建構脫鹵球菌與固氮菌共培養系統促進氮源缺乏環境下的還原脫氯作用

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：1

、訪客IP：52.14.126.74

姓名

陳庭萱(Ting-Hsuan Chen) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

建構脫鹵球菌與固氮菌共培養系統促進氮源缺乏環境下的還原脫氯作用
(Coupling between nitrogen fixation and chloroethene dechlorination in a co-culture of Dehalococcoides mccartyi and Pelobacter SFB93)

相關論文

★ 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現並藉由microRNAs調控DNA修復機制	★ 研究Dicrotophos對HepG2細胞毒性之分子機制：CSA蛋白質在毒性扮演之角色
★ TNT經由ROS介導之內質網壓力及粒線體失衡誘導人類肝臟細胞凋亡	★ Pseudomonas sp. A46全基因組分析與重金屬復育基因工程菌開發
★ 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷	★ 三硝基甲苯之毒理機制及生物降解暨多氯乙烯汙染模場生物整治
★ 探討人類肝癌細胞HepG2經4-氨基聯苯處理過後miRNA-630對於同源重組修復相關蛋白MCM8的調控機制	★ 假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討
★ 開發新穎性包埋Dehalococcoides mccartyi及Clostridium butyricum之長效脫氯膠體	★ 探討DNA損傷反應與慢性暴露4-胺基聯苯產生之肝臟毒性
★ 以Lpp-OmpA工法建構新穎性基因工程菌強化鎘生物復育能力	★ 硒代胱氨酸通過誘導人肝細胞癌中的 DNA 損傷和抑制 DNA 修復途徑來增強順鉑敏感性
★ 轉錄體分析 Acetobacterium woodii 降解1,1,1-三氯乙烷機制並用以協助 Dehalococcoides進行還原脫氯	★ 以宏觀基因體分析新穎 Candidatus Dehalobacterium strain DLY 降解二氯甲烷機制
★ 研究雙特松對HepG2細胞之DNA修復的影響	★ 金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

三氯乙烯(Trichloroethene, TCE)是一種工業用溶劑，也是常見的環境汙染物。一般而言，三氯乙烯被廣泛用於金屬部件的脫脂。在三氯乙烯的整治技術中，零價鐵是具有潛力的技術之一，能夠作為三氯乙烯的還原催化劑，有效促進三氯乙烯通過β-elimination 轉化成無毒的產物，移除地下水或土壤中的三氯乙烯。然而，此一化學反應的主要產物中包含了乙炔，乙炔已被證明會抑制三氯乙烯的脫氯作用。本研究即是針對乙炔對生物性修復三氯乙烯的抑制作用進行探討，藉由將脫鹵球菌(Dehalococcoides mccartyi, Dhc)與乙炔發酵菌(Pelobacter acetylenovorans SFB93)共培養的方式，將造成脫氯抑制的乙炔轉換成脫氯菌生長所需的碳源，此時乙炔則作為共生系統中唯一的碳源。同時，本研究亦利用 SFB93 之固氮酶活性，扮演此共生系統中氮源提供者的角色，使脫鹵球菌在氮源缺乏條件下亦能持續進行還原脫氯。此外，由於SFB93 為一海水分離菌株，為找出最適合的共生條件，本研究中探討了鹽度對於脫氯菌共生系統的影響，以在共生環境中取得最好的脫氯效率。在共生系統中，1.6 mM 乙炔會對 Dhc 還原脫氯造成顯著的抑制作用，且無法在SFB93 消耗乙炔後而得到恢復。然而，當乙炔濃度低於 0.1 mM 以下便不會對還原脫氯造成抑制。結合上述結果，最終本研究以 0.1 mM 乙炔作為共生系統中唯一碳源，並且調整氯化鈉濃度至 10g/L、15g/L 及 20g/L，成功建立 Dhc 與 SFB93 共培養系統，並在無添加額外氮源且僅有乙炔作為唯一碳源的之條件下，共培養系統中的脫鹵球菌能夠持續進行還原脫氯，FL2 能夠將 TCE 還原至氯乙烯(vinyl chloride, VC)並有部分乙烯產生，而 BAV1 能夠將二氯乙烯(cis-1,2-Dichloroethylene, cis-DCE)還原至無毒性乙烯。共培養系統能夠解決透過微生物間的相互作用解決實場整治上脫鹵球菌因生物可利用
性氮源缺乏而導致還原脫氯活性下降之困境，可發展有應用潛力的TCE生物整治方式。

摘要(英)

