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系統識別號 U0026-0407201917560100
論文名稱(中文) 耐高溫及耐鹼油脂分解菌篩選與培養之研究
論文名稱(英文) Isolation and enrichment of thermo- and alkali-tolerant oil-degrading bacteria
校院名稱 成功大學
系所名稱(中) 環境工程學系
系所名稱(英) Department of Environmental Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 陳思宇
研究生(英文) Szu-Yu Chen
學號 P56064063
學位類別 碩士
語文別 英文
論文頁數 88頁
口試委員 指導教授-黃良銘
口試委員-鄭幸雄
口試委員-林財富
口試委員-張嘉修
中文關鍵字 碳氫化合物降解菌  耐高溫菌  耐鹼菌  冷軋油 
英文關鍵字 hydrocarbons degrading bacteria  thermotolerant  alkalitolerant  cold rolling oil 
學科別分類
中文摘要 水是人類生活中不可或缺的資源,因此水資源的保護是一個重要的議題。對於工業廢水的處理程序而言,在傳統物理及化學處理系統中添加生物處理系統,特別是針對污染物濃度較低或具有分散性的情況下,可以提高處理效率並降低成本。然而,對於部分極端環境,特別是許多工業廢水處理廠來說,生物處理系統的穩定性是脆弱、易受影響的。而其中一種應對策略是利用生物添加法,也就是將選定的菌株或混合培養的菌群添加到環境或系統中,以促進微生物對污染物的降解效率。在此研究中,分別利用冷軋廢水處理場生物薄膜反應器的活性污泥,以及混和化肥廠廢水處理系統活性污泥及油污染場址分離出來的菌株,經馴養一段期間後分離出了 Chelatococcus sp. ES-SL-4和Chelatococcus daeguensis strainTAD1,其具有耐熱和耐鹼能力,並且可以利用冷軋油作為唯一的碳源和能源。Chelatococcus sp. ES-SL-4和C. daeguensis strainTAD1可在pH9、45°C下,初始TPH濃度為1450 mg L-1的條件下,於培養的14天中,分別達到80%和77.5%的TPH去除率。在放大培養實驗中,Chelatococcus sp. ES-SL-4可在規模為5 L的生物反應器,於培養6天後,達到75%的COD去除率和69.2%的TPH去除率,並且期間觀察到為生物濃度的增加,因此確定其具有降解冷軋油的能力,並且可以在純種培養的狀況下利用冷軋油作為唯一的碳源和能源生長。在混合培養實驗中,接種馴養污泥的組別具有最佳的降解能力,其可達到CO2產生量38.3 mL及TPH去除率62.0%。證明在馴養污泥中可能存在其他具有冷軋油降解能力更佳的微生物,而這些微生物為存活但不可培養的微生物(viable but nonculturable, VBNC)。
英文摘要 Water is an indispensable resource for human beings, the conservation of water resource is an important issue. Biotreatment process can potentially improve the treatment efficiency of conventional physico-chemical treatment. However, the biological treatment systems in extreme environments, especially in many industrial wastewater treatment plants, are vulnerable. One of the strategies is bioaugmentation, a process of adding selected strains/mixed cultures to environment or system. In this study, isolates B-1, B-4, D-2, E-1 and G-1 that with thermo- and alkali-tolerant ability and can use cold rolling oil as sole carbon and energy source, were isolated. Isolate B-1 and D-2 reached 80 and 77.5% total petroleum hydrocarbons (TPH) removal at pH 9, 45°C, with initial TPH concentration of 1450 mg L-1 within 14 d of incubation. To scale up, Chelatococcus sp. ES-SL-4 removed 75% of COD and 69.2% of TPH in 6 d in a 5-L bioreactor, showing the ability to degrade cold rolling oil and use it as sole carbon and energy source. In the mixed-cultural experiments, the batch inoculating with enriched sludge had the best degrading performance, which had accumulated 38.3 mL of CO2 in headspace and transferred 1053 mg L-1 of TPH (62.0% of removal). The occurrence of unculturable consortia in enriched sludge participate the degradation of cold rolling oil. To sum up, this study demonstrates the potential to degrade wastewater containing cold rolling oil in alkaline and thermophilic environment.
論文目次 摘要 I
Abstract II
Acknowledgements III
Contents IV
List of tables VII
List of figures VIII
Chapter 1 Introduction 1
Chapter 2 Literature Review 3
2.1 Cold rolling wastewater 3
2.1.1 Steel-making process 3
2.1.2 Characteristic of cold rolling wastewater 4
2.1.3 Treatment process of cold rolling wastewater 5
2.2 Biotreatment of wastewater 8
2.3 Oil-degrading bacteria 11
2.3.1 Degradation pathway of oil 11
2.3.2 Biodegradation of hydrocarbon at extreme environment 14
2.4 Molecular biotechnology 21
2.4.1 DNA extraction 22
2.4.2 Polymerase chain reaction (PCR) 23
2.4.3 Sequencing 25
Chapter 3 Materials and Methods 27
3.1 Research framework 27
3.2 Isolation of cold rolling oil degrading bacteria 28
3.2.1 Culture media 28
3.2.2 Sampling and enrichment 29
3.2.3 Isolation 31
3.3 Batch experiment 32
3.3.1 Preliminary screening experiment 32
3.3.2 Growth condition experiment 33
3.3.3 Scale-up experiment 34
3.3.4 Mixed-cultural experiment 34
3.4 Analytical methods 36
3.4.1 General water quality analysis 36
3.4.2 Gas composition analysis 36
3.4.3 Biomass concentration measurement 38
3.4.4 Total petroleum hydrocarbons (TPH) analysis 40
3.5 Molecular biotechnology analysis 42
3.5.1 DNA extraction 42
3.5.2 Polymerase Chain Reaction (PCR) 43
3.5.3 Phylogenetic and molecular evolutionary analysis 45
Chapter 4 Results and Discussion 46
4.1 Isolation and screening for cold rolling oil degrading bacterial 46
4.1.1 Enrichment of cold rolling oil degrading bacterial 46
4.1.2 Preliminary screening experiment 49
4.2 Characterization of isolated degrading bacteria 55
4.2.1 Growth curve 55
4.2.2 Colony morphology 56
4.2.3 Bacterial identification 57
4.3 Growth condition experiment 60
4.3.1 COD analysis result of growth condition experiment 60
4.3.2 TPH analysis result of growth condition experiment 63
4.4 Scale-up experiment 68
4.5 Mixed-cultural experiment 74
Chapter 5 Conclusion and suggestions 78
5.1 Conclusions 78
5.2 Suggestions 79
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