Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil
학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2020. 8. 가종억. BTEX (benzene, toluene, ethylbenzene, and xylene) compounds are the most frequently encountered subsurface contaminants among the various petroleum hydrocarbons. For contamination removal at cold climate sites, bioremediation technology is appe...
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Format: | Doctoral or Postdoctoral Thesis |
Language: | Korean |
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서울대학교 대학원
2020
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Online Access: | http://hdl.handle.net/10371/169661 http://dcollection.snu.ac.kr/common/orgView/000000162711 |
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ftseoulnuniv:oai:s-space.snu.ac.kr:10371/169661 |
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record_format |
openpolar |
institution |
Open Polar |
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Seoul National University: S-Space |
op_collection_id |
ftseoulnuniv |
language |
Korean |
topic |
Massilia aromaticivorans New Taxa BTEX compounds Biodegradation 신종 BTEX 물질 생물학적 분해 630 |
spellingShingle |
Massilia aromaticivorans New Taxa BTEX compounds Biodegradation 신종 BTEX 물질 생물학적 분해 630 손지관 Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
topic_facet |
Massilia aromaticivorans New Taxa BTEX compounds Biodegradation 신종 BTEX 물질 생물학적 분해 630 |
description |
학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2020. 8. 가종억. BTEX (benzene, toluene, ethylbenzene, and xylene) compounds are the most frequently encountered subsurface contaminants among the various petroleum hydrocarbons. For contamination removal at cold climate sites, bioremediation technology is appealing due to their potential to be more economical and efficient than alternative. The aim of this study is to isolate, identify, and characterize novel bacterial strains from the Arctic and Antarctica soil that could degrade the BTEX compounds at low temperature. A novel aromatic hydrocarbon-degrading bacterial strain, designated ML15P13T, was isolated from Arctic soil at the Svalbard Islands, Norway, using enrichment culture technique. Cells were Gram-stain-negative, aerobic, motile with multiple flagella at one polar end, and rod-shaped. Growth was observed at 4-35 ℃, pH 6.0-8.0, and 0-0.5% (w/v) NaCl. According to 16S rRNA gene analysis, strain ML15P13T was grouped with members of the genus Massilia and closely related to Massilia atriviolacea SODT (98.4%), Massilia violaceinigra B2T (98.3%), Massilia eurypsychrophila B528-3T (97.7%), Massilia glaciei B448-2T (97.7%), and Massilia psychrophlia B115-1T (96.6%). Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity between genome sequences of strain ML15P13T and the closely related species ranged from 75.8 to 84.3%, from 19.6±1.0 to 21.6±0.3%, and from 68.8 to 71.0%. The major fatty acids were C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Q-8 was the major ubiquinone. The polar lipid profile showed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, and five unidentified polar lipids. The G+C content of the genomic DNA is 64.2 mol%. The BTEX biodegradation rate was low in MM broth, but adding a small amount of yeast extract, peptone, and tryptone greatly enhanced the biodegradation at low temperature. The ML15P13T utilized 77.3% of BTEX mixture (initial concentration of each BTEX compounds, 30 mg/L) in MM broth with 0.2% yeast extract for 20 days at 15 ℃. Moreover, the BTEX biodegradation rates were assessed at different temperatures (10, 15, 20, and 28 ℃) in