Trichloroethylene (TCE) is an industrial solvent and a common environmental pollutant. Trichloroethylene is widely used for degreasing metal material. Zero-valent iron is one of the potential remediation technologies for trichloroethylene. It can be used as a reduction catalyst for trichloroethylene, which can effectively promote the conversion of trichloroethylene into non-toxic products through β-elimination, and remove trichloroethylene present in groundwater or soil. However, the middle product of this chemical reaction contains acetylene, which has been shown to inhibit the biological dechlorination of trichloroethylene.
The purpose of this research is to investigate the inhibitory effect of acetylene on bioremediation of trichloroethylene. We co-cultured Dehalococcoides mccartyi (Dhc) with Pelobacter acetylenovorans SFB93 by using acetylene as the sole carbon source. At the same time, we also used the nitrogenase activity of SFB93 to support the nitrogen sources in this symbiotic system, so that Dhc can perform reductive dechlorination continuously even under a nitrogen-deficient environment. As SFB93 is a seawater-isolated strain, demonstrated that the effects of salinity on this symbiotic system results revealed that 1.6 mM acetylene caused a significant inhibitory effect on the reductive dechlorination of Dhc and could not be recovered after the consumption of acetylene by SFB93. However, the reductive dechlorination was not inhibited when the acetylene concentration was below 0.1 mM. Combined with the above results, we used 0.1 mM acetylene as the sole carbon source in the coculture system with a salinity of 10 g/L, 15 g/L, or 20 g/L sodium chloride. The co-culture system of Dhc and SFB93 was successfully established. With no additional nitrogen source but only nitrogen gas presented, Dhc in the coculture system can continue to perform reductive dechlorination, FL2 reducing TCE to vinyl chloride(VC) and producing ethene, BAV1 can reduce cis-DCE to non-toxic ethene completely. Thus, the coculture system can solve the dilemma of the reduction of reductive dechlorination activity caused by the lack of bioavailable nitrogen sources in the field remediation through the interaction between microorganisms and we can develop a potential TCE biological remediation technology in the future.

關鍵字(中)

★ 乙炔
★ 脫鹵球菌
★ 乙炔發酵菌
★ 共培養
★ 鹽度

關鍵字(英)

★ Acetylene
★ Dehalococcoides
★ Pelobacter acetylenovorans SFB93
★ Coculture
★ salinity

論文目次

摘要 i
Abstract ii
致謝 iv
目錄 v
圖目錄 viii
表目錄 x
縮寫表(Abbreviations ) xi
第壹章緒論 (Introduction) 1
1.1 三氯乙烯 (Trichloroethene, TCE) 1
1.2 多氯乙烯整治工法 2
1.2.1 化學及物理整治法 2
1.2.2 好氧生物整治及厭氧生物整治法 3
1.2.3生物刺激法及生物強化法(Biostimulation & Bioaugmenatation) 3
1.3實場整治所面臨的困境 3
1.3.1零價鐵整治產物乙炔所造成的抑制 4
1.3.2 生物可利用氮影響還原脫氯速率 4
1.4 微生物群落 (microbial consortia)以及共培養 (coculture) 5
1.4.3 Pelobacter strain SFB93 5
第貳章研究目的 7
1.4.1 脫鹵球菌(Dehalococcoides mccartyi, Dhc) 8
第參章實驗材料與方法(Material & method) 9
3.1 實驗材料 9
3.1.1使用儀器與廠牌(equipment) 9
3.1.2 常用藥品與試劑(Chemical) 11
3.2 實驗方法 15
3.2.1絕對厭氧技術 15
3.2.2 乙炔製備 15
3.2.3 菌株繼代培養與保存 16
3.2.4 共培養的建立 17
3.2.5 DNA 萃取 17
3.2.6 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 18
3.2.7 DNA 膠體純化 19
3.2.8 DNA 接合反應 20
3.2.9勝任細胞的製備(TSS法) 21
3.2.10轉型作用(transformation) 21
3.2.11即時定量連鎖聚合酶反應 (Real-time quantitative polymerase chain reaction, qPCR) 22
3.2.12氣相層析儀 (Gas Chromatography)檢測多氯乙烯及其代謝物 22
第肆章實驗結果 (Result) 24
4.1乙炔抑制Dehalococcoides mccartyi還原脫氯及生長 24
4.2 探討共培養SFB93乙炔抑制的移除及鹽度影響 25
4.3乙炔抑制Dehalococcoides mccartyi還原脫氯的濃度測試 26
4.4乙炔作為唯一碳源共培養Dehalococcoides mccartyi (FL2 & CWV2)與Pelobacter SFB93 27
4.5在氮源缺乏環境下乙炔作為唯一碳源之共培養系統 28
第伍章討論 (Discussion) 31
5.1 乙炔抑制脫鹵球菌還原脫氯 31
5.2 移除乙炔抑制及鹽度影響 31
5.3乙炔為碳源之Dhc/SFB93共培養系統 32
5.4探討Dhc可利用氮來源並利用SFB93生物性固氮以提供Dhc生長及脫氯所需之氮源 32
第陸章結論 (Conclusion) 34
參考文獻 (Reference) 35
圖表 40
附加資料 (Supplementary file) 53

