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研究生:陳世卿
研究生(外文):Shih-Chin Chen
論文名稱:EDTA分解微生物之培養與應用
論文名稱(外文):The cultivation and application of the EDTA-degrading microorganism
指導教授:方鴻源方鴻源引用關係
指導教授(外文):Hung-Yuan Fang
學位類別:博士
校院名稱:國立雲林科技大學
系所名稱:工程科技研究所博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
畢業學年度:92
語文別:中文
論文頁數:151
中文關鍵詞:生物降解非線性迴歸基質抑制動力學生物處理
外文關鍵詞:EDTABurkhol cepaciabiodegradationsubstrate-inhibition
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本研究主要以生物降解二級生物處理系統出流水中仍殘存的有機物質EDTA為目標,在國外之研究文獻僅有三株EDTA降解菌被提出,有鑑於此,本研究進行EDTA本土化分解菌之篩選,經自台灣本土環境篩選,該菌株經以API 20NE鑑定其菌屬結果為Burkhol cepacia YL-6, YL-6 菌株屬好氧性菌種,大小約5∼15 µm。以Fe-EDTA為主要碳源時,其最適添加濃度為1000 mg/L;以硫酸銨及尿素為氮源而按1:1之比例添加時,其最適添加濃度為750 mg/L;以磷酸鉀為磷源時,其最適添加濃度為750 mg/L;最適溫度及pH值分別為30 ℃及7.0。以此生長條件進行降解試驗,YL-6菌降解1000 mg/L Fe-EDTA,經培養18天後其平均比生長速率為0.1352 d-1,EDTA降解率約90 %,COD去除率約77 %,TOC去除率約67 %。在 Cu-EDTA降解試驗中,添加300 mg/L FeSO4.7H2O於培養基中,以Fe+2置換Cu+2後培養20天,EDTA降解率約75 %,COD去除率約76 %,TOC去除率約74 %。在最適生長條件下進行三種不同碳源(Fe-EDTA、醋酸鉀及乙胺)之批式追加醱酵培養,三種不同碳源之醱酵菌液經熱風乾燥後,所得製劑活菌數,以醋酸鉀為碳源培養所得之活菌數較高。三種不同碳源製成之製劑添加於Fe-EDTA或Cu-EDTA進行降解試驗,以乙胺為碳源所製成之製劑對Fe-EDTA及Cu-EDTA降解時間較短, COD去除率分別為89 %及85 %,EDTA去除率為100 %。由比生長速率隨著基質濃度之升高而降低之結果,証明了Fe-EDTA分解遵循著基質抑制動力學。本研究引用文獻上之五種基質抑制模式以非線性迴歸方法,利用統計套裝軟體SPSS,推估計算動力參數,結果最大比生長速率為0.206- 0.150 d-1,飽和常數為80-74 mg/L及基質抑制常數為890-2289 mg/L。預測值與實驗觀測值之最大比生長速率比較,低估約3.7 ﹪至1.5 ﹪。五種不同基質抑制模式中,以Haldane模式為最適宜之比生長速率預測模式。由Fe-EDTA及Cu-EDTA試驗結果顯示,添加YL-6菌液於活性污泥系統中比一般未添加之活性污泥系統,其EDTA及COD之去除率均較高。
EDTA, the target compound of this study from the effluent of secondary biotreatment units, can be biodegraded by special microorganisms. So far , there are three species of microorganisms-Agrobactrium , Gram-negative BNCI , and DSM9103-that can degrade EDTA and are pubished in the literature. We have successfully isolated a bacterial strain that can degrade EDTA. It was identified as Burkhol cepacia, an aerobic species, elliptically shaped with a l ength of 5-15 µm. The growth medium contains 1000 mg/L ferric-EDTA as carbon source, 750 mg/L of (NH4)2SO4 + (NH2)2CO as nitrogen source, and1000 mg/L of KH2PO4 as phosphorus source, and mineral factors such as Fe and Mg. Incubated at pH 7.0 , 30 ℃, and 150 rpm on a shaker for 15 d, the average specific growth rate of this microbe is 0.135 d-1 , which shows that the respective degradation efficiency of Fe-EDTA and Cu-EDTA is 90 % and 75% individually.
Bioaugmentation production of EDTA-degrading bacterium Burkhol cepacia YL-6 was carried out in aerobic fermentor under the optimal conditions. Three different carbon sources (ferric-ethylene-diaminetetraacetate (Fe-EDTA), potassium acetate, and ethylamine) were used. The EDTA-degradation time required for the afore-mentioned bioaugmentation agents made by feeding various carbon sources lay in the following order: ethylamine < potassium acetate < Fe-EDTA. Especially, the most efficient bioaugmentation agent (made by feeding ethylamine) only required 10 and 13 days for complete degradation of EDTA from the Fe-EDTA and Cu-EDTA complexes, respectively. It shows that ethylamine was the best substrate for augmentation production of EDTA-degrading bacterium Burkhol cepacia YL-6.
Evaluation of kinetic parameters showed Fe-EDTA degradation follows substrate inhibition kinetics. This is evident from the decrease in specific growth rate with increase in the initial substrate concentration greater than 500 mg/L. To estimate the kinetic parameters-μmax, KS and KI, five substrate-inhibition models were used. From the results of non-linear regression, the value of μmax ranged from 0.150 to 0.206 d-1, KS from 74 to 87 mg/L, and KI from 890 to 2289 mg/L. The five models were found to underestimate the maximum specific growth rate by 1.5- 3.7 %. Therefore, predictions based on these models would result in lower predicted value than those from the experimental kinetic data.
