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dc.contributor.author주양희-
dc.creator주양희-
dc.date.accessioned2016-08-26T03:08:08Z-
dc.date.available2016-08-26T03:08:08Z-
dc.date.issued2003-
dc.identifier.otherOAK-000000003507-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/194604-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000003507-
dc.description.abstractGenerally, oil spill accidents are often likely to occur in the form of ship wreckage or storage tanker leaks in marine ecosystem, groundwater. For remediation of these contaminated sites, bioremediation is suggested and applied. However, in many cases, the used oil degrading bacteria is not adapted to the real contaminated site condition such as low temperature. In this study, cold-adapted or psychrotrophic oil-degrading bacteria had been isolated from biofilm which was formed in an oil contaminated site, and I characterized these bacteria. From microbial community of the biofilm, Rhodococcus sp., Acinetobacter, Bacillus sp., Enterobacter sp., and Sphaerotilus sp. were isolated. Among these isolate, Rhodococcus sp. strain YHLT-1 and Rhodococcus sp. strain YHLT-2 were tested for the growth ability from 4 to 30℃ and studied their growth patterns and specific growth rates at each temperature. Both of strain YHLT-1 and YHLT-2 could grow at 4, 15, and 30℃. These strains could degrade the crude oil as a sole carbon source. The oil degrading activities of total hydrocarbons and each TPH in crude oil increased over the 90% at 4 ~ 15℃ as well as 30℃ when initial crude oil concentration was added at 8,000 mg/L. These results indicated that these strains were cold-adapted or psychrotrophic crude oil degrading bacteria. Strain YHLT-2, which had better oil degradability than strain YHLT-1, was tested for the resistance against high salt concentration and oil degrading activities on the soil medium. Strain YHLT-2 could grow on LB medium containing 7% (w/v) NaCl at 15℃ and significantly degrade the oil on the soil medium, even though the degradability was lower than liquid medium at 4 and 15℃. Therefore, Rhodococcus sp. strain YHLT-1 and YHLT-2, which are cold-adapted and halotolerance bacteria, would be used at petroleum contaminated sites under various environmental conditions of low temperature and high salt concentration for bioremediation. Microbial operation, which was involved in the process of the pollutant treatment or in the contaminated site, except physicochemical process, has influential in degrading pollutants. However, microorganisms in environmental samples have not been accurately investigated, because typical cultivation-dependent methods can detect only a fraction of total bacteria present in an environment. Thus, it needs to study characteristics of microbial community using molecular analysis methods such as PCR amplification tageting 16S-rDNA and denaturing gradient gel electrophoresis (DGGE) analysis for acquiring more specific information. In this study, three types of environmental samples, which were thermobiofilter, hot compost, and soil samples, had been analyzed using DGGE analysis. In case of thermobiofilter samples which were operated at hot temperature, microbial communities of biofilter where benzene/toluene (BT biofilter) and food-waste odor (FW biofilter) had been treated, were compared with inoculated source (Source). In addition, compost samples were analyzed to assess microbial succession during the composting operation process. Finally, microbial communities of rice field soil (AE) and tidal mud flat (OE), which were enriched under the anaerobic condition adding toluene and nitrate as a sole carbon source and a final electron acceptor, respectively, were compared with control samples (AR and OR). In the thermobiofilter, most isolates of Source belonged to gamma-subdivision. BT biofilter involved