 불가능한 미생물을 확인할 수 있었다. Bacterial communities derived from cheonggukjang and raw rice straw collected from a Mireuksan farm and a Heungup cheonggukjang in Gangwon province were investigated using both culture-based method and denaturing gradient gel electrophoresis (DGGE) analysis. Pure cultures, which were isolated from raw rice straw and cheonggukjang and cultured on tryptic soy agar plates (53-76 colonies per plate), were identified by analysis of 16S rRNA sequences. The traditional culture-based method and analysis of PCR-amplified 16S rRNA by DGGE revealed that for samples collected from the Mireuksan farm, Pantoea agglomerans and Bacillus subtilis were the predominant species in the raw rice-straw and cheonggukjang, respectively. For samples collected from the Heungup cheonggukjang, Bacillus amyloliquefaciens was the predominant species in both raw rice straw and cheonggukjang. Other microorganisms, including members of Pantoea, Bacillus, Enterococcus, Enterobacter, Pseudomonas, Rhodococcus, and Acinetobacter, were also present in the raw rice-straw and cheonggukjang, as were bacteria that could not be cultured.

Detection of Nocardia sp. Hl7-1 by PCR during Bioremediation of Crude Oil-Contaminated Soil

Baek, Kyung-Hwa,Lee, Young-Ki,Lee, In-Sook,Oh, Hee-Mock,Yoon, Byung-Dae,Kim, Hee-Sik 한국미생물 · 생명공학회 2004 한국미생물·생명공학회지 Vol.32 No.1

For the detection of the oil-degrading bacterium, Nocardia sp. Hl7-1, inoculated during the bioremediation of oil-contaminated soil, a species-specific primer was constructed based on the 16S rDNA sequence of this strain. Two forward primers and two reverse primers were designed and tested against both closely and distantly related bacterial strains. All the primers designed were specific to the Nocardia sp. H17-1. Particularly, primer sets NH169F-NH972R and NH575F-NH972R could be used to detect 50 fg of template DNA and $1.2${\times}$10^4$ CFU/g of sandy soil. These two PCR primer sets successfully detected the H 17-1 strain in the oil-con-laminated soil samples containing heterogeneous DNA. We also conformed the primer specificity by restriction-enzyme cleavage of the PCR products and denaturing gradient gel electrophoresis. 원유로 오염된 토양의 생물학적 복원과정 동안 접종된 Nocardia sp. Hl7-1 균주를 확인하기 위해 165 rDNA sequence에 기초하여 균주에 특이적인 primer를 제작하였다 14균주의 16S rDNA sequence비교를 통해 제작된 4개의 primer set는 Hl7-1 균주를 특이적으로 검출할 수 있었다. 특히 NH169F-NH972R과 NH575F-NH972R의 primer set는 50 fg의 DNA와 $1.2${\times}$10^4$ cfu/g-soil의 균체농도까지 민감하게 검출할 수 있었다. 이 두 primer set는 원유로 오염된 토양의 bioremediation과정 동안 접종된 Hl7-1 균주의 특이적 검출을 가능케 하였으며, 이는 사용된 primer set에 의해 증폭된 PCR산물을 제한효소(EcoRI)로 절단한 결과와 DGGE를 통한 Hl7-1 균주의 확인을 통해 본 연구에서 제작된 primer set의 특이성을 검증하였다.

Comparison of Bacterial Community in Soil Using Denaturing Gradient-Gel Electrophoresis and Forensic Application

이준희,한지혜,최미정,안희중 한국과학수사학회 2013 과학수사학회지 Vol.7 No.3

The microbial diversity can be regarded as an integral part for comparative forensic analysis of soil. This diversity may be used as a biological indicator in tracing the original location of the soil or comparing co-identity of soils. In order to evaluate this effectiveness, we examined the bacterial communities of soil by collecting soil samples from nine different sites in Daejeon and applying a 16S rRNA-based cultivation-independent approach. The formation of bacterial community in soil was more influenced by its ecosystem than its geographical location. This study provides a principal data for soil discrimination by comparative analysis of soil microbial community. If this biological analysis will be used with other physical and chemical analysis, which can be a powerful tool for comparing co-identity of soils and this will make it possible to trace the original location of the soil.

