Title Page

Contents

Abstract 13

Chapter 1. Introduction 18

1.1. Background 18

1.2. Thesis Rationale 22

1.2.1. Aim 1 : Examining reductive dechlorination in tidal mudflat microcosms 23

1.2.2. Aim 2 : Developing and characterizing a Dehalococoides-containing microbial consortium that reductively dechlorinates tetrachloroethene to environmentally benign ethene 24

1.2.3. Aim 3 : Exploring bioaugmentation to achieve complete dechlorination of chlorinated ethenes in saline tidal flat environments 25

1.3. References 27

Chapter 2. Literature Review 30

2.1. Tidal flat : features, ecosystems roles, and contamination 30

2.2. Chlorinated ethenes in the environments 31

2.2.1. Terrestrial environments 33

2.2.2. Marine and coastal environments 36

2.3. Biodegradation of chlorinated ethenes 37

2.3.1. Aerobic and anaerobic oxidation 38

2.3.2. Cometabolic degradation 40

2.3.3. Microbial reductive dechlorination 41

2.4. Organohalide-respiring bacteria and reductive dehalogenases 43

2.4.1. Diversity of organohalide-respiring bacteria 43

2.4.2. Reductive dehalogenases 48

2.4.3. Organohalide-respiring bacteria in marine environments 49

2.5. Bioremediation strategies 50

2.6. Molecular biological tools 53

2.6.1. Polymerase chain reaction 55

2.6.2. Quantitative real-time PCR 55

2.6.3. 454 Pyrosequencing 59

2.7. References 61

Chapter 3. Microbial Community Structure and Dynamics in Tetrachloroethene-Dechlorinating Tidal Mudflat Microcosms 77

3.1. Introduction 77

3.2. Materials and methods 80

3.2.1. Sample collection and microcosm setup 80

3.2.2. Analytical methods 81

3.2.3. PCR amplification of 16S rRNA genes, cloning and sequencing 82

3.2.4. Quantitative real-time PCR analysis 83

3.2.5. 454 Pyrosequencing 84

3.2.6. Community analysis 85

3.3. Results 86

3.3.1. PCE reductive dechlorination 86

3.3.2. Changes in microbial diversity during the incubation 87

3.3.3. Community responses to enrichments with PCE as electron acceptor 88

3.3.4. Phylogenetic analysis: comparison of tidal mudflat and freshwater dechlorinating communities 93

3.3.5. Quantification of Desulfuromonas spp. in dechlorinating commumties 95

3.3.6. Quantification of Dehalococcoides mccartyi 16S rRNA genes and reductive dehalogenase genes in microcosms and various sediment samples 97

3.3.7. Phylogenetic analysis: comparison of tidal mudflat and freshwater Dehalococcoides maeeartyi 16S rRNA genes 98

3.4. Discussion 99

3.5. References 103

Chapter 4. Development and Characterization of PCE-to-Ethene Dechlorinating Microcosms with Contaminated River Sediment 108

4.1. Introduction 108

4.2. Materials and methods 110

4.2.1. Site characterization and sampling 110

4.2.2. Microcosm setup and DNA extraction 112

4.2.3. Serial transfers, selection of electron donors, and DNA extraction 113

4.2.4. 454 Pyrosequencing 114

4.2.5. Analysis of sequences obtained from pyrosequencing 116

4.2.6. Quantitative real-time PCR 117

4.3. Results 118

4.3.1. Dechlorination activities of enrichment cultures. 118

4.3.2. Bacterial community shifts during the dechlorination process 119

4.3.3. Identification and sequence analysis of Dehalococcoides populations 121

4.3.4. Quantification of Dehalococcoides mccartyi 16S rRNA and vcrA genes 124

4.3.5. Phylogenetic analysis of vcrA genes 124

4.3.6. Archaeal community shifts during the dechlorination process 125

4.3.7. Dechlorination activities of transfer cultures and substrate range evaluation 127

4.3.8. Microbial community analysis of 7th transfer cultures(이미지참조) 129

4.4. Discussion 130

4.5. References 138

Chapter 5. Bioaugmentation of Tidal Mudflat Microcosms to Achieve Detoxification of Chlorinated Ethenes 144

5.1. Introduction 144

5.2. Materials and methods 147

5.2.1. Sample collection and microcosm setup 147

5.2.2. Salinity effect on KB-1 culture 149

5.2.3. Quantitative real-time PCR 150

5.2.4. 454 pyrosequencing and community analysis 151

5.2.5. Sanger sequencing and phylogenetic analysis 152

5.3. Results 153

5.3.1. Dechlorination activities in the presence of 3% salt 153

5.3.2. Quantification of Dehalococcoides mccartyi 16S rRNA and vcrA genes 157

5.3.3. Pyrosequencing analysis of the bioaugmented microcosm 159

5.3.4. Analysis of Dehalococcoides 16S rRNA genes obtained from nested PCR 161

5.3.5. Analysis of VC-reductase genes 163

5.3.6. Dechlorination in transfer cultures 163

5.4. Discussion 166

5.5. References 172

Chapter 6. Conclusions and Future Research 177

Abstract in Korean 183

Table 2.1. Chemical properties of chlorinated ethenes and ethene, quality standard for drinking water in Korea, and maximum contaminant levels (MCLs) in the U. S. 34