As in the real wastewater test, it showed that the addition of bioaugmentation agents to the activated sludge system can enhance the removal efficiency of EDTA and COD.
中文摘要………………………………………………………………………………Ⅰ
英文摘要………………………………………………………………………………Ⅲ
誌謝……………………………………………………………………………………Ⅴ
目錄……………………………………………………………………………………Ⅵ
表目錄…………………………………………………………………………………Ⅹ
圖目錄…………………………………………………………………………………XI

第一章 緒論 1
1-1 研究背景 1
1-2 研究目標 1
1-3 研究內容 2
第二章 文獻回顧 4
2-1環境微生物 4
2- 2微生物之營養需求 4
2-3培養基 8
2-3-1 培養基之種類 8
2-3-2 EDTA分解菌培養基之成份 8
2-4微生物培養之物理條件 12
2-4-1溫度 12
2-4-2 pH 13
2-4-3 氣體 14
2-4-4 滲透壓(osmotic pressure) 15
2-5 微生物之增殖生長 15
2-6 微生物生長之測定方法 17
2-7 微生物之生長速率 18
2-7-1 單一酵素系之動力學-Michaelis-Menten式 18
2-7-2 單細胞微生物之生長速率模式 19
2-7-3 基質抑制動力學(Substrate inhibition kinetics) 21
2-7-4模式背景及參數說明 22
2-8 EDTA之特性 27
2-8-1 pH對EDTA解離之影響 27
2-8-2 EDTA之錯合反應 30
2-8-3 EDTA之水溶性及金屬置換能力 31
2-8-4 EDTA螯合物之穩定性 32
2-9 EDTA螯合物之毒性及危害 34
2-10 EDTA之應用 34
2-10-1 EDTA在印染上的應用 36
2-10-2 EDTA於醫療上之應用 36
2-10-3 EDTA於食品之應用 36
2-10-4 EDTA於環境復育之應用 37
2-11 EDTA及其螯合物之物化處理 38
2-11-1 熱處理法 38
2-11-2光解法 38
2-11-3電化學法 38
2-11-4 臭氧氣化法 39
2-12 EDTA之生物處理 39
2-12-1 EDTA 之分解菌 39
2-12-2 EDTA之代謝途徑及代謝產物 40
第三章 研究流程與實驗材料、設備 45
3-1研究流程 45
3-2實驗藥品與設備 46
3-2-1實驗藥品 46
3-2-2實驗設備 47
第四章 台灣本土EDTA分解菌之篩選、分離與鑑定 49
4-1 前言 49
4-2 實驗方法 49
4-2-1 菌株之分離、篩選 49
4-2-2 分析方法 51
4-2-3 EDTA降解菌株之篩選分離 51
4-2-4 EDTA分解菌之菌鑑定 52
4-2-5 EDTA分解菌之電子顯微像 53
4-3結果與討論 54
4-3-1 EDTA分解菌之篩選、分離結果 54
4-3-2 YL-6菌株初步降解EDTA測定 55
4-3-3 YL-6菌株之菌相觀察與菌學鑑定 56
第五章 EDTA分解之最適生長條件探討 61
5-1前言 61
5-2實驗方法 61
5-2-1不同碳源添加量之實驗方法 62
5-2-2不同氮源添加量之實驗方法 62
5-2-3不同磷酸鹽添加量之實驗方法 62
5-2-4不同pH值之實驗方法 62
5-2-5 不同培養溫度之實驗方法 63
5-2-6最適生長條件下菌株對EDTA之降解實驗 63
5-3 結果與討論 65
5-3-1不同碳源濃度對菌體生長之影響 65
5-3-2不同氮鹽濃度對菌體生長之影響 68
5-3-3不同磷酸鹽濃度對菌株生長之影響 70
5-3-4不同pH值對菌株生長之影響 72
5-3-5不同溫度對菌株生長之影響 74
5-3-6 YL-6菌降解Fe-EDTA 情形 76
5-3-7 Cu-EDTA降解試驗 78
第六章 EDTA分解菌擴大培養及生物製劑化探討 82
6-1前言 82
6-2實驗方法 82
6-2-1批式醱酵培養(Batch culture) 82
6-2-2批式追加醱酵培養 (Fed-batch culture) 83
6-2-3微生物製劑化 83
6-2-4微生物製劑對EDTA分解率評估 84
6-3結果與討論 85
6-3-1批式醱酵培養(Batch culture) 85
6-3-2批式追加醱酵培養(Fed-batch culture) 86
6-3-3 YL-6菌製劑之效益評估 89
第七章 EDTA分解菌生長動力學探討 95
7-1 前言 95
7-2 實驗方法 96
7-2-1 菌株來源 96
7-2-2 培養基 96
7-2-3 分析方法 97
7-3 結果與討論 99
7-3-1 不同Fe-EDTA(基質)濃度下YL-6菌株生長情形 99
7-3-2最大比生長速率(μmax)及飽和常數(Ks)值 100
7-3-3基質抑制動力參數估算 100
第八章Burkhol cepacia 應用於實際廢水之效能 110
8-1小型生物模擬廠試驗 110
8-2應用於印刷電路板廢水之實驗 111
第九章 結論及建議 115
參考文獻 118
成果發表 126
附 錄 127
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