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研究生:高俊璿
研究生(外文):Chun-hsuan Kao
論文名稱:高濃度石化油污染土壤之不同微生物降解三類石油碳氫化合物研究
論文名稱(外文):Phase study on three-groups hydrocarbon bioremediation of high oil contaminated soil with different microorganisms
指導教授:鄭幸雄鄭幸雄引用關係
指導教授(外文):Sheng-shung Cheng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:環境工程學系碩博士班
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:184
中文關鍵詞:生物刺激生物優植生物復育碳氫化合物真菌
外文關鍵詞:hydrocarbon isolationBiostimulationBioremediationfungiBioaugmentation
相關次數:
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本研究係以提升長期受總石油碳氫化合物(TPH)污染之土壤的生物復育效率為目標,以循序漸進的方式進行對TPH的解析研究,以及找尋能有效降解碳氫化合物之微生物。研究中以一離場土耕法生物復育之模場研究(phase 1)為基礎,探討整治期中遭遇的問題及整治後期殘留碳氫化合物無法降解的原因,以實驗室規模之批次試程調控最佳化之環境因子、解析污染物之組成變化及監測微生物族群的變動,查證受長期污染土壤之碳氫化合物的生物可降解性(phase 2),並進一步透過三類碳氫化合物之離析研究各碳氫化合物之生物可降解性(phase 3),期望藉此尋得有效縮短整治時程、提升整治效率的方式。
在phase 1中,以兩組長期受TPH污染之土壤作為復育對象,利用土耕法結合柱塞迴流法及生物優植、生物刺激等生物復育策略進行離場整治共231天。兩組土壤各有四堆高約2m的20m3土壤,初始污染濃度分別為S:3,500 ~ 4,500 mg/kg dry soil; T:5,000 ~ 7,500 mg/kg dry soil。皆於啟動初期進行第一次生物優植及生物界面活性劑的刺激。S組於初期28天內即完成第一階段之快速降解,隨後持平,殘餘濃度約為1,500 mg/kg dry soil; T組則於啟動70天後完成第一階段之降解,隨後亦持平,殘餘濃度為2,000 ~ 3,500 mg/kg dry soil。期中因降解持平而進行營養鹽的添加及第二次的生物優植,但發現仍無助於殘餘濃度的分解。研究中觀察到兩組土壤的特性化降解差異,包括前期的降解速率及中後期的持平,S組各土堆的降解速率快,殘留濃度低,普遍優於T組各土堆,觀察造成差別的原因在於污染物的組成差異(Biodegradability)及土壤中有機質及土壤質地所造成的生物可及性(Bioavailability)不同。
Phase 2由模場研究中之兩土堆因污染物的組成造成的降解特性差異為基礎,於實驗室進一步延伸至TPH因污染年限造成的組成差異影響生物可降解性(Biodegradability)之驗證,經自然衰減的風化作用程度不同,碳氫化合物的組成就不同,短期污染土壤S組各土堆的降解速率常數k27約為0.025明顯快於L組(k27 = 0.018),而去除率S = 75% > L = 60%,由Sum peaks/UCM值進一步解析碳氫化合物的差異,S組起始值約為0.1,L組 = 0.03,即長期污染土壤經自然衰減後生物可降解性較低。最後S組殘餘濃度約為3,000 mg/kg dry soil,L組則約為4,000 mg/kg dry soil。
由不同的污染年限影響生物復育之研究中觀察到長期污染土壤中的TPH生物可降解性低,因此仍有4,000 mg/kg dry soil的殘餘濃度。Phase 3中再進一步透過對三類碳氫化合物的解析觀察,欲了解何物才是造成降解性差的主因。發現殘留濃度的TPH多由Resins及Asphaltenes累積貢獻。土壤初始污染濃度為14,000 mg/kg dry soil,經過生物優植及營養鹽、碳源強化、添加生物界面活性劑的生物刺激等策略之復育後,以KW的去除率82%最高,除了提供易於利用的碳源之外,KW還提供了高量真菌的優植如Pseudallescheria boydii、Aspergillus fumigatus;其次為NE-L有80%的去除率,除了初期的快速降解之外也在復育中期發現有真菌如Aspergillus versicolor、Candida guilliermondii大量出現,且兩組之Resins及Asphaltenes殘餘率為各組中最低,顯示極性物質的消長、真菌菌相的存在及生物復育的效率三者間的關聯性。本研究也藉此提供兩種微生物菌相的階段性降解之整治經驗,期望能在未來應用於實場整治。
The content of this study mainly targeted on increasing the bioremediation effects, the research on total petrolum hydrocarbon (TPH) analysis and looking for effective TPH degrading microorganisms for aged TPH contaminated soil. In oreder to find out the biodegradability of the residual hydrocarbon (Case 2), it made a soil batch experimental design in this study relying on the results and problems from a pilot scale phase study (ex-situ landfarming bioremediation)(Case 1), and made another for studying the detail chemical composition of the residual hydrocarbon by three-groups hydrocarbon isolation with gas-chromatographic techniques (Case 3). Expected that bioremediation stage could be shorten and the efficiency could be increased.