콩 재배가 토양 미생물 군집 활성도에 미치는 영향

백계령,이계준,김태영 한국환경농학회 2019 한국환경농학회지 Vol.38 No.2

BACKGROUND: For sustainable agriculture, there are various agricultural practices including low input. Over the last few decades high input of chemical fertilizer and compounds results in environmental pollution and deterioration of soil fertility. Soybean (Glycine max L.) is well known eco-friendly crop due to their symbionts. Soybean has a relationship with nitrogen fixation bacteria called rhizobia. In this research work, we investigated effects of soybean cultivation on soil microorganism activities. METHODS AND RESULTS: Experiments were conducted in pots and potato cultivation was used as reference. Soil chemical properties were analyzed considering soil nutrient over cropping period. For the soil microbial community analysis, dehydrogenase activity analysis (DHA) analyzed along with denaturing gradient gel electrophoresis. The results showed that higher soil organic matter in the soybean cultivation soil than in the potato cultivation soil. Available P2O5 concentration increased gradually in both pots but showed higher value in the potato cultivation soil. DHA value implying microbial activities showed higher value in the soybean cultivation soil over all cropping period. CONCLUSION: The cause of high microbial activity in the soybean cultivation soil was considered to the effects of some specific microorganisms related to soybean cultivation. Therefore, the availability of soybean cultivation for sustainable agriculture should be encouraged in terms of microorganism community activity in soil.

PCR-DGGE를 이용한 누룩에서의 미생물 다양성 분석

Seung Jik Kwon(권승직),Jae Hak Sohn(손재학) 한국생명과학회 2012 생명과학회지 Vol.22 No.1

누룩은 탁주와 약주의 제조를 위한 당화효소와 알코올발효를 위한 미생물의 공급원으로서 제품의 맛과 품질을 결정하는 중요한 역할을 한다. 본 연구에서는 산성누룩의 세균과 진균의 다양성을 조사하기 위해 순수분리 종과 16S 및 28S rRNA gene를 대상으로 한 PCR-DGGE를 이용한 분석을 수행하였다. 누룩 내 세균의 수는 2.7×10? CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcous spp., Weissella spp., Staphylococcus spp. 그 외 endophytic bacterium, uncultured gamma-proteobacteria, uncultured Cyanobacteria와 Actinobacteria였다. PCR-DGGE profile에서 주된 우점종은 Pediococcous pentosaceus와 uncultured Cyanobacteria 이었다. 누룩 내 진균의 수는 3.5×10? CFU/g이었으며 순수분리와 PCR-DGGE 분석에서 우점종은 Trichomonascus spp., Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp., Aspergillus spp., Cladosporium spp.였다. PCR-DGGE profile에서 주된 우점종은 Pichia kudriavzevii와 Aspergillus oryzae이었다. PCR-DGGE 기술은 본 연구에서 누룩의 미생물군집을 평가하기 위해 처음으로 사용되었으며 미생물 다양성을 설명하는 데 효과적임을 입증하였다. Nuruk plays a significant role in the flavor and quality of Takju and Yakju, which are produced through saccharification and alcohol fermentation by various microorganisms. In this study, we identified microbial strains isolated from a plate count and PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes, in order to characterize bacterial and fungal diversity in Sansung Nuruk. The numbers of bacteria and fungi in Nuruk were 1.5×10? CFU/g and 2.2×10? CFU/g, respectively. The 16S rRNA gene sequence indicated that the predominant bacteria in the isolates and PCR-DGGE profile of Nuruk were Kocuria spp., Pantoea spp., Lactobacillus spp., Pediococcus spp., Weissella spp., Staphylococcus spp., endophytic bacterium, uncultured Gamma-proteobacteria, uncultured Cyanobacteria, and Actinobacteria. Dominant bacteria from the PCR-DGGE profile were Pediococcous pentosaceus and uncultured Cyanobacteria. The 28S rRNA gene sequence indicated the predominant fungi in the isolates and PCR-DGGE profile to be Trichomonascus spp. Pichia spp., Torulaspora spp., Wickerhamomyces spp., Sacharomycopsis spp., Lichtheimia spp., Mucor spp., Rhizopus spp. Aspergillus spp., and Cladosporium spp. Dominant fungi from the PCR-DGGE profile were Pichia kudriavzevii and Aspergillus oryzae. The PCR-DGGE technique was used for the first time in this study to assess a microbial community in Nuruk and proved to be an effective protocol for profiling microbial diversity.