參考文獻

Akob, Denise M, Shaun M Baesman, John M Sutton, Janna L Fierst, Adam C Mumford, Yesha Shrestha, Amisha T Poret-Peterson, Stacy Bennett, Darren S Dunlap, and Karl B Haase. Detection of diazotrophy in the acetylene-fermenting anaerobe Pelobacter sp. strain SFB93. Applied and Environmental Microbiology. 2017;83(17).
Akob, Denise M, John M Sutton, Janna L Fierst, Karl B Haase, Shaun Baesman, George W Luther III, Laurence G Miller, and Ronald S Oremland. Acetylenotrophy: a hidden but ubiquitous microbial metabolism? FEMS Microbiology Ecology. 2018;94(8).
Baesman SM, Sutton JM, Fierst JL, Akob DM, Oremland RS. Syntrophotalea acetylenivorans sp. nov., a diazotrophic, acetylenotrophic anaerobe isolated from intertidal sediments. Int J Syst Evol Microbiol. 2021;71(3).
Chiu, Weihsueh A, Jennifer Jinot, Cheryl Siegel Scott, Susan L Makris, Glinda S Cooper, Rebecca C Dzubow, Ambuja S Bale, Marina V Evans, Kathryn Z Guyton, and Nagalakshmi Keshava. Human health effects of trichloroethylene: key findings and scientific issues. Environmental Health Perspective. 2013;121(3).
Council NR. Contaminants in the subsurface: Source zone assessment and remediation. 2005. 030909447X.
Dolinová I, Štrojsová M, Černík M, Němeček J, Macháčková J, Ševců A. Microbial degradation of chloroethenes: a review. Environmental Science and Pollution Research International. 2017;24(15).
Elkin ER, Harris SM, Su AL, Lash LH, Loch-Caruso R. Placenta as a target of trichloroethylene toxicity. Environmental Science: Processes & Impacts. 2020;22(3):472-486.
Ensley BD. Biochemical diversity of trichloroethylene metabolism. Annual Reviews in Microbiology. 1991;45(1):283-299.
Ellis, David E, Edward J Lutz, J Martin Odom, Ronald J Buchanan, Craig L Bartlett, Michael D Lee, Mark R Harkness, and Kim A DeWeerd. Bioaugmentation for accelerated in situ anaerobic bioremediation. Environmental Science & Technology. 2000;34(11).
Frascari D, Zanaroli G, Danko AS. In situ aerobic cometabolism of chlorinated solvents: A review. Journal of Hazardous Materials. 2015;283.
Gavaskar A, Tatar L, Condit W. Cost and performance report nanoscale zero-valent iron technologies for source remediation. Naval Facilities Engineering Service Center Port Hueneme CA;2005.
He J, Sung Y, Krajmalnik‐Brown R, Ritalahti KM, Löffler FE. Isolation and characterization of Dehalococcoides sp. strain FL2, a trichloroethene (TCE)‐and 1, 2‐dichloroethene‐respiring anaerobe. Environmental Microbiology. 2005;7(9):1442-1450.
He J, Holmes VF, Lee PK, Alvarez-Cohen LJA, microbiology e. Influence of vitamin B12 and cocultures on the growth of Dehalococcoides isolates in defined medium. Applied and Environmental Microbiology. 2007;73(9):2847-2853.
Hendrickson, Edwin R, Jo Ann Payne, Roslyn M Young, Mark G Starr, Michael P Perry, Stephen Fahnestock, David E Ellis, and Richard C Ebersole. Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe. Applied and Environmental Microbiology. 2002;68(2):485-495.
Huling SG, Pivetz BE. In-situ chemical oxidation. Environmental Protection Agency Washington DC Office Of Water;2006.
Kang D, Jacquiod S, Herschend J, Wei S, Nesme J, Sørensen SJ. Construction of simplified microbial consortia to degrade recalcitrant materials based on enrichment and dilution-to-extinction cultures. Frontiers in Microbiology. 2020;10:3010.
Kaya D, Kjellerup BV, Chourey K, Hettich RL, Taggart DM, Löffler FE. Impact of fixed nitrogen availability on Dehalococcoides mccartyi reductive dechlorination activity. Environmental Science & Technology. 2019;53(24):14548-14558.
Krajmalnik-Brown R, Hölscher T, Thomson IN, Saunders FM, Ritalahti KM, Löffler FE. Genetic identification of a putative vinyl chloride reductase in Dehalococcoides sp. strain BAV1. Applied and Environmental Microbiology. 2004;70(10):6347-6351.