Actinobacteria and FW biofilter involved baillales compared with the other samples. Hot compost sample exhibited fungi during the composting. Thermophilic bacteria, which belonged to bacillales, were detected at final process. In the rice field soil, control sample exhibited very simple band pattern and its isolates were belong to agricultural soil bacteria. However, the treated sample exhibited various bacteria belong to alpha, beta, gamma-subdivision, especially, Denitrificians which used nitrate, was detected. In the tidal mud flat, control sample s isolates were related with marine microorganisms, however, isolates from treated soil sample were belong to various subdivision, especially, Thauera sp. which related with denitrificians, was detected. This molecular analysis method can provided accurate information on important microbial communities s role in environmental samples. Moreover, DGGE could confirm the changes in microbial patterns, visually. ; 대부분의 기름유출사고는 해양이나 지하수 등에서 선박의 침몰이나 저장탱크에서 유출되는 것이 빈번히 발생하고 있다. 이런 오염지역의 복원하는데 있어. bioremediation은 경제적이며 효율적인 방법으로 제시되고 실용화되고 있으나, 적용하는 미생물은 실제 지하 환경의 저온에 적응되어 있지 않은 것을 사용하는 경우가 많이 있다. 본 연구에서는 유류로 오염된 곳에 형성된 biofilm으로부터 지하 환경과 유사한 저온 환경에 적응한 원유 분해능을 가진 미생물을 분리하고 그 특성을 알아보고자 하였다. Biofilm의 microbial community에서 나온 균은 Rhodococcus sp., Acinetobacter, Bacillus sp., Enterobacter sp.와 Sphaerotilus sp.로 밝혀졌으며, 이중 Rhodococcus sp. strain YHLT-1과 Rhodococcus sp. strain YHLT-2로 모두 4℃부터 30℃까지 성장이 가능한가를 확인하고 그 성장 양상과 비성장 속도를 조사하여본 결과 각 온도에서 성장이 가능하였고 각 온도별 원유분해 경향을 확인하였으며, total hydrocarbon과 원유내 각 TPHs별 상대분해활성에 대해서도 확인해본 결과, 원유의 농도를 대략 8,000 mg/L로 초기에 주입하였을 때, 30℃, 15℃, 그리고 4℃에서 원유를 비생물학적 요소를 제외하고 계산한 상대분해활성으로 보았을 때, 90% 이상의 값을 보이며, 모두 분해능을 가지고 있음을 확인할 수 있었다. 이를 통해 볼 때, biofilm으로부터 분리된 원유분해 균주는 psychrotrophic 또는 cold-adapted crude oil degrading bacteria인 것이 확인되었다. 또한 strain YHLT-1보다 빠르고 효율적인 분해능을 보인 strain YHLT-2를 가지고 염도별 실험을 통해 염에 대한 저항성을 조사하였고, 액상 외에 토양에서 갖는 분해능의 정도에 대해서도 알아보았다. 염도에 대한 저항성은 염분이 7% (w/v)까지 15℃ 조건에서 성장하는 것이 확인되었으며, 토양에서 원유의 분해능을 확인해본 결과, 15℃와 4℃에서 액상보다 상대분해활성이 떨어지기는 하지만 분명히 분해가 진행되는 것이 확인되었다. 그러나 이것은 초기 원유 농도가 80,000 mg/L로, 원유에 의한 inhibition이 크게 작용했으리라 추측된다. 본 실험을 통해 확인한 것은 biofilm에서 분리된 Rhodococcus sp. strian YHLT-1과 2, 모두 유류 오염지역에 bioremediation으로써 적용이 가능할 것으로 보이며, 특히 저온 환경과 염도가 있는 환경에 대해서도 원유를 분해하는데 이용될 수 있을 것이다. 오염된 물질을 처리하는 과정에서 사용하는 미생물학적 분해나, 오염된 곳에서의 물리화학적 변화 외에 일어나는 미생물의 변화는 각 process에 상당한 영향을 미치고 있다. 그러나 실제, 미생물 가운데 현재까지 사용되는 배지조건에서 배양되어지는 것과 배양되어지지 않은 것이 존재하므로, 이제까지 배양되는 미생물들이 전체 미생물 군집의 1%미만이란 점을 고려해볼 때, 보다 정확한 고찰이 필요할 것이다. 이런 미생물 군집을 16S-rDNA sequence를 증폭하여 DGGE를 통해 그 변화를 확인하고 각 bands를 isolated하고 identify하는 이용한 분자생물학적 방법으로 보다 정확한 특성을 알아보고자 하였다. 본 연구에서 조사된 환경시료는 총 3종류로, 고온바이오필터(thermobiofilter)와 hot compost, 그리고 토양 sample이다. 고온에서 운전된 thermobiofilter로 각각 benzene/toluene을 분해하는 것(BT biofilter)과 음식물 쓰레기 악취를 처리하는 것(FW biofilter)의 미생물상을 접종원(Source)과 비교 분석하였다. 또한 compost는 공정기간동안 미생물상 변화를 조사하였다. 마지막으로 toluene과 nitrate를 첨가하여 혐기적 조건에서 enrichment culture한 전(control) 후(treated)의 논토양(AR/AE)과 갯벌(OR/OE)의 미생물상을 비교 분석하였다. Thermobiofilter의 경우, 접종원은 gamma-subdivision에 속한 균들이 많이 나왔다. BT biofilter의 경우는 Acinobacteria가 주를 이루고, FW biofilter의 경우는 다른 sample보다 bacillales가 더 많이 나온 것을 확인할 수 있었다. Hot compost의 sample의 경우, 공정중간에 fungi가 나오는 것을 확인할 수 있었으며, 마지막날의 sample에서는 bacillales속에 속하는 것으로 고온성 bacteria가 검출되어졌다. 논토양의 경우, enrichment culture 전에는 단순한 band를 보이고 분석결과도 논토양에서 나오는 균주였으나, enrichment culture 이후에는 다양한 subdivision에 속하는 것들이 나오는 것으로 확인되었다. 특히 nitrate를 이용하는 denitrificians가 검출되어졌으며, alpha, beta, gamma-subdivision으로 속하는 group이 다양하게 나타났다. 갯벌 시료의 경우, enrichment culture 전에는 해양과 관련된 균주들로 구성되었으나, 이후에는 더 다양한 subdivision으로 나뉘며, 역시 denitrificians와 관계가 있는 Thauera sp. 와 같은 종이 검출되어졌다. 이를 이용해서 보다 환경시료 내부에 중요한 역할을 하는 미생물 군상에 대해 보다 정확한 정보를 얻을 수 있었으며, DGGE를 통한 시각적 확인이 가능한 것을 확인할 수 있었다.-