PCR-DGGE를 이용한 막걸리발효에서 미생물 다양성 분석

Seung Jik Kwon(권승직),Tae-Young Ahn(안태영),Jae Hak Sohn(손재학) 한국생명과학회 2012 생명과학회지 Vol.22 No.2

금정산성 막걸리<SUP>®</SUP>는 전통적인 수제누룩과 쌀로부터 발효된 한국의 전통적인 술이다. 본 연구에서는 막걸리 발효기간 동안 세균과 진균의 다양성을 특성화하기 위해 16S와 28S rRNA 유전자를 목적으로 하는 PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) 분석을 수행하였다. 막걸리 발효기간 동안 PCR-DGGE profile에서 검출된 세균은 16S rRNA 유전자 서열에 기초한 동정결과 Lactobacillus spp. (L. curvatus, L. kisonensis, L. plantarum, L. sakei 및 L. gasseri), Pediococcus spp. (P. acidilactici, P. parvulus, P. agglomerans및 P. pentosaceus), Pantoea spp. (P. agglomerans 및 P. ananatis) 그리고 Citrobacter freundii로 총 12종이었으며, 배양2일 이후 L. curvatus가 주된 우점 종을 형성하였다. 반면 PCR-DGGE profile에서 검출된 진균은 28S rRNA 유전자 서열에 기초한 동정결과 Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera 및 Torulaspora delbrueckii로 6종이었으며 주된 우점 진균은 배양0일에서 2일에 P. kudriavzevii에서 배양 3일에서 6일에 S. cerevisiae로 전환되었다. 결과적으로 PCR-DGGE분석은 막걸리발효기간 동안 미생물의 구조와 다양성을 이해하는 데 유용한 도구임을 보여주었다. Kumjungsansung-Makgeolli<SUP>®</SUP> is a traditional Korean rice wine that is fermented from traditional nuruk and rice. In this study, we performed the PCR-denaturing gradient gel electrophoresis (DGGE) analysis targeting the 16S and 28S rRNA genes to characterize bacterial and fungal diversity during Makgeolli fermentation. The predominant bacteria in the PCR-DGGE profile during Makgeolli fermentation were Lactobacillus spp. (Lactobacillus curvatus, L. kisonensis, L. plantarum, L. sakei, and L. gasseri), Pediococcus spp. (P. acidilactici, P. parvulus, P. agglomerans, and P. pentosaceus), Pantoea spp. (P. agglomerans and P. ananatis), and Citrobacter freundii; these were identified on the base of analysis of 16S rRNA gene sequences. The dominant bacterium during Makgeolli fermentation was L. curvatus. The predominant fungi in PCR-DGGE profile during Makgeolli fermentation were Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera, and Torulaspora delbrueckii, and these were identified on the basis of analysis of 28S rRNA gene sequences. The dominant fungal species during Makgeolli fermentation changed from P. kudriavzevii at 0-2 days incubation to S. cerevisiae at 3-6 days incubation. This study suggests that PCR-DGGE analysis could be a suitable tool for the understanding of microbial diversity and structure during Makgeolli fermentation.