Kruse, Stefan, Dominique Türkowsky, Jan Birkigt, Bruna Matturro, Steffi Franke, Nico Jehmlich, Martin von Bergen, Martin Westermann, Simona Rossetti, and Ivonne %J The ISME journal Nijenhuis. Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoides and Sulfurospirillum dehalogenating tetrachloroethene to ethene. The ISME journal. 2021;15(6):1794-1809.
Lee C-Y, Liu W-D. The effect of salinity conditions on kinetics of trichloroethylene biodegradation by toluene-oxidizing cultures. Journal of Hazardous Materials. 2006;137(1):541-549.
Lee, Patrick KH, Brian D Dill, Tiffany S Louie, Manesh Shah, Nathan C VerBerkmoes, Gary L Andersen, Stephen H Zinder, and Lisa Alvarez-Cohen. Global transcriptomic and proteomic responses of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation. Applied and Environmental Microbiology. 2012;78(5):1424-1436.
Löffler FE, Ritalahti KM, Zinder SH. Dehalococcoides and reductive dechlorination of chlorinated solvents. In: Bioaugmentation for groundwater remediation.2013:39-88.
Mao, Xinwei, Ronald S Oremland, Tong Liu, Sara Gushgari, Abigail A Landers, Shaun M Baesman, and Lisa Alvarez-Cohen. Acetylene fuels TCE reductive dechlorination by defined Dehalococcoides/Pelobacter consortia. Environmental Science & Technology. 2017;51(4):2366-2372.
Mao X, Stenuit B, Polasko A, Alvarez-Cohen L. Efficient metabolic exchange and electron transfer within a syntrophic trichloroethene-degrading coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei. Applied and Environmental Microbiology. 2015;81(6):2015-2024.
Men Y, Seth EC, Yi S, Allen RH, Taga ME, Alvarez-Cohen L. Sustainable growth of Dehalococcoides mccartyi 195 by corrinoid salvaging and remodeling in defined lactate-fermenting consortia. Applied and Environmental Microbiology. 2014;80(7):2133-2141.
Miller LG, Baesman SM, Kirshtein J, Voytek MA, Oremland RS. A biogeochemical and genetic survey of acetylene fermentation by environmental samples and bacterial isolates. Geomicrobiology Journal. 2013;30(6):501-516.
Pon G, Hyman MR, Semprini L. Acetylene inhibition of trichloroethene and vinyl chloride reductive dechlorination. Environmental Science & Technology. 2003;37(14):3181-3188.
Rosero-Chasoy G, Rodríguez-Jasso RM, Aguilar CN, Buitrón G, Chairez I, Ruiz HA. Microbial co-culturing strategies for the production high value compounds, a reliable framework towards sustainable biorefinery implementation–an overview. Bioresource Technology. 2021;321:124458.
Russell HH, Matthews J, Sewell G. TCE removal from contaminated soil and ground water. Ground-water issue. Environmental Protection Agency, Washington, DC (United States). Office of Solid Waste and Emergency Response;1992.
Skubal KL, Barcelona MJ, Adriaens P. An assessment of natural biotransformation of petroleum hydrocarbons and chlorinated solvents at an aquifer plume transect. Journal of Contaminant Hydrology. 2001;49(1-2):151-169.
Solis MIV, Abraham PE, Chourey K, Swift CM, Löffler FE, Hettich RL. Targeted detection of Dehalococcoides mccartyi microbial protein biomarkers as indicators of reductive dechlorination activity in contaminated groundwater. Scientific Reports. 2019;9(1):1-11.
Van Der Voort M, Abee T. Transcriptional regulation of metabolic pathways, alternative respiration and enterotoxin genes in anaerobic growth of Bacillus cereus ATCC 14579. Journal of Applied Microbiology. 2009;107(3):795-804.
Wagner A, Segler L, Kleinsteuber S, Sawers G, Smidt H, Lechner U. Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;368(1616):20120317.
Watts RJ, Teel AL. Treatment of contaminated soils and groundwater using ISCO. Practice Periodical of Hazardous. 2006;10(1):2-9.
Wu C, Schaum J. Exposure assessment of trichloroethylene. Environmental Health Perspectives. 2000;108(suppl 2):359-363.
Yin Y, Yan J, Chen G, Murdoch FK, Pfisterer N, Löffler FE. Nitrous oxide is a potent inhibitor of bacterial reductive dechlorination. Environmental Science & Technology. 2018;53(2):692-701.

指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2022-10-18

推文