In the pilot scale phase study KH100 (Case 1), 80 m3 of TPH contaminated soil was divided into four soil piles and each for 20 m3, and another 80 m3 of soil was divided into another fours. One named S series and another named T series. Bioremediation was operated with landfarming, “Plug flow reaction and recirculation seeding,” bioaugmentation and biostimulation in 231 days. The initial TPH concentration of S series is 3,500 ~ 4,500 mg/kg dry soil, and T series is 5,000 ~ 7,500 mg/kg dry soil. Both of them added enriched and mixed culture of four TPH degrading bacteria for bioaugmentation and biosurfactant rhamnolipid for biostimulation at the beginning. S series finished the first step of quickly degradation in 28 days than got stopping and with residual about 1,500 mg/kg dry soil. T series finished the first step of degradation in 70 days than got stopping and with residual about 2,000 ~ 3,500 mg/kg dry soil mg/kg dry soil. Enhanced nutrient and made 2nd bioaugmentation because the degradation got stopping at the middle stage, but it didn’t worked. To observed that S and T series have characterized degrading tendency with a reason about TPH compositions.
Relying on the degrading characteristics of the two series soil in the pilot scale phase study, it made an experimental design about the effect of soil contaminated ages on bioremediation (Case 2). The TPH composition changed after natural attenuation, and became less biodegradable. It could be observed especially in long term contaminated soil (L series). The biodegradability of long and short term contaminated soil could be studied by gas-chromatographic techniques, S series is much beter than L series and the removal of S series (75%) is beter than L series (60%) , too.
According to the biodegradability study by gas-chromatographic techniques, the reason about hardly degrading of residual hydrocarbon is more clear. But it still need to know that what kind of hydrocarbon has less biodegradability. Therefore, three groups hydrocarbon was isolated with silica gel column, and found that Resins & Asphaltenes have less biodegradability. The release of polar metabolic by products like Resins during HC microbial assimilation under bioremediation may occur, and accumulation of polar metabolic byproducts was demonstrated to inhibit the HC degradation. Fungi like Pseudallescheria boydii、Aspergillus fumigatus、Aspergillus versicolor、Candida guilliermondii were found from KW and NE-L soil and demonstrated that could assimilate the polar metabolic byproducts, remove the inhibiting machinism and increase the bioremediation efficiency.
第一章 前言 1
第二章 文獻回顧 3
2-1 油污染土壤概況介紹 3
2-1-1 土壤污染案例介紹 4
2-1-2 土壤污染整治技術應用趨勢 7
2-2 油污染土壤環境研究 11
2-2-1 土壤特性介紹 11
2-2-2 總石油碳氫化合物介紹 18
2-2-3 總石油碳氫化合物之生物降解性研究 24
2-3 離場生物復育技術整治研究 39
2-3-1 影響土壤生物復育之環境因子 39
2-3-2 土壤生物復育技術介紹 42
2-3-3 系統化環境分子生物技術 46

第三章 材料與方法 49
3-1 研究材料 49
3-1-1 總石油碳氫化合物 49
3-1-2 生物製劑 50
3-2 污染土壤系統分析 51
3-2-1 土壤環境因子分析 51
3-2-2 總石油碳氫化合物分析 52
3-2-3 土壤中之微生物菌落計數 - 平盤計數法 (Plate-counts) 55
3-2-4 掃描式電子顯微鏡 (Scanning electron microscope, SEM) 57
3-3 分子生物技術分析 58
3-3-1 總DNA萃取 58
3-3-2聚合酶酵素連鎖反應 (Polymerase chain reaction, PCR) 60
3-3-3尾端修飾限制片段長度多型性 (T-RFLP) 61
3-3-3微矩陣生物晶片之偵測技術 (Microarray Biochip) 62
3-4 生物復育試驗 64
3-4-1 模場規模之土壤反應器KH-100 64
3-4-2 不同污染年限土壤之降解批次試驗 69
3-4-3不同微生物族群之優植配合生物刺激進行之最佳化復育研究 72
第四章 結果與討論 75
4-1 現址離場土耕法生物復育模場研究KH-100 75
4-1-1 復育期間總石油碳氫化合物之變化探討 75
4-1-2 復育期間之土壤微生物種類及數量變化探討 84
4-1-3 復育期間土堆降解特性比較 87
4-2 土壤受油品污染的年限對生物降解的影響研究 92
4-2-1 土壤環境因子之監控 93
4-2-2 生物降解期間微生物菌落數變化趨勢探討 96
4-2-3 總石油碳氫化合物之降解特性探討 100
4-2-4 總結 111
4-3 不同微生物族群之優植及生物刺激對三類碳氫化合物之降解研究 113
4-3-1 土壤環境因子概述 114
4-3-2 生物復育期間TPH之降解曲線分析 118
4-3-3 生物復育期間土壤微生物菌量分析 130
4-3-4 應用分子生物技術監測微生物族群變化之探討 143
4-3-5 生物復育期間TPH三類石油碳氫化合物分析 157
4-3-6 總結 166
第五章 結論與建議 167
5-1 結論 167
5-2 建議 171
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