혐기성 미생물에 의한 toluene과 xylene 분해 특성

조경숙,조원실,류희욱 한국냄새환경학회 2004 실내환경 및 냄새 학회지 Vol.3 No.2

Six kinds of the microbial consortia were obtained from the enrichment culture using the mixture gases of toluene and xylene as sole carbon sources and sulfate as a final electron acceptor. The degradation rates of the mixture gases by the consortia were determined, and the microbial structure in the consortia were characterized using 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE). The enriched consortia could simultaneously degrade toluene and xylene in the mixture gases. The toluene and xylene degradation rates in the mixture gases were 24.6∼49.1 and 2.8∼6.2 μmol L-1 d-1, respectively. Nine clones among 15 clones, isolated from the enriched consortia, were affiliated with the bacteria related to the degradation of hydrocarbons, chlorinated solvent, trichlorobenzene and TCE. These clones are expected to play important roles on the anaerobic degradation of toluene and xylene in the consortium. Toluene과 xylene의 혼합가스를 유일 탄소원으로 하고, sulfate를 최종전자수용체로 공급한 조건에서 농화 배양하여 얻은 6 종류의 미생물 농화배양액의 toluene과 xylene분해 특성을 조사하고 미생물 군집 특성을 분자생물학적 방법을 이용하여 분석하였다. 본 연구에서 얻은 농화배양액은 toluene과 xylene을 동시에 분해할 수 있었고, toluene과 xylene 분해속도는 각각 24.6∼49.1 및 2.8∼6.2 μmol L-1 d-1이었다. 농화배양액으로부터 분리한 15개의 clone 중에서 9개의 clone이 탄화수소화합물, 염소계 용매, trichlorobenzene 및 TCE 분해와 관련된 세균들과 유사성이 높은 것으로 동정되었는데, 이들 clone이 toluene 및 xylene 혼합가스의 혐기성 생분해에 관여하고 있을 것으로 사료되었다.

Perchloroethylene과 Trichloroethylene의 혐기적 탈염소화 및 미생물 군집 분석

이재원,김병혁,안치용,김희식,윤병대,오희목,Lee Jae-Won,Kim Byung-Hyuk,Ahn Chi-Yong,Kim Hee-Sik,Yoon Byung-Dae,Oh Hee-Mock 한국미생물학회 2005 미생물학회지 Vol.41 No.4

울산, 여수 등 공단지역의 토양, 하천의 저니, 해양의 준설토 둥을 이용하여 난분해성 염소화합물인 PCE (perchloroethylene)및 TCE (trichloroethylene)의 혐기성 탈염소화에 관련하는 미생물을 탐색하고 이들의 탈염소화 효율을 조사하였다. 혐기성 상호대사에 의한 탈염소화 효율을 조사하기 위해 전자공여체로 acetate를 사용하여 혐기성 회분식 실험을 실시하였으며, 이와 병행하여 분자생물학적인 기법인 16S rDNA의 PCR-Double Gradient DGGE (DG-DGGE)를 이용하여 미생물의 군집을 분석하였다. 그 결과 울산 태화강 및 여수 하남천의 저니를 접종한 경우 PCE는 $70\%,\;65\%$, TCE는 $50\%,\;45\%$의 높은 탈염소화 효율을 나타내었다. 또한 16S rDNA의 PCR을 이용한 DG-DGGE로 미생물 군집을 분석한 결과, 탈염소화 효율이 높은 지역의 저니에는 Desulfovibrio sup.의 미생물이 주로 존재함을 확인하였다. In this study, the anaerobic enrichment cultivation was performed with the sediments and the dredged soils from the cities of Ulsan, Masan, Yeosu, Gwangyang, Ansan and Seongnam. Acetate as an electron donor and PCE (perchloroethylene) or TCE (trichloroethylene) as an electron acceptor were injected into the serum bottle with an anaerobic medium. After the incubation of 12 weeks, the removal efficiency of PCE was highest at $70\%$ in the treatment with the sediment of Ulsan. Also, the bacterial community structure was analyzed by D-DGGE (double denatured gradient gel electrophoresis) through PCR-based 16S rDNA approaches. The dominant species id the anaerobic enrichment were found to belong to the genus of Desulfovibrio.