dc.description.tableofcontentsCONTENTS = i TABLES = v FIGURES = vii ACKNOWLEDGEMENT = x Part 1. Isolation and characterization of cold-adapted crude oil degrading bacteria ABSTRACT = 1 I. INTRODUCTION = 3 II.THEORY = 6 2.1. Environmental fate of petroleum hydrocarbons = 6 2.2. TPH analysis methods = 10 2.2.1. Petrol range organics (PRO) analysis = 10 2.2.2. Diesel range organic (DRO) analysis = 11 2.3. Treatment of petroleum hydrocarbon pollution = 11 2.3.1. In situ bioremediatin technologies = 14 2.3.2. Ex situ bioremediatin technologies = 15 2.4. Environmental factors affecting biodegradation = 18 2.5. Biodegradation of petroleum hydrocarbons in aerobic condition = 20 2.5.1. Bioavailability = 20 2.5.2. Pathway of hydrocarbon degradation = 21 2.5.3. Oil-degrading bacteria = 23 III. MATERIALS AND METHODS = 25 3.1. Media and crude oil = 25 3.2. Site description and biofilm sampling = 26 3.3. Screening and isolation of cold-adapted crude oil degrading bacteria from biofilm = 29 3.4. Identification of isolated crude oil degrading bacteria = 29 3.4.1. PCR amplification of isolated crude oil degrading bacteria = 29 3.4.2. Sequence PCR and sequencing = 31 3.5. Characterization of cold-adapted crude oil degrading bacteria = 34 3.6. Comparison of crude oil degradability of isolates at different temperatures = 36 3.7. Methods of analysis = 37 IV. RESULTS AND DISCUSSION = 38 4.1. Isolated bacteria from biofilm = 38 4.2. Identification of isolated bacteria from biofilm = 38 4.2.1. The results of isolated bacteria sequence from biofilm = 38 4.2.2. The phylogenetic diagram of isolated bacteria from biofilm = 43 4.3. Specific growth rate and halotolerance of isolates = 46 4.3.1. Specific growth rate at different temperatures = 46 4.3.2. Halotolerance = 50 4.4. Comparison of crude oil degradability at different temperatures = 53 4.4.1. Growth pattern on crude oil at different temperatures = 53 4.4.2. Concentration of residual crude oil at different temperatures = 55 V. CONCLUSION = 70 ABSTRACT (Korean) = 73 Part 2. Characterization of microbial community in environmental samples using denaturing gradient gel electrophoresis ABSTRACT = 75 I. INTRODUCTION = 77 II. THEORY = 80 2.1. Nucleic acid-based methods = 80 2.2. 16S-ribosomal RNA gene (16S-rDNA) = 81 2.2.1. Definition = 81 2.2.2. Advantages for analysis using 16S-rDNA = 84 2.3. Denaturing gradient gel electrophoresis (DGGE) = 86 2.3.1. Theory and concept = 86 2.3.2. Advantages and disadvantages of DGGE = 87 III. MATERIALS AND METHODS = 88 3.1. Description of samples = 88 3.2. The extraction of genomic DNA = 89 3.2.1. Thermobiofilter samples = 89 3.2.2. Hot compost samples = 90 3.2.3. Soil samples = 90 3.3. PCR amplification of 16S-rDNA fragments = 91 3.3.1. Thermobiofilter and hot compost samples = 91 3.3.2. Soil samples = 92 3.4. Denaturing gradient gel electrophoresis(DGGE) analysis = 94 3.4.1. Thermobiofilter samples = 94 3.4.2. Hot compost and soil samples = 94 3.5. DGGE band isolation and analysis = 95 3.6. Sequence PCR and sequencing = 96 IV. RESULTS AND DISCUSSION = 98 4.1. The DGGE patterns of various environmental samples = 98 4.1.1. The DGGE patterns of thermobiofilter samples = 100 4.1.2. The DGGE patterns of hot compost samples = 100 4.1.3. The DGGE patterns of soil samples = 100 4.2. DGGE band sequence analysis = 103 4.2.1. The microbial communities of thermobiofilter samples = 103 4.1.2. The microbial communities of hot compost samples = 109 4.1.3. The microbial communities of soil samples = 114 V. CONCLUSION = 120 ABSTRACT (Korean) = 124 BIBLIOGRAPHY = 126 APPENDIX = 141-
dc.formatapplication/pdf-
dc.format.extent1895701 bytes-
dc.languageeng-
dc.publisher이화여자대학교 과학기술대학원-
dc.titleCharacterization of microbial community in environmental samples by 16S-rDNA analysis-
dc.typeMaster's Thesis-
dc.identifier.thesisdegreeMaster-
dc.identifier.major과학기술대학원 환경학과-
dc.date.awarded2003. 2-
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