MM broth with 0.05% yeast extract. The ML15P13T degraded 63.2, 64.4, and 67.4% of BTEX mixture at 10, 15, and 20 ℃ for 10 days, respectively. In addition, the MP15P13T was characterized by maximal biodegradation ability at 28 ℃ and utilized 72.1% of BTEX mixture for 10 days. Based on the results for genotypic and phenotypic study, it is concluded that strain ML15P13T represents a novel species of the genus Massilia, for which the name Massilia aromaticivorans sp. nov. is proposed. BTEX (벤젠, 톨루엔, 에틸벤젠, 자일렌)은 유류 오염지역에서 가장 자주 발견되는 석유계 탄화수소이다. 추운 기후에서의 환경오염을 제거하기 위해서는 다른 환경정화방법보다 더 효율적이고, 경제적인 생물학적 환경정화 방법이 사용된다. 따라서 본 연구에서는 저온에서 BTEX 물질을 분해할 수 있는 신종 균을 북극과 남극 토양에서 분리, 동정하며, 분해 특성을 분석하였다. 방향족 탄화수소를 분해할 수 있는 신종 미생물 ML15P13T 균주를 북극 노르웨이의 스발바르 제도의 토양에서 enrichment 기법을 이용하여 분리하였다. 균주는 그람 음성균이며 호기성이고, 균주의 한쪽 끝에 여러 개의 편모를 가지면서 운동성을 나타내고, 간균형이었다. 균은 4-35 ℃, pH 6.0-8.0, 0-0.5% (w/v)의 염분농도에서 성장 가능하였다. 16S rRNA 유전자 서열 분석 결과 ML15P13T 균주는 Massilia 속에 속하였으며, Massilia atriviolacea SODT (98.4%), Massilia violaceinigra B2T (98.3%), Massilia eurypsychrophila B528-3T (97.7%), Massilia glaciei B448-2T (97.7%), Massilia psychrophlia B115-1T (96.6%)의 균주들과 가장 밀접하였다. 뉴클레오티드 동일성(average nucleotide identity), 디지털 DNA-DNA 혼성화(digital DNA-DNA hybridization), 아미노산 동일성(average amino acid identity)의 분석 결과는 각각 75.8-84.3%, 19.6±1.0-21.6±0.3%, 68.8-71.0%였다. C16:0, summed feature 3 (C16:1 ω6c 그리고/또는 C16:1 ω7c), summed feature 8 (C18:1 ω7c 그리고/또는 C18:1 ω6c) 가 주요한 지방산으로 검출되었다. Q-8이 주요한 유비퀴논 (ubiquinone)이었다. 주요 극성 지질 검출 결과 포스파티딜에탄올아민 (phosphatidylethanolamine), 포스파티딜글리세롤 (phosphatidylglycerol), 디포스파티딜글리세롤 (diphosphatidylglycerol)과 하나의 밝혀지지 않은 인지질(phospholipid)과 다섯 개의 밝혀지지 않은 극성 지질(polar lipid)이 검출되었다. DNA G+C 함유량은 64.2 mol%이었다. 낮은 온도의 MM 배지에서 BTEX 생분해 능력은 낮았지만, 소량의 효모 추출물, 펩톤, 트립톤을 추가로 넣어준 배지에서 생분해 능력이 향상되었다. ML15P13T 균주는 0.2%의 효모추출물을 추가한 MM 액체배지에서 20일 동안 15 ℃에서 배양한 결과 BTEX 혼합물 (각 BTEX 물질의 초기 농도, 30 mg/L)의 77.3%를 분해하였다. 또한, 0.05%의 효모 추출물을 추가한 MM 액체배지에서 다양한 온도 (10, 15, 20, 28 ℃)에서 BTEX 생분해 능력을 측정하였다. ML15P13T 균주는 10일 동안 10, 15, 20 ℃에서 각각 63.2, 64.4, 67.4%의 BTEX 혼합물을 분해하였다. 게다가, ML15P13T 균주는 28 ℃에서 가장 높은 생분해 특성을 보여주었으며, 10일동안 72.1%의 BTEX 혼합물을 분해하였다. polyphasic taxonomy 분석 결과, ML15P13T균주는 Massilia 속에 속하는 신종 (Massilia aromaticivorans sp. nov.) 균주로 밝혀졌고, 낮은 온도와 온화한 온도에서 BTEX 혼합물을 잘 분해하는 것으로 관찰되어 이 균주는 추운 지역의 오염된 유류 성분 제거에 효율적으로 활용될 수 있다고 판단된다. Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 4 1. Media and culture condition 4 2. Chemicals 4 3. Isolation of BTEX-degrading bacteria 7 4. Colony Repetitive Extragenic Palindromic-PCR 10 5. 16S rRNA gene sequencing and phylogenetic analysis 12 6. Whole genome sequencing, assembly, annotation and analysis 14 7. Phenotypic and biochemical characteristic 16 8. Chemotaxonomic analysis 18 9. Biodegradation of BTEX compounds by ML15P13T 19 10. Effect of biodegradation ability on different media and temperature 20 Ⅲ. RESULTS 21 1. Isolation of BTEX-degrading bacteria 21 2. Strain identification by 16S rRNA gene sequence analysis and colony REP-PCR analysis 22 3. Phylogenetic and whole genome sequencing analysis of ML15P13T 25 4. Morphological and phenotypic characteristics of ML15P13T 30 5. Chemotaxonomic characteristics of ML15P13T 37 6. Biodegradation of BTEX by ML15P13T in mineral medium with different conditions. 41 Ⅳ. DISCUSSION 44 Ⅴ. LITERATURE CITED 49 Ⅵ. ABSTRACT IN KOREAN 58 Master |
author2 |
가종억 농업생명과학대학 농생명공학부 식물미생물학과 |
format |
Doctoral or Postdoctoral Thesis |
author |
손지관 |
author_facet |
손지관 |
author_sort |
손지관 |
title |
Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
title_short |
Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