Table 2.2. Summary of aerobic and anaerobic transformation of chlorinated ethenes 39

Table 2.3. Reductively dechlorinating isolates capable of partial dechlorination of PCE to TCE or cis-DCE 45

Table 2.4. Different Dehalococcoides mccartyi strains and Dehalococcoides-containing consortia involved in reductive dechlorination of chlorinated ethenes 46

Table 2.5. Primer sets targeting 16S rRNA and RDase genes of dechlorinating bacteria 56

Table 2.6. qPCR primers and probes targeting 16S rRNA and RDase genes of Dehalococcoides mccartyi. 57

Table 3.1. Bacterial community composition at the phylum/sub-class level, and the relative abundances of bacterial phylum/sub-class groups (% of total). (N ＝ Number of sequences, TI ＝ Top initial sediments, TM ＝ Dechlorinating microcosm with top sediments, BI ＝ Bottom initial sediments, BM ＝ Dechlorinating microcosm with bottom sediments, and ND inditates 'Not... 90

Table 3.2. Genus level identification of the populations and their relative abundances (% of total) in the initial tidal falt sediments and microcosms maintained with PCE as electron acceptor. Sequences from Titanium pyrosequencing were used.... 91

Table 4.1. Identification of major archaeal populations in culture WJ-2 during incubation with TCE as electron acceptor. OTUs are based upon 3% sequence dissimilarity. 126

Table 4.2. Total mass of chlorinated ethenes remained in the transfer cultures amended with different substrates. The presented data were chosen from triplicate experiments. 127

Table 4.3. Bacterial community composition at the genus level, and the relative abundances of major genera (% of total). Pyrosequencing was performed using technical duplicates.... 129

Table 4.4. Quantification of bacterial 16S rRNA genes, Dehalococcoides mccartyi 16S rRNA genes, and vcrA genes using two detection chemistries: TaqMan and SYBR Green. There were to significant differences in 16S rRNA gene results as previously reported (49), but notable differences were found in vcrA gene results (bold numbers with underlines).... 135

Table 5.1. Total mass of chlorinated ethenes and ethene in the tidal flat microcosms measured at Day 0 and after 110 days. 154

Figure 1.1. Organohalide respiration (microbial reductive dechlorination) of chlorinated ethenes 19

Figure 1.2. Research needs for complete detoxification of chlorinated ethenes in coastal sediments. 20

Figure 1.3. Research aims and tasks derived from research needs. 23

Figure 2.1. A typical scheme of subsurface contamination by DNAPL(s) showing the formation of ganglia in the zone of residual DNAPL, pooled DNAPL above low permeable porous media or barrier, and dissolved phase contaminant plume. 35

Figure 2.2. Detoxification of PCE and TCE via organohalide respiration. 42

Figure 2.3. Previously characterized RDases genes from Dehalococcoides mccartyi strains. 49

Figure 3.1. Sediment sampling from various estuary/tidal flat sites. 81

Figure 3.2. Reductive dechlorination of PCE (●) to TCE (▼) or cis-DCE (■) in the microcosms for top (TM) or bottom (BM) tidal flat sediments. The Y-axis error bar indicates a standard drror form at least two independent microcosm experiments. 87

Figure 3.3. Rarefaction curve analysis (OTUs at sequence dissimilarity cutoff 〈3%) and Shannon index as an indicator of microbial diversity. N indicates the number of sequences used for the rarefaction curve analysis and calculation for Shannon index. 88

Figure 3.4. Populations in 166 OTUs among 13,277 of total OTUs significantly increased during the incubations (0.10% to 44.7% in the top sediments and 0.20% to 51.3% in the bottom sediments). Based upon 100% of sequence similarity, those sequences in 166 OTUs were identified using Greengenes database.... 92

Figure 3.5. Distribution of 16S rRNA sequences obtained from the Titanium pyrosequencing. Desulfuromonas-like clusters were close relatives to Desulforumonas michiganensis strain BB1, and strain BRS1, which were previously characterized PCE-... 94

Figure 3.6. 16S rRNA-based qPCR results for total bacteria, Desulfuromonas and Dehalococcoides in the tidal mudflat sediments (TI, BI) and their dechlorinating microcosms (TM, BM). SYBR green assays were used. 96

Figure 3.7. qPCR results (TaqMan) for total bacterial 16S rRNA, Dehalococcoides mccartyi 16S rRNA, and reductive dehalogenase genes. The gene copy numbers of reductive dehalogenase genes include vcrA and bvcA.... 97

