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研究生(外文):Hao-Yu Lo
論文名稱(外文):Disturbance of Microbial Functional Gene Expression by Controlled Substances in River Sediment
外文關鍵詞:Controlled substanceSedimentReal-time PCRBiogeochemical cyclesFunctional gene
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近年來,人類污染物排放至河川水體而導致對生態環境的影響已經越來越受 到矚目。目前一般的污水處理程序對這些污染物質的移除能力有限, 已有研究 證實,處理過後的放流水以及放流水所流經的河川水體中可以偵測到一些管制藥 品的存在。近年來已有不少研究分別探討各種新興污染物對於土壤及底泥中的微 生物族群所造成的影響,然而這些研究大多偏重於對整體環境微生物群集多樣性 或單一微生物族群的變化,較少研究探討污染物對這些微生物所造成的代謝功能 之影響。因此本篇實驗目地為藉由生態模擬培養槽實驗來探討所選定的四種管制 藥品是否會改變微生物本身的生理反應變化,尤其針對與元素循環相關的代謝基 因為觀察目標。本篇研究中所選用的代謝基因為微生物體中參與氮循環之固氮作 用及脫硝作用、硫循環之硫酸鹽還原作用、磷酸根傳輸作用、甲烷循環之生成及 氧化作用的功能性基因。實驗中架設六組生態模擬培養槽實驗進行六個月的連續 流實驗,包含一組控制組及五組加入不同管制藥品的實驗組。本實驗選擇可用來 代表即時性表現的 mRNA 當作觀察對象,進行定量反轉錄聚合酶鏈鎖反應 (qRT-PCR),觀察所選定之特定功能性基因在實驗組與控制組實驗之間的差異。 根據實驗組培養槽的結果顯示,藉由 16S rRNA 表現量增加表示微生物族群受到 藥物影響下有增長的趨勢,管制藥物可增加微生物對硫酸鹽的還原反應、對於氮 循環之固氮作用及脫硝作用皆有先刺激而後抑制的現象發生,此外,可待因及愷 它命兩種藥物皆有促進底泥微生物進行甲烷氧化及降低甲烷合成的表現。綜合本 實驗結果,底泥微生物族群參與所選定之特定功能性基因的表現會受到管制藥品 的影響,且對於各種不同功能性基因的影響程度會因為四種不同管制藥品而有所 差異。因此,排放至河川水體中的管制藥品可能會對河川底泥中微生物的代謝基 因造成影響。

In recent years, discharge of anthropogenic substances into waters has been drawing a lot of attention from the general public. Since conventional wastewater treatment processes could only remove these pollutants partially, many studies reported detection of several controlled substances in wastewater effluent and nearby water bodies. There are many studies exploring the impacts of emerging contaminants to soil and sediment microorganisms. However, these studies mostly emphasize on individual species or on phylogenetic diversity of the total bacteria community, whereas the changes in microbial metabolic activities are rarely mentioned. Therefore, the objective of this study was to investigate the physical response of microorganism in river sediments from four different controlled substances, with special focus on changes of specific functional gene related to biogeochemical cycles. Representative biogeochemical functional genes (sulfate reduction, ammonia oxidation, nitrogen fixation, denitrification, phosphate uptake regulator, methanogenesis and methanotrophy) were selected for quantitative reverse transcription PCR (qRT-PCR) tests to monitor the changes in the experimental microcosms. Six different microcosms with different controlled substance spiked in the influent were prepared to observe the biogeochemical gene expression changes over a six-month period. The results showed that by comparing the control and target microcosms, the increase in 16S rRNA indicates microbial groups that are responsive to controlled substances incubation. For instance, functional genes responsible for sulfate reduction was amplified under the influence of controlled substances, nitrogen fixation and denitrification gene expressions were first stimulated but slowed down after the initial acceleration. In addition, codeine and ketamine were observed to stimulate methane oxidation and reduce methane generation in the sediment microorganism. In conclusion, specific microbial functional groups in the sediment are susceptible to the presence of controlled substances, and the level of impact for each functional gene expression also varied from different controlled substances.

致謝 i
摘要 iv
1.1 Background 1
1.2 Aims of the study 3
2.1 Occurrence of PPCPs in environment 4
2.1.1 Occurrence of PPCPs in aquatic environment 4
2.1.2 Potential effects to aquatic ecosystem 5
2.1.3 Illicit drugs as emerging contaminants 7
2.1.4 Target compounds 10
2.2 Biogeochemical cycle 13
2.3 Applications of molecular biology technique in the environment: 14
2.3.1 RNA as the target molecule 15
2.3.2 Quantifying gene expression by quantitative reverse transcription PCR 16
3.1 Research framework 18
3.2 Sampling and experimental apparatuses setup 20
3.2.1 Sampling 20
3.2.2 Sediment microcosm setup 20
3.3 RNA sample 21
3.3.1 RNA extraction 21
3.3.2 DNase treatment 23
3.3.3 cDNA synthesis 24
3.4 Q-PCR 25
3.4.1 Standard curves for cDNA quantitation 25
3.4.2 SYBR Green quantitative assay 26
3.4.3 Quantification of target genes 27
3.5 Water sample analysis 29
3.5.1 TOC analysis 29
3.5.2 High Performance Liquid Chromatography Tandem Mass 29
3.6 Data analysis 30
4.1 Control experiments for substrate concentration 32
4.1.1 Total organic carbon (TOC) 32
4.1.2 Target compounds (COD, MOR, KET and METH) 33
4.2 Microcosms sediment RNA extraction 35
4.3 Standard curve and detection limit for real time PCR 35
4.4 Significance of controlled substances to microbial functional group structure 40
4.4.1 Statistical analysis results 40
4.4.2 Sediment 16S rRNA gene expression 45
4.4.3 Sediment biogeochemical functional gene expression 49
4.5 Significance of controlled substances to microbial biogeochemical functional genes performance 58
4.5.1 Statistical analysis results 58
4.5.2 Response of sulfur-cycle functional gene to controlled substances 64
4.5.3 Response of nitrogen-cycle functional genes to controlled substances 67
4.5.4 Response of methane-cycle functional genes to controlled substances 75
4.5.5 Response of phosphate transporter gene to controlled substances 85

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