title_full |
Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
title_fullStr |
Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
title_full_unstemmed |
Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil |
title_sort |
biodegradation of btex compounds by massilia aromaticivorans ml15p13t isolated from arctic soil |
publisher |
서울대학교 대학원 |
publishDate |
2020 |
url |
http://hdl.handle.net/10371/169661 http://dcollection.snu.ac.kr/common/orgView/000000162711 |
geographic |
Arctic Norway Svalbard |
geographic_facet |
Arctic Norway Svalbard |
genre |
Antarc* Antarctica Arctic Svalbard |
genre_facet |
Antarc* Antarctica Arctic Svalbard |
op_relation |
000000162711 http://hdl.handle.net/10371/169661 http://dcollection.snu.ac.kr/common/orgView/000000162711 I804:11032-000000162711 000000000043▲000000000048▲000000162711▲ |
_version_ |
1766264644998004736 |
spelling |
ftseoulnuniv:oai:s-space.snu.ac.kr:10371/169661 2023-05-15T13:57:03+02:00 Biodegradation of BTEX Compounds by Massilia aromaticivorans ML15P13T Isolated from Arctic Soil 북극토양에서 분리한 Massilia aromaticivorans ML15P13T의 BTEX 물질의 생물학적 분해 연구 손지관 가종억 농업생명과학대학 농생명공학부 식물미생물학과 2020 http://hdl.handle.net/10371/169661 http://dcollection.snu.ac.kr/common/orgView/000000162711 kor kor 서울대학교 대학원 000000162711 http://hdl.handle.net/10371/169661 http://dcollection.snu.ac.kr/common/orgView/000000162711 I804:11032-000000162711 000000000043▲000000000048▲000000162711▲ Massilia aromaticivorans New Taxa BTEX compounds Biodegradation 신종 BTEX 물질 생물학적 분해 630 Thesis Dissertation 2020 ftseoulnuniv 2020-10-16T00:24:42Z 학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2020. 8. 가종억. BTEX (benzene, toluene, ethylbenzene, and xylene) compounds are the most frequently encountered subsurface contaminants among the various petroleum hydrocarbons. For contamination removal at cold climate sites, bioremediation technology is appealing due to their potential to be more economical and efficient than alternative. The aim of this study is to isolate, identify, and characterize novel bacterial strains from the Arctic and Antarctica soil that could degrade the BTEX compounds at low temperature. A novel aromatic hydrocarbon-degrading bacterial strain, designated ML15P13T, was isolated from Arctic soil at the Svalbard Islands, Norway, using enrichment culture technique. Cells were Gram-stain-negative, aerobic, motile with multiple flagella at one polar end, and rod-shaped. Growth was observed at 4-35 ℃, pH 6.0-8.0, and 0-0.5% (w/v) NaCl. According to 16S rRNA gene analysis, strain ML15P13T was grouped with members of the genus Massilia and closely related to Massilia atriviolacea SODT (98.4%), Massilia violaceinigra B2T (98.3%), Massilia eurypsychrophila B528-3T (97.7%), Massilia glaciei B448-2T (97.7%), and Massilia psychrophlia B115-1T (96.6%). Average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity between genome sequences of strain ML15P13T and the closely related species ranged from 75.8 to 84.3%, from 19.6±1.0 to 21.6±0.3%, and from 68.8 to 71.0%. The major fatty acids were C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Q-8 was the major ubiquinone. The polar lipid profile showed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified phospholipid, and five unidentified polar lipids. The G+C content of the genomic DNA is 64.2 mol%. The BTEX biodegradation rate was low in MM broth, but adding a small amount of yeast extract, peptone, and tryptone greatly enhanced the biodegradation at low temperature. The ML15P13T utilized 77.3% of BTEX mixture (initial concentration of each BTEX compounds, 30 mg/L) in MM broth with 