開發新穎性包埋Dehalococcoides mccartyi及Clostridium butyricum之長效脫氯膠體

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黎如月(Le Nhu Nguyet) 查詢紙本館藏

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論文名稱

開發新穎性包埋Dehalococcoides mccartyi及Clostridium butyricum之長效脫氯膠體
(Development of Silica Gel with Co-immobilized Dehalococcoides mccartyi and Clostridium butyricum for Long-Term Dechlorination)

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摘要(中)

含氯有機物是一種常見於地下水汙染的致癌物質，尤其是二氯乙烯(cis-DCE)及氯乙烯(VC)最難被完全整治。脫鹵球菌(Dehalococcoides mccartyi, Dhc) 在含氯有機物之汙染整治是關鍵的菌種，本菌在環境中一旦缺乏氫氣及適當的碳源或是環境條件不佳，則會導致現地場址累積二氯乙烯及氯乙烯。丁酸梭菌(Clostridium butyricum, C. butyricum) 被我們選為應用於發酵氫氣及醋酸的主要菌種，同時搭配包埋技術應可有效提升地下水的脫氯降解速率。先前的研究中，應用包埋丁酸梭菌的膠體可以提高地下水汙染中的三氯乙烯脫氯速率，然而，地下水原生的脫鹵球菌數量不高，使得無法達到完全脫氯。本研究中欲開發一種包埋脫鹵球菌BAV1及丁酸梭菌的矽膠膠體，解決現地脫鹵球菌菌數不足之問題，該膠體的成膠利用到矽膠奈米顆粒及矽烷化物。在本研究證明在無提供氫氣及醋酸的環境下，脫鹵球菌BAV1可以成功與丁酸梭菌包埋在一起並保有脫氯活性。在高濃度二氯乙烯(8mM) 培養實驗下，本包埋矽統可以保護脫鹵球菌使其不失去活性，且本膠體擁有超過80天以上的長效脫氯活性因為脫鹵球菌受到馴化可以快速生長。在實際應用方面，厭氧批次實驗結果顯示，本膠體可以在28天將受到二氯乙烯的地下水完全脫氯至無毒的乙烯。因此，本研究成功開發新穎的脫氯包埋膠體並且擁有應用於含氯有機物汙染場地之生物整治潛力。

摘要(英)

Chloroethenes are common groundwater pollutants classified as toxic and carcinogenic to humans especially cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Dehalococcoides mccartyi (Dhc) are key anaerobic bacteria for bioremediation of chloroethene-contaminated sites, but the lack of hydrogen and carbon source (acetate) cause the accumulation of cis-DCE and VC. Clostridium butyricum (C. butyricum) is a priority candidate of H2 and acetate support for practical application. Cell immobilization is an effective and promising technique to enhance dechlorination rate. Previous research demonstrated that immobilized C. butyricum in silica gel showed a great potential to promote the trichloroethene dechlorination efficiency in contaminated groundwater. However, the local Dhc abundances were low resulted in stalling or incomplete dechlorination. In this study, a silica gel with co-immobilized Dhc strain BAV1 and C. butyricum was developed. Silica gel system was formed by silica nanoparticles and silicon alkoxides via a sol-gel process. BAV1 and C. butyricum were successfully co-immobilized in silica gel without the extra addition of H2 and acetate. The immobilized system could protect BAV1 from high cis-DCE concentration (8 mM). Long-term dechlorination activity of the co-immobilized system was maintained for more than 80 consecutive days due to the adaptation and rapid growth of immobilized bacteria. For the practical application, results from batch experiments showed that the developed system completely reduced cis-DCE into ethene, a non-toxic product, in the contaminated groundwater within 28 days. Therefore, this novel co-immobilized system could be a potential approach for bioremediation in the chloroethene-contaminated sites.

關鍵字(中)

★ 脫鹵球菌
★ 丁酸梭菌
★ 包埋
★ 矽膠
★ 生物整治

關鍵字(英)

★ Dehalococcoides mccartyi
★ Clostridium butyricum
★ co-immobilized
★ silica gel
★ bioremediation

論文目次

CHINESE ABSTRACT i
ENGLISH ABSTRACT ii
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES viii
LIST OF APPENDIXES ix
ABBREVIATIONS x
INTRODUCTION 1
1. Background 1
2. Technologies for removal of chlorinated ethenes 1
3. Research objective 3
LITERATURE REVIEW 4
1. Chlorinated ethenes 4
2. Abiotic and biotic degradation of chlorinated ethenes 4
3. Dehalococcoides mccartyi strain BAV1 5
4. Clostridium butyricum 6
5. Silica gel bioencapsulation system 6
MATERIALS AND METHODS 8
1. Equipment and chemicals 8
1.1 Equipment 8
1.2 Chemicals 8
2. Bacterial strains and growth conditions 12
2.1 Dehalococcoides mccartyi strain BAV1 12
2.2 Clostridium butyricum 13
3. Cloning 14
3.1 PCR 14
3.2 Gel extraction 15
3.3 Digestion 15
3.4 Ligation 16
3.5 TSS competent cell 16
3.6 Transformation of E. coli competent cells 17
3.7 Plasmid extraction and purification 17
4. DNA extraction and cell number quantification 19
4.1 DNA extraction 19
4.2 Cell number quantification by using qPCR 20
5. Silica gel encapsulation system 22
5.1 Reagents 22
5.2 Experimental procedure 22
6. Chemical analysis 23
RESULTS AND DISCUSSION 25
1. Dechlorination of cis-DCE by Dehalococcoides mccartyi strain BAV1 25
2. Growth curve of Clostridium butyricum and its ability to produce hydrogen and acetate 25
3. Co-culture of free BAV1 and Clostridium butyricum 26
4. Dechlorination of cis-DCE by immobilized Dhc strain BAV1 in silica gel 26
5. Co-immobilization of BAV1 and Clostridium butyricum for cis-DCE dechlorination 27
6. Protective ability of immobilized BAV1 system in silica gel 27
7. Long-term dechlorination of co-immobilized BAV1 and Clostridium butyricum in silica gel 28
8. Contaminated groundwater application 28
CONCLUSION AND FUTURE WORK 30
1. Conclusion 30
2. Future work 30
FIGURES 31
TABLES 40
REFERENCES 47
APPENDIXES 54

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指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2020-7-9

推文