臺灣博碩士論文加值系統

前人研究以1:1比例添加堆肥進行五氯酚(PCP)污染土生物復育，可達70 %降解但是堆肥添加量過多造成實際應用上的困難。於是本研究分成兩批次土罐實驗，探討堆肥以營養液生物刺激或生物添加法取代的可行性。首先以滅菌/不滅菌堆肥或氮磷營養及降解菌(9703)添加進行PCP污染土復育實驗。發現添加有/無滅菌堆肥的降解率並沒有明顯的差別，堆肥本身的菌群對PCP降解影響很小；而添加氮磷營養塩時雖土壤微生物族群結構和總異營菌菌數變化不大，但降解菌菌數增多，而PCP降解在29天有最佳12%降解率，認為堆肥菌群沒有生物放大的作用，而是傾向於生物刺激的功效。為了減少堆肥用量，設計不同C: N:P比的營養或抽取堆肥濾液當可溶有機質營養，並配合從長期污染土中篩選5株Pseudomonas sp.降解菌、長期污染土培養的混合菌及9703進行生物復育比較PCP降解效果。結果顯示添加氮磷營養塩或堆肥濾液可溶有機質營養液可使PCP降解提升10~20%；隨著添加氮磷營養塩量增加降解效率也逐步提高，氮磷量最高添加比例時(C:N:P=100:20:2)有最佳的PCP降解率32 %；相同氮磷量處理組(C:N:P=100:10:1)添加 9703或長污土可培養混合菌發現，能增強生物刺激效果降解率達到33%。由此研究結果顯示添加適當比例氮磷營養塩或抽取堆肥濾液並配合有效降解菌株可以取代大量堆肥有最好的降解率，提供一個能運用於復育PCP污染土的選擇。

Pentachlorophenol(PCP) contaminated soil amended with compost in a 1:1 ratio are useful for PCP degradation in previous study. However, the bulky composts are not convenient for the practical application. To reduce the usage amount are the purpose of this investigation. We design two batches of bioreactors experiments to realize whether nutritional supplement or degradation microbes are the key parameter of amended compost. First, sterilized/non-sterilized compost or nitrogen are applied to PCP contaminated soil which was inoculated with PCP-degradating-bacteria (9703) in a 4L bioreactor for 29 days. It was found that sterilized/non-sterilized compost had no significant effect on PCP degradation, but changing on microbial quantity. However, the N / P nutrient amendments did have a significant effect on microbial populations and PCP decreased by 12%. Then we amended filtrate of compost as soluble organic carbon comparing with the N/P supplement in PCP contaminated soil and inoculated with five strains Pseudomonas sp., that we isolated, mix culture of long-term PCP contaminated soil or 9703 on the 0.25L bioreactor for 30 days. The results show that nutrient amendment (N and P) or dissolved organic matter amendment can be increased by 10 ~ 20% on PCP degradation. The more N/P nutrient amendments shows the more PCP degradation in soil. C: N: P ratio are 100:20:2 shows best PCP degradation rate of 32%. Biostimulation with nutrient amendment (C: N: P = 100:10:1) can enhance more 5% of PCP removal by augmentation with the 9703 or the mix culture of long-term PCP contaminated soil. The combination of biostimulation with N/P nutrient amendments or dissolved organic matter from compost filtrate and bioaugmentation with efficacy PCP degradation bacteria effectively decrease the PCP in soil. It’s a feasible way to reduce the usage amount of composts in bioremediation.