0.2% yeast extract for 20 days at 15 ℃. Moreover, the BTEX biodegradation rates were assessed at different temperatures (10, 15, 20, and 28 ℃) in MM broth with 0.05% yeast extract. The ML15P13T degraded 63.2, 64.4, and 67.4% of BTEX mixture at 10, 15, and 20 ℃ for 10 days, respectively. In addition, the MP15P13T was characterized by maximal biodegradation ability at 28 ℃ and utilized 72.1% of BTEX mixture for 10 days. Based on the results for genotypic and phenotypic study, it is concluded that strain ML15P13T represents a novel species of the genus Massilia, for which the name Massilia aromaticivorans sp. nov. is proposed. BTEX (벤젠, 톨루엔, 에틸벤젠, 자일렌)은 유류 오염지역에서 가장 자주 발견되는 석유계 탄화수소이다. 추운 기후에서의 환경오염을 제거하기 위해서는 다른 환경정화방법보다 더 효율적이고, 경제적인 생물학적 환경정화 방법이 사용된다. 따라서 본 연구에서는 저온에서 BTEX 물질을 분해할 수 있는 신종 균을 북극과 남극 토양에서 분리, 동정하며, 분해 특성을 분석하였다. 방향족 탄화수소를 분해할 수 있는 신종 미생물 ML15P13T 균주를 북극 노르웨이의 스발바르 제도의 토양에서 enrichment 기법을 이용하여 분리하였다. 균주는 그람 음성균이며 호기성이고, 균주의 한쪽 끝에 여러 개의 편모를 가지면서 운동성을 나타내고, 간균형이었다. 균은 4-35 ℃, pH 6.0-8.0, 0-0.5% (w/v)의 염분농도에서 성장 가능하였다. 16S rRNA 유전자 서열 분석 결과 ML15P13T 균주는 Massilia 속에 속하였으며, Massilia atriviolacea SODT (98.4%), Massilia violaceinigra B2T (98.3%), Massilia eurypsychrophila B528-3T (97.7%), Massilia glaciei B448-2T (97.7%), Massilia psychrophlia B115-1T (96.6%)의 균주들과 가장 밀접하였다. 뉴클레오티드 동일성(average nucleotide identity), 디지털 DNA-DNA 혼성화(digital DNA-DNA hybridization), 아미노산 동일성(average amino acid identity)의 분석 결과는 각각 75.8-84.3%, 19.6±1.0-21.6±0.3%, 68.8-71.0%였다. C16:0, summed feature 3 (C16:1 ω6c 그리고/또는 C16:1 ω7c), summed feature 8 (C18:1 ω7c 그리고/또는 C18:1 ω6c) 가 주요한 지방산으로 검출되었다. Q-8이 주요한 유비퀴논 (ubiquinone)이었다. 주요 극성 지질 검출 결과 포스파티딜에탄올아민 (phosphatidylethanolamine), 포스파티딜글리세롤 (phosphatidylglycerol), 디포스파티딜글리세롤 (diphosphatidylglycerol)과 하나의 밝혀지지 않은 인지질(phospholipid)과 다섯 개의 밝혀지지 않은 극성 지질(polar lipid)이 검출되었다. DNA G+C 함유량은 64.2 mol%이었다. 낮은 온도의 MM 배지에서 BTEX 생분해 능력은 낮았지만, 소량의 효모 추출물, 펩톤, 트립톤을 추가로 넣어준 배지에서 생분해 능력이 향상되었다. ML15P13T 균주는 0.2%의 효모추출물을 추가한 MM 액체배지에서 20일 동안 15 ℃에서 배양한 결과 BTEX 혼합물 (각 BTEX 물질의 초기 농도, 30 mg/L)의 77.3%를 분해하였다. 또한, 0.05%의 효모 추출물을 추가한 MM 액체배지에서 다양한 온도 (10, 15, 20, 28 ℃)에서 BTEX 생분해 능력을 측정하였다. ML15P13T 균주는 10일 동안 10, 15, 20 ℃에서 각각 63.2, 64.4, 67.4%의 BTEX 혼합물을 분해하였다. 게다가, ML15P13T 균주는 28 ℃에서 가장 높은 생분해 특성을 보여주었으며, 10일동안 72.1%의 BTEX 혼합물을 분해하였다. polyphasic taxonomy 분석 결과, ML15P13T균주는 Massilia 속에 속하는 신종 (Massilia aromaticivorans sp. nov.) 균주로 밝혀졌고, 낮은 온도와 온화한 온도에서 BTEX 혼합물을 잘 분해하는 것으로 관찰되어 이 균주는 추운 지역의 오염된 유류 성분 제거에 효율적으로 활용될 수 있다고 판단된다. Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 4 1. Media and culture condition 4 2. Chemicals 4 3. Isolation of BTEX-degrading bacteria 7 4. Colony Repetitive Extragenic Palindromic-PCR 10 5. 16S rRNA gene sequencing and phylogenetic analysis 12 6. Whole genome sequencing, assembly, annotation and analysis 14 7. Phenotypic and biochemical characteristic 16 8. Chemotaxonomic analysis 18 9. Biodegradation of BTEX compounds by ML15P13T 19 10. Effect of biodegradation ability on different media and temperature 20 Ⅲ. RESULTS 21 1. Isolation of BTEX-degrading bacteria 21 2. Strain identification by 16S rRNA gene sequence analysis and colony REP-PCR analysis 22 3. Phylogenetic and whole genome sequencing analysis of ML15P13T 25 4. Morphological and phenotypic characteristics of ML15P13T 30 5. Chemotaxonomic characteristics of ML15P13T 37 6. Biodegradation of BTEX by ML15P13T in mineral medium with different conditions. 41 Ⅳ. DISCUSSION 44 Ⅴ. LITERATURE CITED 49 Ⅵ. ABSTRACT IN KOREAN 58 Master Doctoral or Postdoctoral Thesis Antarc* Antarctica Arctic Svalbard Seoul National University: S-Space Arctic Norway Svalbard |