Figure 3.8. Phylogenetic placement of Chloroflexi group (YCTF Chloroflexi 01 and 02 from this study) with previously characterized Dehalococcoides populations. The scale bar represents 1 % sequence divergence. 99

Figure 4.1. The distributions and concentrations of chlorinated ethenes in the Woosan industrial site in Wonju, South Korea. Suspected sources were an asphalt laboratory (A), a food company (B), and a molding company (C) (modified from Baek and Lee,... 111

Figure 4.2. Transfers of the dechlorinating culture WJ. Each transfer culture was maintained 100~120 days for complete PCE to ethene dechlorination to occur. 114

Figure 4.3. Dechlorination of chlorinated ethenes in the microcosms using sediment sample ＃4. Culture WJ-1 (A) was amended with PCE and Culture WJ-2 with TCE (B). M1, M2, and M3 indicate the periods, when samples were taken for DNA extraction. 119

Figure 4.4. Changes in relative abundance of populations in respond to the dechlorination enrichment. The relative abundances are the mean values of duplicate samples. The genera with the dots on the left side are known PCE/TCE dechlorinators from previous studies.... 120

Figure 4.5. Phylogenetic placement of Dehalococcoides 16S rRNA gene sequences. Approximately 10% of Dehalococcoides sequences formed a separate cluster (Group 2).... 122

Figure 4.6. qPCR results for total bacterial 16S rRNA, Dehalococcoides mccartyi 16S rRNA and dehalogenase genes (tceA, bvcA, vcrA) at each dechlorination stage (WJI, initial sediment; M1, PCE-to-cis-DCE dechlorination; M2, cis-DCE-to-VC dechlorination; M3, VC-to-ethene dechlorination,; M4,... 123

Figure 4.7. The VC dehalogenase genes (vcrA) obtained from pyrosequencing were almost identical to the known dehalogenase genes from Dehalococcoides sp. strains VS and GT.... 125

Figure 4.8. Dechlorination of chlorinated ethenes in the 7th transfer culture. No methane production occurred due to the addition of BES in the 6th transfer cultures.(이미지참조) 128

Figure 4.9. The Chloroflexi populations from this study differed phylogenetically from previously characterized bacterial populations of Chloroloflexi (▼) and Dehalococcoides mccartyi (●) isolates, and uncultured Chloroflexi populations (▲) in... 132

Figure 5.1. Microcosm setups using Yeochari tidal flat sediments, bioaugmentation cultures, PCE, lactate in various combinations. 148

Figure 5.2. Microcosm setups using Third Creek sediments, KB-1 culture, PCE, lactate in combinations. 149

Figure 5.3. Liquid culture setups to examine salinity effects on consortium KB-1 150

Figure 5.4. Dechlorination of chlorinated ethenes in Third Creek (TC) sediment microcosms. (A) TC1 (TC+PCE+lactate), (B) TC2 (3% salinity+TC+PCE+lactate), (C)... 155

Figure 5.5. Dechlorination of chlorinated ethenes by consortium KB-1 (i.e., no addition of NaCl) (A) KS1 (KB-1+PCE+lactate), (B) KS2 (KB-1+VC+H₂+acetate). 156

Figure 5.6. Quantification of bacterial 16S rRNA genes, Dehalococcoides mccartyi 16S rRNA genes and reductive dehalogenase genes (tceA, bvcA, vcrA). The blank boxes represent the amount of inoculated genes. The dotted lines around tceA and vcrA genes indicate that the genes were detectable but not quantifiable. 158

Figure 5.7. Bacterial community structure of YT1 at genus level. The duplicate samples yielded 1,948 and 1,829 sequences, respectively after filtering and chimera removal. The relative abundances indicated on the graph are the mean value of the duplicates. 160

Figure 5.8. Distribution of 16S rRNA gene sequences obtained from Titanium 454 pyrosequencing. YT1 Dehalococcoides cluster belonged to the Pinellas group, while other major populatoins (YT1 Clostridiisalibacter and YT1 Pelobacter cluster) were... 161

Figure 5.9. Phylogenetic analysis of Dehalococcoides populations detected in the tidal flat sediments or tidal flat amended microcosms (YT3) (■), KB-1 culture or KB-1 inoculated microcosms (YT1) (▲), WJ culture or WJ inoculated microcosms (YT7)... 162

Figure 5.10. Two VC dehalogenase genes ((A) bvcA and (B) vcrA) obtained from consortium KB-1 (KB-1), KB-1 inoculated microcosms (YT1), culture (WJ), and WJ inoculated microcosms (YT7).... 164

Figure 5.11. Dechlorination of chlorinated ethenes (A) in YT1 (B) YT7 transfer cultures. 165

Figure 5.12. Dechlorination of PCE in the 3rd transfer of culture YT1.(이미지참조) 166