目錄

中文摘要............................................................................................................I
Abstract.............................................................................................................II
誌謝.................................................................................................................IV
目錄.................................................................................................................Ⅴ
圖表目錄.........................................................................................................IX
第1章 前言.....................................................................................................1
1.1研究緣起.................................................................................................1
1.2研究目的.................................................................................................1
第2章 文獻回顧.............................................................................................3
2.1五氯酚污染介紹.....................................................................................3
2.2五氯酚的危害.........................................................................................3
2.3五氯酚之生物降解.................................................................................4
2.3.1五氯酚之降解菌種..........................................................................4
2.3.2五氯酚之降解途徑..........................................................................8
2.3.3微生物處理污染物之方式..............................................................9
2.3.3.1共代謝.......................................................................................9
2.3.3.2協同作用...................................................................................9
2.4生物復育...............................................................................................10
2.4.1生物復育技術................................................................................10
2.4.1.1添加營養鹽.............................................................................13
2.4.1.2添加微生物.............................................................................13
2.4.1.3添加電子接受者.....................................................................14
2.4.2影響生物復育之因素....................................................................14
2.4.2.1污染物因素.............................................................................14
2.4.2.2微生物因素.............................................................................14
2.4.2.3環境因素.................................................................................16
2.5生物技術在環境科學上的應用...........................................................17
2.5.1分子生物在土壤微生物族群分析上的應用................................17
2.5.2變性梯度膠體電泳........................................................................18
第3章 材料與方法.......................................................................................20
3.1實驗材料...............................................................................................20
3.1.1實驗土壤及菌株來源....................................................................20
3.1.2實驗試劑及設備............................................................................20
3.2實驗架構...............................................................................................21
3.3土罐實驗1評估堆肥及降解菌添加對土壤中五氯酚降解影響.......22
3.3.1實驗設計........................................................................................22
3.3.2實驗流程........................................................................................22
3.3.3菌種活化........................................................................................23
3.3.4試驗土壤製備................................................................................24
3.3.5土壤最大保水力............................................................................24
3.3.6堆肥來源及製備............................................................................24
3.3.7土罐實驗系統................................................................................25
3.4五氯酚降解菌篩選分離.......................................................................26
3.4.1降解菌菌落外觀............................................................................26
3.4.2降解菌的生長曲線........................................................................26
3.4.3液態降解試驗................................................................................26
3.4.4液態降解pH值變化.....................................................................27
3.4.5菌株16S rDNA序列鑑定.............................................................27
3.4.6土壤菌種選殖及clone鑑定.........................................................27
3.5土罐實驗2評估降解菌及營養塩添加對土壤中五氯酚降解影響...28
3.5.1實驗流程........................................................................................28
3.5.2實驗設計........................................................................................29
3.5.3試驗準備........................................................................................29
3.5.4土罐實驗系統................................................................................30
3.6土壤分析...............................................................................................30
3.6.1土壤質地分析................................................................................30
3.6.2土壤pH值測定.............................................................................31
3.6.3含水量............................................................................................31
3.6.4有機質含量測定............................................................................32
3.6.5總氮測定........................................................................................32
3.6.6有效性氮測定................................................................................32
3.7土壤中PCP降解分析..........................................................................32
3.7.1土壤活性測定－CO2釋放量監測.................................................32
3.7.2有機碳含量分析............................................................................33
3.7.3土壤五氯酚萃取............................................................................33
3.7.4五氯酚檢量線製作........................................................................34
3.7.5土壤五氯酚殘留量檢測................................................................34
3.8微生物量及菌群結構分析...................................................................34
3.8.1總異營菌數及降解菌數測定........................................................34
3.8.2土壤中微生物族群結構分析........................................................35
3.8.2.1土壤DNA萃取......................................................................35
3.8.2.2聚合酶連鎖反應.....................................................................36
3.8.2.3瓊酯凝膠電泳實驗.................................................................36
3.8.2.4變性梯度膠電泳.....................................................................37
3.8.2.5膠體染色.................................................................................37
3.8.2.6膠體圖譜之分析.....................................................................38
3.9實驗數據統計分析...............................................................................39
第4章 結果...................................................................................................40
4.1土罐實驗1評估堆肥及降解菌對土壤五氯酚降解試驗…...............40
4.1.1土壤分析........................................................................................40
4.1.2土壤PCP降解力分析...................................................................41
4.1.2.1土壤活性分析.........................................................................41
4.1.2.2 土壤PCP濃度變化...............................................................42
4.1.3生物量分析....................................................................................43
4.1.3.1土壤中總菌及降解菌之變化.................................................43
4.1.3.2土壤微生物菌群菌相變化.....................................................45
4.2五氯酚降解菌篩選及分離...................................................................48
4.2.1降解菌菌落外觀............................................................................48
4.2.2降解菌的生長曲線........................................................................50
4.2.3液態降解試驗................................................................................51
4.2.4液態降解pH值變化.....................................................................52
4.2.5菌株16S rDNA序列鑑定結果.....................................................53
4.2.6非培養之菌種鑑定結果................................................................54
4.3土罐實驗2評估降解菌及營養塩添加對土壤五氯酚降解試驗.......56
4.3.1土壤及營養塩分析........................................................................56
4.3.1.1含水量變化.............................................................................56
4.3.1.2土壤pH值變化......................................................................57
4.3.1.3有效性氮變化.........................................................................58
4.3.2土壤中PCP降解分析...................................................................59
4.3.2.1可溶性有機質含量變化.........................................................59
4.3.2.2土壤PCP殘留量....................................................................60
4.3.2.3五氯酚在土壤中之一階反應式.............................................64
4.3.3微生物量及菌群結構分析............................................................65
4.3.3.1土壤中總菌及降解菌之變化.................................................65
4.3.3.2土壤微生物菌群菌相變化.....................................................67
第5章 討論...................................................................................................71
第6章 總結...................................................................................................74
參考文獻.........................................................................................................75
附錄.................................................................................................................82
作者介紹.........................................................................................................88

本研究利用堆肥、滅菌堆肥或無機營養塩添加，三種不同因素並接種Pseudomonas plecoglossicida (9703)降解菌對土壤五氯酚降解的影響進行29天、4L土罐生物復育實驗。結果顯示堆肥菌群對復育的效果有限，而添加氮磷無機營養塩並能加強長期污染土的降解菌降解PCP。又以不同氮磷營養塩比例、堆肥濾液及添加不同降解菌，三種不同因素對土壤五氯酚降解的影響進行30天、0.25L土罐生物復育實驗。結果顯示隨著添加氮磷營養塩量增加降解效率也著步提高；而添加堆肥濾液刺激土壤微生物及添加菌生長，使微生物量豐富，降解率與氮磷營養塩(100:10:1)相仿。比較篩選的混合菌株、實驗室先前的9703菌及富集長污土的可培養菌發現，單獨添加9703菌株處理組降解率優於其他兩組有最佳的降解率33.92%。由此實驗結果顯示添加適當比例氮磷營養塩或堆肥濾液並配合降解菌株有最好的降解率，且堆肥的使用量也降到污染土重量的3 %，提供一個能運用於復育五氯酚污染土的選擇。
復育方式有很多種，須考慮到成本及急迫性，此實驗生物堆只是提供一種選擇。實場復育條件遠比實驗參數變化多，本研究可提供實場管理運作做參考。未來建議尋找9703菌株最佳降解之生長條件，以便運用於實場生物復育做管理依據。



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